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Haaland, Ingvild, Sigrun M. Hjelle, Håkon Reikvam, André Sulen, Anita Ryningen, Emmet McCormack, Øystein Bruserud, and Bjørn Tore Gjertsen. "p53 Protein Isoform Profiles in AML: Correlation with Distinct Differentiation Stages and Response to Epigenetic Differentiation Therapy." Cells 10, no. 4 (April 7, 2021): 833. http://dx.doi.org/10.3390/cells10040833.
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p53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53 protein isoforms are modulated during epigenetic differentiation therapy in AML, and if p53 isoform expression could be a potential biomarker for predicting a response to this treatment. p53 full-length (FL), p53β and p53γ protein isoforms were analyzed by 1D and 2D gel immunoblots in AML cell lines, primary AML cells from untreated patients and AML cells from patients before and after treatment with valproic acid (VPA), all-trans retinoic acid (ATRA) and theophylline. Furthermore, global gene expression profiling analysis was performed on samples from the clinical protocol. Correlation analyses were performed between p53 protein isoform expression and in vitro VPA sensitivity and FAB (French–American–British) class in primary AML cells. The results show downregulation of p53β/γ and upregulation of p53FL in AML cell lines treated with VPA, and in some of the patients treated with differentiation therapy. p53FL positively correlated with in vitro VPA sensitivity and the FAB class of AML, while p53β/γ isoforms negatively correlated with the same. Our results indicate that p53 protein isoforms are modulated by and may predict sensitivity to differentiation therapy in AML.
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Ghosh, Arnab, Judith Michel, Divya Venkatesh, Riccardo Mezzadra, Lauren Dong, Fadi Samaan, Ricardo Gomez, et al. "Abstract 250: Activating canonical p53 functions in tumor-associated macrophages improves immune checkpoint blockade efficacy." Cancer Research 82, no. 12_Supplement (June 15, 2022): 250. http://dx.doi.org/10.1158/1538-7445.am2022-250.
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Abstract Canonical p53-activated pathways can influence a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages (TAMs). We examined whether p53 activity in the tumor microenvironment (TME) influences antitumor immunity and show that p53 signaling induced pharmacologically with APR-246 (eprenetapopt) can augment the efficacy of immune checkpoint blockade (ICB) in preclinical models, a strategy that is also being tested in patients (NCT04383938). We first investigated the effects of combining APR-246 with ICB in wildtype C57BL6 (B6) mice bearing syngeneic p53 wildtype MC38 colon cancer and B16 melanoma tumors. The combination of an anti-PD-1 antibody (RMP1-14) with APR-246 in mice significantly delayed tumor growth (p < 0.001) and improved survival of tumor-bearing mice, compared to monotherapies (p < 0.01). To further dissect the effects of APR-246 on myeloid and T cells in the TME, we used a conditional knockout of p53 in CSF1R+myeloid cells (CSF1Rcre/p53fl mice), or T cells (CD8cre/p53fl mice). CSF1Rcre/p53fl had loss of tumor control and worse survival with APR-246+anti-PD-1. CD8cre/p53fl had intact tumor control. To study enhanced p53 activity in the TME, we performed flow cytometry, cytokine multiplex and global transcriptional profiling by RNA seq. We found enhanced p53-activity led to increased infiltration of T cells, increased MHC-II expression in TAMs and downregulation of M2-associated cytokines. This was associated with cellular senescence in TAMs and induction of canonical p53-induced senescence-associated secretory phenotype (SASP). Our preclinical findings informed the development of a phase I/II clinical trial using APR-246 with pembrolizumab for patients with advanced solid tumors (NCT04383938). We studied peripheral blood samples from two of the patients with tumor regression and two patients in whom tumors progressed on therapy. We analyzed peripheral blood mononuclear cells (PBMCs) and serum prior to therapy, and at the beginning of cycle 2 and 5 for the patients with tumor control, and at the end of therapy for patients who had progression. Single cell RNA-seq of PBMCs demonstrated a signature consistent with T cell activation and proliferation, and SASP-associated changes in the myeloid compartment as seen in mice. T cell profiling of PBMCs by flow cytometry demonstrated strong proliferation of T cells in patients with tumor control. Serum cytokine analysis demonstrated robust in IL-12, IFN-gamma and Eotaxin-1 in the two responders, which was not seen in the patients whose tumors progressed. Our study illustrates p53-induced SASP in TAMs as a mechanism to reprogram the TME and augment responses to ICB. Ongoing studies will help determine biomarkers that are predictive of response to APR-246+ICB therapy. Citation Format: Arnab Ghosh, Judith Michel, Divya Venkatesh, Riccardo Mezzadra, Lauren Dong, Fadi Samaan, Ricardo Gomez, Nathan Suek, Aliya Holland, Yu-Jui Ho, Mohsen Abu-Akeel, Luis Felipe Campesato, Levi Mark Bala Mangarin, Cailian Liu, Hong Zhong, Sadna Budhu, Andrew Chow, Roberta Zappasodi, Marcus Ruscetti, Scott W. Lowe, Taha Merghoub, Jedd D. Wolchok. Activating canonical p53 functions in tumor-associated macrophages improves immune checkpoint blockade efficacy [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 250.
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Pourebrahimabadi, Rasoul, Zoe Alaniz, Lauren B. Ostermann, Hung Alex Luong, Rafael Heinz Montoya, Peter P. Ruvolo, Steven M. Kornblau, Joseph D. Khoury, Carlos E. Bueso-Ramos, and Michael Andreeff. "p53 Mediated Bone Marrow Mesenchymal Stem Cell Expansion Supports Acute Myeloid Leukemia Development." Blood 134, Supplement_1 (November 13, 2019): 523. http://dx.doi.org/10.1182/blood-2019-127245.
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Acute myeloid leukemia (AML) is a heterogeneous disease that develops within a complex microenvironment. Reciprocal interactions between the bone marrow mesenchymal stem/stromal cells (BM-MSCs) and AML cells can promote AML progression and resistance to chemotherapy (Jacamo et al., 2014). We have recently reported that BM-MSCs derived from AML patients (n=103) highly express p53 and p21 compared to their normal counterparts (n=73 p<0.0001) (Hematologica, 2018). To assess the function of p53 in BM-MSCs, we generated traceable lineage specific mouse models targeting Mdm2 or Trp53 alleles in MSCs (Osx-Cre;mTmG;p53fl/fl and Osx-Cre;mTmG;Mdm2fl/+) or hematopoietic cells (Vav-Cre;mTmG;p53fl/fl and Vav-Cre;mTmG;Mdm2fl/+). Homozygote deletion of Mdm2 (Osx-Cre;Mdm2fl/fl) resulted in death at birth and displayed skeletal defects as well as lack of intramedullary hematopoiesis. Heterozygote deletion of Mdm2 in MSCs was dispensable for normal hematopoiesis in adult mice, however, resulted in bone marrow failure and thrombocytopenia after irradiation. Homozygote deletion of Mdm2 in hematopoietic cells (Vav-Cre;Mdm2fl/fl) was embryonically lethal but the heterozygotes were radiosensitive. We next sought to examine if p53 levels in BM-MSCs change after cellular stress imposed by AML. We generated a traceable syngeneic AML model using AML-ETO leukemia cells transplanted into Osx-Cre;mTmG mice. We found that p53 was highly induced in BM-MSCs of AML mice, further confirming our findings in primary patient samples. The population of BM-MSCs was significantly increased in bone marrow Osx-Cre;mTmG transplanted with syngeneic AML cells. Tunnel staining of bone marrow samples in this traceable syngeneic AML model showed a block in apoptosis of BM-MSCs suggesting that the expansion of BM-MSCs in AML is partly due to inhibition of apoptosis. As the leukemia progressed the number of Td-Tomato positive cells which represents hematopoietic lineage and endothelial cells were significantly decreased indicating failure of normal hematopoiesis induced by leukemia. SA-β-gal activity was significantly induced in osteoblasts derived from leukemia mice in comparison to normal mice further supporting our observation in human leukemia samples that AML induces senescence of BM-MSCs. To examine the effect of p53 on the senescence associated secretory profile (SASP) of BM-MSCs, we measured fifteen SASP cytokines by qPCR and found significant decrease in Ccl4, Cxcl12, S100a8, Il6 and Il1b upon p53 deletion in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) compared to p53 wildtype mice. To functionally evaluate the effects of p53 in BM-MSCs on AML, we deleted p53 in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) and transplanted them with syngeneic AML-ETO-Turquoise AML cells. Deletion of p53 in BM-MSCs strongly inhibited the expansion of BM-MSCs in AML and resulted in osteoblast differentiation. This suggests that expansion of BM-MSCs in AML is dependent on p53 and that deletion of p53 results in osteoblast differentiation of BM-MSCs. Importantly, deletion of p53 in BM-MSCs significantly increased the survival of AML mice. We further evaluated the effect of a Mdm2 inhibitor, DS-5272, on BM-MSCs in our traceable mouse models. DS-5272 treatment of Osx-cre;Mdm2fl/+ mice resulted in complete loss of normal hematopoietic cells indicating a non-cell autonomous regulation of apoptosis of hematopoietic cells mediated by p53 in BM-MSCs. Loss of p53 in BM-MSCs (Osx-Cre;p53fl/fl) completely rescued hematopoietic failure following Mdm2 inhibitor treatment. In conclusion, we identified p53 activation as a novel mechanism by which BM-MSCs regulate proliferation and apoptosis of hematopoietic cells. This knowledge highlights a new mechanism of hematopoietic failure after AML therapy and informs new therapeutic strategies to eliminate AML. Disclosures Khoury: Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding. Bueso-Ramos:Incyte: Consultancy. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy. OffLabel Disclosure: Mdm2 inhibitor-DS 5272
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Pourebrahim, Rasoul, Peter P. Ruvolo, Steven M. Kornblau, Carlos E. Bueso-Ramos, and Michael Andreeff. "Genetic Dissection of p53 Driven Senescence of Bone Marrow Mesenchymal Cells in Acute Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 2625. http://dx.doi.org/10.1182/blood-2018-99-117585.
