Academic literature on the topic 'B cell lymphoma, Chronic Lymphocytic Leukemia, B lymphocytes, mouse models'

Abstract Abstract 3894 p63 is a p53 homolog whose function depends on the cellular context. The full-length TAp63 variant may carry an anti-oncogenic potential in solid tumor models, where it mediates Ras-induced cellular senescence, antagonizes tumorigenesis and suppresses the development of metastases. In hepatoma cells TAp63 is involved in activation of both extrinsic and intrinsic apoptosis pathways. By contrast, ΔNp63, an amino-terminally truncated p63 variant, is oncogenic in tumors of squamous cell origin. TAp63 is the predominantly expressed p63 isoform in lymphoid malignancies. Increased expression of TAp63 in diffuse large B-cell lymphoma confers an unfavorable prognosis. In CLL TAp63 was shown to mediate B-cell homing to the bone marrow, thus possibly contributing to apoptosis evasion. We studied expression of p63 in CLL and determined whether p63 plays a role in CLL B-cell survival and sensitivity to chemotherapy. We enrolled 25 previously untreated subjects with B-CLL at the Norris Cotton Cancer Center (Lebanon, NH). CLL B-cells were isolated from peripheral blood with standard Ficoll-Hypaque technique and purified using a B-cell (CLL) Isolation Kit. Small interfering RNA against p63 were delivered using Lonza Nucleofector with transfection efficiency of 20–50% and viability of 60–80% at 24 h. Viability was enhanced when cells were cocultured with “feeder cells”. To test sensitivity to chemotherapy, cells were treated with 10 μM chlorambucil and 5 μM fludarabine for 48–72 h. For apoptosis analysis cells were stained with Annexin V and 7-AAD and assayed by flow cytometry. Five lymph node tissues were analyzed for p63 expression by immunohistochemistry using a mouse monoclonal p63 antibody (clone 4A4). Of 25 CLL patients 15 were males (60%). Median age was 61 years. Median follow up was 3 years. Most patients presented in Rai stage 0–1 (92%). TAp63 mRNA transcripts were expressed in all CLL samples, while ΔNp63 variant was not detected. TAp63α was the predominantly expressed splicing variant. A C-terminally truncated TAp63γ variant was detected at a low level. TAp63 mRNA transcript levels were higher in CLL B-cells than in normal B-cells, but 10- to 100-fold lower than in high-grade lymphoma cell lines. Of five lymph nodes analyzed, weak nuclear p63 staining was detected in one. In this small cohort, p63 expression in the peripheral blood CLL B-cells did not correlate with ZAP-70 expression. CD38-positive (>30%) CLL samples expressed higher TAp63 mRNA transcript levels (p<0.05). siRNA knockdown of p63 in CLL cells resulted in protection from spontaneous apoptosis at 24–48 h, which persisted when cells were cultured on a “feeder layer”. Furthermore, p63 knockdown conferred protection against chlorambucil and fludarabine. B-cell receptor crosslinking with IgM resulted in a 2.5-fold repression of TAp63 transcription, consistent with the pro-survival role of a B-cell receptor in CLL. Thus, TAp63α is the predominantly expressed p63 variant in the peripheral blood CLL cells. TAp63a contributes to the intrinsic apoptosis program in CLL and may play a role in sensitizing CLL B-cells to standard chemotherapy drugs. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract SCI-27 Targeted therapy in hematologic malignancies has achieved significant therapeutic success when relatively selective inhibition is attainable to a target dispensable to the majority of normal cells. The best appreciated example of this is imatinib in chronic myeloid leukemia (CML), in which a single translocation forms a fusion protein involving the tyrosine kinase ABL that molecularly defines the disease. Kinase inhibition of ABL in this setting by imatinib (or other second- and third-generation kinase inhibitors) promotes durable, long-term remission in CML patients. The great majority of B-cell malignancies lack a characteristic translocation, activating mutation, or other aberration that facilitates such targeted therapy as employed in CML. Nonetheless, a common global signaling pathway involving the B-cell receptor (BCR) pathway has emerged as one that might be important to the control of these diseases. Furthermore, mouse models of select kinase targets (phosphoinositide-3 kinase p110 isoform-delta and Bruton's tyrosine kinase [BTK]) have shown that these are dispensable relative to long-term survival. Indeed, knockout or mutation of BTK and p110 delta each have a modest phenotype outside of diminished B-cell development and function. Based upon these preclinical observations, several molecules targeting BCR signaling have come forward to the clinic with exciting results across a wide range of B-cell malignancies. GS-1101 is a selective phosphoinositide-3 kinase p100 delta inhibitor with a very favorable toxicity profile that has shown promising clinical activity in low-grade lymphoma and chronic