Journal articles: 'Nalm6 cells'

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Ramírez Saldaña, Maricela, Miguel Aguilar Santelises, Martha Moreno Lafont, Leopoldo Santos Argumedo, Vladimir Paredes Cervantes, and Rubén López Santiago. "Human B cells are targets for Brucella abortus infection. (P3130)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 186.12. http://dx.doi.org/10.4049/jimmunol.190.supp.186.12.

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Abstract It has been demonstrated that Brucella abortus is capable to multiply into professional antigen presenting cells such as macrophages and dendritic cells. However, there are a few reports about Brucella infection into B cells. In this work, we evaluated the ability of B. abortus 2308 for infect the human B cell line, NALM6 R. B cells cultured in RPMI medium supplied with L-glutamine and 10% of fetal calf serum were infected with several MOIs of Brucella abortus 2308 transformed by electroporation with the pBBR1MCS-2-gfp-mut3 plasmid to evaluate the entry of green fluorescent Brucella. The percentage of cells containing bacteria was evaluated by flow cytometry at three time points. To evaluate whether Brucella was on the surface or into the B cells, kinetics of infection was performed and analyzed by transmission electron microscopy. Association of Brucella abortus 2308-gfp-mut3 with NALM6 R cells increased in a dose-time dependent manner to reach a maximum of 50% of NALM6 R infected cells. Electronic microscopy revealed that NALM 6R B cells produce filopodia over B. abortus. Once inside, the bacteria resided into small vesicles. Apparently bacteria underwent some bactericidal mechanism, because the electron microscopy showed bacteria degraded into vesicles. It was not observed evidence of replication of bacteria inside B cells. Brucella abortus 2308 is capable to interact with NALM6 R membrane, to induce filopodia formation and to entry into B cells inside vesicles.

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Tang, Yongmin, Lixia Li, Di Wang, Hongqiang Shen, Baiqin Qian, Chunfang Luo, and Haizhong Zhang. "Study on the Preparation of the Immunotoxin 2E8-NCTD and Its Targeting Killing Effect In Vitro." Blood 110, no. 11 (November 16, 2007): 4186. http://dx.doi.org/10.1182/blood.v110.11.4186.4186.

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Abstract Objective: Monoclonal antibody (mAb) conjugated with certain toxin to generate immunotoxin bears an important and promising new therapy for patients with hematopoietic malignancies. However, most toxic moieties conjugated to antibody proteins reported were toxic proteins which presented immunogenicity to patients capable of producing anti-toxin antibody. Norcantharidin (NCTD) is a small molecule of toxin derived from a Chinese medicine Mylabris phalerata Pallas. The chemical name of this agent is 7-Oxabicyclo(2,2,1) heptane-2,3-dicarboxylic anhydride with a molecular weight of 168.15 CAS. It does not have the immunogenicity to human body so that it bears a promising potential for development of new targeting drug. In this study, a new clone of self-made anti-CD19 mAb named ZCH-4-2E8 conjugated with NCTD was used to investigate its targeting efficacy against CD19+ lymphoid malignant Nalm-6 cells in vitro in order to provide the experimental fundamentals for the further development of this new targeting agent. Methods: 2E8 monoclonal antibody was prepared from mouse ascites and purified by gel chromatography. The purity of the antibody protein was checked by SDS-PAGE assay. Immunotoxin 2E8-NCTD was successfully generated through conjugating CD19 mAb protein and Norcantharidin by the activated ester method. The binding activity of the immunoconjugate (2E8-NCTD) to CD19 antigens on cell surface and the expression levels of CD19 antigens on Nalm-6 and K562 cells were examined by flow cytometery. Comparisons of the inhibitory effects among PBS, purified 2E8 antibody, Norcantharidin and immunotoxin 2E8-NCTD groups on cell growth of either Nalm6 cells or K562 cells were made. Results: The purity of the purified 2E8 antibody was more than 99% demonstrated by SDS-PAGE assay. 2E8 antibody in the supernatant reacted with 99.34% of Nalm6 cells, while only 0.98% of K562 cells were reacted with this antibody. The new generated immunotoxin (2E8-NCTD) had a positive rate of 99.90% on Nalm6 cells with little reduction of binding activity. From the in vitro study, both 2E8-NCTD and Norcantharidin were shown to have significant inhibitory effects on the growth of CD19+Nalm-6 cells after 96 h of culture with inhibitory rates of 71.25% and 97.62%, respectively (P < 0.001), while the purified 2E8 antibody (inhibitory rate of 30.29, P > 0.05) didn’t show any significant influences on the growth of Nalm6 cells as compared to that of negative control. No significant inhibitory effects were identified among immunotoxin 2E8-NCTD and 2E8 antibody as compared to that of control group on CD19-K562 cells with inhibitory rates of 15.41% and 4.17%, respectively, P > 0.05, indicating that a significant targeting effect of the 2E8-NCTD against Nalm6 cells was noticed. Conclusions: The immunotoxin 2E8-NCTD was successfully synthesized by activated ester method with an excellent targeting killing efficacy on CD19+ Nalm-6 leukemia cells in vitro, which lays the experimental fundamentals for the further development of this new targeting agent.

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Iwasa, Masaki, Yasuo Miura, Aya Fujishiro, Sumie Fujii, Noriko Sugino, Satoshi Yoshioka, Akihiro Tamura, et al. "Bortezomib Attenuates Adhesion of B Cell Precursor Acute Lymphoblastic Lleukemia Cells to Bone Marrow Mesenchymal Stromal/Stem Cells Via Regulating SPARC Expression." Blood 126, no. 23 (December 3, 2015): 786. http://dx.doi.org/10.1182/blood.v126.23.786.786.

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The prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is poor. Many cases in complete remission experience relapse of leukemia despite intensive chemotherapy or hematopoietic stem cell transplantation. This clinical observation suggests that minimal residual disease (MRD) still exists after these intensive therapies. Cell adhesion-mediated drug resistance (CAM-DR) is one of the mechanisms to support MRD in the bone marrow microenvironment (Clin Cancer Res 14:9, 2008). Mesenchymal stromal/stem cells (MSCs) are a cellular component of bone marrow (BM) and maintain physiological precursor B lymphopoiesis (Nature Rev Immunol 6:107, 2006). We investigated our hypothesis that survival of BCP-ALL cells is supported by their direct adhesion to BM-MSCs in BM microenvironment. First, we confirmed that BCP-ALL cells exhibited their drug resistant phenotype through adhesion to human BM-MSCs using human BCP-ALL cell line, Nalm6. We isolated human BM-MSCs from normal bone marrow samples (AllCells, Emeryville, CA). When Nalm6 cells were co-cultured with human BM-MSCs, they were prone to adhere to BM-MSCs. Nalm6 adhered to BM-MSCs (Nalm6/ad) were more resistant to the treatment with various anti-cancer drugs including doxorubicin than Nalm6 in suspension (Nalm6/su). Immunoblot analysis showed that expression level of anti-apoptotic protein Bcl-2 and phosphorylation level of pro-survival kinase Akt are higher in Nalm6/ad compared with those in Nalm6/su. In cell cycle analysis using Ki67/PI fluorescence-activated cell sorter (FACS) assay, the percentage of populations in G0 phase and S/G2/M phase was higher in Nalm6/ad compared with that in Nalm6/su, which indicated the increase of MRD and the high proliferation of leukemic cells in adhesive population, respectively. Therefore, we considered that detachment of Nalm6 from BM-MSCs is important to restore chemosensitivity of BCP-ALL cells and to eliminate these cells. Accordingly, we screened drugs that are capable of disrupting the adhesion of Nalm6 to BM-MSCs, and found that several proteasome inhibitors had a such activity. BM-MSCs were treated with bortezomib (10nM for 24h or 100nM for 1h), carfilzomib (3 nM for 24 h or 100 nM for 1 h) or oprozomib (10 nM for 24 h or 300 nM for 4 h), and then washed with PBS and co-cultured with Nalm6 cells. We confirmed that the number of BM-MSCs is not affected with these drugs irrespective of their concentration and incubation time by MTT assay. Co-cultured cells were divided into suspension cells and adherent cells. Each population was stained with monoclonal antibodies against CD19 and CD90 to detect Nalm6 and BM-MSCs, respectively, and analyzed by FACS. When BM-MSCs were pre-treated with bortezomib, carfilzomib or oprozomib, the number of Nalm6/ad was significantly decreased, and inversely, the number of Nalm6/su was increased, as compared to that of co-cultures with untreated BM-MSCs. Intriguingly, BM-MSCs treated with bortezomib showed aberrant expression of secreted protein acidic and rich in cysteine (SPARC). Immunoblot analysis showed that SPARC expression of BM-MSCs was transiently increased after bortezomib treatment. In co-cultures with BM-MSCs transfected with siRNA targeting SPARC, the number of Nalm6/ad was increased. In addition, the number of Nalm6 adhered to BM-MSCs pre-treated with recombinant human SPARC was lower than that adhered to control non-treated BM-MSCs. Therefore, SPARC showed anti-adhesive property and was one of the molecules associated with adhesion of BM-MSCs to Nalm6. Finally, we tested whether bortezomib shows therapeutic effects in vivo. Bortezomib administration supported survival of BCP-ALL xenograft model mice utilizing Nalm6, which was concomitant with the decrease in leukemia cells in the femur and in the size of spleen, as compared to those in non-treated control mice. In summary, Nalm6 cells that adhered to BM-MSCs contributed to construction of chemoresistant population similar to MRD. Here, we found that this population could be detached by treatment of BM-MSCs with bortezomib through transient increase in their SPARC expression. Our findings suggest that bortezomib or other proteasome inhibitors might be promising drugs to treat BCP-ALL through attenuating the adhesion of leukemic cells to BM-MSCs, and shed new insight into a therapeutic strategy in BCP-ALL by targeting BM-MSCs. Disclosures No relevant conflicts of interest to declare.

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Hermanova, Ivana, Karel Valis, Karel Fiser, Hana Nuskova, Jan Trka, and Julia Starkova. "L-Asparaginase Strongly Affects Bioenergetics in Leukemic Cells." Blood 120, no. 21 (November 16, 2012): 779. http://dx.doi.org/10.1182/blood.v120.21.779.779.

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Abstract Abstract 779 L-asparaginase (L-asp) is an important component of childhood acute lymphoblastic leukemia (ALL) therapy. Its cytotoxic effect is based on the depletion of extracellular asparagine and glutamine. Leukemic cells are sensitive to this depletion due to the lower activity of asparagine synthetase compared to healthy cells. However, the mechanism of resistance development remains unclear. The aim of this study was to obtain further insights into the mechanisms underlying the cytotoxic effect of L-asp. We used two models: resistant preB ALL leukemic cells derived from the REH (TEL/AML1[+]; very sensitive) and NALM6 (TEL/PDGFRB[+]; medium sensitive) cell lines by long-term incubation with L-asp. As a second model we used REH and NALM6 incubated with L-asp for 24 hrs, mimicking acute phase of treatment. We performed GEP of REH, res-REH, NALM6 and res-NALM6. There was an overlap of 30 genes changed in both cases. Applying the pathway analysis we found that L-asp influences the regulation of lipid metabolism and apoptosis regulated by mitochondrial proteins. Next we merged our data with GEP data published by Holleman et al. (NEJM, 2004) of ALL patients′ samples sensitive/resistant to L-Asp. Two pathways, one regulating protein translation, the other metabolism, scored very highly. Next to glucose, glutamine is the other major cellular energy source, it is also important for activation of PI3K/Akt/mTOR pathway - the key regulator of translation. Since L-asp also depletes glutamine, we focused on the effect of L-asp treatment on bioenergetics and translation in leukemic cells. To confirm the effect of L-asp on PI3K/Akt/mTOR pathway we measured the amount of Akt protein and P70S6K/p-P70S6K (downstream target of mTOR) in REH and NALM6 treated for 24 hours with L-asp. Akt and p-P70S6K expression fell in both cell lines. Next we were interested if PI3K/mTOR inhibition affects the sensitivity of resistant cell lines. We measured the cytostatic effect of co-treatment: L-asp with mTORC1 inhibitor (rapamycin); with dual inhibitor of mTORC1 and PI3K (LY294002); and with specific inhibitor of PI3K (PX866) on REH, res-REH, NALM6 and res-NALM6. The MTS assay proved the L-asp/rapamycin combination the most effective. It strongly diminished viability of all cell lines, even in those resistant to L-asp. c-Myc as an activator of glutamine catabolism showed a significant decrease in REH and NALM6 after incubation with L-asp. In addition, c-Myc was also decreased in res-REH compared with REH. Importantly, c-Myc activates glycolysis which is a main energy generator in cancer cells rather than oxidative phosphorylation (OXPHOS). We showed diminishement of Glucose transporter type 1 protein expression and 2.5-times reduced level of lactate, the product of cancer cell glycolysis, in media after L-asp exposure. Next we measured cell respiration that mirrors oxidative fosforylation (OXPHOS). REH and NALM6 showed increased capacity of respiratory chain after L-asp exposure, which was observed as an increase of endogenous respiration in uncoupled state. REH boosted the capacity of the respiratory chain from 163 to 236 pmol O2/s/mg (p<0.01). NALM6 have increased the capacity of the respiratory chain from 78 to 155 pmol O2/s/mg (p<0.01). Interestingly, REH has a significantly higher capacity of respiratory chain compared with NALM6 and a lower ability to regulate it. Moreover, res-REH had elevated the level of the endogenous oxygen consumption whereas res-NALM6 levels did not change. Our results indicate that leukemic cells, which normally depend on glycolysis for energy generation rather than OXPHOS, are able to swap these two mechanisms of energy generation under amino acid deprivation stress. The data show that L-asp inhibits mTORC1 and strongly affects bioenergetics pathways of leukemic cells. It decreased c-Myc-regulated glycolysis and increased OXPHOS. We believe that these metabolic changes are mainly caused by the effort of the cells to mobilize another mitochondrial pathway as Krebs cycle and β-oxidation, with the aim to supply depleted amino acids. We conclude from these data that resistance of the cells is caused by better biochemical adaptability to the nutrient deprived environment. Support: GAUK 92710, IGA NT1249, UNCE204012 Disclosures: No relevant conflicts of interest to declare.

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Gu, Yan, Chunhua Song, Sinisa Dovat, Qinglong Guo, Qinyu Ge, and Zheng Ge. "Oncogenesis of CRLF2 Overexpression and Effect of JAK2 Inhibitor in CRLF2 Overexpressed B-Cell Acute Lymphoblastic Leukemia." Blood 134, Supplement_1 (November 13, 2019): 2757. http://dx.doi.org/10.1182/blood-2019-125259.

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Backgroud: Cytokine receptor-like factor 2 (CRLF2) plays an important role in the development of normal B lymphocytes, which can mediate the proliferation of early B cells. However, the diect oncogenic effect of CRLF2 overexpression in acute lymhpoblastic leukemia (ALL) is far yet to be clarified. Here, we explored the effect of CRLF2 overexpression on cell proliferation and the effect of the novel JAK2 inhibitor on B-ALL cells with CRLF2 overexpression. Methods: The 83 patients with newly-diagnosed ALL (56 B-cell and 27 T-cell ALL; range from 14 to 77 years old) between June 2008 and June 2016 were studied at Zhongda Hospital Southeast University. The 21 normal bone marrow subjects were enrolled as controls. The qPCR method is developed for detection CRLF2 expression and the CRLF2 overexpression was determined with a cutoff value more than the highest sample of normal bone marrow control. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using uni- and multivariate Cox model. Event-free survival (EFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by log-rank test. CRLF2 F232C gain-of-function mutant which we previously reported or CRLF2 were expressed in Nalm6 and 697 B-ALL cells with lentiviral transduction. WST-1 cell proliferation assay and in vitro clonogenic assay were performed upon JAK2 inhibitor (BBT594) treatment. Nalm6-CRLF2-luc, Nalm6-F232C-luc, and Nalm6-vector-luc cells were injected via tail vein into the NSG mice. The leukemia engraftment was monitored once a week by living imaging. Results: The expression of CRLF2 in patietns with ALL was significantly higher than the normal control (P<0.0001). Patients with CRLF2 overexpression had a significantly higher WBC count (53*10^9/L vs. 29.5*10^9/L, P=0.041). Survival analysis showed that the patients with CRLF2 overexpression had a worse EFS and OS, the differences were statistically significant (11 months vs. 26 months, P=0.043 and 15 months vs. 32 months, P=0.015). Also, the CRLF2 expression is determined with flow cytometry after staining with FITC-CRLF2 antibody in 28 samples. The correlation analysis was performed on the CRLF2 expression detected by qPCR and flow cytometry, respectively. A significant positive correlation of the two methods was observed(r=0.957, P<0.0001). These data not only indicate that CRLF2 overexpression is a marker of poor outcome, but also reveal the qPCR might be a simple and quick method for screening CRLF2 overexpression in the clinic compared to flow cytometry which is commonly used. We further found that expression of CRLF2 or CRLF2 F232C mutant into Nalm6 and 697 B-ALL cells dramatically increase the CRLF2 mRNA level, which is 69 times than vector-only control. Moreover, CRLF2 or CRLF2 F232C significantly promotes the cell proliferation of Nalm6 and 697 cells compared to vector only (P<0.001). In addition, JAK2 inhibitor (BBT594) treatment showed the significant dose-dependent cell proliferation arrest and clonogenic inhibition in CRLF2 or CRLF2 F232C overexpressed Nalm6 and 697 cells compared to vector-only control. Furthermore, in vivo we observed the 5-fold higher signal intensity of leukemia engraftment in the mice injected with Nalm6-CRLF2-luc or Nalm6-F232C-luc compared to that of Nalm6-vector-luc control 1-3 weeks after the injection(P<0.001). The Nalm6-CRLF2-luc and Nalm6-F232C-luc infiltrations were observed in bone marrow, central nervous system, liver and spleen of the mice. Conclusion: We showed that CRLF2 overexpression could enhance the proliferation and infiltration of human B-ALL cells, and for the first time indicated that JAK2 inhibitor could suppress the cell proliferation and clonogenesis of the CRLF2 overexpressed B-ALL cells. Our data provide direct evidence of the oncogenic role of CRLF2 overexpression and the new therapeutic potential for targeting CRLF2 overexpressed B-ALL with JAK2 inhibitor. Disclosures No relevant conflicts of interest to declare.

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Barredo, Julio C., Sanja Altman-Hamandzic, and Guy J. Leclerc. "Differences in Folylpoly-γ-Glutamate Synthetase (FPGS) Expression in Childhood ALL Result from Cell Lineage of Origin and Presence of Non-Random Translocations." Blood 104, no. 11 (November 16, 2004): 2087. http://dx.doi.org/10.1182/blood.v104.11.2087.2087.

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Abstract Methotrexate (MTX) is a universal component of chidlhood ALL therapies and its conversion to long chain polyglutamates (PG) by folylpoly-γ-glutamate synthetase (FPGS) is essential for its antileukemic acitivity. Expression of FPGS appears to be controlled by tissue/lineage specific and proliferation-dependent mechanisms. Levels of FPGS mRNA, protein, and enzyme activity are 2-3 fold higher in B-precursor (Bp) ALL cells when compared to T-lineage ALL, and these differences correlate with intracellular accumulation of long chain MTX-PG and lymphoblast sensitivity to MTX. To characterize these lineage differences in FPGS expression between B and T lymphoblasts we examined its mRNA expression in Nalm6 (Bp-ALL) and CCRF-CEM (T-ALL) cells during cellular growth and cell cycle checkpoints. During early exponential growth (24 hrs), FPGS expression was 6-fold higher in Nalm6 when compared to CCRF-CEM but decreased significantly after 72 hrs while it was unchanged in CCRF-CEM cells. During G1/G0 phase we found that FPGS expression was 15-fold higher in Nalm6 when compared to CCRF-CEM cells. Taken together, these data suggest that during proliferation and cell cycle progression FPGS gene expression is regulated differently in Bp-ALL and T-ALL cells. To determine whether this lineage-specific regulation occurs at the transcriptional level we performed nuclear run-on assays. We found that FPGS mRNA transcription initiation rate was 1.7-fold higher in Nalm6 when compared to CCRF-CEM cells, indicating that differences in promoter regulation lead to the observed lineage differences in FPGS expression in Bp- vs. T-ALL. We then used a methylation specific PCR assay to investigate the methylation status of the FPGS proximal promoter region from both Nalm6 and CCRF-CEM cells. Our data indicate that the FPGS proximal promoter region is unmethylated in both cell lines and therefore can not explain the observed lineage-specific differences. 5′-RACE experiments demonstrated that Nalm6 and CCRF-CEM have the same FPGS transcriptional start sites, but a reporter gene assay indicated that the minimal promoter region that directs FPGS transcription in CCRF-CEM cells is insufficient to drive FPGS mRNA transcription in NALM6 cells. In order to identify potential regulatory regions directing FPGS transcription in Bp-lymphoblasts, we used DNaseI hypersensitivity assays. We identified a hypersensitive region located 8.5 kbp upstream to exon 1 in Nalm6 cells suggesting that tissue-specific regulatory elements responsible for lineage-specific FPGS expression in Bp-ALL cells may be localized within this region. Finally, we detected reduced levels of FPGS mRNA expression in RCH-ACV (Bp-ALL, t(1:19)/E2A-PBX1) and REH (Bp-ALL, t(12:21)/TEL-AML1) cells expressing chromosomal translocated fusions when compared to control (Nalm6). To characterize the molecular basis of the E2A-PBX1 and TEL-AML1 interactions with FPGS mRNA expression in Bp lymphoblasts we used antisense and RNAi technology to downregulate these two genetic fusions. Our data lead us to hypothesize that in addition to lineage-specific regulatory differences in FPGS expression, molecular mechanisms associated with non-random translocations may alter FPGS mRNA expression and influence MTX sensitivity in Bp-ALL lymphoblasts.