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Abstract Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy characterized by bone marrow infiltration of abnormally proliferating leukemic blasts which results in fatal anemia, bleeding and infectious complications due to compromised normal hematopoiesis. Patients with complete remission (CR) but incomplete blood cell count recovery (CRi) have significantly shorter survival compared to CR patients. Although there is a correlation between CRi and minimal residual disease (MRD), the two variables were shown to be independent risk factors for relapse development (1). The mechanism by which AML induces bone marrow failure in patients is largely unknown. Here, we demonstrate that AML derived MSCs highly express p53 and p21 proteins and are more senescent compared to their normal age-matched controls as demonstrated by high β-galactosidase staining (figure 1. A, B&C). Emerging evidence indicates that the aging of endosteal niche cells results in lower reconstitution potential of hematopoietic stem cells (2). To functionally evaluate the effects of AML on bone marrow MSCs, we utilized a murine leukemia model of the AML microenvironment. We transplanted Osx-Cre;mTmG mice with AML cells and compared the senescence of MSCs in normal bone marrow (Figure 1.D) with AML (Figure 1.E). Consistent with our initial findings in human, AML strongly induced senescence of osteoblasts. This suggests that AML suppresses normal hematopoiesis by inducing senescence in the hematopoietic niche. To address the role of p53 signaling in senescence of MSCs we generated a traceable conditional p53 gain/loss model specifically in bone marrow MSCs using Osx-Cre;mTmG; Mdm2fl/+ and Osx-Cre;mTmG;p53fl/fl mice respectively (Figure 1.F). Deletion of p53 in bone marrow MSCs resulted in an increased population of osteoblasts (GFP+) in Osx-Cre;mTmG;p53fl/fl mice in comparison to Osx-Cre;mTmG mice suggesting that p53 loss in osteoblasts inhibits senescence of osteoblasts. In order to evaluate p53 activity after recombination of p53fl alleles in the osteoblasts, we isolated MSCs from bone marrows and analyzed the expression of p21.P21 was significantly down regulated in osteoblasts (GFP+) derived from Osx-Cre;mTmG;p53fl/fl mice whereas its expression in the hematopoietic cells from same tissue (tdTomato+) remained comparable to p53 wild type suggesting that p21 as the master regulator of senescence is regulated by p53 in bone marrow mesenchymal cells. To evaluate the effect of p53 loss in osteoblasts and its impact on hematopoietic cells, we isolated the GFP+ cells (osteoblasts) and RFP + cells (hematopoietic) by FACS. Senescent cells, non-cell autonomously, modulate the bone marrow microenvironment through the senescence-associated secretory phenotype (SASP). We analyzed the expression of fifteen SASP cytokines by QPCR. Deletion of p53 in bone marrow mesenchymal cells strongly abrogated the expression of several SASP cytokines. Interestingly several Notch target genes such as Hey1 and Hey2 were highly induced in MSCs following p53 deletion suggesting a role for Notch signaling in hematopoietic failure following AML induced MSCs senescence. Our data suggest that AML induces senescence of endosteal niche resulting in hematopoietic failure. These findings contribute to our understanding of the role of p53 in leukemia MSCs and could have broad translational significance for the treatment of hematopoietic failure in patients with AML.Chen X, et al. (2015) Relation of clinical response and minimal residual disease and their prognostic impact on outcome in acute myeloid leukemia. J Clin Oncol 33(11):1258-1264.Li J, et al. (2018) Murine hematopoietic stem cell reconstitution potential is maintained by osteopontin during aging. Sci Rep 8(1):2833. Disclosures Andreeff: Astra Zeneca: Research Funding; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SentiBio: Equity Ownership; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Jazz Pharma: Consultancy; Reata: Equity Ownership.
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Verdeil, Gregory, Marine Leblond, Claire Imbratta, and Daniel E. Speiser. "Combination of anti-CD40 and anti-PD1 revert M2 polarization to limit tumor growth in a genetically engineered blander cancer mouse model." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 194.3. http://dx.doi.org/10.4049/jimmunol.202.supp.194.3.
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Abstract Bladder cancer (BCa) represents the fourth most common cancer in men with a poor patient prognosis for advanced disease. About 75% of patients present non-muscle invasive BCa (NMIBC) and 25% present muscle invasive BCa (MIBC) or metastatic disease. Although BCG immunotherapy is a common treatment of BCa, the dynamics of immune cell infiltration and its relevance for disease outcome are still not well understood. We took advantage of a genetic based mouse model of MIBC to obtain a profound understanding of the immune contexture over tumor development. P53Fl/Fl/PTENFl/Fl mice were injected with Cre recombinase-expressing adenoviral vectors directly into bladder by micro-surgery. We observed the presence of NMIBC, MIBC and the development of metastases at 5, 9 and 13 weeks after vector injection, respectively. As tumor evolves, we observed a transition from an anti-tumoral microenvironment (MHCIIhigh TAM, CD4 and CD8 TILs) to a pro-tumoral one (MHCIIlow TAM and MDSC), going along with an increased expression of PD-1 by T cells and accumulation of regulatory T cells. After testing various immunotherapy tretaments on mice with MIBC, we found out that combination of αCD40+αPD1 increases CD8 TILs infiltration and IFNg production, re-polarized TAM toward a M1-like phenotype and increased the survival of the mice.
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Guo, Xiaofeng, Yan Liu, Jessica L. Kim, Emily Y. Kim, Edison Q. Kim, Alexandria Jansen, Katherine Li, et al. "Effect of cyclical intermittent hypoxia on Ad5CMVCre induced solitary lung cancer progression and spontaneous metastases in the KrasG12D+; p53fl/fl; myristolated p110fl/fl ROSA-gfp mouse." PLOS ONE 14, no. 2 (February 27, 2019): e0212930. http://dx.doi.org/10.1371/journal.pone.0212930.
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Du, Ziwei, Miltiadis Tsesmelis, and Thomas Wirth. "Abstract A059: NF-κB inducing kinase (NIK) deletion accelerates the progression of pancreatic cancer." Cancer Research 82, no. 22_Supplement (November 15, 2022): A059. http://dx.doi.org/10.1158/1538-7445.panca22-a059.
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Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and the leading cause of cancer-related deaths. The canonical NF-κB pathway displays critical functions in the development of PDAC. However, the relationship between the non-canonical NF-κB pathway and PDAC is still obscure. Here, we analyzed the role of the non-canonical NF-κB signaling in precancerous lesions and PDAC development by deleting NF-κB inducing kinase (NIK), an essential kinase for the activation of the non-canonical pathway. We used KC mice (Pdx1-cre/LSL-KrasG12D) that develop pancreatic intraepithelial neoplasias (PanINs) already at the age of 8 weeks. To evaluate the role of the non-canonical NF-κB signaling, KC mice were crossed with NIK-floxed mice (NiC) to generate the KNiC (Pdx1-cre/KRASG12D/NIKfl/fl) mouse model. Mice were injected with cerulein to promote the development of pancreatitis and support the development of PanINs. At the age of 8 weeks, mice were sacrificed and analyzed. Furthermore, to analyze the role of the non-canonical pathway in PDAC development, we crossed -the rapidly PDAC developing- KPC mice (Pdx1-cre/LSL-KrasG12D/p53fl/fl) to NiC mice and generated the KPNiC (Pdx1-cre/KRASG12D/NIKfl/fl/p53fl/fl) mouse model. Mice were analyzed at the age of 8 weeks or when they reached their humane endpoint. Analysis of the pancreata revealed that deletion of NIK supported the development of higher-grade precancerous lesions and an increased remodeling area. Importantly, NIK deletion accelerated the establishment of PDAC already by the age of 8 weeks. In addition, the pancreatic/body weight ratio was significantly higher in the KNiC group compared to the KC group (Mean: KC 0.018 vs KNiC 0.030, P<0.05). These results were further verified by Ki67 staining, where we detected more proliferating cells in KNiC pancreata (Mean: KC 66.53/mm2 vs KNiC 205.60/mm2, P<0.05). Moreover, FN1 expression level (Mean: KC 1.00 vs KNiC 2.14, P<0.05) and the number α-SMA+ cells (Mean: KC 6.73% vs KNiC 17.47%, P<0.05) were dramatically elevated in KNiC pancreata, indicating increased desmoplastic reaction after NIK deletion. Interestingly, KNiC pancreata displayed stronger activation of the STAT3 pathway which is associated with PanIN development and progression (Mean: KC 1.00 vs KNiC 1.73, P<0.05). With respect to the KPC and KPNiC mice, NIK deletion strongly accelerated the development of PDAC at the age of 8 weeks (83.3% of KPNiC mice, 25% of KPC mice). Finally, the median survival of KPNiC mice (71 days) was remarkably shorter than the survival of KPC mice (85 days). Our data demonstrate that deletion of NIK in KC mice promoted the progression of precancerous lesions, a stronger fibrotic reaction, and activation of the STAT3 pathway. Further, deletion of NIK in KPC mice accelerated cancer development and shortened the lifespan of the mice. These results indicate that inactivation of the non-canonical NF-κB pathway leads to PDAC deterioration. Analysis on later time points and further examination of the mechanism is ongoing. Citation Format: Ziwei Du, Miltiadis Tsesmelis, Thomas Wirth. NF-κB inducing kinase (NIK) deletion accelerates the progression of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A059.
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Lanfranca, Mirna Perusina, Ilona Kryczek, Andrew Rhim, Alexander Girgis, Jenny Lazarus, Marina Pasca Di Magliano, Weiping Zou, and Timothy Frankel. "IL-22 Promotes Pancreatic Cancer Tumorigenesis through Induction of Stemness and Epithelial to Mesenchymal Transition." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 66.22. http://dx.doi.org/10.4049/jimmunol.198.supp.66.22.
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Abstract Elevated levels of Interleukin 22 (IL-22) and its receptor (IL-22R) are associated with poor prognosis in pancreatic ductal adenocarcinoma (PDAC) and the underlying mechanism is currently unknown. Our aim is to investigate the role of the IL-22 axis in PDAC initiation and progression. Impact of IL-22 on tumor initiation was assayed by subcutaneous and intravenous inoculation of PDAC cells into wild-type and IL-22−/− mice. The PKCY (Pdx1-Cre; KrasG12D; p53fl/+; RosaYFP) model of pancreas cancer was used to study the in-vivo presence and significance of IL-22 in spontaneous tumors. Results were confirmed in human specimens of surgically resected pancreas cancer. IL-22R was present in all tested PDAC lines and IL-22 treatment led to STAT3 phosphorylation and subsequent increased expression of EMT (Epithelial to Mesenchymal Transition) transcription factors. Cells transitioned to a mesenchymal phenotype and robust tumor sphere formation was observed. While tumors readily formed in wild-type mice, initiation, establishment and growth were impaired in IL-22−/− mice and PKCY-IL-22−/− mice. Increased levels of IL-22 were found in both spontaneous murine tumors and surgical specimens compared to control tissue. IHC of tumors showed diffuse IL-22R staining with increasing intensity of pSTAT3 and EMT markers as tumors progressed to invasive cancer. FACS analysis identified type 3 innate lymphoid cells and TH22 cells as the source of IL-22 in both human and murine PDAC. Our data suggests that IL-22 is integral in the initiation, progression and establishment of pancreatic cancer, positioning it as an attractive target for cancer therapy.
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Shimazu, Yosuke, Agila Somasundaram, Daniel Brat, and Oren Becher. "PDTM-13. A NOVEL DIFFUSE MIDLINE GLIOMA MODEL INITIATED IN OLIG2-EXPRESSING PROGENITORS OF THE NEONATAL BRAIN." Neuro-Oncology 21, Supplement_6 (November 2019): vi189. http://dx.doi.org/10.1093/neuonc/noz175.789.