lymphocytic leukemia (CLL). The toxicity of this orally administered agent is quite modest in the majority of patients and allows sustained continuous dosing. Similarly, ibrutinib is an irreversible inhibitor of BTK and has shown promising clinical potential in an even broader range of B-cell malignancies, including diffuse large B-cell lymphoma, mantle cell lymphoma, low-grade lymphoma, and CLL. Toxicity with ibrutinib has also been modest, allowing long-term continuous dosing. Notably, each of these agents also produces an atypical mobilization of malignant lymphocytes into the blood soon after treatment. This treatment lymphocytosis is BCR-target-related due to diminished CXCR4/SDF-1 interface between tumor cells and stromal cells in the bone marrow, with egress of these cells to the blood. GS-1101 and ibrutinib are now entering phase III studies for regulatory approval and offer great potential to change the treatment paradigm of both CLL and B-cell non-Hodgkin lymphoma (NHL). Questions moving forward with these agents will include molecular predictors of response, feasibility and efficacy of combining with other effective therapies, and mechanisms of resistance. The scientific session presentation will provide an overview of the most promising BCR signaling agents in CLL and NHL clinical trials. Disclosures: No relevant conflicts of interest to declare.

Abstract Overexpression of antiapoptotic members of the Bcl-2 family is observed in approximately 80% of B-cell lymphomas, contributing to intrinsic and acquired drug resistance. Nullifying the antiapoptotic influence of these proteins can potentially overcome this resistance, and may complement conventional chemotherapy. ABT-737 is a BH3-only mimetic and potent inhibitor of the antiapoptotic Bcl-2 family members Bcl-2, Bcl-XL, and Bcl-w. In vitro, ABT-737 exhibited concentration-dependent cytotoxicity against a broad panel of lymphoma cell lines including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). ABT-737 showed synergism when combined with the proteasome inhibitors bortezomib or carfilzomib in select lymphoma cell lines and induced potent mitochondrial membrane depolarization and apoptosis when combined with either. ABT-737 plus bortezomib also induced significant apoptosis in primary samples of MCL, DLBCL, and chronic lymphocytic leukemia (CLL) but no significant cytotoxic effect was observed in peripheral blood mononuclear cells from healthy donors. In severe combined immunodeficient beige mouse models of MCL, the addition of ABT-737 to bortezomib enhanced efficacy compared with either drug alone and with the control. Collectively, these data suggest that ABT-737 alone or in combination with a proteasome inhibitor represents a novel and potentially important platform for the treatment of B-cell malignancies.

Abstract Introduction & Objectives: Efforts to characterize the heterogeneity of advanced hematologic malignancies using large-scale genomic studies have identified recurrent monoallelic mutations affecting the E571 residue of the essential nuclear exporter, Exportin-1 (XPO1; E571K in ~80% of cases, E571G in ~15% of cases). E571-XPO1 mutations alter the charge and structural basis of the cargo-binding region, disrupting critical biophysical interactions between XPO1 and its' cargos. Enriched in hematologic malignancies, E571-XPO1 mutations are predominantly reported in chronic lymphocytic leukemia (CLL; 5-10% of cases), classical Hodgkin's lymphoma (~25% of cases), and primary mediastinal B cell lymphoma (PMBCL; 25-30% of cases). The subsequent change in XPO1-cargo localization alters the transcriptional profile and overall phenotype of the leukemic cell, with evidence suggesting hyper-active NF-κB and NFAT signaling pathways as leading leukemogenic mechanisms. Moreover, while overall immune dysfunction in CLL leads to infections as a major cause of morbidity and mortality, CLL patients with E571-XPO1 mutations are more susceptible to death by infection, suggesting these mutations may exacerbate the leukemia-induced immunosuppressive phenotype. Similarly, inactivating mutations/deletions to A20 (TNFAIP3 gene), the master regulator of NF-κB, are recurrently reported in several B cell malignancies but most frequently observed in PMBCL (~30% of cases). E571-XPO1 mutations and TNFAIP3 deletions/mutations have been found as co-occurring genetic abnormalities in PMBCL, and while TNFAIP3 mutations in CLL are rare, functional convergence on NF-κB and immune signaling suggests altered XPO1 and A20 activity may have unreported pathogenic significance in CLL. Thus, we aimed to explore the oncogenic and immunologic consequence of co-occurring XPO1 and A20 abnormalities by evaluating a novel in-vivo model recapitulating this scenario. Methods: To explore concurrent aberrations to XPO1 and A20, we developed a novel mouse model to recapitulate this event (Eµ-XPO1xA20 KO). This model was generated by crossing the Eµ-XPO1 transgenic