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Borges, Christopher M., Kevin Wasko, Jared M. Nasser, Kelly Donahue, Amanda Pfautz, Lincy P. Antony, Glenn Leary, Steven Sexton, Richard A. Morgan, and Karrie K. Wong. "Preclinical Development of Edit-201, a Multigene Edited Healthy Donor NK Cell with Enhanced Anti-Tumor Function and Superior Serial Killing Activity in an Immunosuppressive Environment." Blood 136, Supplement 1 (November 5, 2020): 33. http://dx.doi.org/10.1182/blood-2020-139988.

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Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For example, KIR mismatching enables allogenic NK cells to respond to MHC positive tumors in a similar manner to MHC negative tumors, making allogeneic NK cell therapy a promising approach for broad oncology indications. Accordingly, allogenic human HD-NK cells, including gene-modified cells, have demonstrated an impressive safety and efficacy profile when administered to patients with advanced hematologic malignancies. However, effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β. To this end, we developed a next-generation allogeneic NK cell therapy using CRISPR-Cas12a gene editing to enhance NK cell function through knockout of the genes CISH and TGFBR2. Both single and simultaneous targeting (DKO) of TGFBR2 and CISH in NK cells using CRISPR-Cas12a produced in/dels at both targets in greater than 80% of NK cells, with greater than 90% of edited NK cells viable at 72 hours post-editing. Importantly, we find that DKO NK cells do not phosphorylate the SMAD2/3 protein downstream of the TGF-b receptor complex and demonstrate increased phosphorylation of pSTAT3 and pSTAT5 upon IL-15 stimulation, consistent with protein level knockout of TGFBR2 and CISH. To determine whether DKO NK cells exhibited superior function relative to control NK cells, we first measured the ability of DKO NK cells to kill Nalm6 cells (adult B cell ALL) relative to unedited control NK cells. We find that in the presence of exogenous TGF-b, DKO NK cells demonstrate improved cytotoxicity against Nalm6 tumor targets by delaying tumor re-growth in comparison to control NK cells. To better characterize the ability of DKO NK cells to kill tumor cells, we developed an in vitro serial killing assay. In this long-duration assay, up to 30 days, control and DKO NK cells (grown in the presence of IL-15) were challenged every 48 hours with a new bolus of Nalm6 tumor targets. Both DKO and unedited NK cells control Nalm6 target cell growth for greater than 18 days (9 additions of new Nalm6 target cells), demonstrating a surprising ability for the same NK cells to serially kill new Nalm6 target cells for a prolonged period of time in vitro. We find that DKO NK cells produce higher levels of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay, suggesting that DKO NK cells can continue to produce these inflammatory cytokines even after serial killing. As many tumors, including hematologic malignancies, have high concentrations of TGF-β in their microenvironments, we next tested the ability of DKO NK cells to control the growth of Nalm6 cells in our serial killing assay in the presence of TGF-b. 10ng/mL TGF-β was added at the start of the assay as well as at each addition of new Nalm6 target cells. We observed that control NK cells fail to restrict Nalm6 target cell growth beyond 4 days (after 1 addition of new Nalm6 target cells) whereas DKO NK cells control Nalm6 target cell growth for greater than 18 days (after 9 additions of new Nalm6 target cells). Similar to the serial killing assay without TGF-b, we find that DKO NK cells produce higher concentrations of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay. Broadening our repertoire of target cells beyond B cell malignancies is now in progress, including the AML-like cell lines HL-60 and THP-1, the multiple myeloma cell line RPMI 8226, and various solid tumor targets. In summary, using CRISPR-Cas12a we demonstrated highly efficient gene editing of primary human NK cells at two unique targets designed to augment NK cell anti-tumor activity across a variety of malignancies. Most significantly, we demonstrate sustained anti-tumor serial-killing activity in the presence of the potent immunosuppressive cytokine TGF-β. Together, the increased overall effector function of CISH/TGFBR2 DKO primary human NK cells and their ability to serial kill, support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine, termed EDIT-201, is being advanced to clinical study. Disclosures Borges: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Donahue:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pfautz:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Leary:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

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Wang, Lingyan, Yue Zhang, Eden Anderson, Rajesh Kumar, Adam J. Lamble, and Rimas J. Orentas. "CD22 CAR-T Induces Both CD19 and CD22 Surface Down-Modulation: Defining a Mechanism of Generalized Immune Evasion and the Effects of Epigenetic Modifiers." Blood 136, Supplement 1 (November 5, 2020): 22–23. http://dx.doi.org/10.1182/blood-2020-134930.

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The success of chimeric antigen receptor modified T lymphocyte (CAR-T cell) therapy for pediatric and adult leukemia and lymphoma has been impressive, but far from universal. Even for the most responsive disease, pediatric pre-B-ALL, one-year remission rates are 50% or less. This brings fundamental questions as to how leukemia resists CAR-T activity front and center. CD19- and CD22-specific CAR-T escape mechanisms appear to differ. For CD19, antigen loss and RNA-splicing variants are common. For CD22, decreasing the number of target molecules on the surface of the leukemia appears to be a primary means of escape (Fry, et al., 2018). Murine models have demonstrated that bryostatin increases CD22 density and improves CAR-T activity (Ramakrishna, et al., 2019). We explored the activity of bryostatin and four FDA-approved epigenetic modifiers (panobinostat, vorinostat, 5-azacytidine, ATRA) for the ability to impact CD19 and CD22 expression in Raji, NALM6, and REH cell lines. Dose ranges tested were not toxic. For CD22: a) bryostatin, increased expression for all lines (surface molecules expressed per cell), b) 5-azacytidine, increased expression only in NALM6, and c) ATRA, increased expression only in Raji. For CD19: a) only bryostatin, and only in NALM6, increased expression. When normal B cells from peripheral blood were tested (n=3) bryostatin did not increase the number of CD19 or CD22 molecules on the cell surface. When cytolysis assays with CD22 CAR-T were carried out, bryostatin potentiated NALM6 killing, but paradoxically inhibited killing of Raji cells. To see if this effect was due to differential target molecule expression, we quantified the number of target (CD22) and non-target (CD19) molecules on the surface of each leukemia line during CTL assays. At high effector to target cell ratios, (E:T 1:1 or greater), culturing of NALM6 or Raji with CD22-CAR-T resulted in a marked reduction in both CD19 and CD22 surface expression at 24 hours, indicating a general mechanism of immune evasion. Even though CD19 and CD22 were elevated in bryostatin-treated NALM6, and CD19 was elevated in bryostatin-treated Raji, CD22 CAR-T activity still decreased target antigen expression 40% to greater than 100%. To see if this effect was due to permanent clonal selection, CAR-T were removed and purified leukemia cells re-cultured on their own. For Raji leukemia cells surviving CD22 CAR-T, the number of CD19 molecules per cell recovered within 24 hours. Full recovery was not seen for CD22, unless Raji cells had also been treated with bryostatin during the CTL assay. Our data highlights that unless bryostatin is included, CAR-T exposure induces a durable down-modulation of CD22 levels on Raji cells. We are currently defining epigenetic control of CD19 and CD22 expression upon exposure to CD22 CAR-T, bryostatin and other modifiers. These findings still do not explain why Raji cell killing is not potentiated by bryostatin, while NALM-6 is. The exposure of leukemia lines to epigenetic modifiers may also induce the expression of other molecules on the cell surface that either potentiate or inhibit CAR-T mediated killing. Preliminary data support that hypothesis that bryostatin may not only modify CD19 and CD22 expression, it may also induce both negative checkpoint molecule and positive T cell activating ligand expression on the leukemia cell surface. Thus, CAR-T induce both general and target-antigen specific changes in leukemia, depending in part on the leukemia subtype analyzed. Bryostatin increased CD22 expression in leukemia cell lines but not in normal B cells, indicating that control of CD22 levels on the cell surface is altered by leukemic transformation. A 24-hour exposure to CD22 CAR-T can induce dramatic transient changes in CD19 and CD22 expression levels, and durable changes in CD22 expression in Raji cells that can be surmounted by the inclusion of bryostatin. The difference in Raji and NALM6 sensitization to killing may be due to the origin of these cells as Burkitt lymphoma (Raji) is more differentiated with respect to B cell lineage than pre-B ALL (NALM6). We can conclude that analysis of CAR-T target ligand number is not sufficient to predict the efficacy of CAR-T therapy. We have demonstrated that mechanisms beyond alteration in target antigen number on the cell surface, such as the induction of positive and negative T cell ligands, must also be considered in the context of the leukemia types targeted by CAR-T therapy. Figure Disclosures Orentas: Lentigen Technology, a Miltenyi Biotec Company: Research Funding.

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Barredo, Julio C., Tingting Kinser, and Guy J. Leclerc. "TEL/AML1 and E2A/PBX1 Tranlsocations Lead to Altered Folylpoly-γ-Glutamate Synthetase (FPGS) Expression in ALL." Blood 106, no. 11 (November 16, 2005): 539. http://dx.doi.org/10.1182/blood.v106.11.539.539.

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Abstract Expression of the human folylpoly-γ-glutamate synthetase (FPGS) gene is controlled by tissue/lineage specific and proliferation-dependent mechanisms. Levels of FPGS mRNA, protein, and enzyme activity are 2–3 fold higher in B-precursor (Bp) ALL cell lines and primary cells when compared to T-lineage ALL. These differences correlate with intracellular accumulation of long chain methotrexate (MTX) polyglutamates (PG), and more important with clinical sensitivity to MTX. However, significant heterogeneity of FPGS expression exists within hematopoietic cells of the same lineage, suggesting additional factors influence FPGS expression in Bp- and T-ALL. cDNA microarray data was analyzed for expression of folate-related genes (DHFR, FPGS, RFC, MTHFR, and γ-GGH) in six prognostic leukemia subtypes (T-ALL, hyperdiploid (> 50 chromosomes), BCR-ABL, E2A-PBX1, TEL-AML1, and MLL) (Yeoh et al, Cancer Cell, 2002). It was found that only the FPGS gene exhibited significant heterogeneity of expression compared the others analyzed. Therefore, we investigated the effect of distinct non-random genomic translocations on FPGS gene expression to determine whether gene fusions may be responsible for the heterogeneity of FPGS expression in ALL cells. Using quantitative fluorescence real-time RT-PCR, we first detected reduced levels of FPGS mRNA expression in the RCH-ACV (Bp-ALL, t(1:19)/E2A-PBX1) and REH (Bp-ALL, t(12:21)/TEL-AML1) human leukemia cell lines expressing non-random chromosomal gene fusions when compared to control (NALM6). To determine if expression of fusion proteins encoded by the E2A-PBX1 and TEL-AML1 translocations lead to decrease FPGS promoter activity and mRNA expression, we used a FPGS-luciferase reporter gene assay. The CHO FPGS null mutant cell line AUXB1, was triple transfected with constructs expressing luciferase (pFPGS-luc), β-galactosidase (pCMVβ), and E2A-PBX1 (pCMVKJ7) or TEL-AML1 (pTEL-AML1) and assayed for luciferase and β-galactosidase activity. Expression of E2A-PBX1 and TEL-AML1, determined by quantitative RT-PCR and Western blot, resulted in 45% and 30% decrease in the level of normalized FPGS-luciferase activity, respectively. These data indicate that FPGS gene transcription is decreased by both translocations, either by direct binding to regulatory elements or indirectly via downstream pathways influenced by E2A-PBX1 and TEL-AML1. Therefore, to confirm these findings and to identify genes or pathways in Bp-ALL regulated by E2A-PBX1 and TEL-AML1 gene fusions, we constructed NALM6 stable cell lines expressing E2A-PBX1 or TEL-AML1 using vectors pCI-neo/E2A-PBX1 and pCI-neo/TEL-AML1. In both NALM6 stable cell lines the level of FPGS mRNA expression was decreased by more than 50% compared to wild type (NALM6). Total RNA was then extracted from NALM6, NALM6/E2A-PBX1, and NALM6/TEL-AML1 cells and analyzed by cDNA microarray. Gene expression profile analysis from existing databases and our NALM6/E2A-PBX1 and NALM6/TEL-AML1 stable cell lines identified genes differentially expressed compared to NALM6 control. These included transcription factors (CSDA, AKAP12, ARL2, PBX1, TEL), cell cycle regulators (CDK2P1), signal transduction (PIK3C3), proliferation and cell migration (PTPRK), zinc finger binding protein (PHF2), among others. This study is the first to demonstrate the causal relationship between the presence of E2A-PBX1 and TEL-AML1 gene fusions and altered FPGS expression and may explain the differential sensitivity to MTX exhibited by these childhood ALL phenotypes.

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Tukaramrao, Diwakar Bastihalli, Arati Sharma, Dhimant Desai, and Sinisa Dovat. "Abstract 6052: Metabolic consequences of casein kinase 2α inhibition in lymphoid leukemia." Cancer Research 83, no. 7_Supplement (April 4, 2023): 6052. http://dx.doi.org/10.1158/1538-7445.am2023-6052.

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Abstract Overexpression of casein kinase 2α (CK2α) is a common feature in lymphoid leukemias. Constitutively active CK2α can disable transcriptional activity of lymphoid transcription factors like IKAROS that act as metabolic gatekeeper and limit the energy supply needed for oncogenic transformation of B cells. Our studies have shown that pharmacological inhibition of CK2α can restore the transcriptional activity of IKAROS and ablate leukemia. However, the role of CK2α in glucose metabolism has not been fully studied in B and T acute lymphoblastic leukemia (ALL). Therefore, in the present study we explored the metabolic alterations induced by the pharmacological inhibition of CK2α in B and T ALL. We studied the effect of CK2α inhibition using a specific inhibitor - CX4945 on cell proliferation, glucose utilization, lactate production, and intracellular ATP levels using established methods in human B-ALL (NALM6) cell line. Effect of CX4945 on glycolysis was studied using the Seahorse cell analyzer in human B (NALM6 and 697) and T (MOLT4 and 697) ALL cell lines. Metabolomics study was undertaken to analyze differential metabolite profiling in NALM6 cells treated with CX4945 compared to vehicle using LC-MS/MS based methods. Results showed that CX4945 induced apoptotic cell death in ALL cell lines with IC50 concentrations ranging between 4-10 µM. CX4945 treatment significantly affected the glucose consumption in NALM6 cells. Similarly, substantial decrease in intracellular ATP and lactate levels compared to vehicle was recorded. CK2α inhibition significantly decreased the glycolytic activity in B- and T-ALL cell lines. It was observed that glycolytic reserves were significantly decreased in 697 (3-fold), MOLT4 (4-fold), CEM (3-fold) cells incubated with CX4945 in comparison to vehicle. The principal component analysis from metabolomics study showed a clear separation between CX4945 and vehicle-treated NAML6 cells. Sixty-four statistically significant, differentially expressed metabolites were recorded in the study. Analyte classes included TCA cycle intermediates, nucleic acids and their precursors as well as glycolysis intermediates that were significantly affected by CK2α inhibition. In conclusion, our study shows that selective inhibition of CK2α by CX4945 caused energy deficiency and cell death in ALL cell lines. CK2 inhibition targeted the key energy dependent pathway by rendering ALL cells inefficient in utilizing glucose and operating glycolysis for generation of cellular energy. These results offer a new mechanistic understanding of CK2α inhibition mediated ablation of ALL. Citation Format: Diwakar Bastihalli Tukaramrao, Arati Sharma, Dhimant Desai, Sinisa Dovat. Metabolic consequences of casein kinase 2α inhibition in lymphoid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6052.

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Benito, Juliana, Yuexi Shi, Barbara Szymanska, Hernan Carol, Ingrid Bohem, Hongbo Lu, Sergej Konoplev, et al. "Targeting the Leukemia-Associated Hypoxic Microenvironment with Hypoxia-Activated Prodrug PR-104." Blood 116, no. 21 (November 19, 2010): 868. http://dx.doi.org/10.1182/blood.v116.21.868.868.

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Abstract Abstract 868 Interactions between leukemia cells and the bone marrow (BM) microenvironment are known to promote leukemia cell survival and confer resistance to drugs commonly used in the management of this disease. We investigated the protective role of hypoxia in the BM microenvironment. We observed a marked expansion of hypoxic niches in the bone marrow of immunodeficient mice engrafted with the acute lymphoblastic leukemia (ALL) cell line Nalm6 and with primary ALL cells, as detected by the reductive 2-nitroimidazole compound pimonidazole (PIM), which forms stable adducts in hypoxic regions. We further demonstrated induction of the chemokine receptor CXCR4, and of the enzyme carbonic anhydrase 9 (CAIX), both targets of Hypoxia-Inducible Factor 1α (HIF-1a), in hypoxic areas of BM from mice harboring the Nalm6 xenografts. Furthermore, we used a CML blast crisis model to evaluate the time course of hypoxia expansion in the BM. In C57Bl6/J mice engrafted with murine HSCs co-expressing BCR/ABL and Nup98 we observed a time-dependent increase in PIM positive areas which coincided with the presence of GFP positive cells. In line with these findings, HIF-1α was highly expressed in BM biopsies from newly diagnosed ALL patients (n=15) but was significantly reduced when the patients achieved complete remission (CR). Culture under hypoxic conditions (1% O2) conferred resistance of pre-B ALL cells REH, Nalm-6, and of AML OCI-AML3 leukemic cells against several chemotherapeutic agents including vincristine, methotrexate and idarubicin (% of Annexin V(+) cells at 21% vs 1%O2; REH plus 1ng/ml vincristine:88.5+/−2.3 vs 18.1+/−10.3; REH plus 0.25uM etoposide: 86.4 +/−7 vs 14.4+/−8.6; Nalm6 plus 10ng/ml methotrexate: 33.4+/−2.7 vs 4.6+/−1; OCI-AML3 plus 50ng/ml idarubicin: 28.45+/−5 vs 12.76+/−1) . Taken together, these results provide rationale for examining the potential of hypoxia-activated pro-drugs to eliminate leukemia progenitor cells within hypoxic niches. To this end, we tested the hypoxia-activated prodrug PR104, a dinitrobenzamide nitrogen mustard that is reduced to its active metabolites under hypoxic conditions (Patterson et al., Clin Can Res 2007). In vitro, PR-104 induced cell death in three different leukemia cell lines (Nalm6, REH and the AML line Molm13) selectively under hypoxic (pO2 1%) conditions. The anti-leukemic efficacy of PR-104 as a single agent was next examined in several in vivo leukemia models. Administration of PR-104 prolonged survival and decreased leukemia burden of 1) NOD/Scid/IL2Rg-KO (NOG) mice injected with cells from primary refractory FLT3-mutated AML; 2) NOG mice injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL; and 3) NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice injected with Nalm6-luciferase ALL cells. Next, we evaluated anti-tumor effects of PR-104 at clinically relevant doses (200, 100 and 50 mg/kg) and at maximal tolerated dose (550 mg/kg) in two ALL xenograft models (a T-lineage ALL and a B-cell precursor ALL) (Figure 1, studies supported by NCI NO1CM42216 and by PPTP contract NO1-CM91001-03). Compared to vehicle control, PR-104 significantly delayed progression of the T-ALL xenografts at all doses tested and at three of the four doses (550, 200 and 100 mg/kg) of B-cell pre-ALL, which resulted in significantly increased event-free survival (EFS) of mice in the treatment groups. Altogether, these findings strongly suggest that targeting hypoxia is feasible. If successful, this approach may significantly impact leukemia therapy and ultimately improve patient survival. This concept is currently being tested in an ongoing Phase I clinical trial of PR-104 in relapsed/refractory AML patients. Disclosures: Wilson: PROACTA: Equity Ownership. Konopleva:PROACTA: PI on clin trial funded by Proacta.

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Tanaka, Yosuke, Masahito Kawazu, Satoshi Inoue, Shinya Kojima, Ueno Toshihide, Toshiaki Uno, Moe Tamura, et al. "Chromatin Architecture Modulation in B-Cell Acute Lymphoblastic Leukemia Carrying DUX4 Fusions." Blood 134, Supplement_1 (November 13, 2019): 1240. http://dx.doi.org/10.1182/blood-2019-125207.