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Abstract Diffuse intrinsic pontine glioma (DIPG) is an incurable brain tumor that arises in the pons of children. Recent studies using single cell RNA-seq and enhancer analysis of DIPG tumor cells, together with analysis of the developing human pons, strongly suggest that an oligodendrocyte progenitor cell is the most likely cell-of-origin for DIPG. Here we describe a novel mouse model by expressing PDGF-B, with H3.3K27M or H3.3 wild-type in Olig2-expressing progenitors via injection into the 4th ventricle using Olig2-tva-cre;p53fl/fl mice. H3.3K27M tumors have high rate of Ki-67, Sox2, and Olig2 positivity and show a higher rate of leptomeningeal dissemination than H3.3 wild-type tumors (95.2％ vs 68.8%, p=0.0303) and mice harboring H3.3K27M tumors demonstrate a significantly shorter survival period than those harboring H3.3 wild-type tumors (31 days vs. 37 days, p=0.0473). While there is not any difference in survival between mice harboring PDGF-B; p53 wild-type; H3.3K27M tumors and those harboring PDGF-B; p53 wild-type; H3.3 wild-type tumors at the 6-month endpoint, we found that H3.3K27M tumors show a higher rate of high-grade glioma than H3.3 wild-type tumors (100% vs. 41.7%, p=0.0017). RNA-seq analysis identified 25 significantly differentially expressed genes with 23 upregulated and 2 downregulated genes in the PDGF-B; p53 null; H3.3K27M tumors compared with the H3.3 wild-type tumors. Phox2b, which is the most upregulated gene in the PDGF-B; p53 null; H3.3K27M tumors, was validated by qRT-PCR and expressed only in brainstem tumors and not expressed in tumors located in any other areas. IHC with Phox2b also revealed positivity in PDGF-B; p53 null; H3.3K27M tumors located only in the brainstem. Ongoing work includes validation of other significant differentially expressed genes as well as elucidation of their role in K27M-mediated gliomagenesis.
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Singh, Ranjodh, Vanessa Bellat, Melinda Wang, Melanie E. Schweitzer, Y. Linda Wu, Ching-Hsuan Tung, Mark M. Souweidane, and Benedict Law. "Volume of distribution and clearance of peptide-based nanofiber after convection-enhanced delivery." Journal of Neurosurgery 129, no. 1 (July 2018): 10–18. http://dx.doi.org/10.3171/2017.2.jns162273.
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OBJECTIVEDrug clearance may be a limiting factor in the clinical application of convection-enhanced delivery (CED). Peptide-based nanofibers (NFPs) have a high aspect ratio, and NFPs loaded with drugs could potentially maintain effective drug concentrations for an extended period sufficient for cancer therapy. The objective of this study was to assess the volume of distribution (Vd) and clearance of variable lengths of NFPs when administered using CED.METHODSNFPs composed of multiple methoxypolyethylene glycol (mPEG)-conjugated constructs (mPEG2000-KLDLKLDLKLDL-K(FITC)-CONH2, for which FITC is fluorescein isothiocyanate) were assembled in an aqueous buffer. The NFPs were approximately 5 nm in width and were formulated into different lengths: 100 nm (NFP-100), 400 nm (NFP-400), and 1000 nm (NFP-1000). The NFP surface was covalently conjugated with multiple Cy5.5 fluorophores as the optical reporters to track the post-CED distribution. Forty-two 6- to 8-week-old Ntv-a;p53fl/fl mice underwent CED to the striatum. Animals were killed immediately, 24 hours or 72 hours after CED. The brains were extracted and sectioned for assessing NFP Vd to volume of infusion (Vi) ratio, and clearance using fluorescence microscopy.RESULTSCED of NFPs was well tolerated by all the animals. The average Vd/Vi ratios for NFP-100, NFP-400, NFP-1000, and unconjugated positive control (free Cy5.5) were 1.87, 2.47, 1.07, and 3.0, respectively, which were statistically different (p = 0.003). The percentages remaining of the original infusion volume at 24 hours for NFP-100, -400, and -1000 were 40%, 90%, and 74%, respectively. The percentages remaining at 72 hours for NFP-100, -400, and -1000 were 15%, 30%, and 46%, respectively. Unconjugated Cy5.5 was not detected at 24 or 72 hours after CED.CONCLUSIONSCED of NFPs is feasible with Vd/Vi ratios and clearance rates comparable to other nanocarriers. Of the 3 NFPs, NFP-400 appears to provide the best distribution and slowest clearance after 24 hours. NFP provides a dynamic theranostic platform, with the potential to deliver clinically efficacious drug payload to brain tumor after CED.
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Cockle, Julia, Lynn Bjerke, Alan Mackay, Yura Grabovska, Anna Burford, Valeria Molinari, Rita Pereira, et al. "IMMU-12. Exploring and modulating the tumour immune microenvironment to facilitate the selection of immunotherapies for paediatric-type diffuse high-grade glioma." Neuro-Oncology 24, Supplement_1 (June 1, 2022): i83—i84. http://dx.doi.org/10.1093/neuonc/noac079.305.
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Abstract Immune cells have the potential to selectively eradicate high-risk brain tumours such as paediatric-type diffuse high-grade glioma (PDHGG). We aim to characterize the tumour immune microenvironment (TIME) of intra-cranial syngeneic mouse models of diffuse hemispheric glioma, H3G34 (DHG-H3G34) and diffuse midline glioma, H3K27 (DMG-H3K27). We also demonstrate how an oncolytic reovirus (Reolysin) can “heat-up” the TIME of our syngeneic models. Orthotopic immunocompetent mouse models of DHG-H3G34 (C57BL/6, NRASG12V + shp53 + shATRX +/- H3.3G34R) and DMG-H3K27 (Nestin-Tv-a/p53fl/fl, RCAS-ACVR1R206H + RCAS-H3.1K27M) were profiled using single-cell RNA-sequencing (scRNA-seq) (10x genomics), a 22-colour custom flow cytometry immune panel and spatial transcriptomics. Differential marker expression was validated with immunohistochemistry and immunofluorescence in tissue sections. Syngeneic mouse tumours treated systemically with Reolysin were also profiled to evaluate the effects of the oncolytic virus on the TIME. Cell type predictions in scRNA-seq using singleR, ssGSEA and expression of individual marker genes suggested that the predominant immune cell types within hemispheric tumours were monocytes (11-21%) and macrophages (10-19%) with much smaller proportions of CD4+ and CD8+ T-cells (4-10%). By contrast, much smaller proportions of monocytes (2%) and macrophages (3%) were observed in the H3.1K27M pontine model. Flow cytometry, immunohistochemistry and immunofluorescence validated scRNA-seq immune profiles and characterised signalling of the PD-1/PD-L1 checkpoint pathway. Spatial transcriptomics allowed immune cell populations to be positioned within tumour sections and showed significant co-localization of CD4+ and CD8+ lymphocytes at tumour margins. Treatment of syngeneic mouse tumours with Reolysin resulted in reduced tumour volumes and altered the TIME, in particular increasing cytotoxic T-cell tumour infiltration. Our results highlight immunological heterogeneity within molecular subgroups of PDHGG and demonstrate ability of a systemically delivered oncolytic virus, Reolysin, to “heat-up” the TIME, contributing to a more immune actionable profile. Future work will help to identify optimal combinations for the next generation of immunotherapies in PDHGG.
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Chattenton, Dani, Ian Rivens, Zheng Jiang, Diana M. Carvalho, Krit Sujarittam, Jessica K. R. Boult, Simon P. Robinson, Chris Jones, Gail ter Haar, and James Choi. "DDEL-06. Drug Delivery to the Pons Using Short-Pulse Focused Ultrasound and Microbubble Exposure for the Treatment of Diffuse Midline Glioma." Neuro-Oncology 24, Supplement_1 (June 1, 2022): i35. http://dx.doi.org/10.1093/neuonc/noac079.127.
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Abstract Despite advances in understanding diffuse midline glioma (DMG-H3K27), including DIPG, there are still no effective treatments available, and the dismal clinical prognosis remains. This is partly because of tumour spread behind an intact blood brain barrier (BBB), preventing drug delivery and the reason for many drugs failing in the clinic. The use of focused ultrasound and intravenous microbubbles enables temporary increases in BBB permeability, allowing drugs to enter the targeted brain region. Building on recent research demonstrating that short pulses (<5 µs) of ultrasound can deliver drugs safely and uniformly to the hippocampus, we evaluated whether a similar result was achievable in the pons of mice. Mice were exposed to ultrasound (peak-negative pressure: 0.4 MPa, pulse length: 5 cycles, centre frequency 1 MHz) emitted in bursts of 38 pulses. During exposure mice received an intravenous injection of SonoVue(R) microbubbles and a fluorescently-tagged tracer (dextran, 3 kDa), acting as a drug mimic. Dextran was successfully delivered to the pons of non-tumour-bearing mice assessed by fluorescence microscopy immediately post-treatment. Dextran delivery was repeatable and confined to the targeted pons region with a homogenous distribution, typical of short pulse ultrasound, and important for treating DMG to ensure all tumour cells receive an equal drug dose. No damage to the brain was observed after H&E staining. Panobinostat has shown promise in vitro but tolerated doses have not shown therapeutic benefit in vivo as it does not cross the BBB. The in vitro toxicity of panobinostat was confirmed in a Nestin-Tv-a/p53fl/fl, RCAS-ACVR1R206H + RCAS-H3.1K27M murine cell line, with a GI50 of 15.56 nM. The ability of focused ultrasound to deliver panobinostat across the BBB to these tumours grown orthotopically will be assessed. Overall, we hope to develop a drug delivery system, that enables therapeutics to cross the BBB, expanding treatment options for DMG.
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Seblani, Maggie, Markella Zannikou, Joseph Duffy, Rebecca Levine, Qianli Liu, Craig Horbinski, Oren Becher, and Irina Balyasnikova. "EXTH-63. A NOVEL MOUSE MODEL OF DIFFUSE MIDLINE GLIOMA FOR TARGETED IMMUNOTHERAPY." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi177. http://dx.doi.org/10.1093/neuonc/noab196.702.
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Abstract In children, diffuse midline gliomas retain poor outcomes, failing to have a durable response to conventional therapies. Immunotherapies hold promise, with the integration of the host's immune system fundamental to their design. Here, we describe a novel genetically engineered immunocompetent model that incorporates interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen, to evaluate the antitumor activity of IL13Rα2-CAR T cell and bispecific T cell engager (BiTE) therapies in preclinical studies. The RCAS-Tva delivery system was utilized to induce gliomagenesis through p53 loss and the constitutive expression of PDGFB and human IL13Rα2 in Nestin-Tva;p53fl/fl mice. Pups were injected with virus-producing DF1 cells, encoding either for RCAS-Cre and PDGFB+IL13Rα2 or RCAS-Cre and PDGFB. Kaplan-Meier survival curves established and compared tumor growth dynamics in both models. Tumor tissue was characterized through immunohistochemistry and H&E staining. Cell lines generated from tumor-bearing tissue were used for orthotopic injection and in vitro studies. Expression of PDGFB and IL13Rα2 was confirmed by flow cytometry and western blot. In both groups, de novo tumors developed without significant difference in median survival between RCAS:PDGFB (n=25, 40 days) and RCAS:PDGB+IL13Rα2 (n=32, 39 days). Tumors demonstrated characteristics of high-grade glioma such as infiltration, pseudopalisading necrosis, microvascular proliferation, high Ki-67 index, heterogenous IL13Rα2 expression, with notable presence of CD11b+ macrophages and low count of CD3+ T cells. Orthotopic tumors from developed cell lines were histologically similar to de novo tumors. Treatment of generated cell lines with IL13Rα2-targeting BiTE protein resulted in a loss of glioma cell viability and target-specific activation of T cells. Engineered de novo tumors possess histopathologic features common to diffused midline gliomas. IL13Rα2-positive cell lines derived from de novo tumors were responsive to targeted treatment, opening the opportunity for preclinical assessment of IL13Rα2-directed immunotherapies, with the potential for clinical translation.