mouse - which overexpresses wildtype (WT), E571K, or E571G-XPO1 under control of a VH promoter-IgH-Eµ enhancer to target transgene expression to immature and mature B cells - with a B cell-specific A20 inactivation mouse (A20 KO) - which lacks functional A20 as a result of Cre recombinase-mediated excision of TNFAIP3 exon 3 via loxP recombination sites flanking this region and Cre recombinase expressed under CD19 promoter/enhancer elements. Eµ-XPO1 and Eµ-XPO1xA20 KO mice were aged and followed, and their B and T cell repertoire was assessed via flow cytometry. Results & Conclusion: We previously demonstrated Eµ-XPO1 mice develop a CLL-like disease (CD19+/CD5+/B220dim B lymphocytes), but leukemia development is significantly delayed - evident between 20-30 months of age. Preliminary analysis in adolescent animals revealed irregular lymphocyte populations as early as 6 months of age in the blood and spleen of Eµ-XPO1xA20 KO mice when compared to non-transgenic and Eµ-XPO1 mice; highlighted by elevated populations of CD93+/CD23+ transitional B cells and CD3+ T cells, and reduced populations of CD21+/IgM+ marginal zone B cells. Moreover, development of a circulating CLL-like disease accompanied by palpable lymphadenopathy and splenomegaly was observed in Eµ-XPO1xA20 KO mice as early as 17-20 months of age, again presenting a distinct immunophenotype inconsistent with that observed in Eµ-XPO1 mice. Additionally, progressive accumulation of CD3+/CD19- T cell leukemia-like populations were observed in a subset of Eµ-XPO1xA20 KO and A20 KO mice, indicating these aberrations may further disrupt and stimulate uncontrolled proliferation affecting the overall immune repertoire. Significance: We report that simultaneous disruption of essential regulators XPO1 and A20 in murine B cells encourages development of irregular B and T cell populations, and can stimulate a progressive CLL-like or T cell leukemia-like expansion. Continued investigation with these models can further our understanding of the relationship between overall immune function and these critical regulatory molecules, and can provide considerable insight to identifying pathways for selective targeting as a personalized therapy in several high-risk cancer types. Disclosures Byrd: AstraZeneca: Consultancy; Takada: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Syndex: Consultancy; Trillium: Consultancy; Vincera Pharmaceuticals: Current equity holder in publicly-traded company.

Abstract Targeting B-cell receptor (BCR) downstream pathways is of therapeutic importance in eradicating chronic lymphocytic leukemia (CLL) cells. Members of the protein kinase C (PKC) family play an important role in B-cell activation. PKC-β has recently been shown to be over-expressed in CLL and essential to CLL development in the TCL1 mouse model. Mice deficient in PKC-β exhibit a survival defect in response to BCR stimulation, correlating with an inability to induce the NF-κB-dependent anti-apoptotic proteins as Bcl-xL and A1. Moreover, PKC-β-dependent activation of NF-κB in stromal cells is pivotal for the survival of B-CLL cells in vivo; wherein PKC-β inhibition was shown to prevent microenvironment protection of CLL. Additionally, PKC-β lies downstream of PLC-γ2 where activating mutations have been noted in BTK (ibrutinib) resistant patients, which conveys a potential mechanism to target resistance related to mutations in this target protein. Therefore exploration of a PCK-β inhibitor in CLL is highly justified and innovative. Sotrastaurin (AEB071) is an orally administered potent inhibitor of classical and novel PKC isotypes; with strong and specific activity on PKC-α, PKC-β and PKC-θ and lesser activity on PKC-δ, PKC-ε, and PKC-η. Pre-clinically, AEB071 has demonstrated in vivo pre-clinical activity in activated B-cell diffuse large B-cell lymphoma (DLBCL) models and is currently being tested for efficacy in CD79b mutated DLBCL. Since PKC-β is indispensable for BCR-induced NF-κB activation and B-cell survival, herein we evaluate the impact of AEB071 on CLL cell survival as a promising therapeutic to target this pathway. Our preliminary work demonstrated that AEB071 was markedly cytotoxic to CLL cells in a dose-dependent (≤6.25uM, p<0.001) and time-dependent manner (p=0.011) as measured by MTS analysis. In a whole blood assay, AEB071 exhibits a retained selective cytotoxicity against tumor cells with a modest reduction in B-CLL cells whereas no effect on T-cells or natural killer cells was detected in CLL patient samples. Notably, upon treatment of blood from healthy subjects, AEB071 showed no toxic effects on normal B-cells, T-cells and natural killer cells. AEB071 inhibits CPG-induced survival of CLL cells in vitro (p<0.01), and effectively blocks the protection induced by soluble factors such as CD40L, IL-4, and TNF (p<0.01), which are known to reduce the spontaneous apoptosis associated with CLL cells. Similar effects were observed with stromal