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B-cell acute lymphoblastic leukemia (B-ALL) carrying DUX4 fusions is a novel cluster of B-ALL. DUX4 fusions are generated from insertions of wild- type (WT) DUX4, mainly into the IGH locus.The translocation replaces the 3′ end of the WT DUX4 coding region with a fragment of IGH or another gene, producing DUX4 out-of-frame fusion proteins devoid of the C terminus of WT DUX4. Usually, WT DUX4 is expressed in germ cells in testis, while its expression is epigenetically repressed in somatic tissues. Recently, it is identified to plays a critical role in transcriptional programs at the cleavage of human fertilized egg. In B-ALL, DUX4-IGH (D-I) is shown to be essential for leukemic transformation; however, little is known about the mechanistic basis. Here in this study, we extensively investigated the biological effects of D-I. First, we assessed the role of D-I using in vitro cell culture assays with human cord blood (CB) CD34+ cells. Introduction of D-I significantly caused retention of the CD34+ cell population compared with the mock vector, even though it failed to preferentially promote differentiation toward B cell lineage in vitro. To analyze the epigenetic and transcription control by D-I, we performed chromatin immunoprecipitation coupled with sequencing (ChIP-seq) using cell lines. In NALM6, a B-ALL cell line carrying D-I, a subset of D-I binding sites is accompanied by H3K4me3 and H3K27ac. We also assessed the histone modification status in Reh cells, a B-ALL cell line without DUX4 fusions, and observed that active histone marks are detected after binding of ectopically expressed D-I. Nevertheless, RNA sequencing of NALM6 and Reh overexpressing D-I showed minimal activation of genes near the D-I binding sites compared with those of NALM6 overexpressing WT DUX4. WT DUX4 is known to preferentially bind and activate repeat elements, especially human endogenous retroviral (HERV) elements in embryonic cells. NALM6 cells overexpressing WT DUX4 showed a drastic increase in the expression of HERV elements, while NALM6 and Reh overexpressing D-I did not. The expression of HERV elements was not altered by D-I in all the genomic regions, and we did not observe increased expression of HERV elements in patient leukemia samples with DUX4 fusions as well. Furthermore, Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) showed that chromatin status was not affected by the binding of D-I at the D-I bound HERV element, indicating that transcriptional and insulating ability of WT DUX4 in these areas are lost in D-I. Next, we performed ATAC-seq using NALM6 cells, comparing the status between pre- and post- D-I knockdown. Genomic areas with decreased ATAC signal after knockdown of D-I are enriched in D-I binding sites, and ATAC signal was increased when we compared the status between pre- and post- induction of D-I in Reh cells. Through the immunoprecipitation of endogenous D-I in NALM6 cells, we identified SWI/SNF complex elements as binding partners of D-I, further highlighting the chromatin opening ability of D-I. Motif analysis of the genomic areas with decreased ATAC signal after knockdown of D-I identified only DUX4 motif as a significant motif, suggesting that D-I is not apparently cooperating with other transcription factors. On the other hand, ATAC signal was increased in substantial genomic areas after knockdown of D-I, and motif analysis identified SPI1, TCF3, and EBF1 motifs. Integrated analysis of transcriptome data also supports the idea that transcription factors related to B cell differentiation are repressed in the presence of D-I, and derepressed after knock down of D-I. Despite the attenuated transcriptional activity, B-ALL carrying DUX4 fusions manifests a characteristic expression pattern. D-I binding sites are not always relevant to the gene areas with increased transcriptions. Therefore, we compared the genomic areas where ATAC signal is raised by D-I, and genes whose expression is affected by D-I. We identified genes with ATAC signal change both in NALM6 cells with D-I knockdown and in Reh cells with D-I induction. We identified D-I binding in some of these genes, and the pharmacological inhibition of one of the genes caused cell death in NALM6 cells in vitro and in vivo, suggesting that this gene is the genuine target of D-I. In summary, our study elucidated the detailed difference of function between WT DUX4 and DUX4-IGH, and demonstrated the ability of DUX4-IGH as a chromatin modulator. Disclosures No relevant conflicts of interest to declare.

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Charkhizadeh, Samira, Mehdi Imani, Nematollah Gheibi, Fateme Shabaani, Akbar Nikpajouh, and Mohammad R. Rezvany. "In Vitro Inhibitory Effect of Recombinant Human Calprotectin on Nalm6 Leukemia Cell Line." Anti-Cancer Agents in Medicinal Chemistry 20, no. 8 (July 24, 2020): 951–62. http://dx.doi.org/10.2174/1871520620666200331101209.

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Background & Purpose: In evaluating new drugs for the treatment of various types of cancer, investigations have been made to discover a variety of anti-tumor compounds with less side effects on normal cells. Investigations have shown that the heterodimers S100A8 and S100A9 inhibit the enzyme casein kinase 2 and then prevent the activation of the E7 oncoprotein. Therefore, the aim of this study was to evaluate the effect of calprotectin as an antitumor compound on the Nalm6 (B cell precursor leukemia cell line). Material & Methods: Transformation of genes encoding S100A8 and S100A9 human, designed in the pQE32 plasmid, was performed by the thermal shock method into E. coli M15 bacteria. After bacterial growth in LB medium, the expression of two S100A8 and S100A9 subunits, the solubility of the protein by SDS-PAGE method was determined. Finally, the S100A8 / A9 complex was equally placed in the microtube. In the next step, the cytotoxic effects of calprotectin produced on the Nalm6 cell line were evaluated using the wst1 test. Then, the apoptosis in these cells was measured using flow cytometry methods with Annexin-V coloration. Results: In the current study, the results showed that the cytotoxic effects of Calprotectin are time and concentration- dependent. Therefore, it can reduce the tumor expression and had a beneficial effect by induced apoptosis in Nalm6 cell line. Conclusion: Calprotectin has an anti-tumor effect on the Nalm6 cell line by increasing apoptosis.

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Karalewitz, Andrew, Natalie Czeryba, Yewei Xing, Derrik Germain, Philip Lapinski, Sheri Barnes, Mark Cameron, et al. "Abstract 2835: Chimeric antigen receptor (CAR) T-cell generation for therapeutic testing in the disseminated NALM6 human B-cell acute lymphoblastic leukemia mouse model." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2835. http://dx.doi.org/10.1158/1538-7445.am2022-2835.

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Abstract Cancer immunotherapies reprogram the patient’s immune system to mount a coordinated response against a malignant target. T cells engineered to express Chimeric Antigen Receptors (CARs) through transduction with a lentiviral vector represent an effective strategy to specifically eliminate cancerous cells from a patient. Currently, five CAR T cell therapies are approved by the FDA for the treatment of hematological malignancies. With the recent clinical and regulatory success of CAR T cell therapies, the next generation of CAR T cells are undergoing preclinical development. Labcorp Drug Development produces CAR T cells to support the development of new preclinical strategies. Here, the CAR T Cell Generation Service is demonstrated using an anti-CD19 CAR T cell as an example. Using peripheral blood mononuclear cells from healthy, human donors as a T-cell source, CAR T cells were produced by lentivirus transduction. Flow cytometry featuring a CAR-specific monoclonal antibody was used to determine transduction efficiency. To assess in vitro activity, co-cultures of T cells and CD19-expressing NALM6 cells were used to measure cytotoxicity. Proinflammatory cytokine production was analyzed using a Meso Scale Discovery V-PLEX assay. For evaluation of in vivo efficacy, a disseminated NALM6-Luc-mCh-Puro human acute B cell lymphoblastic leukemia model in female NSG mice was conducted. After T cells were treated with lentivirus, approximately 25% of total cells were CD3+/CAR+. Greater than 95% of all target NALM6 cells were killed by a high dose of anti-CD19 CAR T cells. In contrast, only 33% of target cells were killed by untransduced (UTD) T cells at the highest concentration tested. Cytotoxicity against the negative control MV-4-11 cells was equal for a high dose of UTD and a high dose of anti-CD19 CAR T cells. When co-cultured with CD19-expressing NALM6 cells, anti-CD19 CAR T cells produced proinflammatory cytokines including IFN-γ. Relative to mock-treated control mice, treatment with anti-CD19 CAR T cells at high, medium, and low doses increased the time of in vivo disease progression by 175%, 125%, and 16%, respectively. Anti-CD19 CAR T cells generated in our lab were specifically active against CD19-expressing target cells. As a premier contract research organization, Labcorp Drug Development is experienced in producing, handling and culturing T cells and CAR T cells for in vitro and in vivo early discovery studies. Preclinical screening of multiple CAR T cell candidates, alone or in combination with other agents, would facilitate the identification and selection of CARs with the most favorable activity profile for progression through the drug development pipeline. By providing a reliable source of CAR T cells, we are supporting early discovery studies and the development of therapeutic strategies in oncology. Citation Format: Andrew Karalewitz, Natalie Czeryba, Yewei Xing, Derrik Germain, Philip Lapinski, Sheri Barnes, Mark Cameron, Daniel Saims, Amber Rowse, Heidi Nielsen, Scott Wise. Chimeric antigen receptor (CAR) T-cell generation for therapeutic testing in the disseminated NALM6 human B-cell acute lymphoblastic leukemia mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2835.

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Leclerc, Gilles M., Guy J. Leclerc, Jeffim N. Kuznetsov, and Julio C. Barredo. "PIM2 Upregulation Leads to Physiological Buffering of Metformin-Induced Cell Death Mediated by ER Stress/UPR in Acute Lymphoblastic Leukemia,." Blood 118, no. 21 (November 18, 2011): 3496. http://dx.doi.org/10.1182/blood.v118.21.3496.3496.

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Abstract Abstract 3496 Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children and adolescents. Despite significant overall improvements in cure rates, outcome remains dismal for patients with resistant phenotypes or after relapse. Therefore, novel treatment strategies are warranted. Recently, we identified the AMP activated protein kinase (AMPK), a regulator of energy homeostasis in eukaryotic cells, as a potential target for ALL therapy due to its effects on cell growth, proliferation, and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We showed that activation of AMPK using metformin (1-5 mM) induced significant cell growth inhibition and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cell line models. Western blot analysis revealed that metformin led to activation of p-AMPK (Thr172) and its downstream target p-ACC (Ser79), the cell proliferation regulator p-Akt (Ser473), and the protein translation regulator p-4EBP1 (Thr70), suggesting that protein translation may be an important determinant in the mechanism of metformin-induced cell death. Indeed, we demonstrated that blocking protein translation with the mTOR inhibitor rapamycin (1 μg/ml) rescued ALL cells from metformin-induced cell death (p < 0.01). In addition, knockdown of AMPK1α expression using shRNAs (shAMPK) abrogated metformin-induced growth inhibition and apoptosis in ALL cells as compared to control cells expressing scramble shRNAs (shCTRL), indicating that AMPK mediates metformin's cytotoxicity in our models. Western blots demonstrated that ALL cells expressing shAMPK exhibit decreased expression of total AMPK, p-p38MAPK (Thr180), p-mTOR (Ser2448), and p-4EBP1 (Thr70) compared to shCTRL cells, implicating regulation of protein translation in the mechanism of cell death induced by metformin. In addition, metformin-induced p-Akt (Ser473) activation observed in shCTRL cells is blocked in shAMPK expressing cells, suggesting that the contextual crosstalk between AMPK and Akt is relevant for metformin's cytotoxicity. Indeed, experiments co-targeting Akt and AMPK using perifosine (6 μM) or the Akt inhibitor X (AIX, 5 μM) plus metformin (5 mM) for 72 h induced synergistic cell death in NALM6 cells (Combination Index (CI) values of 0.21 for perifosine + metformin, and 0.19 for AIX + metformin). Our studies uncovered that apoptotic death in NALM6 and CCRF-CEM cells treated with metformin correlated with metformin's induction of ER stress/UPR in ALL cells, as demonstrated by increased expression of the UPR markers IRE1α and CHOP. More important, rapamycin rescued metformin-treated ALL cells by relieving ER stress/UPR as demonstrated by decreased IRE1α and CHOP. These observations support our previous findings that ER stress/UPR mediates cell death in ALL cells under metabolic stress, and is tightly coupled to regulation of protein translation (Mol Cancer Ther 10:437, 2011). To further investigate the relationship between protein translation and ER stress/UPR, we examined the role of PIM1/2 kinases, particularly PIM2 known to regulate CAP protein translation, in metformin-induced ALL cell death. Our results indicate that expression of PIM2 is significantly increased in NALM6 cells treated with metformin (5–10 mM) for 72 h. We also observed concomitant decrease in the expression of the UPR markers IRE1α, ATF6, and CHOP, raising the possibility that PIM2 upregulation may be a compensatory survival mechanism to regulate protein translation and suppress metformin-induced ER stress/UPR. To test this hypothesis, we co-treated NALM6 cells with the small molecule PIM1/2 kinase inhibitor V (80 μM) and metformin (5 mM) and found that inhibition of PIM2 in metformin-treated NALM6 cells induced synergistic cell death (CI = 0.28). Taken together, our data indicate that PIM2 plays a role in buffering cell death in metformin treated cells, and that regulation of protein translation modulates ER stress/UPR induced apoptosis in ALL cells. Consequently, our data support strategies that exploit synthetic lethality by combining activators of AMPK such as metformin and compounds that target regulation of protein translation or protein degradation as suitable for clinical translation in patients with ALL. Disclosures: No relevant conflicts of interest to declare.

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Kohler, M. Eric, Zachary Walsh, Kole Degolier, and Terry J. Fry. "Enhancing the Antigen-Sensitivity of the CD22 CAR through Modulation of the Affinity and Linker-Length of the Single-Chain Fragment Variable." Blood 136, Supplement 1 (November 5, 2020): 41–42. http://dx.doi.org/10.1182/blood-2020-143372.

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The advent of chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL). CD19 directed CAR T cells have demonstrated the ability to induce complete remissions in up to 90% of r/r ALL patients. Despite this remarkable upfront success, relapse after CAR T cell therapy remains a major obstacle to long term remissions. A major mechanism for relapse after CD19-directed CAR T cell therapy is the recurrence of antigen-negative ALL cells. In recent years, CD22 CAR T cell therapy has emerged as an effective salvage therapy for patients with CD19-negative ALL. In a phase I clinical trial, CD22 CAR T cells were able to induce remission in up to 80% of patients with CD19-negative ALL. Patients achieving remission, who did not undergo a consolidative hematopoietic stem cell transplant, were found to be at high risk of relapse due to downregulation of the CD22 antigen below the threshold required for effective CD22 CAR T cell activity. Thus, strategies to increase the antigen-sensitivity of CD22 CAR T cells have the potential to enhance the induction and duration of remission in ALL patients. As the properties of a CAR that influence sensitivity to antigen are not well defined, we began by testing the impact of increasing the affinity of the single-chain fragment variable (scFv) for the CD22 antigen. T cells from healthy donors were activated and transduced with a second-generation, 4-1BB CAR containing either the standard affinity (SA)-m971 scFv used in the prior clinical trial, or a high affinity (HA) scFv generated by affinity maturation of the m971 scFv. SA- and HA-CD22 CAR T cells were evaluated in vitro and in vivo against clones of the pre-B ALL cell line, NALM6, which express CD22 at wild type levels (CD22WT), sub-physiologic levels (CD22Lo), supra-physiologic levels (CD22Hi) or in which CD22 was deleted (CD22Neg). We found that the amount of CD22 expressed on the leukemia cells resulted in dose-dependent expression of activation markers, such as CD69 and CD25 (p<0.05) on CD22 CAR T cells. Similarly, CAR T cell functions, such as the secretion of interferon-gamma (IFNg, p<0.0001) and interleukin-2 (IL-2, p<0.0001) as well as cytotoxic degranulation (p<0.0001) were all significantly impacted by the amount of CD22 on the surface of NALM6. A similar pattern of antigenic dose-response was seen in the signaling of CAR T cells, with phosphorylation of ERK reflecting the level of CD22 antigen (p<0.001) and correlating with the increased in vivo efficacy of the CAR T cells against CD22WT NALM6, relative to CD22Lo NALM6. Increasing the affinity of the CD22 CAR did not impact the in vivo efficacy against CD22WT NALM6 at either a therapeutic or subtherapeutic dose, however, HA-CD22 CAR T cells significantly prolonged the survival of NSG mice with CD22Lo NALM6, relative to SA-CD22 CAR T cells (p<0.01). The enhanced activity of HA-CD22 CAR T cells against CD22Lo leukemia did not correlate with improved in vitro functionality, as the HA-CD22 CAR T cells surprisingly demonstrated lower IL-2 secretion (p<0.01), lower proliferation (p<0.05) and diminished in vitro lysis of CD22Lo NALM6 (p<0.05), relative to SA-CD22 CAR T cells. ERK phosphorylation, however, was significantly increased in HA-CD22 CAR T cells (p<0.01) and was the only in vitro marker which correlated with the enhanced in vivo activity seen with the affinity-matured CAR. Previous clinical experience has demonstrated the importance of using a short linker (consisting of a single G4S sequence) between the heavy and light chains of the m971 scFv, therefore we next evaluated the impact of linker length on the activity of the HA-CD22 CAR. HA-CD22 CARs were generated with either a short- or long-linker (G4S x1 vs G4S x3, respectively) and evaluated in vitro and in vivo. While the short linker improved proliferation in vitro, there was no significant impact of linker length on cytokine production or lysis of CD22Lo NALM6. In a xenograft model, HA-CD22 CAR T cells with the long-linker demonstrated slower progression of CD22Lo leukemia and significantly prolonged survival of NSG mice with CD22WT leukemia relative to HA-CD22 CAR T cells with the short-linker (p<0.01). Taken together, these studies suggest that increasing the affinity of a scFv is a promising strategy for enhancing CAR sensitivity to low levels of target antigen, with the potential to decrease post-CAR T cell relapses due to antigen downregulation. Disclosures No relevant conflicts of interest to declare.

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Leclerc, Guy J., Jianfeng Du, Joanna DeSalvo, Gilles M. Leclerc, and Julio C. Barredo. "The BCR-ABL Fusion Protein Sensitizes Acute Lymphoblastic Leukemia to Apoptotic Death Induced by the Dual Glycolytic and Glycosylation Inhibitor 2-Deoxy-D-Glucose." Blood 120, no. 21 (November 16, 2012): 2439. http://dx.doi.org/10.1182/blood.v120.21.2439.2439.

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Abstract Abstract 2439 Tumor metabolism has emerged as a hallmark of cancer by which the oncogenic profile of cancer cells pairs energy availability with cell growth and survival. BCR-ABL positive acute lymphoblastic leukemia (BCR-ABL+ ALL) is a highly resistant phenotype, and the BCR-ABL fusion protein has been correlated with alterations in glucose metabolism. The glucose analogue 2-deoxy-D-glucose (2DG) has been found to be an effective antitumor agent in both animal models and human clinical trials for solid tumors based on the dependency of hypoxic tumor cells on anaerobic glycolysis to generate ATP. 2DG inhibits two key glycolytic enzymes, hexokinase (HK) and phosphoglucose isomerase (PGI), and interfers with N-linked glycosylation by its incorporation in place of mannose into lipid-linked oligosaccharide (LLO) chains which leads to premature termination of LLO synthesis. Unlike hypoxic solid tumors, we recently demonstrated that 2DG induces cell death in ALL under normoxia preferentially by inhibiting N-linked glycosylation resulting in ER stress/UPR-mediated apoptosis, although inhibition of glycolysis also contributed to cell death in a phenotype specific-manner (Mol Cancer Res 10:969, 2012). Among ALL subtypes, BCR-ABL+ ALL t(9;22) cell lines SupB15 and TOM1 exhibited the highest sensitivity to 2-DG suggesting that BCR-ABL expression may be linked to sensitivity to 2DG. To investigate the role of BCR-ABL expression in this process, we constructed NALM6 (Bp-ALL) stable cell lines expressing BCR-ABL p190 fusion, and assessed their level of sensitivity to 2DG under normoxia. Expression of the BCR-ABL p190 fusion in NALM6 cells significantly increased 2DG-induced apoptosis compared to mock transfected NALM6 cells, indicating that BCR-ABL expression results in higher sensitivity to 2DG in this experimental system. Western blot analysis of BCR-ABL+ ALL (SUPB15 and TOM1) and Bp-ALL (NALM6) cell lines demonstrated that BCR-ABL expression induced alterations in the PI3K/Akt/p70S6K, GSK-3β/Mcl-1, p-STAT3 (Ser727), p-PKCδ (Thr505), and GRP78/p-eIF2α (Ser51) signaling pathways. Treatment with 2DG resulted in upregulation of p-AMPK (Thr172) and p-GSK-3β (Ser9), and downregulation of p-mTOR (Ser2448), p-p70S6K (Thr389), p-STAT3 (Ser727) and Mcl-1 in both BCR-ABL+ ALL (SUPB15 and TOM1) and Bp-ALL cells (NALM6). As we have previously described, 2DG induces ER stress and the expression of the unfolded protein response (UPR) markers (Mol Cancer Res 10:969, 2012). In BCR-ABL+ ALL cells, 2DG led to upregulation of IRE1α, GRP78 and CHOP (a marker of UPR induced cell death) and resulted in apoptotic cell death (>60%), which correlated with lower level of Mcl-1 expression. To further investigate the role of Mcl-1 on 2DG's sensitivity we used pharmacological inhibitors and shRNA. We found that Mcl-1 down-regulation sensitized Bp-ALL cells to 2DG (36% ± 4% cell death; p<0.001 for NALM6/shMcl-1 vs. NALM6/shScramble treated with 2DG 0.5mM). We then examined the role of GSK-3β/Mcl-1 signaling in K562, a highly 2DG resistant CML cell line expressing BCR-ABL p210 fusion. We found that K562 cells expressed high levels of Mcl-1. Inhibition of PI3K/Akt/GSK-3β pathway using LY294002 sensitized K562 cells to 2DG, suggesting that downregulation of Mcl-1 by GSK-3β activation leads to 2DG sensitivity. Finally, we co-targeted BCR-ABL+ ALL cells with TKIs (Imatinib) plus the glycolytic/glycosylation inhibitor 2DG and found significant increase in cell death for the combination compared to single drug (62.6% ± 0.8% cell death; p<0.001 for 2DG 4mM + Imatinib 0.5μM vs. single drug). Further, no cross-resistance was detected between 2DG and Imatinib in TKI-resistant BCR-ABL+ ALL cells. Taken together, our data demonstrate that BCR-ABL p190 expression sensitizes Bp-ALL cells to 2DG by altering the GSK-3β/Mcl-1 signaling pathway and inducing ER stress/UPR mediated cell death. This study supports further consideration of strategies combining inhibitors of glucose metabolism such as 2DG with BCR-ABL TKIs for future clinical translation in BCR-ABL+ ALL. Disclosures: No relevant conflicts of interest to declare.

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Sipol, Alexandra, Thomas Grünewald, Guenther Richter, Caroline Wernicke, Hans-Jochem Kolb, Torsten Haferlach, and Stefan Burdach. "Overexpression of the Metabolic Stress Sensor and Proglycolytic Transcription Factor ΜLΧΙΡ Mediates Malignancy of Common Acute Lymphoblastic Leukemia in Vivo and Is Targetable By Allorestricted Peptide Specific T Cells." Blood 124, no. 21 (December 6, 2014): 2436. http://dx.doi.org/10.1182/blood.v124.21.2436.2436.