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Jaeger, Alex M., Andrew Weeden, and Anika Ali. "Abstract LB351: A novel mouse model to interrogate the functional relationship between protein synthesis and antigen presentation in cancer." Cancer Research 83, no. 8_Supplement (April 14, 2023): LB351. http://dx.doi.org/10.1158/1538-7445.am2023-lb351.
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Abstract Antigen presentation is a fundamental component of cancer immunity. Cytotoxic CD8+ T cells, the primary mediators of cell killing, recognize peptide antigens presented in the context of Major Histocompatibility Complex Class I (MHC-I). MHC-I presents an astoundingly diverse array of peptides, collectively known as the immunopeptidome. The biosynthesis of the immunopeptidome, which integrates gene expression, protein synthesis, and proteolytic processing, is not well understood. Additionally, most studies interrogating the immunopeptidome use cultured cells in vitro, which lack physiologically relevant stimuli. In contrast, in vivo studies utilize bulk tumor or tissue lysates, in which heterogeneous cell mixtures obscure the ability to understand cell type or disease specific patterns of antigen presentation. The technical limitations have hampered our understanding of the cancer immunopeptidome in physiologically relevant tumor microenvironments in vivo. Here we present a novel genetically engineered mouse model (GEMM) of cancer, that includes KrasLox-Stop-Lox-G12D; p53fl/fl; a Cre conditionally tagged MHC-I (KbStrep), and a Cre conditionally tagged ribosome (Rpl22HA/HA), termed the “KP/RiboMHC” mouse model. The KP/RiboMHC model enables simultaneous purification of ribosomes and peptide MHC complexes to functionally interrogate translational dynamics and antigen presentation in vivo. We developed optimized workflows to perform simultaneous ribosome profiling and immunopeptidomics from lung and pancreas cancer cell lines in vitro and specifically from malignant cells in vivo. Thus, the KP/RiboMHC model provides an unprecedented opportunity to ask fundamental questions regarding the influence of physiological stimuli in the tumor microenvironment on the biosynthesis of the immunopeptidome. Insights gained using this mouse will pave the way forward for the development of context specific, peptide-centric cancer immunotherapies. Citation Format: Alex M. Jaeger, Andrew Weeden, Anika Ali. A novel mouse model to interrogate the functional relationship between protein synthesis and antigen presentation in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB351.
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Hwang, Elizabeth E., Alex Lee, Stanley Leung, Marcus Breese, and Alejandro Sweet-Cordero. "Abstract 1107: Notch activation drives resistance to Kras(G12C) inhibition in lung adenocarcinoma." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1107. http://dx.doi.org/10.1158/1538-7445.am2022-1107.
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Abstract Approximately one-third of lung adenocarcinoma (LUAD) tumors contain driver mutations in the Kras GTPase, most commonly occurring as Kras(G12C) activating mutations. Despite the promising development of covalent G12C small molecule inhibitors, early-phase clinical trial data indicate most patients will likely demonstrate disease progression through either intrinsic or acquired resistance. Thus, there is an urgent need to identify such resistance mechanisms to targeting Kras. Our lab previously discovered a unique requirement for Notch3 in tumor-propagating cells from the KrasLSL-G12D p53fl/fl LUAD mouse model as well as in patient-derived xenografts. Here we report activated Notch3 as a novel driver of resistance to Kras(G12C) inhibition in LUAD cell lines. Aiming to study the Notch3 transcriptional signature in LUAD, we developed an inducible model of Notch3 intracellular domain (NICD) expression in Kras-mutant LUAD cell lines. Our data demonstrate that activated NICD modulates downstream RTK signaling pathways to suppress both cell cycle arrest and apoptosis in response to G12C inhibitor treatment. We subsequently performed RNA-sequencing to identify Notch-dependent genes driving resistance and in parallel, used Cut&Run technology to interrogate the global chromatin binding profiles of Notch3 and associated histone marks in NICD-expressing cell lines. We integrate our transcriptomic and epigenomic data to reveal that NICD acts as a key regulator of Rho family genes, which in turn support cell survival upon Kras inhibition. We additionally find enrichment of cell division and morphological signatures in inhibitor treated NICD cell lines and we elucidate the mechanism of direct Notch target genes in mediating the apoptotic response. Finally, we conduct a focused in vitro CRISPRi screen in the H358 LUAD cell line to identify the subset of Notch target genes required for NICD-induced resistance. Our results define a Notch3 transcriptional landscape in LUAD and demonstrate a role for Notch signaling in promoting resistance to small molecule Kras inhibitors. Citation Format: Elizabeth E. Hwang, Alex Lee, Stanley Leung, Marcus Breese, Alejandro Sweet-Cordero. Notch activation drives resistance to Kras(G12C) inhibition in lung adenocarcinoma [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 1107.
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Zhang, Jingfang, Yangang Liu, Guangyao Kong, Yuan-I. Chang, Erik A. Ranheim, Jinyong Wang, Juan Du, Alisa Damnernsawad, and Jing Zhang. "Loss Of p53 Promotes Transformation Of Oncogenic Nras-Induced Chronic Myelomonocytic Leukemia To An Acute Phase." Blood 122, no. 21 (November 15, 2013): 3790. http://dx.doi.org/10.1182/blood.v122.21.3790.3790.
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Abstract Chronic myelomonocytic leukemia (CMML) primarily occurs in the elderly with the median age ranging from 65 to 75 years. As defined by WHO, CMML is characterized by persistent monocytosis in peripheral blood, hepatosplenomegaly, and the absence of BCR-ABL fusion gene. CMML is a devastating cancer for multiple reasons, one of which is that approximately 20% of CMML cases evolve into acute myeloid leukemia (AML) soon after their first diagnosis. However, little is known about the cellular and molecular mechanisms underlying this malignant transformation. Recently, our lab developed a CMML mouse model induced by oncogenic NrasG12D/+ expressed from its endogenous locus. Above 90% of recipient mice with NrasG12D/+ bone marrow cells developed CMML-like phenotypes with a median survival of ∼56 weeks. Interestingly, none of these mice spontaneously transform to AML. To identify the pathogenetic origins underlying CMML transformation to AML, we further deleted p53 expression in NrasG12D/+ bone marrow cells using p53fl/fl allele and Mx1-Cre because deletion of p53 is a common genetic event observed in oncogenic Ras-driven cancers. We found that ERK1/2 is significantly hyperactivated in NrasG12D/+; p53-/- hematopoietic stem/progenitor cells (enriched for myeloid progenitors) in the absence of cytokines or in the presence of low concentration of GM-CSF. Concomitantly, the mutant myeloid progenitors show significantly increased self-renewal in a serial replating assay in vitro. We transplanted NrasG12D/+, p53-/-, or NrasG12D/+; p53-/- bone marrow cells into lethally irradiated mice. Unlike recipients with p53-/- cells that died of a T-cell disease with 100% penetrance and a median survival of 24 weeks, ∼70% of recipients with NrasG12D/+; p53-/- cells died of AML or acute myeloid sarcoma with a median survival of 16 weeks. These malignant myeloid diseases are transplantable in secondary recipients. Interestingly, only mutant hematopoietic stem cells (HSCs) could initiate and maintain leukemia phenotypes in the NrasG12D/+ induced CMML model, whereas both NrasG12D/+; p53-/- HSCs and myeloid progenitors could initiate AML or acute myeloid sarcoma. Our results indicate that deletion of p53 cooperates with NrasG12D/+ mutation to transform CMML into an acute phase. This malignant transformation is initiated by mutant myeloid progenitors, which show increased self-renewal and potentially serve as leukemia initiating cells. Disclosures: No relevant conflicts of interest to declare.
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Churchhouse, A. M., C. V. Billard, M. J. Arends, and K. B. Myant. "P036 A novel p53-mediated mouse model of inflammatory bowel disease-associated colorectal cancer." Journal of Crohn's and Colitis 14, Supplement_1 (January 2020): S150. http://dx.doi.org/10.1093/ecco-jcc/jjz203.165.
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Abstract Background Current mouse models of colitis-associated cancer often utilise a combination of APC mutation, Dextran sulphate sodium (DSS) and azoxymethane administration. However, only 13% of human inflammatory bowel disease (IBD)-associated cancers is associated with an APC mutation1. Instead, they are frequently associated with mutation of p53 (occurring in 63% of human cancers1). Whilst some models of IBD-associated cancer using either global p53 loss or mutant p53 have been described, we felt that a more pathophysiologically relevant model would be represented using p53 loss solely in the colonic epithelium. Methods Colonic epithelial p53 floxing in Vil-CreERT2p53fl/fl mice was achieved through intraperitoneal injection of 120 and 80 mg/kg tamoxifen on two consecutive days. After 16 days, mice were started on a chronic DSS treatment protocol. Chronic DSS-induced colitis involved three 5-day cycles of low-dose DSS (1.5% initially and subsequently 1% for the remaining two cycles), interspersed with normal drinking water for 2 weeks to facilitate recovery. After three cycles of DSS, mice were aged to 164 days. Results At 164 days post induction of DSS, 100% of mice had developed adenomata with low-grade dysplasia (n = 4). Adenomata in 50% of these mice (n = 2) also had features of high-grade dysplasia. There were between one and three adenomata in each animal. No features of invasive adenocarcinoma were demonstrated. There was partial β-catenin nuclear localisation in these tumours. Background chronic colitis was evident, even though the experiment ended over 3 months after the DSS treatment finished. Conclusion To the best our knowledge, this is the first mouse model of colitis-associated cancer using p53 loss in the colonic epithelium together with chronic colitis induction using DSS. The experiment ended over 3 months after the DSS treatment finished, showing that the resultant chronic colitis can persist long after the initial insult is given. The fact that β-catenin is partially, but not fully nuclear localised suggests an alternative pathway to tumour formation in this p53fl/fl model, and may therefore be more pathophysiologically relevant to human IBD-associated colorectal cancers. Further work is now being carried out to both validate and further evaluate this novel mouse model, including assessment of stem cell dynamics in response to colitis in the context of p53 loss. Reference
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Date, Yuki, Tomoya Ueno, and Kosei Ito. "Abstract 5732: p53 deficiency causes Myc dysregulation through a novel TGFβ-facilitated promoter switching mechanism to instigate osteosarcoma development." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5732. http://dx.doi.org/10.1158/1538-7445.am2022-5732.