cell contact; wherein AEB071 showed enhanced cytotoxic potency on CLL cells under co-culture conditions with stromal cells compared to CLL alone (p<0.05). Additionally, AEB071 attenuated anti-IgM-induced survival of CLL cells with a modest induction of apoptosis (p<0.001). Furthermore, treatment of PMA- or BCR-activated CLL cells with AEB071 could effectively abrogate downstream survival pathways including ERK1/2, p38MAPK, AKT, GSK3β, and NF-κB as revealed by immunoblot analysis. Collectively, this data indicate that therapeutic strategies to inhibit PKC-β have the potential to disrupt signaling from the microenvironment that lead to in vivo CLL cell survival and potentially drug resistance. Current studies are ongoing to evaluate the in vivo tolerability and therapeutic efficacy of AEB071 in the Eμ-TCL1 transgenic mouse model of CLL. In conclusion, PKC-β represents an innovative target for CLL and therefore, future efforts targeting PKC with the PKC inhibitor AEB071 as monotherapy in clinical trials of relapsed and refractory CLL patients may be warranted. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 360 In CLL, the bone marrow (BM) represents a typical site of involvement and relapse, suggesting a preferential homing of leukemic cells to this anatomical site compared to other lymphoid organs, though the mechanisms controlling CLL cell migration and accumulation within the BM are unclear. In order to define the rules driving in vivo CLL cell re-circulation between the blood and tissutal compartments, we specifically generated two different mouse models and investigated the role played by HS1; this molecule, other than being a putative prognostic factor in CLL, is also involved in cytoskeleton reorganization of lymphocytes, and, potentially, in the control of cellular shape, migration and homing. First, we established a novel transplantable xenograft murine model of CLL by engrafting the cell line MEC1 into RAG2-/-γc-/- mice, at a variance with previous studies in nude mice where MEC cells failed to engraft. Likely due to the lack of B, T and NK cells (while nude mice retain NK cells), RAG2-/-γc-/- animals were successfully transplanted with the CLL cell line through either subcutaneous or intravenous routes, resulting in a systemic blood and tissutal involvement. When subcutaneous MEC1 cells silenced for HS1 expression were injected in these animals, we observed a preferential localization in the tumor draining axillary and inguinal lymph nodes and especially in the BM, when compared to controls. As we have previously demonstrated that CLL cases with hyper-phosphorylated HS1 show a worse clinical outcome, we took advantage of this mouse model to investigate the in vivo homing ability of primary CLL cells from patients showing different HS1 phosphorylation patterns. Purified leukemic cells from 4 patients with hyper-phosphorylated HS1 were labeled with high concentration of the dye CSFE, and each sample was paired and admixed with purified CLL cells obtained from patients with low levels of HS1 phosphorylation and separately labeled with low CSFE concentration. Each pair of samples was injected i.v. into RAG2-/-γc-/- mice recipients. When we analyzed the different organs of the animals by flow-cytometry, the differential expression of CFSE fluorescence (CFSE-high vs CFSE-low) allowed us to distinguish between the two leukemic cell populations with opposite HS1 phosphorylation status. In 3/4 experiments, CLL cells with hyper-phosphorylated HS1 revealed a preferential homing to the BM. Based on these results and on the in vitro evidence that B lymphocytes from HS1-/- mice have an impaired spontaneous migration, we have crossed HS1-/- (H-/-) mice with the Eμ-TCL1 transgenic (Ttg) mouse, an animal model that between 13 and 18 months of age develops a disease resembling human CLL. In the H-/-/Ttg mice, monoclonal CD19+CD5+ cells became evident earlier (at 7-13 months of age) and in significantly higher proportion as compared to Eμ-TCL1 transgenic mice. Cells preferentially localized in the BM where leukemic cells are usually observed at low frequencies in the Eμ-TCL1 mouse (mean value: 28%±16 vs 5%±2, respectively, p=0,008). These findings suggest that HS1 may have a relevant role in both normal and leukemic B-cells and in particular is crucial for cell migration, through its involvement in cytoskeleton organization. Accordingly, we also provide evidence that, in the absence of HS1, cells fail to form actin-myosin complexes, leading to an instability of the cell signalling complex. Our findings suggest a relationship between the expression of HS1 and the development and progression of CLL, most notably in terms of BM involvement, indicating that specific abnormalities in the cytoskeleton organization may be pivotal in regulating leukemic migration and infiltration in selected anatomical sites. This points at HS1 as a target for development of novel cancer treatments, aiming at interfering with the lymphoid tissue infiltration and invasion which is characteristic of the disease. In addition, these animal models could become very useful for evaluating the biological basis of CLL growth and dissemination as well as the efficacy of new therapeutic agents. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Ibrutinib, a first-in-class, once-daily oral inhibitor of Bruton's tyrosine kinase (BTK), is indicated by the US FDA for the treatment of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma, including patients with deletion 17p, patients with mantle cell lymphoma who have received at least 1 prior therapy, and patients with Waldenström's macroglobulinemia. In addition to blocking B-cell activation via inhibition of BTK signaling, ibrutinib was reported to modulate immune function by driving TH1 response through IL-2-inducible T-cell kinase (Dubovsky, Blood 2013). Ibrutinib was also shown to reprogram macrophages toward a TH1 phenotype that fostered CD8+ T-cell cytotoxicity in pancreas ductal adenocarcinoma-bearing mice (Gunderson, Cancer Discov 2016). Both the direct and indirect effects on TH1 response may play a role in the therapeutic efficacy of ibrutinib in models of lymphoma and solid tumors (Sagiv-Barfi, PNAS 2015). Another essential component of the immune system with a role against cancer is natural killer (NK) cells involved in the recognition and elimination of tumor cells. In this study, we evaluated the effects of ibrutinib on NK cell-mediated cytotoxicity in mouse models of B-cell lymphoma. Methods: Experiments in TMD8 xenograft models were performed in CB17.SCID and NSG mice, and experiments in an A20 syngeneic model were performed in BALB/c mice. Mice were orally administered ibrutinib (once daily) for a total duration of 2 weeks starting from the time tumors reached a volume of 100-150 mm3. Tumor BTK occupancy was determined by a gel-based probe assay 4 hours after the last dose of ibrutinib, and was normalized to the total BTK level. NK cell-mediated cytotoxicity was also evaluated in mice carrying X-linked spontaneous mutation in BTK, Btk(xid) mice (009361; Jackson Laboratory). For the NK cell cytotoxicity assay, mouse splenocytes were added to PKH67-labeled YAC-1 cells at different effector-to-target ratios and incubated at 37°C for 4 hours. Propidium iodide was added and flow cytometry was performed to determine target cell viability. Human or mouse cytokines/chemokines were quantified using MILLIPLEX® MAP Kit (HCYTMAG-60K-PX38 and MCYTMAG-70K-PX32). Results: In TMD8 ABC-DLBCL models, tumor suppression after ibrutinib treatment was only observed in CB17.SCID mice but not in NSG mice despite tumor BTK occupancy of >90% in both models. As these 2 strains of mice differ in their NK cell profile (NSG mice lack mature T, B, and NK cells whereas CB17.SCID mice are severely deficient in T and B cells), we were interested in studying the immune modulation function of ibrutinib on NK cells. Interestingly, elevated NK cell cytotoxicity was seen in Btk(xid) mice compared to Btk wild-type mice, suggesting a potential role of BTK depletion in NK cell function. Treatment with ibrutinib increased NK cell-mediated cytotoxicity in both syngeneic A20 B-cell lymphoma and TMD8 xenograft models (Figure 1), but did not affect NK cell population (% of NKp46+ or CD3-CD49b+). A20 tumor-bearing mice had lower NK cell cytotoxicity compared to non-tumor-bearing mice, suggesting suppression of NK cell function by tumors. In addition, ibrutinib reduced tumor-derived cytokines IL-6 and IL-10, which are negative regulators of NK cells. IFN-gamma secreted by nontumor cells was increased in the sera of CB17.SCID but not in NSG mice after ibrutinib treatment, providing further evidence of the increased NK function with ibrutinib. The detailed mechanisms of modulation of NK cell function by ibrutinib is currently under investigation. Conclusions: We report herein that ibrutinib enhanced NK cell-mediated cytotoxicity in mouse models of B-cell lymphomas. In addition to its direct effect on BTK inhibition in tumor cells, these data provide further evidence of the immune modulation function of ibrutinib. The role of ibrutinib in NK cell activation as observed in the current study may further expand the potential application of ibrutinib therapy. Disclosures Kuo: Pharmacyclics, LLC, an AbbVie Company: Employment, Other: Travel, Accommodations, and Expenses, Patents & Royalties: Pharmacyclics, LLC, an AbbVie Company; AbbVie: Equity Ownership. Hsieh:Pharmacyclics, LLC, an AbbVie Company: Employment. Whang:Pharmacyclics, LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. Huang:Juno: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment; Five Prime: Equity Ownership; Merrimack: Equity Ownership. Sirisawad:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment. Chang:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment, Patents & Royalties: Pharmacyclics, LLC, an AbbVie Company.