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Abstract Oncogene addiction provides ideal targets for successful immunotherapy. MLXIP (MAX like protein X interacting protein, also known as MondoA) is a metabolic stress sensor and a proglycolytic transcription factor potentially involved in metabolic addiction features of leukemia, the Warburg effect and anoikis. MLXIP dimerizes with MLX within the MYC interactome and promotes longevity in C. elegans (Johnson et al. 2014). The MYC interactome comprises the MYC/MAX/MAD/MLX/MLXIPtranscriptionfactornetwork: Its key players MYC, MAD and MLXIP differentially mediate proliferation, differentiation or metabolism by heterodimerization with MAX or MLX: While MAX is available for partnering with MYC vs. MAD, MLX may partner with MAD or MLXIP alternatively. In contrast to the perception of MLXIP as a tumor suppressor gene in some solid tumors such as breast and colon cancer (O'Shea and Ayer, 2013), we have extended our previous notion of up regulation of MLXIP in B cell acute lymphoblastic leukemia(ALL, Burdach & Richter 2007, Haferlach et al. 2010) by demonstrating that MLXIP is highly overexpressed and induces stemness, proliferation and B cell receptor signaling pathway signatures in common ALL (cALL, Wernicke et al. 2012). Here we report on the role of MLXIP in malignancy of cALL in vivo. Given the non accessibility of transcription factors by chimeric antigen receptor transgenic T cells (CARs) and the superiority of allorestricted T cells in T cell receptor (TCR) based immunotherapy of leukemia (Burdach & Kolb 2013), we also tested the targetability of MLXIP by allorestricted peptide specific T cells to ultimately generate MLXIP specific allorestricted TCR transgenic T cells (ATRs, instead of CARs). Our human/murine xenotransplantation model with immunodeficient RAG2-/-gc-/- mice was used (Richter et al. 2009). NALM6 and 697 cALL lines were retrovirally transduced with MLXIP short hairpin RNA (shRNA). Upon successful MLXIP knock down (kd), kd and control lines were injected into the mice; CD10+ blasts in blood, spleen and marrow were assessed. MLXIP specific T cells were generated by priming of donor HLAA0201 negative (A2-) T-cells with A2+ dendritic cells bearing MLXIP peptides, multimer-based sorting and subcloning of A2-CD8+ T-cells. For priming of T cells, five MLXIP peptides were chosen by SYMPEITHI, BIMAS and NetCTL1.2. analyses. Peptide 428 stabilized best A2 expression on TAP-deficient T2 cells. Specificity and functionality of T cell clones were tested by ELISpot interferon g (IFg) and granzyme B assays with six MLXIP+ leukemia lines (A2+, A2-). Off target effects of MLXIP specific T-cell clones were assessed by IFg reactivity against the MLXIP expressing A2+ NALM6 cell line vs. A2+ and A2- EBV immortalized lymphoblastoid cell lines from six donors. Peptide homology was assessed with BLAST algorithms in SWISSPROT. We found MLXIP to be overexpressed in both ALL and AML, while it was low in solid tumors including breast and colon cancer. MLXIP kd in the cALL cell line NALM-6 reduced the transcript by 80%. Importantly, in vivo MLXIP maintained 90-99% of CD10+ leukemic blasts in blood, marrow and spleen. Spleen size and weight normalized by MLXIP kd: While signs of leukemia engraftment were observed in all mice, the differences of CD10+ blasts in blood, marrow and spleens in the control vs. the MLXIP kd group were highly significant (p=0.008). The decrease of leukemic splenomegaly after MLXIP kd was impressive. Median spleen weight was 0.22g vs. 0.08g in the control vs. the kd group (Figure 1: The upper spleen displays the leukemic infiltration in control mice. The two lower spleens are from mice injected with MLXIP kd NALM6 cells). MLXIP peptide 428 specific T cell clones successfully recognized and killed A2 positive MLXIP expressing NALM6 and 697 cALL lines. In contrast, MLXIP specific T cell clones did not release IFg and granzyme B under stimulation with A2- REH, RS4;11 cALL lines. We identified the nucleotide sequence of alpha and beta variable chains of MLXIP specific A2 restricted TCR to finally obtain MLXIP specific ATRs. Peptide dependent and independent off target alloreactivity was very low compared to reactivity against NALM-6 cALL. In conclusion, these findings demonstrate that MLXIP maintains leukemic burden and malignancy of cALL in vivo. Moreover, we identified MLXIP as a promising target for immunotherapy of cALL and potentially other MLXIP expressing leukemias, including AML. Figure 1: Figure 1:. Disclosures Haferlach: MLL: Equity Ownership. Burdach:PDLI: Equity Ownership.

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Ueno, Shikiko, Jiayun Lu, He Jie, Ailing Li, XiaoXian Zhang, Leslie E. Silberstein, Jerome Ritz, and Li Chai. "SALL4 Is a Key Survival Factor in Acute B-Cell Lymphoblastic Leukemia." Blood 118, no. 21 (November 18, 2011): 2428. http://dx.doi.org/10.1182/blood.v118.21.2428.2428.

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Abstract Abstract 2428 SALL4 is a zinc-finger transcriptional factor and a member of the SALL gene family. It plays an essential role in the maintenance of ESC pluripotent and self-renewal properties by interacting with other two key regulators in ESCs, Nanog and Oct4. We previously have shown that stem cell factor SALL4 is aberrantly expressed in 75% of acute B-cell lymphoblastic leukemia (B-ALL). We have also shown that SALL4 is aberrantly expressed in AML, and down-regulation of SALL4 in AML leads to significant cell death. In this study, we focused on investigating the functional role of SALL4 in human B-ALL leukemogenesis. We first assessed the SALL4 mRNA level in four B-ALL cell lines (REH, Nalm6, 697, Blin-1) and five primary patient samples by qRT-PCR. We observed SALL4 mRNA in these four cell lines increased when compared to normal human CD34 negative BM cells. Moreover 4 of 5 primary samples showed high level expression of SALL4, suggesting that SALL4 might play a role in B-ALL pathogenesis. Then, we selected a SALL4 expressing B-ALL cell line (REH and Nalm6) and attenuated SALL4 expression through GFP-labeled shRNA approach in these cell lines. We monitored the growth of SALL4 knockdown and control REH and Nalm6 cells through MTS assay. SALL4 knockdown cells had a decreased growth rate compared to that of the control cells. We also stained SALL4 knockdown and control cells with Annexin V and 7-AAD by flow cytometric quantitation of apoptotic cells. The percentages of apopotic cells in SALL4 knockdown cells were much higher than these in controls. These data demonstrated that inhibition of SALL4 in REH cells and Nalm6 cells led to reduced proliferation and increased apoptosis. We then examined the oncogenesis ability of SALL4 knockdown REH cells in a mouse xenotransplantation model. SALL4 knockdown or control REH cells were injected intravenously into immunodeficient mice. All the recipients succumbed to fatal leukemia within 4 to 6 weeks post transplantation. In both BM and spleen of SALL4 knockdown recipients the engrafted proportion of GFP+ cells was significantly decreased compared to the initial donor cells. Whereas, in both BM and spleen control recipients the percentage of GFP+ REH cells engrafted was similar to that of initial donor cells. This suggests that down-regulation of SALL4 is essential for B-ALL engraftment. To rule out the observed engraftment defect was due to homing, we next performed homing assay. SALL4 knockdown or control cells were injected intravenously into immunodeficient mice as well. Three hours of the injection, mice were sacrificed and analyzed the percentage of GFP+ cells in BM and spleen by flow cytometry. There was no difference among SALL4 knockdown and the control. Furthermore, we performed gene expression profiling on apoptosis-related genes in SALL4 knockdown and control REH cells. The result showed that in SALL4-knockdown REH, TNF mediated cell apoptosis pathways was up-regulated as well as multiple caspase members. The expression of Caspase 3, Caspase 8, FADD was up-regulated in both SALL4 knockdown REH and Nalm6 when compared to these controls, and was verified by real time RT-PCR. This suggests that SALL4 could repress apoptosis through the TNF signal pathway. In summary, we report a novel SALL4/TNF pathway in maintaining cell survival in B-ALL. Disclosures: No relevant conflicts of interest to declare.

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Xu, Bing, Manman Deng, Zhiwu Jiang, Jie Li, Kai Chen, and Peng Li. "Role of Mitochondrial Apoptotic Pathway in Disulfiram/Copper Mixture-Induced Cell Apoptosis in Human B-Lineage Acute Lymphoblastic Leukemia in Vitro." Blood 126, no. 23 (December 3, 2015): 4920. http://dx.doi.org/10.1182/blood.v126.23.4920.4920.

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Abstract Backgrounds: The long term prognosis of adult B-lineage acute lymphoblastic leukemia (B-ALL) is poor when compared with pediatric B-ALL. The current therapeutic regimen for adult B-ALL often results in refractory and relapsing diseases. Therefore, it is urgently needed to explore novel approaches to treat adult B-ALL. Disulfiram (DS) has been used clinically as a safe anti-alcoholism drug for over 6 decades. Recent studies demonstrated that disulfiram/cooper mixture (DS/Cu) was cytotoxic to multiple solid cancers, but its effects on B-ALL cells are still unclear. Moreover, the molecular mechanism of the cytotoxicity of DS/Cu to tumor cells was poorly defined. In this study, we investigated the effects of DS/Cu on B-ALL cells in vitro and its related cytotoxic mechanism. Results: Firstly, CCK8 assay indicated that DS/Cu markedly inhibited Nalm6 cell proliferation in a dose-dependent manner. Secondly, colony-forming assay showed that DS/Cu also abolished the clonogenicity of Nalm6 cells (P<0.001). Thirdly, FACS analyses revealed that DS/Cu mixture could induce apoptosis of Nalm6 cells, as well as primary B-ALL cells (n=16) in a dose-dependent manner. We additionally analyzed the relationship between clinical characteristics of B-ALL patients, including age, WBC counts, immunophenotype, cytogenetics, risk stratification and Ph chromosome, with the efficacy of DS/Cu on B-ALL cells. The apoptosis isolated from pro-B and cytogenetic abnormality B-ALL pastients was higher. Therefore, our results demonstrated that DS/Cu mixture could induce significant cytotoxicity against B-ALL cells in vitro. To decipher the cytotoxic mechanism of DS/Cu mixture, JC-1 staining was done and the results showed that DS/Cu mixture could significantly reduce the mitochondrial membrane potential in Nalm6 cells (P<0.01) and 7 cases of primary B-ALL cells (P<0.05). Consistently, Western Blot analysis showed that DS/Cu induced B-ALL cell apoptosis by down-regulating the expression of anti-apoptotic protein Bcl2 and Bcl-XL, as well as activating caspase-3 and its substrate PARP. Hence, our results indicated that DS/Cu induced apoptosis of B-ALL cells at least partly through the intrinsic mitochondrial apoptotic pathway. Conclusion: Our results demonstrated that DS/Cu not only significantly inhibit proliferation and clonogenicity, but also induce apoptosis of B-ALL cells in vitro.The mitochondrial apoptotic pathway might be the molecular mechanism of DS/Cu-induced apoptosis of B-ALL cells. Disclosures No relevant conflicts of interest to declare.

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Zanjirband, Maryam, Soheila Rahgozar, and Narges Aberuyi. "miR-16-5p enhances sensitivity to RG7388 through targeting PPM1D expression (WIP1) in Childhood Acute Lymphoblastic Leukemia." Cancer Drug Resistance 6, no. 2 (2023): 242–56. http://dx.doi.org/10.20517/cdr.2022.113.

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Aim: Given the encouraging results of the p53-Mdm2 inhibitor RG7388 in clinical trials and the vital function of miR-16-5p in suppressing cell proliferation, the aim of the present study was to investigate the combined impact of RG7388 and miR-16-5p overexpression on the childhood acute lymphoblastic leukemia (chALL). Methods: miRTarBase and miRDB, along with KEGG and STRING databases, were used to predict miR-16-5p target genes and explore protein-protein interaction networks, respectively. B- and T-lymphoblastic cell lines, in addition to patient primary cells, were treated with RG7388. Ectopic overexpression of miR-16-5p in Nalm6 cell line was induced through cell electroporation and transfection of microRNA mimics was confirmed by qRT-PCR. Cell viability was evaluated using the MTT assay. Western blot analyses were performed to evaluate the effects of RG7388 and miR-16-5p upregulation on the protein levels of p53 and its downstream target genes in chALL cells. Paired sample t-test was employed for statistical analyses. Results: MTT assay showed RG7388-induced cytotoxicity in wild-type p53 Nalm6 cell line and p53 functional patient primary cells. However, CCRF-CEM and p53 non-functional leukemic cells indicated drug resistance. Western blot analyses validated the bioinformatics results, confirming the downregulation of WIP1, p53 stabilization, as well as overexpression of p21WAF1 and Mdm2 proteins in Nalm6 cells transfected with miR-16-5p. Moreover, enhanced sensitivity to RG7388 was observed in the transfected cells. Conclusion: This is the first study indicating the mechanistic importance of miR-16-5p overexpression in chALL and its inhibitory role in leukemia treatment when combined with the p53-Mdm2 antagonist, RG7388. These findings might be useful for researchers and clinicians to pave the way for better management of chALL.

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Mansilla-Soto, Jorge, Justin Eyquem, Sascha Haubner, Mohamad Hamieh, Judith Feucht, Noémie Paillon, Andres Zucchetti, et al. "132 HLA-independent T cell receptors effectively target low abundance antigens." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A142. http://dx.doi.org/10.1136/jitc-2021-sitc2021.132.

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BackgroundChimeric antigen receptors (CARs) engage antigen independently of HLA and enable sustained T cell proliferation when they are endowed with both activating and costimulatory functions. While remission rates have been noticeably elevated in numerous clinical trials targeting CD19, CD22 or BCMA, relapses are common. One of the several underlying relapse mechanisms is antigen escape, which refers to a relapsing tumor that is either negative for the targeted antigen or expresses the latter at a low level. Failure to eliminate antigen-low tumors raises questions about the sensitivity of CARs and the minimum antigen density that is required for effective tumor eradication. Unlike CARs, TCRs engage antigen in an HLA-dependent manner, and they do so with high sensitivity. We hypothesized that a TCR/CD3 complex containing the same heavy and light immunoglobulin chains as a CAR will display increased sensitivity to the target antigen.MethodsWe edited the TRAC locus in human primary T cells to establish a novel antigen receptor structure, termed HLA-independent TCR or HIT receptor, by incorporating into the TCR/CD3 complex the same heavy and light chains as those of a corresponding CAR. We assessed their antigen sensitivity against a panel of cell lines expressing different antigen levels, analyzing their cytotoxicity, cytokine secretion, signaling response and degranulation activity. HIT and CAR T cells were further evaluated for their anti-tumor response using established ALL and AML mouse models.ResultsCD19-TRAC-HIT and CD19-TRAC-CAR T cells lysed wild-type NALM6 (~27,000 CD19 molecules) and NALM6 variants with 100-fold less CD19. As CD19 levels decreased further, CAR T cells no longer killed their target, in contrast to HIT T cells. HIT T cells showed increased expression of IFN-gamma, IL-2 and TNF-alpha upon exposure to NALM6 cells expressing ~20 CD19 molecules per cell, compared to CAR T cells. This increased sensitivity of HIT receptors correlated to their greater signaling response, upon exposure to the low-antigen-density NALM6. Phospho-proteomic analyses further confirmed this increased response of HIT T cells to low antigen levels. Altogether, these results confirm that HIT receptors endow T cells with greater antigen sensitivity than canonical CARs. We further showed that HIT T cells have higher in vivo anti-tumor activity compared to CAR T cells in mice bearing low-antigen-density ALL or AML.ConclusionsHIT receptors consistently afford high antigen sensitivity and mediate tumor recognition beyond what current CARs can provide. HIT receptors open new prospects for targeting cell surface antigens of low abundance.Ethics ApprovalEight- to 12-week-old NOD/SCID/IL-2Rgamma-null (NSG) male mice (Jackson Laboratory) were used under a protocol approved by the MSKCC Institutional Animal Care and Use Committee.

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Kawamata, Norihiko, Fabienne Isken, Stefanie Goellner, C. Müller-Tidow, and H. Phillip Koeffler. "ChIP-on-Chip Analysis Using Dominant Negative Form of PAX5 Fusion Gene Revealed Genes Associated with Tumorigenesis Were Directly Regulated by PAX5 in a Human B Cell Line, NALM6." Blood 112, no. 11 (November 16, 2008): 3585. http://dx.doi.org/10.1182/blood.v112.11.3585.3585.

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Abstract PAX5 is a transcriptional factor playing an important role in B-cell development. Overexpression of PAX5 induced by translocation to the enhancer region of immunoglobulin heavy chain gene occurs in non-Hodgkin lymphomas (NHL), suggesting that PAX5 can be also associated with development of NHL. To identify genes associated with tumorigenesis in malignancies overexpressing PAX5, we performed ChIP-on-chip analysis using PAX5 specific antibody. Non-specifically immunoprecipitated DNA by antibodies can cause false positive results using ChIP-on-chip analysis (background). To reduce the background in ChIP-on chip analysis, we used a dominant negative form of PAX5 and a wild-type PAX5 specific antibody for our ChIP-on-chip analysis. We have previously found a PAX5 chimeric protein expressed in acute lymphoblastic leukemia in which the C-terminal end of PAX5 was replaced by C20ORF112 protein (Kawamata N et al, Proc Natl Acad Sci U S A. Aug. 12, 2008). We have also found that this chimeric protein behaved in a dominant negative fashion over the wild-type PAX5 and suppressed expression of target genes of wild-type PAX5. PAX5 chimeric protein can compete with wild-type PAX5 for binding on the promoter region of direct down-stream target genes. To identify the genes directly regulated by PAX5 in human B-cells, we transfected the dominant-negative form of PAX5 chmeric protein, PAX5-C20ORF112 (PAX5-C20S) into NALM6 human B-cell leukemia cells which constitutively express abundant PAX5. Transfected cells were collected and chromatin immunoprecipitation (ChIP) assay was performed using PAX5 C-terminal specific antibody which can recognize only wild-type PAX5, but not the chimeric PAX5 protein, PAX5C20S. As a control, we also performed ChIP assay using NALM6 cells transfected with an empty vector. Immunoprecipitated DNA was recovered and amplified using the whole genome amplification technique. The DNAs were hybridized with oligonucleotide probes containing the promoter regions of the human genome. The levels of hybridized DNA were quantified and genes directly bound by PAX5 were identified. Comparison between NALM6 cells transfected with the empty vector and PAX5C20S significantly reduced the background and allowed identification of genes directly regulated by PAX5 in NALM6, including BUB1B, SSSCA1, CEP68, and BAG1. BUB1B, CEP68 and SSSCA1 are proteins involved in mitosis; BAG1 is a protein associated with apoptosis. Dysregulation of these genes by overexpressed PAX5 may be associated with development of B-cell malignancies.

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Mladenov, Emil, Katja Paul-Konietzko, Veronika Mladenova, Martin Stuschke, and George Iliakis. "Increased Gene Targeting in Hyper-Recombinogenic LymphoBlastoid Cell Lines Leaves Unchanged DSB Processing by Homologous Recombination." International Journal of Molecular Sciences 23, no. 16 (August 16, 2022): 9180. http://dx.doi.org/10.3390/ijms23169180.

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In the cells of higher eukaryotes, sophisticated mechanisms have evolved to repair DNA double-strand breaks (DSBs). Classical nonhomologous end joining (c-NHEJ), homologous recombination (HR), alternative end joining (alt-EJ) and single-strand annealing (SSA) exploit distinct principles to repair DSBs throughout the cell cycle, resulting in repair outcomes of different fidelity. In addition to their functions in DSB repair, the same repair pathways determine how cells integrate foreign DNA or rearrange their genetic information. As a consequence, random integration of DNA fragments is dominant in somatic cells of higher eukaryotes and suppresses integration events at homologous genomic locations, leading to very low gene-targeting efficiencies. However, this response is not universal, and embryonic stem cells display increased targeting efficiency. Additionally, lymphoblastic chicken and human cell lines DT40 and NALM6 show up to a 1000-fold increased gene-targeting efficiency that is successfully harnessed to generate knockouts for a large number of genes. We inquired whether the increased gene-targeting efficiency of DT40 and NALM6 cells is linked to increased rates of HR-mediated DSB repair after exposure to ionizing radiation (IR). We analyzed IR-induced γ-H2AX foci as a marker for the total number of DSBs induced in a cell and RAD51 foci as a marker for the fraction of those DSBs undergoing repair by HR. We also evaluated RPA accretion on chromatin as evidence for ongoing DNA end resection, an important initial step for all pathways of DSB repair except c-NHEJ. We finally employed the DR-GFP reporter assay to evaluate DSB repair by HR in DT40 cells. Collectively, the results obtained, unexpectedly show that DT40 and NALM6 cells utilized HR for DSB repair at levels very similar to those of other somatic cells. These observations uncouple gene-targeting efficiency from HR contribution to DSB repair and suggest the function of additional mechanisms increasing gene-targeting efficiency. Indeed, our results show that analysis of the contribution of HR to DSB repair may not be used as a proxy for gene-targeting efficiency.

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Xing, Dongxia, Wendy Fang, William K. Decker, Sufang Li, Simon N. Robinson, Hong Yang, David Steiner, et al. "Ex Vivo Expansion of Cord Blood NK Cell Have In Vivo Efficacy Against Leukemia." Blood 110, no. 11 (November 16, 2007): 2741. http://dx.doi.org/10.1182/blood.v110.11.2741.2741.