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Abstract p53 deficiency and Myc dysregulation, driven by genetic and epigenetic alterations, respectively, are frequently associated with cancer. Previously, we reported that the combination of p53 inactivation and Myc overexpression, mediated by Runx transcription factors (TFs), leads to osteosarcoma (OS), the most common primary bone cancer. However, the mechanisms leading to Myc upregulation by Runx in a p53-deficient microenvironment remain unclear. TGFβ is a main driver of the epithelial-to-mesenchymal transition and its signal-dependent TFs, Smads, are known to associate with lineage-determining TFs including Runx. As TGFβ paracrine signalling is reportedly increased during human OS development, we immunodetected increased co-expression of TGFβ and phospho-Smad2 in the bone marrow of osteoprogenitor-specific p53 knockout mice, Osterix (Osx)/Sp7-Cre; p53fl/fl mice, which are widely used as an animal model of human OS. Heterozygous deletion of the type II TGFβ receptor in those mice weakened their susceptibility to OS, phenocopying the overall life-extending effect of Myc depletion in the same model. p53 disruption caused TGFβ to aberrantly induce Myc in osteoprogenitors, rendering the cells tumorigenic upon transplantation into immunocompromised mice. Within the 3-Mb Myc regulatory region, we identified a TGFβ-responsive element dubbed m340, which contains binding sites for multiple TFs including Runx. The m340 enhancer caused Myc upregulation upon p53 disruption by switching its associated promoter from the lncRNA Pvt1 to Myc. In support of this promoter switching mechanism, a specific disruption of the evolutionarily conserved p53 site within the Pvt1 promoter recapitulated TGFβ-induced Myc overexpression. At m340, TGFβ promoted its occupancy by a transcriptional complex consisting of Runx, its cooperative factors Smads and AP1, and CBP, further activating the enhancer by increasing H3K27ac deposition. Runx inhibition not only repressed the aberrant upregulation of Myc by reducing Smad occupancy at m340, but also suppressed OS development in vivo. These results suggest that p53 deficiency causes Myc dysregulation through a novel promoter switching programme that is facilitated by TGFβ signalling and reveals a novel epigenetic mechanism underlying the interaction between cancer-predisposing genetic alterations (p53 deficiency) and environmental cues (TGFβ) in tumorigenesis. Considering the mediating role Runx plays in m340 activation, our study may provide a clinical rationale for targeting Runx in p53-deficient malignancies. Citation Format: Yuki Date, Tomoya Ueno, Kosei Ito. p53 deficiency causes Myc dysregulation through a novel TGFβ-facilitated promoter switching mechanism to instigate osteosarcoma development [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 5732.
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Lankadasari, Manendra B., Yuan Liu, Di He, Samhita Bapat, Brooke Mastrogiacomo, Harry B. Lengel, and David R. Jones. "Abstract 3142: BRMS1 alters the tumor inflammatory signature and immune infiltration in lung adenocarcinoma." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3142. http://dx.doi.org/10.1158/1538-7445.am2022-3142.
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Abstract Background: Lung adenocarcinoma (LUAD) is the leading cause of cancer-related deaths. Metastatic cancer is highly fatal which contributes to at least 90% of cancer-associated morbidities and mortalities. During metastasis, tumor cells alter their signaling cascade and interact with the immune cells in the tumor microenvironment Therefore, it is necessary to characterize and understand how these cancer cells influence tumor-infiltrating immune cells to facilitate and even enhance their ability to metastasize. BRMS1 is a metastasis suppressor gene which is frequently downregulated in LUAD. We, and others, have shown that loss of BRMS1 results in distant metastatic disease. Given the strong correlation between immune suppression, metastasis, and therapy resistance we sought to elucidate if BRMS1 contributes to these processes. Specifically, we hypothesized that LUAD cells downregulate BRMS1 which influences the immune cell composition in the tumor. This creates an immune-suppressive tumor microenvironment aiding in metastasis. Methods: To test our hypothesis, we generated KrasG12D P53fl/fl Brms1-/- mice which spontaneously develop tumors with Ad-Cre intratracheal inoculation. To elucidate the role of BRMS1 in the context of cancer, we performed RNA sequencing on tumors from Brms1 wild type and knockout background. We used MCP counter, an in silico cellular deconvolution algorithm on our bulk RNA seq data to identify various subsets of tumor-infiltrating immune cells. Furthermore, our observations were validated by immunofluorescence and flow cytometry. Results: Differential gene expression analysis using RNA seq data revealed a reduction in CXCL9, CCL5, CLEC1B, CCL9, CCL7 and other proinflammatory molecules in the Brms1-/- tumors. Gene ontology and gene set enrichment analysis highlighted a diminished immune response signature in Brms1-/- tumors. Hallmarks like interferon and IL6 signaling, complement and inflammation are highly enriched in Brms1+/+ tumors. MCP counter also suggested a significant increase in NK cells and reduction in CD8+ T cell population in the Brms1-/- tumors. To validate our observation, we performed immunofluorescence and flow cytometry to assess the number of infiltrating immune cells in the tumor microenvironment. Immunofluorescence data showed reduced cytotoxic T cells (CD8+ T cells) in Brms1-/- mice. The flow cytometric analysis also revealed a reduction in the proliferative potential of these CD8+ T cells along with the increased presence of myeloid-derived suppressor cells in the Brms1-/- mice. Conclusion: Our data suggest for the first time that reduced BRMS1 expression which is generally observed in multiple tumors not only influences metastasis but also alters tumor-infiltrating immune cell composition. Thus, BRMS1 downregulation presents as one of the potential immune evasion mechanisms that could be targeted for an improved therapy outcome in LAUD. Citation Format: Manendra B. Lankadasari, Yuan Liu, Di He, Samhita Bapat, Brooke Mastrogiacomo, Harry B. Lengel, David R. Jones. BRMS1 alters the tumor inflammatory signature and immune infiltration in lung adenocarcinoma [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 3142.
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Yang, Xiaojie, Mohamad Zoabi, Simone Hausmann, Natasha M. Flores, Xiaoyin Lu, Jibo Wu, Ana Morales-Benitez, Or Gozani, and Pawel K. Mazur. "Abstract A080: METTL21A inhibits pancreatic ductal adenocarcinoma tumorigenesis through methylation of HSPA1/8." Cancer Research 82, no. 22_Supplement (November 15, 2022): A080. http://dx.doi.org/10.1158/1538-7445.panca22-a080.
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Abstract Background: Our work focuses on the role of METTL21A, a member of the little studied seven β-strand family of candidate lysine methyltransferases (KMTs), and its role in the regulation of pancreatic ductal adenocarcinoma (PDAC) development. Using a meta-analysis of publicly available gene expression datasets, we found that METTL21A is significantly downregulated in PDAC versus normal tissue and the reduced expression predicts poor patient survival. Thus, there are intriguing correlations connecting METTL21A to PDAC pathogenesis; however, to date, there are no data on the role of METTL21A in cancer and an enzymatic function for METTL21A is poorly recognized. Methods: To test the hypothesis that METTL21A plays a role in PDAC initiation and progression, we generated conditional Mettl21a knockout mice and crossed to PDAC models: p48Cre/+ KrasG12D/+ (Kras vs. Kras;Mettl21a) and p48Cre/+ KrasG12D/+ p53fl/fl (Kras;p53 vs. Kras;p53;Mettl21a). Furthermore, we studied the impact of METTL21A depletion on human PDAC cell lines and PDX growth in vivo and in vitro. To identify the substrate of METTL21A enzymatic activity we performed methylation assays followed by mass spectrometry analysis. Results: Histopathological analysis of pancreatic tissues of Kras and Kras;Mettl21a mice at 6 months of age revealed that loss of METTL21A significantly accelerates pancreatic cancer initiation. Additional studies using Kras;p53 and Kras;p53;Mettl21a mice demonstrated that METTL21A depletion accelerates PDAC progression and results in a significantly shorter overall survival. In congruence with those observations, knockdown of METTL21A in human PDAC cell lines leads to the robust increase in cell proliferation, enhances colony formation ability in vitro and xenograft growth in vivo. In contrast, cells rescued with ectopic expression of wildtype METTL21A but not with enzyme-dead mutant METTL21A showed significant growth impairment in vitro and in vivo. Next, our in vivo methylation assays identified HSPA1 and HSPA8 (members of the HSP70 protein family) as substrates of METTL21A in PDAC cells. Our subsequent analysis showed that METTL21a specifically tri-methylates HSPA1 and HSPA8 at lysine 561 (K561me3). Next, to directly evaluate the role of METTL21A mediated methylation of HSPA1/A8 at K561 in regulating cancer phenotype, we knocked out HSPA1/A8 and complemented with expression of wildtype or K561A mutant HSPA1/A8 in PDAC cell lines. We found that only methylation-resistant HSPA1/A8 increased cell proliferation and tumor growth in vivo. Together, these results argue that in PDAC, the principal physiologic activity of METTL21A is generation of HSPA1/A8 K561me3, which suppresses cancer growth. Conclusion: Our research revealed that METTL21A is a novel tumor suppressor that inhibits the initiation and progression of PDAC through methylation of the heat-shock proteins HSPA1/8 substrate-binding domain. Citation Format: Xiaojie Yang, Mohamad Zoabi, Simone Hausmann, Natasha M. Flores, Xiaoyin Lu, Jibo Wu, Ana Morales-Benitez, Or Gozani, Pawel K. Mazur. METTL21A inhibits pancreatic ductal adenocarcinoma tumorigenesis through methylation of HSPA1/8 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A080.
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Nutt, W. Sam, Takahiro Miyazaki, Mario Marcondes, Stanley R. Riddell, and Shivani Srivastava. "Abstract 1152: NKTR-255, a polymer-conjugated IL-15, dramatically improves ROR1 CAR-T cell persistence and anti-tumor efficacy in an autochthonous model of ROR1+ lung cancer." Cancer Research 83, no. 7_Supplement (April 4, 2023): 1152. http://dx.doi.org/10.1158/1538-7445.am2023-1152.
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Abstract CAR-T cells have mediated remarkable clinical responses against hematological malignancies, but this success has yet to extend to solid tumors, where many factors in the tumor microenvironment (TME) inhibit their persistence and function. In a phase 1 trial at our Center, CAR-T cells targeting the tumor-associated antigen ROR1 in patients with non-small cell lung cancer (NSCLC) and triple negative breast cancer rapidly became dysfunctional and persisted poorly at tumor sites, ultimately failing to mediate objective responses. To evaluate strategies to improve ROR1 CAR-T efficacy, we adapted the KrasLSL-G12D/+;p53fl/fl (KP) autochthonous model of lung adenocarcinoma to express ROR1 (KPROR1). This aggressive model mimics the initiation, progression, and suppressive TME of human NSCLC. ROR1 CAR-T cells in KPROR1 mice rapidly declined in number, lost the ability to produce pro-inflammatory cytokines, and failed to significantly enhance tumor control, recapitulating the major barriers to CAR-T efficacy we observed in patients. NKTR-255 is a novel immunotherapeutic consisting of a polymer conjugated recombinant human interleukin-15 (rhIL-15) that could enhance the activity of CAR-T cells. NKTR-255 binds to the IL-15 receptor and IL-2/IL-15 receptor, thus maintaining the full potential spectrum of IL-15 biology while exerting a sustained effect on CD8+ memory T cell and NK cell expansion and persistence. We hypothesized that the combination of CAR-T cells and NKTR-255 would be feasible and safe and may extend the duration of efficacy of ROR1 CAR-T cells in ROR1+ lung tumors by improving their expansion and persistence. To test this, we treated tumor-bearing KPROR1 mice with control or ROR1 CAR-T cells with or without weekly injections of NKTR-255. NKTR-255 treatment resulted in a robust expansion of ROR1 CAR-T cells in the peripheral blood relative to vehicle-treated mice, which correlated with increased CAR-T cell infiltration into lung tumors. CAR-T cells in NKTR-255-treated mice also showed reduced signs of T cell exhaustion, including reduced expression of inhibitory receptors and enhanced ability to produce effector cytokines upon ex vivo re-stimulation. Importantly, combination therapy with both NKTR-255 and ROR1 CAR-T cells resulted in significantly improved tumor control relative to either treatment alone. Together, our work shows that NKTR-255 dramatically improves the persistence, function, and anti-tumor activity of ROR1 CAR-T cells in an aggressive autochthonous model of ROR1+ lung cancer. Our findings provide rationale to combine NKTR-255 with CAR-T cell therapy as a strategy to enhance the anti-tumor efficacy of CAR-T cells in patients with solid tumors. Citation Format: W. Sam Nutt, Takahiro Miyazaki, Mario Marcondes, Stanley R. Riddell, Shivani Srivastava. NKTR-255, a polymer-conjugated IL-15, dramatically improves ROR1 CAR-T cell persistence and anti-tumor efficacy in an autochthonous model of ROR1+ lung cancer [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 1152.