Abstract Background: Targeted monoclonal antibodies (mAbs) such as the anti-CD20 rituximab, are proven therapies in lymphoma, yet these diseases remain incurable because of primary or acquired resistance. Using a eukaryotic expression system to produce antigen closely representing endogenous protein, we developed a new therapeutic antibody against an alternative lymphoma target. B cell activating factor receptor (BAFF-R/TNFRSF13C) is a tumor-necrosis factor receptor superfamily member specifically involved in B lymphocyte development and mature B cell survival. Although earlier attempts to target the BAFF/BAFF-R axis therapeutically for B cell tumors yielded limited success, BAFF-R remains an attractive target for B cell lymphoma therapeutic antibody development, particularly for rituximab-resistant tumors. Methods and Results: We generated 2 mAbs to human BAFF-R expressed as a natively folded, eukaryotically glycosylated cell-surface immunogen on engineered mouse fibroblast (L) cells. Both mAbs specifically bound BAFF-R-expressing L cells, but not the parental counterparts. Each of the complementarity determining regions (CDRs) of the 2 mAbs are unique, suggesting different binding epitopes. Both mAbs bound with high affinity to the human B cell lymphoma cell lines JeKo-1 (mantle cell lymphoma; MCL), SU-DHL6 (diffuse large B cell lymphoma; DLBCL), Raji (Burkitt's lymphoma) and RL (follicular lymphoma). Because our goal is to develop antibodies for clinical use, we substituted in human IgG1 Fc to generate the chimeric mAbs C55 and C90. The chimeric mAbs retained the binding specificity and affinity of the mouse antibodies to their target cells. By immunohistochemistry C55 and C90 staining was specific to the B cell-containing organs tonsil and spleen. No detectable staining was observed in heart, lung, brain, liver, and kidney. Using primary human natural killer (NK) cells as effectors, we demonstrated the chimeric antibodies induced potent antibody-dependent cellular cytotoxicity (ADCC) against BAFF-R-expressing L cells and the JeKo-1, SU-DHL6, Raji and RL human lymphoma cell lines. C55 and C90 were also able to elicit ADCC in primary human lymphomas; they efficiently killed tumor cells from patients with MCL, DLBCL, follicular lymphoma and chronic lymphocytic leukemia (CLL) (n=8). Notably, 5 of these primary lymphomas were from patients who had relapsed after rituximab treatment (2 MCL, 3 CLL). We next determined the activity of C55 and C90 in models of drug-resistant lymphoma. Both ibrutinib and rituximab are effective anti-lymphoma agents, however, primary or acquired resistance to these drugs is common. We derived a rituximab-resistant JeKo-1 variant (JeKo-1 CD20KO) using the CRISPR/HDR system to knock-out CD20 gene expression, and also used the naturally ibrutinib-resistant (Z-138) lymphoma cell line. We confirmed that the C55 and C90 anti-BAFF-R antibodies induced ADCC in both drug-resistant cell lines in vitro. Using xenogenic tumor models in NOD scid gamma (NSG) mice we observed remarkable in vivo anti-tumor effects of both the C55 and C90 chimeric antibodies. We found our antibodies significantly inhibited growth of implanted Z-138 and JeKo-1 CD20KO lymphomas (P<0.001; Figure). Conclusion: In contrast to previously reported BAFF-R antibodies, our in vitro and in vivo results strongly support the translational development of our novel BAFF-R-specific monoclonal antibodies, especially as an alternative immunotherapy against ribuximab- or ibrunitib-resisitant B cell maglinancies. Other preliminary data also suggest BAFF-R may be an effective target of CAR T cells. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Aggressive B-cell lymphomas occurring in the setting of Chronic Lymphocytic Leukemia (CLL) as a large cell transformation are an important clinical problem, and improved mouse models to test novel and targeted therapeutics are needed. The Eµ-Myc mouse overexpresses c-Myc gene which is placed under control of the Myc promoter and lymphoid-specific IgH enhancer (Eµ), resulting in c-Myc overexpression and spontaneous B-cell lymphoma development. The Eµ-Myc mice have been used in drug development, however malignancy develops at variable ages and with differing genetics and response to therapeutic agents, making drug studies difficult. The Eµ-TCL1 transgenic mouse overexpresses the human TCL1 oncogene, under the control of the B-cell specific IgVH promoter and Eµ enhancer. Mice develop a spontaneous mature B-cell leukemia after a long latency period and represent a well-established model of human CLL. We crossed the Eµ-Myc and TCL1 mice to create a new model of aggressive B-cell lymphoma to test novel therapeutics that would be more homogeneous than the Eµ-Myc model. Methods: Eµ-Myc and TCL1 mice on C57BL/6 background strain were crossed and resulting genotypes were verified by PCR. All mice were kept in standard pathogen-free housing until death or removal from study. All mice born within a 24 month time period were followed for survival. At the time of death, spleen, lymph nodes, bone marrow, and liver were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4uM, and stained with hematoxylin and eosin for histologic evaluation. For engraftment studies, adoptive cell transfer was done by injecting fresh splenic white blood cells (1e7 cells, >75% CD5+/CD19+) into the tail vein of C57BL/6 wild type recipient mice. In the drug treatment experiments ibrutinib (~30mg/kg/day) was administered in drinking water and KPT-8602 (15mg/kg) by daily oral gavage. Survival was shown using Kaplan-Meier curves and survival curves compared statistically using a log-rank test. Results: The Eµ-Myc/TCL1 mice had significantly worse survival (median 45.5, range 29-74 days) compared to Eµ-Myc (median 118, range 42-520 days) or TCL1 (median 359.5, range 188-531 days) mice (p=<0.0001). Survival curves are show in Figure 1. Pathologic examination revealed aggressive B-cell histology similar to Burkitt lymphoma in both the Eµ-Myc mice and Eµ-Myc/TCL1 mice, whereas the TCL1 mice showed mature B-cell morphology. Infiltration of the bone marrow of Eµ-Myc and Eµ-Myc/TCL1 mice was extensive in comparison with TCL1 mice. In all three mouse genotypes the normal architecture in the spleen was effaced by neoplastic cells. Adoptive transfer of Eµ-Myc/TCL1 splenic lymphocytes produced similar pathologic findings to the Eµ-Myc/TCL1 transgenic mice with less severe splenic enlargement in the engrafted mice. Median survival of the engrafted mice was short (38 days). There was a differential response to targeted therapeutics between the groups. Ibrutinib, an irreversible inhibitor of Bruton tyrosine kinase (BTK) previously demonstrated to improve survival in TCL1 mice, had no survival advantage over vehicle treatment in either Eµ-Myc or Eµ-Myc/TCL1 mice (p=0.876 and P=0.83 respectively). KPT-8602, a second generation selective inhibitor of exportin 1 (XPO1) was tested in Eµ-Myc/TCL1 engrafted mice, and demonstrated a significant improvement in survival compared to vehicle alone (p=<0.0001). A first generation agent (selinexor) targeting XPO1 is currently in human clinical trials for aggressive lymphomas, including large cell transformation of CLL. Conclusions: Mice with both c-Myc and TCL1 transgenes develop an aggressive B-cell malignancy and have decreased survival compared to Eµ-Myc or TCL1 mice. The histology of the disease is similar to that of Eµ-Myc mice, but with shorter survival with less variability. The resulting mouse model has differential responses to targeted therapeutics, with a poor response to ibrutinib more similar to aggressive lymphoma than CLL. The malignant B cells can be adoptively transferred into immunocompetent mice for experimental drug treatment studies. These qualities make Eµ-Myc/TCL1 mice a useful tool to test new therapies for aggressive lymphoma where there is an unmet clinical need. Work is currently underway to further define the genetic characteristics of this mouse in comparison to human lymphoma sub-types and responsiveness to targeted agents. Disclosures Byrd: Acerta Pharma BV: Research Funding.