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Abstract Introduction Allogeneic stem cell transplantation has demonstrated the ability to prevent leukemic relapse via an immune-mediated graft-vs.-leukemia effect. Natural killer (NK) cells have been shown to comprise a significant component of this anti-leukemia effect and have been reported to enhance engraftment and reduce graft-vs.-host disease. Ex-vivo expansion of peripheral blood-derived NK cells has been demonstrated. Cord blood (CB) is a promising alternate source of NK cells with enhanced cytokine/antigen-responsiveness, proliferation and increasing availability. We studied expansion conditions for the generation of CB NK cells for clinical use, and evaluated them in vivo in NOD-SCID/IL2Rgnull mice engrafted with human leukemia. Methods Two expansion protocols were compared: CD56+ NK cells were isolated and cultured with irradiated CD56− feeder cells in RPMI-1640 supplemented with 100ng/mL IL-2 for 21 days. CB mononuclear cells were depleted of CD3+ cells prior to culture. In vivo NK cytolytic activity was measured via chromium release assays on leukemia cell lines (K562, Nalm6) and fresh patient leukemia blasts. Functionality was confirmed in vivo as well by injecting 106 K562 cells or 2.5 ×104 Nalm6 cells into NOD-SCID/IL2Rgnull mice. 2 ×106 CB-expanded NK cells were administered on day +1, and an additional 5×106 CB NK cells were administered on day +7. NK killing of leukemic targets was then confirmed by flow cytometry. Results Feeder cells mediated >30-fold expansion of CB NK cells, generating a nearly pure population that contained 97% CD3−CD56+ cells. Expanded CB NK could lyse >90% of K562 targets at an E:T ratio of 10:1, as well as >40% of patient leukemia blast. In CD3− depleted cultures, similar expansions were observed, however only 55% of these cells were CD3−CD56+ with the remainder being 10% CD3+CD56+ NK/T and 35% CD3+ T lymphocytes. In the NOD-SCID~/IL2Rgnull model, ex vivo expanded CB NK cells demonstrated the ability to reduce the leukemia burden of both AML (K562) and ALL (Nalm6) cells >50%. Conclusions We have demonstrated the feasibility of expanding CB NK ex vivo to clinically-relevant doses with minimal manipulation. These expanded CB NK cells demonstrated cytotoxic activity in vitro and in vivo against a variety of human leukemic cell lines and patient leukemia blasts. These results provide rationale for immunotherapy of leukemia with CB-derived NK cells.

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Ma, Xiao-Tong, Ya-Kun Mou, Yang-Yang Zhao, Nan Wang, Xiao-Yan Liu, Jing Yin, Yan-Ping Ma, and Dan Guo. "Set7 Is Highly Expressed in B-Lineage Acute Lymphoblastic Leukemia Cells and Overexpression of Set7 Exhibits Antileukemia Effect." Blood 128, no. 22 (December 2, 2016): 5088. http://dx.doi.org/10.1182/blood.v128.22.5088.5088.

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Abstract Set7 is a member of protein lysine methyltransferase family that is highly conserved in vertebrates. Set7 can regulate the maintenance of chromosome structure, cell cycle and apoptosis, and plays an important role in many kinds of cancer, metabolism and inflammatory process. However, studies concerning its pathogenic role in leukemia are scarce. We analyzed the expression of Set7 in different types of leukemia and healthy human bone marrow cells or peripheral blood from the Oncomine databases (http://www.oncomine.org), and showed that Set7 was highly expressed in acute lymphoid leukemia (ALL) patients, especially in patients with B-ALL. In this study, we examined the expression of Set7 in various hematopoietic tumor cell lines, bone marrow samples from patients and healthy volunteers. We found that Set7 was highly expressed in B-ALL cell lines Nalm6 and REH compared with other hematopoietic malignant cell lines. Furthermore, Set7 was overexpressed in bone marrow mononuclear cells of adult and pediatric patients with B-ALL than in healthy human bone marrow samples, and sorted CD19+ cells, as well (P<0.001). To investigate Set7 gene function, retroviral-mediated overexpression or removal experiments were performed. Gain-of-function and loss-of-function analyses in Nalm6 and REH cells demonstrated that Set7 inhibited cell growth, colony formation and enhanced the chemosensitivity of B-ALL cells to Vincristine (VCR), Arabinocytidine (Ara) and Daunorubicin (DNR). Protein sequence analysis showed that Ebf1 might be a potential substrate for Set7. The expression of Rad51 and Tnsf11, the downstream target genes of Ebf1, were changed as expected when Set7 was overexpressed in Nalm6 and REH. Our study shows for the first time that overexpression of Set7 has an inhibitory effect on B-ALL. These findings suggest that Set7 may be a promising molecular therapy target for B-ALL treatment and provide new clues for understanding the molecular mechanisms of the leukemogenesis of B-ALL. Disclosures No relevant conflicts of interest to declare.

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Mardiana, Sherly, Olga Shestova, Stephan A. Grupp, Marco Ruella, David M. Barrett, and Saar Gill. "Repurposing Bi-Specific Chimeric Antigen Receptor (CAR) Approach to Enhance CAR T Cell Activity Against Low Antigen Density Tumors." Blood 138, Supplement 1 (November 5, 2021): 1727. http://dx.doi.org/10.1182/blood-2021-146995.

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Abstract BACKGROUND Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies, as highlighted by high complete remission rates and FDA approval of CD19-specific CAR T cell products. However, depth and duration of remission are limited by antigen loss/downregulation on tumors, as observed in clinical trials using CAR T cells targeting the CD19 or CD22 in leukemia and lymphoma, BCMA in multiple myeloma, and EGFRvIII in glioblastoma. This observation forms the basis of current efforts to develop multi-targeting CAR T cells to prevent antigen-negative escape. Antigen density is an important factor modulating CAR T cell response, since antigen expression below a certain threshold fails to trigger the full range of T cell functions. Given that signal strength induced upon antigen encounter determines CAR T cell activity, we hypothesized that simultaneous targeting of two dimly-expressed antigens will result in enhanced CAR T cell signaling and anti-tumor function, approaching that seen in response to one highly-expressed antigen. This is important given the heterogeneity of antigen expression in various cancers. Therefore, the bi-specific CAR T cells currently being developed to prevent antigen-negative escape could also be used to enhance efficacy against low antigen density (LAD) tumors. Results from this study will provide a novel rationale for using multi-specific CAR T cells and illuminate the mechanisms of successful CAR T cell therapy. METHODS Lentivirus transduction was performed to generate CAR T cells from healthy human T cells, using second generation 4-1BBz CARs specific for either human CD19 or CD22, or both in cis, herein referred to as CAR19, CAR22, or CAR19/22, respectively (Figure 1A). For in vitro functional characterization, we performed co-culture assay of T cells and B cell leukemia cell line NALM6, which is known to express high levels of both CD19 and CD22. To assess T cell function against LAD tumor cells, primary patients' B-ALL samples expressing low antigen density in comparison to the NALM6 cell line were used (Figure 1B). CAR T cell anti-tumor potency was determined by assessing CAR T cell cytotoxicity and cytokine production. For in vivo therapeutic study, primary patients' B-ALL samples with dimly expressed CD19 and CD22 were used to evaluate and compare the therapeutic efficacy of mono- versus bi-specific CAR T cells. Additionally, we generated a LAD tumor model by deleting the highly expressed CD19 and CD22 from the ALL cell line NALM6 using CRISPR/Cas9, transducing the now antigen-negative cell line with CD19 and CD22, followed by single cell cloning to generate a cell line expressing low antigen density for both the CD19 and CD22. We engrafted tumor cells in NSG mice, followed by administration of CAR19, CAR22, CAR19/22 or untransduced T cells. Therapeutic efficacy was assessed by measuring tumor burden using either flow cytometry or bioluminescent imaging. RESULTS Cytotoxicity assay revealed that the bi-specific CAR19/22 T cells killed tumor cells more rapidly than CAR19 or CAR22 T cells. Further, compared to mono-specific CAR T cells, the bi-specific CAR19/22 T cells produced significantly more pro-inflammatory cytokines including IL-2 and IFNg, in response to stimulation with LAD primary samples or NALM6 cells. This increased cytokine-producing capacity compared to mono-specific CAR T cells was maintained following repeated antigen stimulation when in vitro exhaustion assay was performed. In vivo, enhanced tumor elimination was observed in mice receiving bi-specific CAR19/22 T cells compared to either of the mono-specific CAR T cells, in both low antigen density primary ALL and NALM6 tumor models. This translated to increased survival rates seen in mice treated with the bi-specific CAR19/22 T cells (Figure 1C-D). CONCLUSIONS Here we showed that bi-specific CAR19/22 T cells are superior to mono-specific CAR19 or CAR22 T cells, not only against LAD tumors but also tumor cells expressing high antigen density, NALM6. This was demonstrated by their enhanced cytokine-producing function, cytotoxic capacity, and therapeutic efficacy in vivo. Results from this study provide a novel rationale for repurposing multi-specific CAR T cells as a strategy to improve efficacy against LAD tumors, in addition to the recognized benefit of reducing antigen-negative escape. Figure 1 Figure 1. Disclosures Shestova: Hemogenyx Pharmaceuticals LLC: Research Funding. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Ruella: viTToria biotherapeutics: Research Funding; Novartis: Patents & Royalties; BMS, BAYER, GSK: Consultancy; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties. Gill: Novartis: Other: licensed intellectual property, Research Funding; Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

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Hlozkova, Katerina, Alpesh Thakker, Natividad Alquézar-Artieda, Marketa Zaliova, Jan Trka, Daniel A. Tennant, and Julia Starkova. "Separate Roles of Asparagine and Glutamine in Cytostatic Effect of L-Asparaginase - Stable Isotope Tracing Approach." Blood 134, Supplement_1 (November 13, 2019): 2575. http://dx.doi.org/10.1182/blood-2019-127911.

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L-asparaginase (ASNase) is one of the crucial components of acute lymphoblastic leukemia (ALL) therapy. Although we have previously shown that ASNase triggers metabolic reprogramming of leukemic cells, the significance and interconnections of the changes have not yet been elucidated. Metabolic reprogramming is an accompanying feature in therapy response and is also triggered by commonly used cytostatic drugs. ASNase hydrolyzes two non-essential amino acids asparagine (Asn) and glutamine (Gln). Therapeutic ASNase concentration used in vitro transforms all Asn and Gln to aspartate (Asp) and glutamate (Glu), respectively. We employed stable isotope tracing to understand the complexity and interconnection of metabolic processes altered in leukemia cells exposed to ASNase. Stable isotope tracing is a unique method enabling the quantification of metabolic flux. When substrate with 13C is metabolized by cells, enzymatic reactions rearrange 13C atoms resulting in specific labeling patterns in downstream metabolites. Therefore, this method allows us to determine exact pathways which are used to metabolize labeled nutrient. Glucose and Gln media concentrations were adjusted to 8mM and 1mM, respectively, to more resemble physiological conditions. All experiments were done with 0.8 IU/ml ASNase which is close to the plasma ASNase level in the induction phase of ALL treatment. We studied the effect of ASNase on two B-ALL cell lines with different sensitivity to this cytostatic drug: REH (IC50=0.000231 IU/ml) and NALM6 (IC50=0.328894 IU/ml). U13C glucose tracing revealed that although pyruvate and lactate intracellular concentrations are not changed after ASNase treatment, the relative flux from glucose to these metabolites (expressed as M+3 which represents 3 13C atoms in 1 molecule) is lowered (e.g. lactate (M+3): NALM6 - (0.5898±0.0026) vs (0.4473±0.0031), p<0.0001); REH - (0.3417±0.0023) vs (0.2411±0.0024), p<0.0001). Unlike glycolytic intermediates, the levels of tricarboxylic acid (TCA) cycle intermediates are lowered after ASNase administration (e.g. relative quantification of malate: NALM6 - (1.6120±0.1202) vs (0.4358±0.0247), p<0.0001; REH - (0.8231±0.0689) vs (0.2484±0.0573), p<0.0001). Interestingly, the relative flux from glucose to TCA cycle (expressed as e.g. malate (M+1-M+4) which represents at least 1 13C atom in a malate molecule) is increased in NALM6 ((0.0947±0.0021) vs (0.3605±0.0070), p<0.0001) and lowered in REH cells ((0.3109±0.0100) vs (0.1449±0.0185), p<0.0001) after ASNase. Since in our setting ASNase depletes both Asn and Gln, we tested what are the separate roles of Asn and Gln depletion in ASNase treatment. We discovered that omitting Gln from the media caused that TCA cycle intermediates are lowered in both cell lines to the same extent as after ASNase. Moreover, the flux from glucose to TCA cycle resembles the one after ASNase in both cell lines. In contrast the results of Asn withdrawal showed that level of TCA cycle intermediates are unchanged. The relative flux from glucose to TCA cycle is mildly downregulated without Asn in the media in both cell lines (e.g. malate (M+1-M+4): NALM6 - (0.1485±0.0014) vs (0.1158±0.0004), p<0.0001; REH - (0.2662±0.0031) vs (0.2024±0.0015), p<0.0001). Many studies emerged that highlight TCA cycle importance in Asp production and hence in sustaining cell viability and proliferation. Although TCA cycle is diminished after ASNase treatment, our results showed that both NALM6 and REH cells are able to maintain Asp and Glu levels. Using U13C Asp and U13C Glu, we discovered that, unexpectedly, both cell lines are able to import Asp and Glu from the media in a dose-dependent manner. Altogether, our results demonstrate different consequences of Asn and Gln depletion during ASNase treatment. According previous studies it is probably only Asn that is completely depleted after ASNase administration in ALL therapy with unchanged or lowered Gln concentration. Therefore, this fact should be considered in in vitro studies where Gln depletion could blur the effect of Asn depletion. Above that, we showed that leukemia cells are able to uptake Asp and Glu. This indicates the possible way how leukemic cells could cope with ASNase treatment. This work is supported by NV18-07-00129 and Charles University Grant agency 79421. Disclosures No relevant conflicts of interest to declare.

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Du, Jianfeng, Joanna DeSalvo, and Julio C. Barredo. "Mechanisms of Sensitivity of B-Precursor, T-Lineage and BCR/ABL Positive Acute Lymphoblastic Leukemia (ALL) to the Glycolytic Inhibitor 2-Deoxy-D-Glucose (2-DG)." Blood 114, no. 22 (November 20, 2009): 3081. http://dx.doi.org/10.1182/blood.v114.22.3081.3081.

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Abstract Abstract 3081 Poster Board III-18 Novel treatment strategies are needed for patients with ALL diagnosed with resistant phenotypes or after relapse. The glucose analogue 2-deoxy-D-glucose (2-DG) is a glycolytic inhibitor that induces growth arrest and cell death by inhibiting the key glycolytic enzymes phosphoglucose isomerase (PGI) and hexokinase (HK). Cancer cells and hypoxic cells are more sensitive to 2-DG due to their reliance on glycolysis for ATP generation. In this study, we evaluated the antileukemic activity of 2-DG in T- and Bp-ALL subtypes characterized by non-random translocations. The cytotoxicity of 2-DG was determined on CCRF-CEM (PTEN mutant T-ALL), NALM6 (Bp-ALL), REH (TEL/AML1+ Bp-ALL), and the BCR/ABL+ ALL cell lines SupB15 and TOM1. Cells were treated with 4mM 2-DG for 72 h under normoxic vs. hypoxic (0.5% O2) conditions using a hypoxia box chamber (BioSpherix), and growth arrest and cell death were assessed. 2-DG induced 2-13 fold higher cell death in all ALL cells tested under normal O2 conditions compared to hypoxia. Similarly, growth inhibition was greater under normoxia. Both BCR/ABL+ ALL cell lines TOM1 and SupB15 were among the most sensitive, while the PTEN mutant CCRF-CEM (T-ALL) cell model was the least. To analyze the mechanisms of higher sensitivity of ALL cells under normoxia, we first used mannose to assess the role of glycosylation in 2-DG induced ALL cell death. Mannose reversed 2-DG induced cell death in CCRF-CEM cells but only partially in NALM6 cells, indicating inhibition of glycosylation mainly mediated death in CCRF-CEM cells, while inhibition of both glycolysis and glycosylation mediated cell death in NALM6 cells. To further assess other mechanisms leading to cell death in ALL, we evaluated mitochondrial integrity in representative Bp- and T-ALL models by determining cellular respiration with a Clark electrode (Hansatech Instruments). Our data show that cellular respiration in NALM6 was 50% lower compared to CCRF-CEM cells, suggesting that an intrinsic mitochondrial dysfunction was also responsible for 2-DG induced cell death in NALM6 cells. We then evaluated the observed higher sensitivity of BCR/ABL+ ALL cells to 2-DG. The AMPK and PI3K/Akt/mTOR pathways regulate protein, fatty acid and glucose metabolism, and key glycolytic enzymes are known to be upregulated in BCR/ABL+ cells. Therefore, we determined changes on AMPK and PI3K/Akt/mTOR signaling following exposure to 2-DG in SupB15 (BCR/ABL+) and in CCRF-CEM cells, the least sensitive ALL model examined. Western immunoblotting showed that 2-DG led to upregulation of p-AMPK (Thr172) and downregulation of p-mTOR (S2448) and p-p70S6K (Thr389). In both cell lines, 2-DG also led to cell death by triggering an unfolded protein response (UPR) evidenced by CHOP expression and PARP cleavage. We previously demonstrated that activation of AMPK leads to upregulation of Akt as a compensatory survival mechanism in ALL cells (Mol Cancer 6: 46, 2007). On this basis, we then tested induction of cell death by the combination of 2-DG (4 mM) plus Akt inhibitor X (AIX) (12 μM) in CCRF-CEM vs. SupB15 cells under normoxic conditions. Again, SupB15 cells were more sensitive to 2-DG, while AIX alone at low dose (12 μM) had minimal effect on either cell line. When both drugs were used in combination at these same concentrations, significant synergism was seen in CCRF-CEM cells but not in SupB15 cells. Analysis of AMPK, mTOR and Akt in CCRF-CEM cells treated with the combination of 2-DG plus AIX showed higher p-AMPK activation and mTOR downregulation compared to 2-DG alone, while these signaling changes were lower in SupB15 cells. Consistent with these findings, this combination led to higher induction of UPR and PARP cleavage in CCRF-CEM vs. SupB15 cells. We conclude that the greater sensitivity of ALL cells to 2-DG under normoxia is due to concomitant inhibition of glycolysis and/or glycosylation, an inherent mitochondrial dysfunction in some phenotypes, and 2-DG induced changes in AMPK and PI3K/Akt/mTOR signaling leading to the induction of UPR. Concomitant Akt inhibition sensitizes the relatively 2-DG resistant CCRF-CEM cells to death with 2-DG. Chemotherapy resistant BCR/ABL+ ALL cells exhibit significant sensitivity to 2-DG, likely due to their increased reliance on glycolysis for ATP generation. On this basis, we propose that glycolytic inhibitors represent a promising novel therapeutic strategy for ALL, particularly for BCR/ABL+ ALL. Disclosures No relevant conflicts of interest to declare.

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Qin, Haiying, Sang M. Nguyen, Sneha Ramakrishna, Samiksha Tarun, Lila Yang, Nicholas Peter Verdini, Waleed Haso, and Terry J. Fry. "Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia." Blood 130, Suppl_1 (December 7, 2017): 810. http://dx.doi.org/10.1182/blood.v130.suppl_1.810.810.

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Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

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Schramm, Joseph W., Melanie Ehudin, Bing He, Chingakham Singh, Daniel Bogush, Jeremy Hengst, Diwakar Bastihalli Tukaramrao, Arati Sharma, Dhimant Desai, and Sinisa Dovat. "Abstract 3833: Leonurine derivatives as a potential novel therapeutic approach to acute lymphoblastic leukemias (ALL)." Cancer Research 83, no. 7_Supplement (April 4, 2023): 3833. http://dx.doi.org/10.1158/1538-7445.am2023-3833.

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Abstract Background: Pre-B cell Acute lymphoblastic leukemia (B ALL) high risk (HR) subgroups continue to result in significant mortality and morbidity of pediatric oncology patients. T cell ALL is higher risk and therapy has made less improvement than B ALL with a higher rate of significant poor outcomes. Novel treatment strategies are required to overcome chemotherapy resistance and improve mortality/morbidity for HR B ALL. Leonurine is a bioactive alkaloid that is naturally occurring only in Herbra Leonuri which has been used in traditional herbal medicine. Traditionally, it has been used for menstrual disorders. Research has further described its ability to scavenge oxygen free radicals, anticoagulation properties, and other anti-inflammatory properties[1]. There are investigations in its role for myocardial infarction, stroke, chronic kidney disease, and other inflammatory disorders[2]. Our research data that suggests that leonurine and its derivatives have antileukemic effects.Methods/Results: We analyzed multiple derivatives of leonurine and selected a potent candidate based on cell viability assays use for further testing designated as investigational leonurine derivative (ILD). WST1 proliferation studies comparing ILD to vehicle were performed in cell lines Nalm6, 697, Molt4, CEM, and JM1 at multiple time points. The half maximal inhibitory concentration (IC50) values was variable depending on the treatment time and cell line although all values were consistently between 1.2-4.4 uM. Apoptosis activity was determined by flow cytometry Anexin/7AAD assays showed increased apoptosis in cell lines treated with ILD for Nalm6, Molt4, and 697 cell lines. Caspase 3/7 activity was increased in cells treated with ILD when compared to vehicle treatment. DNA damage assays were performed which revealed only an increased frequency in single strand breaks and not double strand breaks. Western blot was performed to determine levels of PI3K, p-AKT, BCL2/BCL-XL and caspases. The blots suggest that apoptosis may be a result of increased activation of PI3K/AKT signaling. We performed RNAseq on cells treated with ILD at the 24-hour time point and present gene ontology data for this analysis. Nalm6, 697, and Molt4 cell lines expressing GFP and Luciferase were injected into NRG mice as means for in vivo pharmacologic testing. NRG mice injected with these cell lines were treated with ILD five days a week for a total of 3 weeks. Nalm 6 leukemia cells showed minimal differences between treatment and control groups.Conclusion: In summary, leonurine derivatives have a promising impact on apoptosis and cell survival. Further investigation into mechanisms and pharmacokinetics/dynamics will be more revealing. Study of leonurine derivatives will result in further translational and therapeutic applications. Citation Format: Joseph W. Schramm, Melanie Ehudin, Bing He, Chingakham Singh, Daniel Bogush, Jeremy Hengst, Diwakar Bastihalli Tukaramrao, Arati Sharma, Dhimant Desai, Sinisa Dovat. Leonurine derivatives as a potential novel therapeutic approach to acute lymphoblastic leukemias (ALL). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3833.