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Maldonado, Ana M., Natalia de la Fuente, and Francisco Portillo. "Characterization of an Allele-Nonspecific Intragenic Suppressor in the Yeast Plasma Membrane H+-ATPase Gene (PMA1)." Genetics 150, no. 1 (September 1, 1998): 11–19. http://dx.doi.org/10.1093/genetics/150.1.11.
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Abstract We have analyzed the ability of A165V, V169I/D170N, and P536L mutations to suppress pma1 dominant lethal alleles and found that the P536L mutation is able to suppress the dominant lethality of the pma1-R271T, -D378N, -D378E, and -K474R mutant alleles. Genetic and biochemical analyses of site-directed mutants at Pro-536 suggest that this amino acid may not be essential for function but is important for biogenesis of the ATPase. Proteins encoded by dominant lethal pma1 alleles are retained in the endoplasmic reticulum, thus interfering with transport of wild-type Pma1. Immunofluorescence studies of yeast conditionally expressing revertant alleles show that the mutant enzymes are correctly located at the plasma membrane and do not disturb targeting of the wild-type enzyme. We propose that changes in Pro-536 may influence the folding of the protein encoded by a dominant negative allele so that it is no longer recognized and retained as a misfolded protein by the endoplasmic reticulum.
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Shingu, Takashi, and Jian Hu. "CBMT-17. DEFECTIVE QKI-DEPENDENT LIPID METABOLISM INDUCES GENOMIC INSTABILITY AND SENSITIZES GLIOBLASTOMA TO IMMUNOTHERAPY." Neuro-Oncology 21, Supplement_6 (November 2019): vi36. http://dx.doi.org/10.1093/neuonc/noz175.139.
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Abstract Despite transformative effects on the therapy of cancers such as melanoma and lung adenocarcinoma, blockade of the T cell immune checkpoints has generated limited impact on glioblastoma. Identifying genetic/genomic alterations that could potentially sensitize the patients to immunotherapy will significantly improve the efficacy of immunotherapy on glioblastoma patients. As part of our effort to identify novel glioma suppressors that affect the interaction of GSCs with their microenvironment, we discovered that the RNA-binding protein Quaking (QKI) is a key regulator of cellular endocytosis. QKI is mutated or deleted in ~34% of human glioblastomas. Supporting QKI’s tumor suppresser function, 92% of the Nestin-CreERT2;QkiL/L;PtenL/L;p53L/L mice developed glioblastoma with a median survival of 105 days, however, the Nestin-CreERT2;PtenL/L;p53L/L mice did not develop any glioma up to a year. Mechanistically, QKI regulates the RNA stability and alternative splicing of numerous protein and lipid components of endolysosomes, particularly the unsaturated fatty acids (UFAs). Functionally, deletion of Qki and inhibition of UFA biosynthesis both decrease endolysosome-mediated receptor degradation, thereby enriching receptors on the cytoplasmic membrane (e.g., Frizzled and Notch1) that are essential for maintaining stemness. This enrichment of receptor signaling enables GSCs to cope with the low ligand levels during their invasion. On the other hand, lower lysosomal activity induced by Qki deletion and UFA loss led to defective mitophagy. We also found that insufficient UFAs in mitochondrial membrane significantly compromised mitochondrial membrane integrity and function. These two mechanisms concomitantly led to accumulation of damaged mitochondria and higher levels of reactive oxygen species (ROS), and consequently genomic instability. Lastly, we found that the higher level of genomic instability induced by Qki loss rendered cells more sensitive to anti-CTLA4 and anti-PD1 antibodies. Taken together, our data suggest that Qki/UFA loss-induced endolysosomal and mitochondrial defects promote gliomagenesis yet render cells vulnerabilities that could be harnessed for therapeutic purposes.
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Nakeesathit, Supatchara, Naowarat Saralamba, Sasithon Pukrittayakamee, Arjen Dondorp, Francois Nosten, Nicholas J. White, and Mallika Imwong. "Limited Polymorphism of the Kelch Propeller Domain in Plasmodium malariae and P. ovale Isolates from Thailand." Antimicrobial Agents and Chemotherapy 60, no. 7 (April 25, 2016): 4055–62. http://dx.doi.org/10.1128/aac.00138-16.
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ABSTRACTArtemisinin resistance inPlasmodium falciparum, the agent of severe malaria, is currently a major obstacle to malaria control in Southeast Asia. A gene named “kelch13” has been associated with artemisinin resistance inP. falciparum. The orthologue of thekelchgene inP. vivaxwas identified and a small number of mutations were found in previous studies. Thekelchorthologues in the other two human malaria parasites,P. malariaeandP. ovale, have not yet been studied. Therefore, in this study, the orthologouskelchgenes ofP. malariae,P. ovale wallikeri, andP. ovale curtisiwere isolated and analyzed for the first time. The homologies of thekelchgenes ofP. malariaeandP. ovalewere 84.8% and 82.7%, respectively, compared to the gene inP. falciparum.kelchpolymorphisms were studied in 13P. malariaeand 5P. ovaleisolates from Thailand. There were 2 nonsynonymous mutations found in these samples. One mutation was P533L, which was found in 1 of 13P. malariaeisolates, and the other was K137R, found in 1 isolate ofP. ovale wallikeri(n= 4). This result needs to be considered in the context of widespread artemisinin used within the region; their functional consequences for artemisinin sensitivity inP. malariaeandP. ovalewill need to be elucidated.
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Meloni, Alessandra, Roberto Perniola, Valeria Faà, Enrico Corvaglia, Antonio Cao, and Maria Cristina Rosatelli. "Delineation of the Molecular Defects in the AIRE Gene in Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Patients from Southern Italy." Journal of Clinical Endocrinology & Metabolism 87, no. 2 (February 1, 2002): 841–46. http://dx.doi.org/10.1210/jcem.87.2.8209.
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In this study, we have carried out molecular analysis of the AIRE (autoimmune regulator) gene in 11 patients (from 8 families) affected by autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, originating from a restricted area of Southern Italy (the Salento peninsula in Puglia). Of the 16 mutant AIRE alleles from the 8 probands studied, 12 carried a missense mutation (W78R in 9, P539L in 2, and P252L in 1), 2 carried the Q358X nonsense mutation, and 2 carried the 1058delT frameshift mutation. All these mutations except the 1058delT are novel. Each of the detected mutations either predicts a premature termination of the protein or results in a nonconservative amino acid change, most likely adversely affecting the function of the protein. The W78R missense mutation is relatively common in these patients, having been detected (in homozygosity or compound heterozygosity) in 6 of the 8 probands tested, indicating the presence of a founder effect. The results of this study contribute to the delineation of the molecular pathology of the AIRE gene and enhance our ability to perform a molecular diagnosis in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients from Southern Italy.
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Ghevaert, Cedric, Alexandre Salsmann, Nicholas A. Watkins, Elisabeth Schaffner-Reckinger, Angela Rankin, Stephen F. Garner, Jonathan Stephens, et al. "A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the αIIbβ3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia." Blood 111, no. 7 (April 1, 2008): 3407–14. http://dx.doi.org/10.1182/blood-2007-09-112615.
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AbstractWe report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the β3 integrin and a P53L mutation in glycoprotein (GP) Ibα. We show that GPIbα-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The β3-H723 causes constitutive, albeit partial, activation of the αIIbβ3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the αIIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO αIIbβ3-H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, αIIbβ3-H723, but not wild-type αIIbβ3, generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34+ stem cell–derived megakaryocytes. We conclude that the constitutive activation of the αIIbβ3-H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree.
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ROMERO, Irma, Ana M. MALDONADO, and Pilar ERASO. "Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists." Biochemical Journal 322, no. 3 (March 15, 1997): 823–28. http://dx.doi.org/10.1042/bj3220823.
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Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitroeffect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of ≈1 μM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein.
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Montoya, Rafael Heinz, Rasoul Pourebrahim, Zoe Alaniz, Lauren B. Ostermann, Jared K. Burks, and Michael Andreeff. "Osterix Marks a Distinct Population of Circulatory Bone Marrow Mesenchymal Stem Cells That Is Increased upon Oncogenic Stress." Blood 136, Supplement 1 (November 5, 2020): 1. http://dx.doi.org/10.1182/blood-2020-142713.
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Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that can differentiate into a variety of lineages and play a critical role in tissue homeostasis upon injury and repair. Previous studies using Osx-Cre transgenic mice have demonstrated that the expression of Sp7 (Osterix) marks a population of mesenchymal progenitor cells that can differentiate to osteoblasts as well as bone marrow stromal cells (Mizoguchi et al., 2014). Using a lineage-tracing system, we show that in addition to marking mesenchymal progenitor cells in the bone marrow, Osx-Cre also marks a population of BM-MSCs that circulate throughout the body and home in different tissues such as lung, spleen, intestine and muscle. Osx-Cre mice crossed with R26-mTmG reporter mice were analyzed at E14.5 and adulthood by fluorescence microscopy and flow cytometry. At E14.5, GFP+ cells were exclusively located in bone tissues. At two months of age, GFP+ cells were detectable in blood and many other tissues such as lung, liver, spleen and intestine. Flow cytometric profiling indicated that the GFP+ cells were positive for BM-MSC markers CD105, CD73 and CD140a. In order to exclude the possibility of non-specific recombination of the reporter in the non-osteoblast-lineage, as previously reported, we performed a pulse chase experiment utilizing Osx-CreER;mTmG mice. Fluorescence microscopy of the bone marrow upon Tamoxifen injection revealed that Cre activity was primarily limited to the osteoblast-lineage and bone tissue, whereas GFP+ cells were undetectable in lung and spleen, indicating that the GFP+ cells in the lung migrated from the bone marrow. Given that previous reports identified p53 as a negative regulator of osteoblast differentiation (Lengner et al., 2006), we further sought to determine the effect of p53 on circulatory BM-MSCs. Flow cytometric analysis of Osx-Cre;p53Fx/Fx;mTmG peripheral blood cells revealed a significant reduction of circulatory GFP+ cells as compared to p53 wild type mice (p<0.0001) suggesting a role for p53 in expansion of circulatory BM-MSCs. To further characterize the population of circulatory BM-MSCs in a cancer model, we analyzed the population of GFP+ cells in a syngeneic leukemia using fluorescence microscopy and flow cytometry. We transplanted p53 wildtype (Osx-Cre;mTmG) and p53 mutant (Osx-Cre;p53Fx/R172H;mTmG) reporter mice with AML-ETO-Turquoise leukemia cells and the population of GFP+ cells were analyzed three weeks after transplant. The population of GFP+MSCs were significantly increased in bone marrow and spleen, indicating the recruitment of circulatory BM-MSCs. Conclusion: We present the Osx-Cre;mTmG mouse as a faithful model to study circulatory BM-MSCs in vivo and identified a role for p53 in the regulation of circulatory BM-MSCs. We previously reported that BM-derived MSCs home to solid tumors and their metastases and can be successfully used as gene-delivery vehicles, both in murine models and in patients (Studeny et al. JCI 2001, Andreeff et al. AACR 2018). This model is the first to conduct studies of circulating MSCs and to further analyze their role in tumor biology and therapy. Lengner, C.J., Steinman, H.A., Gagnon, J., Smith, T.W., Henderson, J.E., Kream, B.E., Stein, G.S., Lian, J.B., and Jones, S.N. (2006). Osteoblast differentiation and skeletal development are regulated by Mdm2-p53 signaling. J Cell Biol 172, 909-921. Mizoguchi, T., Pinho, S., Ahmed, J., Kunisaki, Y., Hanoun, M., Mendelson, A., Ono, N., Kronenberg, H.M., and Frenette, P.S. (2014). Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development. Dev Cell 29, 340-349. Figure Disclosures Andreeff: Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Amgen: Research Funding.