Abstract Previously, we identified and validated PIM-1 as a differentially expressed gene in mantle cell lymphoma (MCL) patient samples. Further, we have shown PIM-1 to be a significant prognostic biomarker in MCL. PIM-1 is an oncogenic serine/threonine kinase that is transcriptionally regulated by cytokines, mitogens, and numerous growth factors. It cooperates with other oncogenes in tumorigenesis and has been implicated in the development of leukemias, lymphomas, late progression events, and most recently in prostate cancer. PIM-1 is overexpressed in aggressive lymphomas, such as the blastoid variant of MCL, and the ABC-type of DLBCL. Here we tested the in vivo cooperation of PIM-1 with TCL1 in murine lymphomagenesis by producing double transgenic murine strains. PIM-1 transgenic mice overexpress murine PIM-1 under the control of the immunoglobulin enhancer Eμ. TCL1 transgenic mice (pEμ -B29-TCL1) fail to downregulate TCL1 expression in mature B and T cells and provide a unique model for mature B-cell malignancies, including Burkitt-like lymphoma (BLL), DLBCL, marginal zone lymphoma, and B-cell chronic lymphocytic leukemia. We hypothesized that PIM-1 would either accelerate TCL1-driven lymphomagenesis, result in the development of immature lymphomas, or both. Lymphoid malignancies were examined by immunohistochemistry and flow cytometry and classified according to the mouse models of human cancer consortium (MMHCC) ‘Bethesda’ classification scheme. Forty double transgenic mice (PIM-1/TCL1) have been generated and observed for a median follow-up of 9 months. To date, 8/40 (20 %) of the PIM-1/TCL1 mice developed lymphomas, in contrast to 9/88 (10%) PIM-1 and 11/49 (22%) TCL1 transgenic mice, with a median follow-up of 7 and 15 months, respectively. A Kaplan-Meyer plot demonstrated statistically significant acceleration of lymphomagenesis in the PIM-1/TCL1 transgenic mice when compared with single TCL1 transgenic mice (p=0.037). PIM-1 transgenic mice developed early (&lt; 7 months of age) T-cell lymphoblastic lymphomas and late (&gt; 20 months of age) DLBCL. TCL1 transgenic mice developed DLBCL, with single occurrences of lymphoblastic, lymphocytic and Burkitt lymphomas. PIM-1/TCL1 transgenic mice developed DLBCL, frequently with extranodal involvement (spleen, liver and lung). A single case of follicular lymphoma was seen. In addition, endogenous expression of PIM kinase family members was investigated in a human lymphoma cell line bank (n=40) by quantitative real-time PCR. PIM-1, PIM-2, and also PIM-3 were found to be overexpressed in cell lines derived from human lymphoid malignancies of multiple histologies. In summary, aberrant PIM-1 overexpression in TCL1 transgenic mice accelerated the development of mature B-cell lymphomas. To date, the classification of lymphomas in PIM-1/TCL1 mice revealed similar histologies as in TCL1 single transgenic mice, mainly DLBCL. The expression of all 3 PIM kinase family members in lymphomas implies that pan-PIM kinase inhibitors should be developed as a potential mechanism of resistance to more restricted PIM inhibitors could be compensatory overexpression of the non-targeted family members.

Despite several recent advancements in the treatment of B lymphoproliferative disorders, still a considerable number of lymphoma cases either cannot be cured or become incurable when relapsing. This issue reflects the need for better and more effective therapies that can be designed once novel pathogenic mechanisms have been designed and suitable preclinical models have been established. Therefore, aim of this thesis was to understand the molecular mechanisms leading to mature B lymphoid malignancies by using different mouse models, in order to identify novel potential therapeutic targets and obtain useful models for preclinical studies. In chapter 2 we show the establishment of a new xenograft model obtained by injecting the CLL cell line MEC1 into immunodeficient Rag2‒/‒gammac‒/‒ mice. This model resembles the aggressive form of human CLL and is conceivably useful to test the efficacy of new therapeutic agents. In chapter 3 we show that HS1 is involved in the trafficking and homing of leukemic B cells and that its deficiency is responsible for an earlier onset of the disease and a reduced survival in the Emu-TCL1 mouse model of CLL. The same animal model was used to investigate the role of TLR pathway in CLL. In chapter 4 we report that the absence of TIR8 accelerates the appearance of the disease and favors the progression into an aggressive form characterized by the accumulation of “prolymphocytoid” cells. Finally in chapter 5 we demonstrate that mice lacking the negative regulator SIGLEC-G are susceptible to B cell lymphoma development with age indicating that the downregulation of SIGLEC10 may be involved in the malignant transformation of human B lymphocytes.