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Jain, Nayan, Zeguo Zhao, Richard Koche, Yosi Gozlan, David Brocks, Tali Raveh-Sadka, Danny Wells, et al. "SUV39H1 disruption imparts functional persistence to CD28-costimulated human CAR T cells." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 122.05. http://dx.doi.org/10.4049/jimmunol.208.supp.122.05.

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Abstract Retrovirally encoded CD19-specific CARs that incorporate CD28 and CD3z signaling motifs (Rv-1928z) have induced remarkable responses in patients with refractory leukemia and lymphoma. These CARs, however, induce a strong effector differentiation program in T cells that can limit their persistence and result in T cell dysfunction. Multiple studies have shown that effector differentiation and eventual dysfunction are associated with transcriptional and epigenetic changes. In this study, we examine the effect of disrupting the histone methyl transferase, SUV39H1 on the functional persistence of human Rv-1928z CAR T cells. SUV39H1 has recently been implicated in regulating memory to effector transition in murine T cells (Pace et al. 2018). To assess the impact of SUV39H1 in human Rv-1928z CAR T cells, we treated immune deficient mice bearing the human ALL cell line, NALM6, with limiting doses of SUV39H1-edited (SUV39H1etd) T cells. SUV39H1etd Rv-1928z CAR T cells have improved anti-tumor efficacy as compared to WT Rv-1928z CAR T cells, with 9/10 NALM6 bearing mice treated with SUV39H1etd Rv-1928z surviving as compared to 1/12 mice treated with WT Rv-1928z over the duration of observation (90 days). This enhanced tumor control in SUV39H1etd Rv-1928z CAR T cells was associated with improved CAR T cell persistence (> day 50). Paired genome accessibility (ATACseq) and transcriptional analysis on SUV39H1etd and WT Rv-1928z CAR T cells that have undergone multiple rounds of tumor rechallenges in vivo revealed epigenetic changes associated with SUV39H1 loss in Rv-1928z CAR T cells that promote expression of memory associated transcription factors and curtail T cell dysfunction in Rv-1928z CAR T cells.

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Singh, Nathan, Noelle V. Frey, Boris Engels, David M. Barrett, Olga Shestova, Pranali Ravikumar, Amy Shyu, et al. "Single Chain Variable Fragment Linker Length Regulates CAR Biology and T Cell Efficacy." Blood 134, Supplement_1 (November 13, 2019): 247. http://dx.doi.org/10.1182/blood-2019-131024.

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We recently conducted a clinical trial of CD22-directed chimeric antigen receptor (CAR) T cells in children and adults with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL). While we did observe some transient responses, overall outcomes were inferior to another recent trial of CD22 CAR T cells in ALL performed at the NCI (Fry, T.J. et al. Nat Med, 2018). Intriguingly, these trials used a CAR that employed the same antigen-binding and intracellular signaling domains, and differed only in the length of linker connecting the variable regions of the single chain variable fragment (scFv). Based on these clinical observations, we sought to identify how the scFv linker impacts CAR biology and regulates CAR-driven T cell activity. The University of Pennsylvania's CD22 CAR contained a long 20 amino acid scFv linker ("CAR22-L") while the NCI's CAR had a 5 amino acid linker ("CAR22-S"). We began by investigating the impact of linker length on CAR biochemistry. Both CAR22-L and CAR22-S had similar antigen-binding affinities (KD of 1.67nM and 6.05nM, respectively). Chromatography revealed that CAR22-L remained monomeric in solution while CAR22-S formed homodimers. To explore how dimerization influenced surface-membrane biology, we developed GFP-tagged versions of each CAR and performed confocal microscopy on CAR+ T cells. CAR22-L exhibited homogenous surface membrane expression, while CAR22-S appeared to self-aggregate and cluster (Fig. 1a). We investigated the impact of this clustering on receptor signaling and found that CAR22-S demonstrated high levels of signaling molecule activation (i.e. Akt, p70-S6 and STAT3) in the absence of antigen engagement. This is consistent with previous reports establishing that CAR clustering can lead to tonic signaling (Long, A.H. et al. Nat Med, 2015). Importantly, this tonic signaling did not lead to autonomous T cell proliferation. We proceeded to evaluate how clustering and tonic signaling impacted CAR function upon antigen engagement. Microscopic evaluation of CAR T cells combined with CD22+ Nalm6 cells revealed greater actin and microtubule organizing complex polarization (P = 0.02 and 0.01, respectively) in CAR22-S cells, consistent with superior immune synapse formation. This was accompanied by increased phosphorylation of PI3K, MAPK and calcium signaling proteins (Fig. 1b) after CAR engagement. RNA sequencing revealed significantly greater activation of immune response gene programs in CAR22-S cells as compared to CAR22-L after overnight exposure to Nalm6. We next investigated the impact that this enhanced receptor-driven activity had on CAR T cell anti-tumor function. CAR T cells were combined with Nalm6 in vitro and residual Nalm6 was serially quantified, revealing that CAR22-S mediated greater tumor control than CAR22-L, particularly at later time periods (P < 0.001). This was associated with greater secretion of IFNg, IL-2 and TNFa (all P < 0.001). Finally, we compared anti-tumor efficacy in xenograft models of systemic Nalm6. NOD/SCID/cg-/- mice were engrafted with Nalm6 and received 1x106 CAR T cells 7 days later. CAR22-S demonstrated greater in vivo expansion (P < 0.0001) and enhanced control of systemic disease (Fig. 1c,P = 0.017), resulting in prolongation of animal survival (Fig. 1d,P = 0.013). Based on these observations, we have designed a novel, affinity-enhanced CD22 CAR and confirmed that shorter linker length improves anti-tumor activity of this CAR. T cells expressing this CAR are currently undergoing evaluation in a phase I clinical trial (ClinicalTrials.org Identifiers NCT03620058 and NCT02650414). Thus far, 4 children and 2 adults have been infused with manageable toxicity. Early outcomes are promising, with 67% achieving complete remission at day 28, compared to 50% in our original CART22 trials. In summary, by investigating the potential mechanisms for an apparent discrepancy in outcomes between two different clinical trials, we demonstrate that reducing the length of the scFv linker results in significant changes to CAR biochemistry that directly lead to antigen-independent receptor activity. In contrast to previously published data demonstrating that tonic signaling of CD28-costimulated CARs is detrimental to T cell function (Long, A.H. et al. Nat Med, 2015), we found that tonic signaling of 4-1BB-costimulated CARs may be beneficial, possibly by priming T cells for rapid response to antigen. Disclosures Singh: University of Pennsylvania: Patents & Royalties. Frey:Novartis: Research Funding. Engels:Novartis: Employment. Zhao:Novartis: Employment. Peng:Novartis: Employment. Granda:Novartis: Employment. Ramones:Novartis: Employment. Lacey:Novartis: Research Funding; Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding. Young:novartis: Research Funding. Brogdon:Novartis: Employment. Grupp:Roche: Consultancy; GSK: Consultancy; Novartis: Consultancy, Research Funding; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards; Cure Genetics: Consultancy. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Maude:Novartis: Consultancy; Kite: Consultancy. Gill:Novartis: Research Funding; Tmunity Therapeutics: Research Funding; Carisma Therapeutics: Research Funding; Amphivena: Consultancy; Aro: Consultancy; Intellia: Consultancy; Sensei Bio: Consultancy; Carisma Therapeutics: Equity Ownership. Ruella:AbClon: Membership on an entity's Board of Directors or advisory committees; Nanostring: Consultancy, Speakers Bureau; Novartis: Patents & Royalties: CART for cancer.

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Singh, Nathan, Olga Shestova, Katharina Hayer, Albert Hong, Pranali Ravikumar, Atushi Hoshino, Ophir Shalem, et al. "CAR T Cell Cytotoxicity Is Dependent on Death Receptor-Driven Apoptosis." Blood 132, Supplement 1 (November 29, 2018): 698. http://dx.doi.org/10.1182/blood-2018-99-117328.

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Abstract Background: T cells engineered to express chimeric antigen receptors targeting the B-cell antigen CD19 (CART19) have demonstrated impressive results in the treatment of lymphoid cancers. Despite these promising outcomes, a significant subset of patients relapse after initial response. To investigate the molecular pathways that drive relapse, we performed an unbiased, CRISPR/Cas9-mediated genome-wide knockout screen in the acute lymphoblastic leukemia (ALL) cell line Nalm6, and found that loss of CD19 was the primary driver of relapse after initial response. This finding is consistent with clinical observations that antigen loss is a primary driver of late disease recurrence, however it fails to address the molecular etiology of intrinsic resistance, which affects ~50% of patients with non-Hodgkin lymphoma and ~20% of patients with ALL, or of late antigen-independent relapse, which accounts for ~40% of relapses in ALL. Identification of the mechanisms regulating CART19 susceptibility is an essential first step in overcoming resistance to this powerful therapy. We hypothesized that genetic alteration in ALL cells were responsible for mediating intrinsic, CD19-independent resistance. To investigate this, we conducted a genome-wide loss of function screen in a model designed to evaluate intrinsic resistance to CART19. Methods: Using a lentiviral guide RNA (gRNA) library containing four distinct gRNAs targeting each human gene (~80,000 gRNAs total), we enabled genome-wide knockout in Nalm6, whereby each target cell lost function of only one gene. This gene-modified cell pool was then exposed to either CART19 or control T cells at a low effector:target ratio (0.25:1) to model the expected in vivo E:T ratio. At 24h, surviving Nalm6 cells were collected and gRNA from these cells underwent next-generation sequencing. Sequenced samples were processed using three distinct genome-scale knockout screen algorithms (MAGeCK, permutation-based non-parametric analysis and ScreenBeam). This pipeline allowed identification of (i) significantly enriched gRNA, postulated to mediate loss of gene function that confers resistance to CART19, and (ii) significantly depleted guides, postulated to mediate loss of gene function that confers sensitivity to CART19. The role of identified genes was then validated in in vitro and in vivo studies. Results: Analysis of gRNA sequencing data from our screen (Figure 1) revealed that the three genes whose loss of function most significantly promoted resistance to CART19 were BID, FADD and CASP8, all of which are key regulators of death receptor-driven apoptosis. TNFRSF10B, encoding the death receptor TRAIL-R2, was also significantly enriched. Interestingly, amongst the 10 genes whose loss most significantly sensitized to CART19 were TRAF2, BIRC2 and CFLAR, all negative regulators of death receptor activity. Pathway analysis of the top 50 genes (25 enriched, 25 depleted) demonstrated significant enrichment in the death receptor pathway, with a false discovery rate of 3.79x10-7. We proceeded to functionally validate the role of BID and FADD in mediating resistance to CART19 by deleting these genes in Nalm6 using de novo designed gRNAs. Strikingly, BIDKO and FADDKO cells were highly resistant to CART19 cytotoxicity in vitro as compared to wild-type Nalm6. Resistance was evident as early as 6 hours after co-culture and was maintained for at least 7 days. Observed resistance to CART19 directly correlated to fraction of KO cells present, suggesting that gene loss was mechanistically responsible for failed CART19 cytotoxicity. We further evaluated the impact of BID or FADD loss on anti-leukemic activity of CART19 in our Nalm6 xenograft model. We observed that BIDKO or FADDKO significantly impaired the anti-leukemic activity of CART19 in vivo. Conclusions: CART19 can cure select patients with B-cell cancers, while others experience transient or no clinical benefit. Using a genome-wide loss of function screen, we identified that death receptor-associated proteins are centrally involved in regulating CART19 cytotoxicity, and that loss of these molecules leads to intrinsic resistance to CART19. These findings are, to our knowledge, the first to characterize the role of death receptors as critical regulators of CART19 cytotoxicity, and suggest that tumor cell modulation of death receptor signaling may drive both inherent resistance and antigen-independent relapse. Disclosures June: Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Gill:Novartis: Research Funding; Carisma Therapeutics: Equity Ownership; Extellia: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ruella:University of Pennsylvania: Patents & Royalties.

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Kitanaka, Akira, Hiroyuki Mano, Mary Ellen Conley, and Dario Campana. "Expression and Activation of the Nonreceptor Tyrosine Kinase Tec in Human B Cells." Blood 91, no. 3 (February 1, 1998): 940–48. http://dx.doi.org/10.1182/blood.v91.3.940.

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Abstract The tyrosine kinase Tec belongs to a new group of structurally related nonreceptor tyrosine kinases that also includes Btk and Itk. Previous studies have suggested that these kinases have lineage-specific roles, with Tec being involved mainly in the regulation of cytokine-mediated myeloid cell growth and differentiation. In this study, we investigated expression and activation of Tec in human B-lymphoid cell lines representing different stages of B-cell maturation, including pro-B (RS4;11, 380, REH), pre-B (NALM6), and mature B (Ramos, and one Epstein-Barr virus [EBV]-transformed lymphoblastoid line) cells. Like Btk, Tec protein was expressed in all B-cell lines tested. Tec was also highly expressed in two EBV-transformed lymphoblastoid lines derived from patients with X-linked agammaglobulinemia (XLA) lacking Btk expression, as well as in tonsillar lymphoid cells. In surface immunoglobulin-positive B cells (Ramos), ligation of the B-cell antigen receptor (BCR) with anti-IgM antibodies caused marked tyrosine phosphorylation of Tec and increased Tec tyrosine kinase activity. Likewise, cross-linking of CD19 with a monoclonal antibody in BCR-negative pro-B (RS4;11, 380) and pre-B (NALM6) cells induced Tec tyrosine phosphorylation and increased Tec autophosphorylation, as well as Btk activation. Tyrosine phosphorylation of Tec, but not of Btk, was detectable in RS4;11 cells after CD38 ligation, suggesting that these kinases are regulated differently. We conclude that Tec is expressed and can be stimulated throughout human B-cell differentiation, implying that this tyrosine kinase plays a role in B-cell development and activation.

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Kitanaka, Akira, Hiroyuki Mano, Mary Ellen Conley, and Dario Campana. "Expression and Activation of the Nonreceptor Tyrosine Kinase Tec in Human B Cells." Blood 91, no. 3 (February 1, 1998): 940–48. http://dx.doi.org/10.1182/blood.v91.3.940.940_940_948.

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The tyrosine kinase Tec belongs to a new group of structurally related nonreceptor tyrosine kinases that also includes Btk and Itk. Previous studies have suggested that these kinases have lineage-specific roles, with Tec being involved mainly in the regulation of cytokine-mediated myeloid cell growth and differentiation. In this study, we investigated expression and activation of Tec in human B-lymphoid cell lines representing different stages of B-cell maturation, including pro-B (RS4;11, 380, REH), pre-B (NALM6), and mature B (Ramos, and one Epstein-Barr virus [EBV]-transformed lymphoblastoid line) cells. Like Btk, Tec protein was expressed in all B-cell lines tested. Tec was also highly expressed in two EBV-transformed lymphoblastoid lines derived from patients with X-linked agammaglobulinemia (XLA) lacking Btk expression, as well as in tonsillar lymphoid cells. In surface immunoglobulin-positive B cells (Ramos), ligation of the B-cell antigen receptor (BCR) with anti-IgM antibodies caused marked tyrosine phosphorylation of Tec and increased Tec tyrosine kinase activity. Likewise, cross-linking of CD19 with a monoclonal antibody in BCR-negative pro-B (RS4;11, 380) and pre-B (NALM6) cells induced Tec tyrosine phosphorylation and increased Tec autophosphorylation, as well as Btk activation. Tyrosine phosphorylation of Tec, but not of Btk, was detectable in RS4;11 cells after CD38 ligation, suggesting that these kinases are regulated differently. We conclude that Tec is expressed and can be stimulated throughout human B-cell differentiation, implying that this tyrosine kinase plays a role in B-cell development and activation.

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Jain, Nayan, Zeguo Zhao, Richard Koche, Yosi Gozlan, David Brocks, Tali Raveh-Sadka, Danny Wells, et al. "Abstract 5583: SUV39H1 disruption enhances the persistence and anti-tumor efficacy of CD28-costimulated human CAR T cells." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5583. http://dx.doi.org/10.1158/1538-7445.am2022-5583.

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Abstract Retrovirally encoded CD19-specific CARs that incorporate CD28 and CD3z signaling motifs (Rv-1928z) have induced remarkable responses in patients with refractory leukemia and lymphoma. These CARs induce a strong effector differentiation program in T cells that can limit their persistence and result in T cell dysfunction. This induction of terminal effector differentiation is accompanied by transcriptional and epigenetic changes resulting in induction of effector transcriptional factors, inhibitory receptors, and suppression of memory associated genes. In this study, we examine the effect of modulating the epigenome of human Rv-1928z CAR T cells by disruption of the histone methyl transferase, SUV39H1, which has been implicated in regulating memory to effector transition in murine T cells (Pace et al. 2018). To assess the impact of SUV39H1 on human Rv-1928z CAR T cells, we treated immune deficient mice bearing the human ALL cell line, NALM6, with limiting doses of SUV39H1-edited T cells. SUV39H1 editing (SUV39H1etd) significantly enhanced the anti-tumor efficacy of Rv-1928z CAR T cells relative to non-edited counterparts, with 9/10 NALM6 bearing mice treated with SUV39H1etd Rv-1928z surviving over the duration of observation (90 days) as compared to 1/12 mice treated with WT Rv-1928z. This enhanced tumor control in SUV39H1etd Rv-1928z CAR T cells was associated with greater initial CAR T cell proliferation upon tumor encounter and enhanced long-term CAR T cell persistence (> day 50). To assess whether the persisting SUV39H1etd Rv-1928z CAR T cells can mount an effective effector response upon tumor rechallenge, we modified the stress test model such that post primary tumor clearance (day 17), mice were rechallenged by tumor 5 times over 70 days. SUV39H1etd Rv-1928z CAR T cells outperformed WT Rv-1928z CAR T cells in eliminating NALM6 upon rechallenge. Paired genome accessibility (ATACseq) and transcriptional analysis revealed epigenetic changes associated with SUV39H1 loss in Rv-1928z CAR T cells that promote expression of memory associated transcription factors and receptors while reducing expression of inhibitory receptors and transcriptional factors associated with T cell dysfunction. In summary, we find that loss of SUV39H1 in human Rv-1928z CAR T cells enhances their anti-tumor efficacy by bringing about changes in their epigenome that enhance their functional persistence. Citation Format: Nayan Jain, Zeguo Zhao, Richard Koche, Yosi Gozlan, David Brocks, Tali Raveh-Sadka, Danny Wells, Anton Dobrin, Yuzhe Shi, Michael Lopez, Gertrude Gunset, Michel Sadelain. SUV39H1 disruption enhances the persistence and anti-tumor efficacy of CD28-costimulated human CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5583.

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Saunders, Philip O., Kenneth F. Bradstock, and Linda J. Bendall. "Dose Escalated RAD001 (Everolimus) Enhances Chemosensitivity in Precursor-B Acute Lymphoblastic Leukemia, through a JNK Dependent Suppression of Cell Cycle Checkpoint Regulation." Blood 112, no. 11 (November 16, 2008): 1604. http://dx.doi.org/10.1182/blood.v112.11.1604.1604.

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Abstract Five year survival for patients with relapsed pre-B ALL remains less than 10%, requiring new approaches to therapy. We sought to evaluate the potential of mTOR inhibitor RAD001 to enhance pre-B ALL cell killing by agents that induce DNA damage or microtubule disruption and identify interactions that may indicate novel approaches to therapy. Combining 16μM RAD001 with agents that cause DNA damage or microtubule disruption in vitro, enhanced caspase-dependent killing (p<0.05) of pre-B ALL cells. We observed 16μM RAD001 suppressed p53 and markedly attenuated p21 responses to DNA damage or vincristine. Lentiviral siRNA knock down of p53 in Nalm6 cells led to significantly increased (p<0.05) cell kill by vincristine relative to luciferase knockdown cells with an intact p53 response. This data indicates enhanced killing by combining RAD001 with DNA damage or vincristine does not require p53. Intracellular flow cytometry revealed that combining 16μM RAD001 with DNA damage or vincristine activates the JNK pathway. c-Jun has been reported to promote proliferation, apoptosis, suppress p53 and p21 promoters and prolong the half-life of p53 analogue, p73. Concordantly, we observed up regulation of p73, puma, bax, bim and cleaved caspase 3, associated with enhanced cell death. This data indicates that p73 provides an alternate pathway to apoptosis. We hypothesized that 16μM RAD001 enhances chemosensitivity through a JNK dependent suppression of cell cycle checkpoint regulation. We observed 1.5μM RAD001 inhibited pRb, Ki67 and PCNA expression, increasing G0/1 cell cycle arrest in response to DNA damage or vincristine, however 16μM RAD001 increased pRb, cyclin D1, Ki67, active CDC2 and PCNA expression. Increased DNA content, BrdU uptake and PCNA expression indicate cell cycle progression occurs in the presence of DNA damage or vincristine, when combined with 16μM RAD001. To validate the role of the JNK pathway in enhancing chemosensitivity we evaluated the impact of JNK inhibition on cell cycle regulation and cell survival. We observed enhanced cell cycle checkpoint regulation, indicated by reduced expression of c-jun, pRb, PCNA and Ki67 in Nalm6 cells. Furthermore, JNK inhibition enhanced G0/1 or G2 arrest in response to DNA damage in Nalm6 and REH cell lines respectively and enhanced G2 arrest in response to vincristine. JNK inhibition led to reduced cell kill by DNA damage or microtubule disruption in Nalm6 and REH cell lines. This data strongly suggests that impaired cell cycle regulation by 16μM RAD001 is mediated through a JNK dependent mechanism. We conclude that dose escalated RAD001 enhances chemosensitivity independently of p53, through a JNK dependent impairment of cell cycle regulation, in response to DNA damage or microtubule disruption. Our data indicates that dose escalated RAD001 has the potential to enhance chemosensitivity in patients with pre-B ALL and provides a rationale for combining agents which induce JNK activation with DNA damage or microtubule disruption, as a therapeutic strategy in pre-B ALL.