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Padariya, Monikaben, Robin Fahraeus, Ted Hupp, and Umesh Kalathiya. "Molecular Determinants and Specificity of mRNA with Alternatively-Spliced UPF1 Isoforms, Influenced by an Insertion in the ‘Regulatory Loop’." International Journal of Molecular Sciences 22, no. 23 (November 25, 2021): 12744. http://dx.doi.org/10.3390/ijms222312744.
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The nonsense-mediated mRNA decay (NMD) pathway rapidly detects and degrades mRNA containing premature termination codons (PTCs). UP-frameshift 1 (UPF1), the master regulator of the NMD process, has two alternatively-spliced isoforms; one carries 353-GNEDLVIIWLR-363 insertion in the ‘regulatory loop (involved in mRNA binding)’. Such insertion can induce catalytic and/or ATPase activity, as determined experimentally; however, the kinetics and molecular level information are not fully understood. Herein, applying all-atom molecular dynamics, we probe the binding specificity of UPF1 with different GC- and AU-rich mRNA motifs and the influence of insertion to the viable control over UPF1 catalytic activity. Our results indicate two distinct conformations between 1B and RecA2 domains of UPF1: ‘open (isoform_2; without insertion)’ and ‘closed (isoform_1; with insertion)’. These structural movements correspond to an important stacking pattern in mRNA motifs, i.e., absence of stack formation in mRNA, with UPF1 isoform_2 results in the ‘open conformation’. Particularly, for UPF1 isoform_1, the increased distance between 1B and RecA2 domains has resulted in reducing the mRNA–UPF1 interactions. Lower fluctuating GC-rich mRNA motifs have better binding with UPF1, compared with AU-rich sequences. Except CCUGGGG, all other GC-rich motifs formed a 4-stack pattern with UPF1. High occupancy R363, D364, T627, and G862 residues were common binding GC-rich motifs, as were R363, N535, and T627 for the AU-rich motifs. The GC-rich motifs behave distinctly when bound to either of the isoforms; lower stability was observed with UPF1 isoform_2. The cancer-associated UPF1 variants (P533L/T and A839T) resulted in decreased protein–mRNA binding efficiency. Lack of mRNA stacking poses in the UPF1P533T system significantly decreased UPF1-mRNA binding efficiency and increased distance between 1B-RecA2. These novel findings can serve to further inform NMD-associated mechanistic and kinetic studies.
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Tien, Jean Ching-Yi, Seema Chugh, Andrew E. Goodrum, Yunhui Cheng, Rahul Mannan, Yuping Zhang, Lisha Wang, et al. "AGO2 promotes tumor progression in KRAS-driven mouse models of non–small cell lung cancer." Proceedings of the National Academy of Sciences 118, no. 20 (May 10, 2021): e2026104118. http://dx.doi.org/10.1073/pnas.2026104118.
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Lung cancer is the deadliest malignancy in the United States. Non–small cell lung cancer (NSCLC) accounts for 85% of cases and is frequently driven by activating mutations in the gene encoding the KRAS GTPase (e.g., KRASG12D). Our previous work demonstrated that Argonaute 2 (AGO2)—a component of the RNA-induced silencing complex (RISC)—physically interacts with RAS and promotes its downstream signaling. We therefore hypothesized that AGO2 could promote KRASG12D-dependent NSCLC in vivo. To test the hypothesis, we evaluated the impact of Ago2 knockout in the KPC (LSL-KrasG12D/+;p53f/f;Cre) mouse model of NSCLC. In KPC mice, intratracheal delivery of adenoviral Cre drives lung-specific expression of a stop-floxed KRASG12D allele and biallelic ablation of p53. Simultaneous biallelic ablation of floxed Ago2 inhibited KPC lung nodule growth while reducing proliferative index and improving pathological grade. We next applied the KPHetC model, in which the Clara cell–specific CCSP-driven Cre activates KRASG12D and ablates a single p53 allele. In these mice, Ago2 ablation also reduced tumor size and grade. In both models, Ago2 knockout inhibited ERK phosphorylation (pERK) in tumor cells, indicating impaired KRAS signaling. RNA sequencing (RNA-seq) of KPC nodules and nodule-derived organoids demonstrated impaired canonical KRAS signaling with Ago2 ablation. Strikingly, accumulation of pERK in KPC organoids depended on physical interaction of AGO2 and KRAS. Taken together, our data demonstrate a pathogenic role for AGO2 in KRAS-dependent NSCLC. Given the prevalence of this malignancy and current difficulties in therapeutically targeting KRAS signaling, our work may have future translational relevance.
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Shen, Jieli, Dat P. Ha, Genyuan Zhu, Daisy F. Rangel, Agnieszka Kobielak, Parkash S. Gill, Susan Groshen, Louis Dubeau, and Amy S. Lee. "GRP78 haploinsufficiency suppresses acinar-to-ductal metaplasia, signaling, and mutant Kras-driven pancreatic tumorigenesis in mice." Proceedings of the National Academy of Sciences 114, no. 20 (May 1, 2017): E4020—E4029. http://dx.doi.org/10.1073/pnas.1616060114.
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Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease in critical need of new therapeutic strategies. Here, we report that the stress-inducible 78-kDa glucose-regulated protein (GRP78/HSPA5), a key regulator of endoplasmic reticulum homeostasis and PI3K/AKT signaling, is overexpressed in the acini and PDAC of Pdx1-Cre;KrasG12D/+;p53f/+ (PKC) mice as early as 2 mo, suggesting that GRP78 could exert a protective effect on acinar cells under stress, as during PDAC development. The PKC pancreata bearing wild-type Grp78 showed detectable PDAC by 3 mo and rapid subsequent tumor growth. In contrast, the PKC pancreata bearing a Grp78f/+ allele (PKC78f/+ mice) expressing about 50% of GRP78 maintained normal sizes during the early months, with reduced proliferation and suppression of AKT, S6, ERK, and STAT3 activation. Acinar-to-ductal metaplasia (ADM) has been identified as a key tumor initiation mechanism of PDAC. Compared with PKC, the PKC78f/+ pancreata showed substantial reduction of ADM as well as pancreatic intraepithelial neoplasia-1 (PanIN-1), PanIN-2, and PanIN-3 and delayed onset of PDAC. ADM in response to transforming growth factor α was also suppressed in ex vivo cultures of acinar cell clusters isolated from mouse pancreas bearing targeted heterozygous knockout of Grp78 (c78f/+) and subjected to 3D culture in collagen. We further discovered that GRP78 haploinsufficiency in both the PKC78f/+ and c78f/+ pancreata leads to reduction of epidermal growth factor receptor, which is critical for ADM initiation. Collectively, our studies establish a role for GRP78 in ADM and PDAC development.
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Bui, Khac Cuong, Mai Ly Thi Nguyen, Samarpita Barat, Xi Chen, Vikas Bhuria, Jun Xing, Linh Toan Nguyen, et al. "Effect of AdipoR agonist in cholangiocarcinoma." Journal of Clinical Oncology 36, no. 4_suppl (February 1, 2018): 323. http://dx.doi.org/10.1200/jco.2018.36.4_suppl.323.
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323 Background: Adiponectin is the key adipokine, which plays an important role in health and disease such as obesity, diabetes, and cancer. Adiponectin is reduced in different tumor types, especially in obesity-related cancer, and recent studies showed that Adiponectin had a potential anti-cancer effect. Obesity is a risk factor for various tumor diseases including cholangiocarcinoma (CC), the second most common primary hepatic cancer. The aim of this study is to investigate for the first time the anti-cancer effect of AdipoR agonist in CC cell lines and a CC engineered mouse model. Methods: Human CC cell lines (TFK-1 and SZ-1) and CC engineered mice (Alb-Cre/KRASG12D/p53L/L) were used to investigate the anti-cancer effects of an AdipoR agonist (2-(4-Benzoylphenoxy)-N-[1-(phenylmethyl)-4-piperidinyl]-acetamide). Cell proliferation, migration, invasion, colony formation, apoptosis assay were applied to evaluate the effect of AdipoR agonist in vitro. In addition, important cancer signalling pathways and targets were analysed by Western Blot. Mice (n = 12) were treated with AdipoR or verhicle and tumor burden and survival were monitored. Results: AdipoR agonist suppressed proliferation, migration, invasion, colony formation and apoptosis in CC cells. AdipoR agonist regulated the expression of different proteins such as EMT markers, pAMPK, pSTAT3, and PARP, which have pivotal functions in cholangiocarcinogenesis. AdipoR agonist also prolonged survival time in a CC engineered mouse model. Conclusions: Our data revealed that AdipoR agonist inhibited successfully cell proliferation, migration, invasion, colony formation and apoptosis in vitro, and prolonged mice survival in vivo through regulation of crucial signaling pathways in cholangiocarcinogenesis. These results suggested that AdipoR agonist might become a new potential candidate for CC treatment in the future.
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Zamler, Daniel, Er-Yen Yen, Takashi Shingu, Jiangong Ren, Cynthia Kassab, Jintan Liu, Amy Heimberger, Jian Hu, Giulio Draetta, and Michael Curran. "IMMU-10. ESTABLISHING EFFECTIVE MODELS FOR IMMUNOTHERAPY IN GBM." Neuro-Oncology 21, Supplement_6 (November 2019): vi121. http://dx.doi.org/10.1093/neuonc/noz175.504.
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Abstract The introduction of immunotherapies has been paradigm shifting for cancers that were previously a death sentence. However, preclinical/clinical studies on glioblastoma (GBM) have generated mixed outcomes in patients, likely due to its great heterogeneity of immune microenvironment, particularly the myeloid cell populations. Primary patient studies have been limited by a difficulty in performing longitudinal studies, uncontrolled environmental conditions, and genetic variability. There is also, unfortunately, a paucity of mouse models that effectively re-capitulate the immune microenvironment of the human disease. To address these difficulties, we have established the Qk/p53/Pten (QPP) triple knockout mouse model established in our lab. The QPP model uses a cre-lox system to induce Qk deletion on a Pten−/−; p53−/− background which helps NSCs maintain their stemness outside the SVZ in Nes-CreERT2;QkiL/L PtenL/L p53L/L mice, which develops glioblastoma with survival of ~105 days. We have preliminarily assessed the QPP tumors as a faithful model to study the immune response to GBM and found them to recapitulate human GBM with respect to differential response to checkpoint blockade therapy and myeloid and T-cells histopathologically, particularly regarding upregulation of Arginase-1 (Arg1). Arg1 is the canonical marker for tumor-associated macrophages (TAMs), which is a major population of myeloid cells that greatly infiltrate in human GBM, sometimes making up more than ~30% of all GBM cells. Given TAMs’ prevalence in the tumor microenvironment and their upregulation of Arg1 in both human GBM and our QPP model, we are testing whether manipulation of Arg1 will impact TAM function and influence GBM growth. We are also evaluating arginine metabolism in TAMs effect on T cell function in GBM. Lastly, we have developed a genetically engineered mouse model to study the role of Arg1 knockout in a GBM context in-vivo. Our studies suggest that Arg1 plays an important role in GBM immune interaction.