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Morisot, Nadege, Sarah Wadsworth, Tina Davis, Nicole Dailey, Kyle Hansen, Denise Gonzalez, Nafees Rahman, et al. "127 Preclinical evaluation of NKX019, a CD19-targeting CAR NK Cell." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A140. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0127.

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BackgroundNatural killer (NK) cells are highly effective and fast-acting cytolytic cells capable of eradicating target cells with limited adverse effects such as cytokine release syndrome (CRS) or graft-versus-host disease. Chimeric antigen receptors (CARs)-engineered NK cells have been recently used against leukemia with encouraging clinical outcomes.1 The surface antigen CD19, expressed by B-lymphoblasts, represents an ideal CAR target against B cell acute lymphoblastic leukemia (B-ALL). We developed a highly potent CD19 -directed CAR NK cell therapy, NKX019, with an extended in vivo half-life aimed at killing CD19-expressing target.MethodsNK cells isolated from healthy PBMCs were expanded in the presence of NKSTIM cells, IL-2, IL-12, IL-18 and transduced with both a CD19-targeted CAR construct and a membrane-bound form of IL-15 (mbIL-15). Control (non-engineered) NK cells were produced in parallel. Cytotoxic activity of NKX019 against CD19+ B-ALL cell line (REH), pre-B ALL cell line (Nalm-6), allogeneic PBMCs was assessed using Incucyte® or flow cytometry. NSG mice bearing either Nalm-6.fluc (Nalm6) or REH.fluc (REH) tumor received different concentrations of NKX019 or control NK cells. In-life analysis of tumor-bearing and naïve NSG mice include: 1) bioluminescence imaging, 2) clinical observations, 3) serum cytokines and 4) CAR+ NK cell persistency.ResultsNKX019 showed enhanced cytolytic activity against REH and Nalm-6 tumor cells compared to control NK cells and CAR19+ T cells. The superiority of NKX019 over CAR19+ T cells was more pronounced at the earlier time point (24 hours) with near identical calculated EC50 observed at 72 hours for both cell types. Increased cytolytic activity of NKX019 was limited to CD19+ cells in bulk PBMCs. Consistent with our in vitro observations, NKX019 controlled Nalm-6 and REH tumor growth in doses as low as 2 × 106 cells/kg for up to 30 days with no apparent increase in cytokines commonly associated with CRS. Increased Nalm-6 tumor growth coincided with an apparent decrease in measurable NKX019 in the periphery. In tumor-naïve NSG mice, NKX019 was detectable in the blood for up to 9 weeks post-infusion consistent with its extended half-life.ConclusionsNKX019 expresses mbIL-15 and is produced in the presence of IL-12 and IL-18, resulting in enhanced in vitro expansion and longer in vivo half-life than non-engineered NK cells. NKX019 also exhibited advantages compared to CAR19+ T cells including faster cytotoxic kinetics and limited production of cytokines associated with CRS. A first-in-human trial of NKX019 in B cell malignancies is planned for 2021.Ethics ApprovalThe animal procedures described in this abstract were conducted in accordance with Explora BioLabs Animal Care and Use Protocol approved by Explora BioLabs Institutional Animal Care and Use Committee.ReferenceLiu, et al. 2020 NEJM

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Ito, Chihiro, Takuya Matsui, Eri Noda, Nijsiri Ruangrungsi, and Masataka Itoigawa. "Biphenyl Derivatives from Garcinia Schomburgkiana and the Cytotoxicity of the Isolated Compounds." Natural Product Communications 8, no. 9 (September 2013): 1934578X1300800. http://dx.doi.org/10.1177/1934578x1300800921.

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Study of the chemical constituents of the stems of Garcinia schomburgkiana Pierre (Guttiferae), collected in Thailand, led to the isolation and identification of five known compounds and two new biphenyl derivatives, schomburgbiphenyl A (1) and B (2). Six phenolic compounds isolated from this plant were screened for their cell growth inhibition activity using several human leukemia cell lines. One compound, oblongifolin C (7), showed significant cytotoxic activity towards Jurkat, NALM6, K562 and HPB-ALL cells.

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Sarno, Jolanda, Pablo Domizi, Yuxuan Liu, Milton Merchant, Dorra Jedoui, Astraea Jager, Andrea Biondi, Giuseppe Gaipa, Alessio Felice Bava, and Kara L. Davis. "Glucocorticoid-Resistant B-Cell Acute Lymphoblastic Leukemic Cells Can be Targeted By BCR-Signaling Inhibition." Blood 138, Supplement 1 (November 5, 2021): 617. http://dx.doi.org/10.1182/blood-2021-150356.

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Abstract Glucocorticoids (GCs) remain a backbone component of therapeutic regimens for childhood B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is a strong prognostic marker of relapse making its understanding an important challenge to be addressed to improve overall patients outcome. Healthy B-cell development is characterized by checkpoints where critical regulatory signaling influences the fate of developing B-cells. These stages are vulnerable for leukemic transformation and as we previously demonstrated, the developmental and functional state of B-ALL cells are of critical importance in treatment failure. Using this developmental framework, we examined the effect of GCs on healthy and malignant B-cells to better understand mediators of GC resistance and ways to overcome it. To define the dynamics of the transcriptional networks surrounding B-cell developmental checkpoints, we performed transcriptomic analysis of sorted pre-proB, pro-B and pre-B cells from 3 healthy donors. We found a coordinated upregulation of B cell receptor (BCR) and Glucocorticoid Receptor (GCR) pathways in healthy B cells during their development. Single cell proteomic analysis of these healthy populations confirmed the coordinated expression of GCR with early B-cell phenotypic markers. Furthermore, in vitro treatment of healthy B-cells with the GC dexamethasone (dex) demonstrated cycling pro-B/pre-B cells to be the most sensitive to GC-induced cell death. Given the importance of GCs in B-ALL treatment, we investigated GCs effects and resistance in NALM6 and REH cell lines. NALM6, expressing high levels of GCR, were significantly sensitive to dex treatment in terms of cell cycle arrest and cell death. By contrast, REH cells were resistant to dex treatment as they lack the GCR. Retroviral transfection of NR3C1 (GCR gene) in REH cells (REH GCR) resulted in acquired sensitivity to dex. To explore the potential crosstalk between GCR and BCR pathways, we also treated cells with BCR signaling inhibitor, dasatinib (das), alone or in combination with dex. While both cell lines survived to treatment with das, the combined treatment increased apoptosis compared to dex alone in NALM6 cells (p=0.0179) and REH GCR cells (p=ns). Whole transcriptome sequencing of dex-resistant cells revealed upregulation of BCR downstream signaling as one of the main pathways associated with resistance. The cell line data implicated active BCR signaling as a path to GCs resistance, so we next analyzed 19 B-ALL primary samples by mass cytometry, after in vitro exposure for 48 hrs to same treatments. Across the entire cohort, dex induced a significant reduction in viability (p=0.0007), compared to vehicle. Treatment with das and dex+das also decreased cell viability compared to vehicle (p=0.0038 and p=0.0002) although was not significant to dex alone. Interestingly dex-resistant cells showed a phenotypic modulation compatible with a late pre-B phenotype with increased CD45 and CD20 expression. In addition, surviving cells showed an activation of downstream targets of BCR signaling such as pSYK, pRPS6 and pCREB that was partially blunted by dasatinib treatment. To understand whether the phenotype and signaling modulation induced by dexamethasone also occurs in patients after treatment with GCs, we analyzed minimal residual disease (MRD) cells following 8 days of treatment with GCs from 9 B-ALL patients. This analysis confirmed our previous findings with MRD cells having same late pre-B cells phenotype and signaling profile as in vitro treated cells. Finally, we tested whether targeting of pre-BCR signaling via dasatinib, could overcome resistance in vivo. We evaluated engraftment of luciferase-expressing NALM6 cells at different timepoints after tail vein injection in mice treated with vehicle, dex, das or dex + das. Bioluminescence analysis revealed a significant reduction of early and late engraftment in the dex + das group compared to vehicle-treated mice. Furthermore, mice receiving the combined treatment also experienced a significant survival advantage as assessed by log rank test (p=0.0002). Taken together these data suggest a coordinated interplay between BCR and GCR pathways in healthy and leukemic B cells. GCs-resistant leukemic cells showed a mature pre-B phenotype that is vulnerable to BCR signaling inhibition in vitro and in vivo suggesting new therapeutic options to overcome GC resistance in childhood B-ALL. Disclosures Biondi: Bluebird: Other: Advisory Board; Novartis: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria. Bava: 10x Genomics: Current Employment. Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.

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Jain, Nayan, Zeguo Zhao, Archana S. Iyer, Michael Lopez, Judith Feucht, Richard P. Koche, Yingqian Zhan, and Michel Sadelain. "Loss of TET2 Uncouples Proliferative and Effector Functions in CAR T Cells." Blood 136, Supplement 1 (November 5, 2020): 1. http://dx.doi.org/10.1182/blood-2020-142957.

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Chimeric antigen receptor (CAR) T cells have opened a new paradigm for the treatment of leukemia and lymphoma. Their production, however, is laborious, requiring tens of millions of CAR T cells per infusion. This constraint could be significantly alleviated if safe and more efficacious T cells could be generated. In a patient with chronic lymphocytic leukemia, treated with anti-CD19 CAR T cells, a recent report described the emergence of a single T cell clone that at its expansion peak accounted for 94% of circulating CAR T cells, coinciding with the development of cytokine release syndrome and tumor regression (Fraietta et. al. Nature 2018). Insertional mutagenesis in this T cell had disrupted an allele of TET2, an epigenetic regulator mediating the oxidation of 5-methylcytosine. The other allele appeared to bear an inherited hypomorphic variant, resulting in the near complete loss of TET2 function in this clone. To understand the mechanisms accounting for this chance clinical finding, we investigated the effect of TET2 loss in human T cells engineered to express different chimeric receptors. Using CRISPR/Cas9, we edited TET2 in T cells engineered to express a CD19-specific second-generation CAR encompassing the costimulatory domain of either CD28 or 4-1BB (Rv-1928z and Rv-19BBz). TET2 disruption enhanced the in vivo anti-tumor activity of Rv-19BBz but not Rv-1928z CAR T cells tested under stress test conditions using limiting CAR T cell doses (as previously described in a human B cell acute lymphoblastic leukemia (B-ALL) NALM6 model, Zhao et. al. Cancer Cell 2015). Since Rv-1928z induces potent effector differentiation but limited persistence compared to Rv-19BBz, we hypothesized that loss of TET2 could amplify the expansion and persistence of 4-1BB-costimulated T cells but not override the differentiation program imparted by Rv-1928z. To test this hypothesis, we utilized two orthogonal approaches known to limit exhaustion and increase persistence of CD28-costimulated CAR T cells, Rv-1928z co-expressed with 4-1BB ligand (Rv-1928z-41BBL) and 1928z driven by the TRAC promoter (TRAC-1928z). Disruption of TET2 enhanced the anti-tumor efficacy of both these CAR T cells and promoted acquisition of a central memory phenotype. However, over time (50-200 days), TET2-edited TRAC-1928z and Rv-1928z-41BBL attained a hyper-proliferative phenotype ultimately requiring euthanasia due to splenomegaly and extensive CAR T cell accumulation in various organs. Post-mortem analysis found no evidence of NALM6 in these mice. This was in contrast to stress test studies with Rv-1928z and Rv-19BBz where most mice succumbed to NALM6 progression. These observations established an essential role for CAR signaling in determining the phenotypic outcome of TET2 loss in T cells. To examine the long-term effects of TET2 disruption in the context of all 4 receptors, we treated human B-ALL bearing mice with curative doses of all 4 CAR T cells and followed them for up to 200 days. We found that all 4 CAR expressing TET2-edited T cells could eventually attain a hyper-proliferative phenotype, but with varying frequency depending on the CAR design (Rv-1928z-41BBL and TRAC-1928z > Rv-19BBz > Rv-1928z). To assess their effector function, NALM6-bearing mice were infused with adoptively transferred hyper-proliferative TET2-edited CAR T cells. Strikingly, these T cells were unable to elicit any tumor control, despite their maintaining a central memory phenotype as assessed by flow cytometry. This loss of effector function was observed for all 4 CAR T cell types, suggesting a discrepancy between function and flow cytometric phenotype. Transcriptional, methylation and genome accessibility studies revealed a unique T cell state wherein the proliferative program is uncoupled from effector response. We identified a unique transcriptional and epigenetic signature that is manifested in a loss of effector function while maintaining robust proliferation. This state stands in contrast to the classically described T cell exhaustion state where loss of effector function is preceded by loss of proliferative ability. TET2 disruption thus promotes a CAR T cell proliferative program that depends on the CAR design but does not in itself enhance anti-tumor activity. Disclosures Sadelain: Atara: Patents & Royalties, Research Funding; Mnemo: Patents & Royalties; Minerva: Other: Biotechnologies , Patents & Royalties; Fate Therapeutics: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding.

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Arnaud, Marie-Pierre, Audrey Vallée, Guillaume Robert, Anne-Gaelle Rio, Elisabetta Dondi, Jacinthe Bonneau, Christine LE ROY, et al. "Disruption Of CD9 Expression Affects Adhesion, Migration and Engraftment Of Pre-B Lymphocytes." Blood 122, no. 21 (November 15, 2013): 1222. http://dx.doi.org/10.1182/blood.v122.21.1222.1222.

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Abstract Introduction CD9 is a membrane protein, member of the tetraspanin family. Recent publications have reported the role of CD9 on engraftment of hematopoietic stem cells, and on cancer stem cell potential. The expression of CD9 has been correlated to the risk of metastases and to a poor clinical outcome in various types of cancer. Surprisingly, CD9 protein is downregulated in ETV6/RUNX1 pre-B acute lymphoblastic leukemia. The purpose of our study is to investigate the effect of CD9 expression on migration and engraftment abilities of pre-B lymphocytes. Materials and Methods The CD9-positive Nalm6 and REH (ETV6/RUNX1) pre-B cells were used. By lentiviral transduction of shRNA targeting mRNA, we generated Nalm6 and REH cell lines depleted in CD9 protein. Engraftment tests were performed in vivo using Nod Scid Gamma immunodeficient mice. REH and Nalm6 cells were detected in bone marrow by CD10 and respectively CD19 or HLA-DR labelling. Ability of the different cell lines to adhere on fibronectin and to migrate through double chambers system in response to CXCL12 were measured in vitro. We also investigated the presence of membrane villosities on REH and REH shCD9 cell surface by scanning electron microscopy. Finally, F-actin polymerization after CXCL12 stimulation was measured by rhodamin-phalloidin labelling. Results In vivo engrafments tests showed that the human cells detected in bone marrow is strongly enriched in CD9 positive cells compared to the initially injected population. This result suggests that CD9 facilitates pre-B lymphobasts homing. An in vitro analysis of adhesion on fibronectin demonstrated that cellular adhesion is dependent on membrane expression of CD9. As well, the more CD9 is expressed, the higher the migration rate in response to CXCL12 chemokine is. The analysis of membrane villosities on REH cell surface revealed that cells over expressing CD9 had longer villosity than shCD9 transducted cell lines. Moreover, F-actin labelling after CXCL12 stimulation showed an increased F-actin polymerization in CD9-positive cells and the formation of actin extensions. Conclusion We provide novel evidence that CD9 is a key player of pre-B lymphoblasts engraftment, adhesion and CXCL12 dependant migration. CD9 expression is related to actin remodelling. We are now investigating a potential link between CD9 and RAC1 activation in response to CXC12. Therefore, the expression level of CD9 could impact leukemic blasts abilities to spread and be responsible of relapses. This work is supported by CNRS, University of Rennes 1, University Hospital of Rennes, la Ligue Régionale contre le Cancer (committee 22, 35 and 56) (MPA, VG, MBT), SFR Biosit UMS 3480 (VG, MBT), Association Laurette Fugain (VG) and Europe Career Integration Grant (MBT). Disclosures: No relevant conflicts of interest to declare.

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Iwanski, Gabriela B., Nils H. Thoennissen, Joy Nakitandwe, Patricia Lin, Norihiko Kawamata, Rahul Nahar, Parham Ramezani-Rad, et al. "Dominant-Negative Impact of PAX5/TEL on Downstream Targets of PAX5 and Essential Pre-B Cell Receptor Genes." Blood 116, no. 21 (November 19, 2010): 3231. http://dx.doi.org/10.1182/blood.v116.21.3231.3231.

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Abstract Abstract 3231 B cell lineage acute lymphoblastic leukemia (ALL) is a common malignancy in childhood, and the pre-B cell receptor (pre-BCR) signalling pathway was previously demonstrated to function as a tumor suppressor. The transcription factor PAX5, a key regulator of B cell development, is frequently involved in chromosomal rearrangements of leukemic blasts. Using high resolution single nucleotide polymorphism (SNP) genomic microarray by us and other groups, several candidate partner genes fused to PAX5 have been detected in samples of pediatric ALL, such as TEL, FOXP1, AUTS2, and C20orf112. Recently, we studied the fusion gene PAX5/TEL and its role in leukemic evolution by Affymetrix HG-U133 plus 2.0 Array of the ALL cell line Nalm6 transfected with a PAX5/TEL construct. PAX5/TEL reduced the expression of PAX5 and its downstream target genes (e.g. CD79A, BACH2, CD19). Moreover, we demonstrated a dominant negative impact of the PAX5/TEL-fusion protein on the binding affinity of wild-type PAX5 to the promoter of CD79A (Iwanski et al., 2009 ASH Abstract No. 3455). To expand our findings, we analyzed the gene expression profile of pediatric ALL samples carrying PAX5/TEL (PAX5/TEL+, n=2) compared to samples with normal PAX5 (n=7) from a genomic ALL study. Samples with normal PAX5 were selected from among 95 B-ALL patients with normal PAX5 status, based on characteristics that most closely matched the two PAX5-TEL+ patients including cytogenetics and current risk stratification. Gene expression data were compiled using the Affymetrix HG-U133A Array, and a heatmap based on the Top 200 probes with the highest expression levels from both sample sets was generated (TIBCO Software Inc.). Notably, the downregulated genes included Bruton agammaglobulinemia tyrosine kinase (BTK; -2.8 fold, FDR < 0.2), an important regulator of pre-BCR signaling, Spleen tyrosine kinase (SYK, -2.3 fold, FDR < 0.2), and IGHM (-5,9 fold; FDR < 0.1), but also significantly up-regulated expression of genes involved in myeloid differentiation, namely Myeloperoxidase (MPO, +24.2 fold, FDR < 0.2), and CCAAT/enhancer binding protein alpha (CEBPA, +3.2 fold, FDR < 0.3), as well as the erythroid genes Aminolevulinate delta-dehydratase (ALAD, +12.2 fold, FDR < 0.2) and the Erythropoietin receptor (EPOR, +7.0, FDR < 0.3). Additionally, we performed a meta-analysis comparing deregulated genes detected in the Nalm6-microarray (PAX5/TEL-MIGR vs. empty vector) to the data from the human B-ALL samples (PAX5/TEL+ vs. normal PAX5). Overall, we identified a set of 35 overlapping genes (FDR < 0.3) that were deregulated in both data sets (21 downregulated, 14 up-regulated). Notably, 10 out of the 21 (47%) downregulated genes are known to be involved in B cell development and BCR signaling, some of them well-recognized as direct PAX5 target genes (e.g. CD79A, CD19, BACH2). Moreover, reporter gene assay with a luciferase reporter construct containing cDNA of the CD19 promoter with PAX5 binding sites (luc-CD19) was performed in Nalm6 cells. Since these cells already express a high level of endogenous PAX5, transcriptional activity of the luc-CD19 reporter plasmid was relatively high in the Nalm6 cells transfected with empty vector, as compared to 293T cells. In contrast, PAX5/TEL-transduced Nalm6 cells displayed a significantly reduced transcriptional activation of the reporter construct (P < 0.01). We also explored if mutation and/or deletion of PAX5 (mut/del PAX5) may have an impact on genes involved in B cell development and the pre-BCR/BCR pathway. Hence, expression files from human ALL samples with mut/del PAX5 (n=50) and B-ALL samples with normal PAX5 (n=95) were analyzed. Notably, only two genes that are known to be involved in B cell development and the pre-BCR pathway were significantly downregulated in samples with mut/del PAX5 compared to normal PAX5, namely CD72, a B cell specific repressor gene activated by PAX5 (-1.51 mean fold, FDR = 0.05), and immunoglobulin heavy constant delta (IGHD), a gene involved in pre-BCR signalling (-1.6 mean fold, FDR = 0.18). These findings suggest no strong influence of mut/del PAX5 on the expression of downstream genes involved in pre-BCR signaling. In conclusion, our results provide further insights into the dominant-negative role of PAX5/TEL and link this fusion gene with the pre-BCR pathway. Disclosures: No relevant conflicts of interest to declare.

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Park, S., A. Register, H. Y. Lee, G. Lutzny-Geier, M. Aigner, M. Andreas, G. Schett, D. Borie, J. Chung, and C. Kaplan. "POS0007 KYV-101, A FULLY HUMAN CD19 CAR T CELL GENERATED FROM AUTOIMMUNE PATIENT LYMPHOCYTES, DEMONSTRATES CAR-MEDIATED AND CD19-DEPENDENT ACTIVITY AGAINST AUTOLOGOUS B CELLS." Annals of the Rheumatic Diseases 82, Suppl 1 (May 30, 2023): 208.2–209. http://dx.doi.org/10.1136/annrheumdis-2023-eular.6228.

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BackgroundA significant unmet medical need remains in the treatment of relapsed and/or refractory B cell-driven autoimmune diseases, including lupus nephritis (LN). The presence of autoantibodies is a hallmark of such diseases and implicates dysregulated B cell function in their pathogenesis. The central role of B cells in these diseases is also supported by the presence of B cells in diseased tissues and the efficacious responses reported with biologic therapies that target B cells. KYV-101 is an autologous CD19 CAR-T cell therapy that depletes pathogenic B cells. Importantly, the CD19 CAR utilized in KYV-101 was previously tested in B-cell lymphoma patients and demonstrated efficacy with an improved safety profile [1]. Since CD19 CAR-T cells target and lyse B cells in both circulation and tissues, a more complete depletion of autoreactive B cells is expected with KYV-101, resulting in better disease control and clinical remission than the current immunotherapies.ObjectivesTo demonstrate the CAR-mediated and CD19-dependent activity of KYV-101 against autologous, patient-derived primary B cells.MethodsAutologous CD4+and CD8+T cells were enriched from healthy donors (HD), systemic lupus erythematosus (SLE), LN or other autoimmune patients. KYV-101 CAR T cells were transduced with a lentiviral vector encoding a fully human single-chain variable fragment (scFV) CD19-targeting domain, a CD8α hinge and transmembrane domain, a CD28 cytoplasmic costimulatory domain, and a CD3ζ cytoplasmic domain. The CAR-mediated and CD19-dependent activity of KYV-101 was monitoredin vitroin a set of cytotoxicity, cytokine release and proliferation studies, in response to either CD19+target cell lines or autologous, patient-derived primary CD19+B cells.ResultsFollowing a 24-hour incubation, KYV-101 generated from HDs or autoimmune patients induced greater and dose-dependent cytotoxicity of both the human CD19+NALM6 cell line and autologous, patient-derived primary B cells than their respective untransduced control T cells. Moreover, an effector cell dose-dependent increase in the production of cytokines such as IFNγ was also observed following co-culture. In contrast, no differences in cytotoxicity nor cytokine production were observed when CD19-target cells (K562 or U937 cells) were co-cultured with KYV-101 or untransduced control T cells. In addition, following a 96-hour incubation, KYV-101 generated from HDs or autoimmune patients proliferated when co-cultured with the NALM6 cells and autologous, patient derived primary B cells, whereas substantially lower levels of proliferation were observed in the untransduced control T cells co-cultured with NALM6 or autologous, patient-derived primary B cells or in KYV-101 and untransduced control T cells co-cultured with the CD19-cell lines K562 and U937.ConclusionKYV-101 generated from autoimmune disease patient lymphocytes demonstrates CAR-mediated and CD19-dependent activity against autologous, patient-derived primary B cells and thus represents a novel therapeutic option for the depletion of pathogenic B cells in autoimmune patients.Reference[1]Brudno JN, et al.,Nat Med, 2020, 26(2):270-280Figure 1.KYV-101-mediated A) cytotoxicity, B) cytokine release and C) proliferation when co-cultured with autologous, patient-derived CD19+human B cells.Acknowledgements:NIL.Disclosure of InterestsSoo Park Employee of: Kyverna Therapeutics, Ames Register Employee of: Kyverna Therapeutics, Ho-Young Lee Employee of: Kyverna Therapeutics, Gloria Lutzny-Geier Grant/research support from: Kyverna Therapeutics, Michael Aigner Grant/research support from: Kyverna Therapeutics, Mackensen Andreas Grant/research support from: Kyverna Therapeutics, Georg Schett Grant/research support from: Kyverna Therapeutics, Dominique Borie Employee of: Kyverna Therapeutics, James Chung Employee of: Kyverna Therapeutics, Charles Kaplan Employee of: Kyverna Therapeutics.

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Khan, Naveed I., Kenneth Francis Bradstock, and Linda J. Bendall. "The Wnt Pathway Modulates Expression of Growth and Survival Genes in Acute Lymphoblastic Leukemia Cells." Blood 108, no. 11 (November 16, 2006): 1850. http://dx.doi.org/10.1182/blood.v108.11.1850.1850.

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Abstract Wnt proteins are important bone marrow-derived growth factors known to support normal hematopoietic progenitor and stem cell development. Here we report that B cell progenitor acute lymphoblastic leukemia (pre-B ALL) cells express Wnt proteins, including Wnt-2b in 33%, Wnt-5a in 42%, Wnt-10b in 58% and Wnt-16b in 25% of cases. The Wnt receptors, Frizzled (Fz)-7 and -8 were also expressed in most cases while Fz-3, -4 and -9 were occasionally detected. Stimulation of pre-B ALL cells with Wnt-3a activated canonical Wnt signaling with increased expression and nuclear translocation of β-catenin. This resulted in a 1.7 to 5.3-fold increase in cell proliferation, which was associated with enhanced cell cycle entry. Wnt-3a also significantly increased the survival of pre-B ALL cells under conditions of serum deprivation. To determine the mechanisms involved we examined the effects of Wnt-3a on gene expression using the leukemic pre-B ALL cell line NALM6 and a cancer specific microarray (GEArray® OHS-802), which contains 440 known cancer genes. Expression of 83 genes (19%) could be detected on the array. Exposure to Wnt-3a for 24 hours resulted in increased (>1.5 fold) expression of 29 genes and reduced (<50% of control) expression of 3 genes. The most highly regulated genes in response to Wnt-3a were MYBL2, E2F1, CD10, VDAC1, CDC25B (upregulated) and TRAIL-R2 (downregulated). Using qRT-PCR, we confirmed regulation of these genes in NALM6 cells and/or in another leukemic cell line LK63. These genes play important roles in the control of cell cycle (MYBL2, E2F1 and CDC25B), apoptosis (VDAC1 and TRAIL-R2) and motility (CD10) in cancer cells. Our results suggest that Wnt signalling regulates cell growth and proliferation in leukemic cells by modulating the expression of a number of genes. To our knowledge this is the first study examining the gene expression profile following Wnt stimulation in leukemic cells and potentially identifies new therapeutic targets for treatment.

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Liu, Xiaojun, David M. Barrett, Shuguang Jiang, Chongyun Fang, Stephan A. Grupp, Carl H. June, and Yangbing Zhao. "Improved Anti-Leukemia Activities of Adoptively Transferred T Cells Expressing Bites." Blood 126, no. 23 (December 3, 2015): 4431. http://dx.doi.org/10.1182/blood.v126.23.4431.4431.

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Abstract Bispecific T cell engagers (BiTEs) that are specific for both a surface target antigen on cancer cells and CD3 on T cells have been used to engage T cells for cancer therapies. However, the development of BiTEs to treat cancer patients still faces enormous challenges. BiTEs need to penetrate well into tumor tissue, where the reactivate tumor resident T cells might have already been tolerized or energized by tumor microenvironments. In addition, the therapeutic potential of exogenously administered BiTEs could be limited by their short half-life and their dissociation from the triggering receptor within a relatively short period of time. RNA electroporation or lentiviral transduction of BiTEs into T cell could potentially enhance cancer treatment efficiency of both BiTEs and T cell adoptive immunotherapy of leukemia. In this study, we examined whether BiTEs could be produced by aCD3/aCD28 Dynal bead stimulated and expanded T cells that were electroporated with mRNA encoding BiTEs (Bis-RNA) and engage T cells specifically targeting tumors, both in vitro and in an aggressive leukemia animal model. We found that T cells with Bis-RNA encoding a CD19-CD3 BiTEs (Blinatumomab) could as efficiently recognize tumor as those T cells with CD19 CAR. We also discovered that the BiTEs secreted by Blinatumomab Bis-RNA transfected T cells could enable non-transfected T cells to specifically recognize CD19 positive tumors in vitro. Moreover, T cells with Bis-RNA showed increased sensitivity of tumor lysis and prolonged functional persistence, comparing to the CD19 CAR T cells. Upon specific antigen stimulation, T cells transfected with Bis-RNA secreted higher cytokines, showed more potent proliferation and less co-stimulation dependency, as well as more resistance to Treg and PD1 induced T cell suppression, than the T cells expressing CD19 CAR. In a Nalm6 leukemia mouse model, the anti-tumor activity of Blinatumomab Bis-RNA T cells showed more sustained anti-tumor activity comparing with CD19 RNA CAR T cells. A fully human CD19 RNA BiTE construct was successfully generated utilizing scFvs of both CD19 and CD3 fully human antibodies and the T cells transferred with this fully human CD19 BiTEs showed similar anti-tumor activities both in vitro and in Nalm6 leukemia model as Blinatumomab BiTEs. To maximize the dose of Bis-RNA electroporated T cells, we used the rapid T cell expansion protocol (REP) that uses OKT3 and IL-2 to stimulate T cells with irradiated allogeneic PBMCs as feeders. We found that this approach expands T cells up to 1000-fold in 10 days, which is 10 times more than aCD3/aCD28 Dynal bead stimulation. The T cells expanded by REP showed enhanced anti-tumor activities in vitro for both CD19 RNA CAR T or Bis-RNA T cells, as evidenced by increased CD137 up-regulation when the T cells were stimulated by CD19+ cell lines, and increased lytic ability, especially when the RNA input dose was limited. Moreover, CAR expression was higher for REP T cells than aCD3/aCD28 Dynal Beads T cells at different times after CAR RNA electroporation, indicating stronger and more persistent anti-tumor activities of REP T cells. To test if the improved anti-tumor activity of REP T cells evidenced in vitro correlates with their in vivo activity, T cells from the same normal donor were stimulated and expanded by either aCD3/aCD28 Dynal beads or REP and electroporated with CD19 CAR RNA or Blinatumomab Bis-RNA. The therapeutic results of those T cells in the NSG/Nalm6 model showed that better control of tumor growth was achieved by using REP CD19 CAR RNA T cells compared to CD19 CAR RNA T cells prepared by bead stimulation (P<0.01). Furthermore, using REP Bis-RNA T cells further improved the treatment results (P<0.05). It was found that treating NALM6 leukemia using REP Bis-RNA T cells led to significantly prolonged survival of the treated mice, which was comparable to what we have seen in the same mouse model treated with CD19 CAR lentivirally transduced T cells. In sumary, BiTEs could be efficiently produced and delivered by T cells to overcome the limitation of short half-life when using exogenously administered BiTEs. Introducing Bis-RNA into T cells for adoptive cell therapy, which not only takes the advantages of CAR based tumor antigen recognition that avoids most common tumor immune evasion mechanism, but also avoids the potential CAR based T cell activation defects, could be developed into a next generation adoptive immunotherapy. Disclosures Liu: University of Pennsylevania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Grupp:Novartis: Consultancy, Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Zhao:Novartis: Patents & Royalties, Research Funding.

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Draper, David W., Derrik Germain, Stacey Roys, Olivia Nelson, and Scott Wise. "Abstract 2749: Preclinical assessment of chimeric antigen receptor (CAR) T persistence and functionality in the disseminated NALM6-Luc human B cell acute lymphoblastic leukemia (ALL) model." Cancer Research 83, no. 7_Supplement (April 4, 2023): 2749. http://dx.doi.org/10.1158/1538-7445.am2023-2749.

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Abstract Preclinical in vivo models are used to profile or refine CAR T therapies before advancing to human studies. Establishing long-term CAR T persistence and efficacy continues to challenge progress in the CAR T space, thus development of robust platforms that can provide longitudinal assessments of CAR T persistence and functionality is paramount. To this end, we used the NALM6-Luc ALL model to develop a flow cytometry platform that provides quantitative analysis of CAR T cells over time as well as surface markers that are documented to correlate with sustained T cell persistence and activation in vivo. Tumored NSG mice were enrolled into treatment groups based on tumor burden calculated from bioluminescence imaging (BLI) data. T cells transduced to express a CD19 CAR (or left untransduced (UTD)) were injected intravenously at 1.0E+07 cells/mouse. Tumor burden was monitored by BLI and flow cytometry was performed weekly on survival bleeds to measure CAR T persistence and examine phenotype. Treatment with CD19 CAR T cells delayed tumor growth resulting in an increase in time to progression of 107.1% compared to UTD controls. However, no regressions were observed. On day 1 post-transfer, CD3+ T cells were detectable in mice that received both CAR T and UTD cells (140+/-26 and 162+/-56 cells/uL blood respectively). T cells declined to near undetectable levels by Day 20, a point when all animals in the UTD treatment group had reached euthanasia criteria. After Day 20, T cells expanded in circulation in the CD19 CAR T treatment group reaching 695+/-327 cells/uL blood by day 40. T cell expansion coincided with exponential tumor outgrowth in the treatment group. To assess T cell functionality, flow cytometry was used to measure the expression of biomarkers for T cell activation (CD25, 4-1BB, and ICOS) and exhaustion markers (TIM-3, PD-1, and LAG-3). CD25, 4-1BB, and ICOS expression did not exceed positivity on more than 15% of CD8+ T cells and peaked by day 30 before downregulation was observed. Notably, PD-1 and LAG-3 expression levels continued to increase throughout the study, suggesting T cells were taking on an exhausted phenotype. Similar trends were observed on CD4+ T cells. To investigate whether the late phase T cell expansion was a graft vs. host response, CAR T cell measurements were compared to non-tumored animals. Expansion as well as PD-1/LAG-3 expression was only observed in tumor-bearing mice indicating the responses were tumor-specific. Taken together, these data demonstrate that CD19 CAR T cells can inhibit NALM6-Luc tumor growth in vivo and expand in circulation in an antigen-specific manner. Furthermore, CAR T cell failure to control tumor growth may be due to onset of an exhausted phenotype. Finally, we demonstrate that flow cytometry can be used to characterize T cell persistence and functionality in murine xenograft tumor models. Citation Format: David W. Draper, Derrik Germain, Stacey Roys, Olivia Nelson, Scott Wise. Preclinical assessment of chimeric antigen receptor (CAR) T persistence and functionality in the disseminated NALM6-Luc human B cell acute lymphoblastic leukemia (ALL) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2749.

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Dolinski, Brian, Vandana Chaturvedi, Daryl Humes, Christina Proano, Mariliis Ott, Jesus Moreno, Garrett Zipp, et al. "Abstract 2734: Modulation of resting T cell status to enhance transduction and CAR T expansion following exposure to CD8-targeted fusosomes." Cancer Research 83, no. 7_Supplement (April 4, 2023): 2734. http://dx.doi.org/10.1158/1538-7445.am2023-2734.

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Abstract Introduction: Fusosomes are novel viral vectors pseudotyped with a modified paramyxovirus envelope targeting specific cell types. We have generated a CD8-targeted fusosome to deliver a CD19-directed chimeric antigen receptor (CAR) transgene to CD8+ T cells in vivo following intravenous (i.v.) injection. Although fusosomes are capable of genetically modifying resting T cells, modulation with chemical or biological agents may increase transduction and facilitate CAR T expansion. Here, we demonstrate that rapamycin and IL-7 enhance resting T cell transduction, and IL-7 supports the expansion of in vivo generated CAR T cells to increase anti-tumor efficacy. Methods: CD8a-targeted fusosome encoding GFP or CD19CAR transgene were generated. To measure the effect on transduction, T cells were pre-treated with IL-7 (15 ng/ml) or rapamycin (30 μM) prior to fusosome exposure. Transduction was assessed by flow cytometry and vector copy number after 7-10 days in culture. CAR T cell function and expansion were assessed in coculture assays with CD19+ Nalm6 tumor cells using live cell imaging. To measure anti-tumor efficacy with a combined fusosome and IL-7 approach, immunodeficient NSG mice were challenged i.v. with Nalm6 tumors expressing firefly luciferase (5E5 cells/mouse) on day -4, PBMC (1E7 cells/mouse) on day -1 followed by CD8/CD19CAR fusosome (1E7 IU/mouse) on day 0. IL-7 (1-5 μg/mouse) was administered subcutaneously twice weekly. Tumor growth was monitored by bioluminescence imaging. Results: CD8/CD19CAR fusosome were effective at transducing resting T cells. Pre-treatment with IL-7 alone resulted in a moderate increase in transduction efficiency (1.5x). In contrast, pre-treatment with both IL-7 and rapamycin further increased transduction efficiency (>5x). As IL-7 could also be used to support CAR T expansion, we tested whether IL-7 post-fusosome exposure could increase CAR T cell numbers and anti-tumor efficacy. In the absence of tumor targets, IL-7 promoted the expansion of CD8+ CAR T cells as well as non-transduced T cells. However, in the presence of tumor antigen, IL-7 treatment resulted in selective expansion of CAR T cells over bystander T cells (40x vs 10x, respectively) in vitro. Importantly, systemic IL-7 treatment following CD8/CD19CAR fusosome administration increased efficacy in Nalm6 tumor bearing NSG mice compared to those treated with fusosome only. Conclusion: T cell-targeted fusosomes encoding a CAR transgene potentially represent a novel, off-the-shelf therapeutic approach for cancer. This study demonstrates that modulation of the resting T cell state can increase susceptibility to fusosome-mediated transduction. The potency of CD8/CD19CAR fusosomes can be increased by pre-treatment of resting T cells with IL-7 and rapamycin to increase transduction or post-treatment with IL-7 to expand CAR T cells to increase anti-tumor efficacy Citation Format: Brian Dolinski, Vandana Chaturvedi, Daryl Humes, Christina Proano, Mariliis Ott, Jesus Moreno, Garrett Zipp, Aaron Lampano, Hadega Aamer, Ouwen Liang, Neal van Hoeven, Kutlu G. Elpek, Aaron E. Foster, Terry J. Fry. Modulation of resting T cell status to enhance transduction and CAR T expansion following exposure to CD8-targeted fusosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2734.

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Leclerc, Guy J., and Julio C. Barredo. "Histone Deacetylase Inhibitor Induces FPGS mRNA Expression in Childhood B-Precursor and T Acute Lymphoblastic Leukemia: Implication of Combination Therapy with Methotrexate To Enhance Cytotoxicity." Blood 110, no. 11 (November 16, 2007): 864. http://dx.doi.org/10.1182/blood.v110.11.864.864.

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Abstract Methotrexate (MTX) is an antifolate widely used to treat childhood acute lymphoblastic leukemia (ALL). MTX is retained within cells as long-chain polyglutamates (MTX-PGs), after metabolism by the enzyme folylpoly-γ-glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR), and the additional inhibition of thymidylate synthetase (TS). The FPGS gene was shown to be regulated by the transcription factors Sp1 and NFY. We performed DNaseI hypersensitive assays and identified a hypersensitive site mapping closely upstream of exon 1 suggesting that chromatin remodeling may contribute to FPGS gene regulation. Using co-immunoprecipitation and Western blotting we investigated the role of histone modifications and chromatin remodeling on the expression of FPGS and uncovered interactions between NFY, Sp1 and HDAC1. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assay (DAPA) revealed that the HDAC1-NFY and HDAC1-Sp1 complex binds to the NFY and Sp1 binding sites in the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by acetylation of NFY and Sp1 factors and interaction with HDAC1, and/or chromatin remodeling. We then examined the effect of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaBu) on the expression of FPGS and other folate-related genes. The level of FPGS, ATP-binding cassette subfamily C (ABCC1), ATP-binding cassette subfamily G (ABCG2), DHFR, γ-glutamyl hydrolase (GGH), solute carrier family 19/folate transporter (SLC19A1), and TS mRNA gene expression was determined by qRT-PCR in NALM6 (Bp-ALL), REH (Bp-ALL, t(12,21)/TEL-AML1), SupB15 (Bp-ALL, t(9,22)/BCR-ABL), and CCRF-CEM (T-ALL) cells treated with NaBu [2mM-5mM]. In all cell lines examined, treatment with NaBu induced 2- to 5-fold the level of FPGS and ABCC1 mRNA expression whereas the level of DHFR, SLC19A1, and TS mRNA expression was decreased. Expression of GGH and ABCG2 mRNAs was increased 2-fold in CCRF-CEM but remained unaltered in Bp-ALL NaBu treated cells. Promoters of butyrate-responsive genes have been shown to contain genetic elements such as Sp1/Sp3 binding sites which interact with HDAC1 to mediate the action of NaBu. On this basis, we hypothesized that pre-treatment of ALL cells with NaBu should lead to induction of FPGS expression and subsequently, higher synthesis of MTX-PG and enhanced MTX cytotoxicity in ALL cells. To test this hypothesis, CCRF-CEM, NALM6, REH, and SupB15 cells were treated sequentially with NaBu (24h) and MTX (4h), and assessed for cell viability. Treatment of NaBu and MTX increased cell death by ∼40% in NALM6, REH, and SupB15 Bp-cells, and ∼60% in CCRF-CCEM cells when compared to treatment with each drug alone. These data suggest that combination of HDACi and MTX may represent a novel therapeutic strategy for treatment of ALL. This strategy may be particularly useful to overcome MTX resistance in patients diagnosed with phenotypes that accumulate low levels of MTX-PGs and for patients after relapse.

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Journal articles on the topic 'Nalm6 cells'

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