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Mehta, Anita, Madeline Townsend, Madisson Oliwa, Patrice Lee, Nicholas Saccomano, Filipa Lynce, Geoffrey Shapiro, Elizabeth Mittendorf, and Jennifer Guerriero. "614 Investigating immune mediated mechanisms of PARPi resistance in BRCA1-associated triple negative breast cancer (TNBC)." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A644. http://dx.doi.org/10.1136/jitc-2021-sitc2021.614.
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BackgroundPoly(ADP-ribose) polymerase inhibitors (PARPi) have improved the outcomes of BRCA-associated breast cancer; however, treatment responses are often not durable. Our preclinical studies demonstrated that PARPi activates the cGAS/STING pathway and recruitment of anti-tumor CD8+ T-cells that are required for tumor clearance [1]. These studies contributed to development of clinical trials testing PARPi plus immune checkpoint blockade (ICB). Unfortunately, early phase trials of PARPi + ICB have not yet suggested efficacy will be superior to PARPi monotherapy. Lack of demonstrated clinical synergy between PARPi + ICB underscores the need to study the tumor microenvironment (TME) during PARPi therapy to identify optimal strategies to enhance T-cell activation. We recently showed that PARPi induces CSF-1R+ suppressive tumor associated macrophages (TAMs) that restrict antitumor immune responses, contributing to PARPi resistance [2]. Removing TAMs with anti-CSF-1R therapy in combination with PARPi significantly enhanced overall survival (OS) compared to PARPi monotherapy in preclinical models [2]. Here, we investigate how modulating TAMs can enhance PARPi + ICB.MethodsMice bearing BRCA1-deficient TNBC (K14-Cre;Brca1f/f;p53f/f) tumors were treated for 98 days with PARPi (Talazoparib) ± small molecule inhibitor of CSF-1R (ARRAY-382; CSF-1Ri) ± anti-PD-1 and then followed for survival. Flow cytometry was employed to elucidate changes in the TME after treatment.ResultsPARPi conferred a significant survival advantage over vehicle treated mice (median OS 33 v. 14 days; p=0.0034) and 2/8 PARPi-treated mice experienced complete tumor clearance at day 98. PARPi + CSF-1Ri treated mice (median OS 140 days) remarkably cleared 7/10 tumors by day 98. The addition of anti-PD-1 to PARPi did not enhance OS compared to PARPi monotherapy. The triple combination of anti-PD-1 + PARPi + CSF-1Ri has not yet significantly enhanced the median OS compared to PARPi + CSF-1Ri (ongoing; 168 v. 140 days); nor did it increase clearance of tumor by day 98 (7/10). However, the triple combination led to superior long term tumor clearance. At day 161 the triple combination exhibited 5/10 tumor free mice compared to 2/10 treated with PARPi + CSF-1Ri. To elucidate how CSR-1Ri enhanced PARPi + ICB responses, flow cytometry was performed and revealed increased expression of the co-stimulatory molecule CD80, reduced tissue resident macrophages (CX3CR1+) and lower CSF-1R expression compared to PARPi + ICB.ConclusionsThese data suggest that targeting immunosuppressive macrophages may induce a favorable anti-tumor immune response and enhance responses to PARPi plus ICB. We are currently evaluating the adaptive immune response in this context.ReferencesPantelidou, C., et al., PARP inhibitor efficacy depends on CD8+ T cell recruitment via intratumoral STING pathway activation in BRCA-deficient models of triple-negative breast cancer. Cancer Discovery, 2019: p. CD-18-1218.Mehta, A.K., et al., Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer. Nat Cancer, 2021. 2(1): p. 66–82.
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Nirala, Bikesh Kumar, Lyazat Kurenbekova, Tajhal Patel, Ryan Lane Shuck, Atreyi Dasgupta, Nino Carlo Rainusso, and Jason T. Yustein. "Abstract 6713: Myc-regulated miR17, 20a modulate RANK expression in osteosarcoma." Cancer Research 83, no. 7_Supplement (April 4, 2023): 6713. http://dx.doi.org/10.1158/1538-7445.am2023-6713.
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Abstract Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Approximately 25-30% of these tumors carry amplification of chromosome 8q24, which harbors the oncogene c-Myc, and correlates with a poor prognosis in patients with OS. To understand the mechanisms that underlie the ability of Myc to alter both the tumor and its surrounding tumor immune microenvironment (TiME), we generated and molecularly characterized an osteoblast-specific Cre-Lox-Stop-Lox;(LSL)-c-MycT58A;p53f/+ knockin genetically engineered mouse model (GEMM). Phenotypically, the Myc knockin-GEMM had rapid tumor development with a high incidence of metastasis. Myc-dependent gene signature in our murine model demonstrated significant homology to the human Myc-amplified OS.Interestingly, we noticed a significant reduction in the osteoclast (OCL) cell population in the Myc knockin OS tumor compared to the p53-driven. We found the expression of RANK was significantly downregulated in the Myc knockin tumor compared to the Non-knockin p53 heterozygous tumors. The RANK/RANKL pathway is vital in OCL maturation and bone modeling/remodeling. To understand the involvement of Myc in RANK regulation, we used murine-derived OS cell lines and transiently knocked down of Myc expression using siRNA. We observed a significant upregulation in RANK expression after Myc knockdown. To decipher the molecular mechanism behind the Myc-dependent regulation of RANK expression in OS, we looked into the Myc-mediated microRNAs. Myc regulates the expression of several microRNAs, including the polycistronic miR-17-92 cluster. The expression of miR17-5p and Mir20a-5p was significantly higher in the GEMM tumor tissue samples isolated from the Myc knockin compared to the p53-driven. Further, we validated the Myc-dependent regulation of miR-17-5p/20a-5p expression using transient knockdown of Myc in mouse Myc knockin-derived cell lines. To examine the role of miR17-5p/20a-5p on the RANK regulation, we performed both gain and loss-of-function studies using microRNA-17/20a mimics and inhibitors. After the treatment with miR-17-5p/20a-5p inhibitors, the expression of RANK was significantly upregulated whereas in the case of miR17/20a mimics reversed these effects and led to a downregulation of RANK expression. We established that miR-17-5p/20a-5p is causally responsible for at least part of the mechanism by which Myc regulates the RANK expression in OS.We concluded that the Myc-regulated miR17/20a modulates the RANK expression that is involved in the OCL cell population regulation and function in the OS. Citation Format: Bikesh Kumar Nirala, Lyazat Kurenbekova, Tajhal Patel, Ryan Lane Shuck, Atreyi Dasgupta, Nino Carlo Rainusso, Jason T. Yustein. Myc-regulated miR17, 20a modulate RANK expression in osteosarcoma [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 6713.
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Barade, Aruna, Arun Kumar Arunachalam, Deborah Arul, Anu Korula, Anup J. Devasia, Fouzia NA, Kavitha M. Lakshmi, et al. "Germline Variants Contribute Significantly to the Pathogenesis of Aplastic Anemia in India." Blood 138, Supplement 1 (November 5, 2021): 1105. http://dx.doi.org/10.1182/blood-2021-148348.
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Abstract Introduction Aplastic anemia (AA) is characterized by a hypoplastic marrow and bone marrow failure (BMF), leading to peripheral pancytopenia. The treatment for AA currently consists of either hematopoietic stem cell transplant (HSCT) or immunosuppressive therapy (IST) with anti-thymocyte globulin and cyclosporine. We have previously reported poor responses to IST in Indian children with AA while adults (>15 years) show responses of 60%. Therefore, we wanted to study if we could identify constitutional variants in children and young adults with AA. Methods We included 102 young patients (≤40 years of age) diagnosed to have AA between year Jan 2006 to April 2021. Genomic DNA was extracted from peripheral blood and relative telomere length (rTL) was measured using quantitative real-time PCR (qPCR). The DNA was used to perform targeted gene capture using a custom capture kit which covered 2196 genes associated with various hematological disorders. Among the 2196 genes, the analysis was restricted to 96 genes associated with AA/IBMFS. Bioinformatics analysis was carried out using Genome Analysis ToolKit (GATK) best practices pipeline. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, the variants were labelled as pathogenic/likely pathogenic/variant of uncertain significance (VUS)/likely benign/benign. The candidate variants were validated by Sanger sequencing. Results The median age of patients was 9 (0-40) years, including 56 males (55%) and 46 females (45%). There were 15 patients with non-severe AA (NSAA) (15%), 63 with severe AA (SAA) (62%) and 24 with very severe AA (VSAA) (23%). The median relative telomere length of the entire cohort was 0.85 (0.21-3.10). The characteristics of patients age-wise are mentioned in Table 1. Genetic variants were identified in 34 patients (33.3%). This included germline pathological (PAT) genetic variants in 12.7%, variants of limited significance in 9.8% and variants of uncertain significance (VUS) in 10.7% patients. Of the 13 patients who had PAT variants, 6 patients had variants in telomere associated genes. This includes 2 patients with splice site and missense mutation in TINF2 (c.1221+5G>A & R282H); 2 patients with missense mutation in TERT (C828W & S947C); and 2 patients with frameshift and nonsense mutation in RTEL1 (L962S & R1010X). Homozygous MPL mutations [L79Q, R522T & P530L(n=3)] were observed in 5 patients. Two patients had PAT variants in DNAJC21 and NLRP12. Eleven VUS variants were present in the following genes, ATM (n=3), TINF2 (n=1), WRAP53 (n=1), BRCA2 (n=1), AK2 (n=1), FANCA (n=1), FANCN (n=1), ERCC6L2 (n=1) & PRF1 (n=1). The incidence of mutations in the age group ≤5, 6-10, 11-15 and 16-30 years were 51.8%, 25.6%, 27.8% & 33.4% respectively. There was significant difference in median rTL between patients with variants and no variants in the age-group 11-15 years (p=0.043). Discussion Genetic analyses studies in aplastic anemia patients were confined to single genes or limited gene sets. Keel et al., reported 5.1% of AA patients carried pathogenic variants in inherited BMF/MDS genes. We observed a high frequency of causative genetic variants (37%) in children (<10 years). Our study results highlight the importance of recognizing pathogenic and VUS genetic variants contributing to the pathogenesis of AA in the lower age group. Figure 1 Figure 1. Disclosures Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.
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Tan, Xiaohong, Lu Tong, Lin Li, Jinjin Xu, Shaofang Xie, Lei Ji, Junjiang Fu, et al. "Loss of Smad4 promotes aggressive lung cancer metastasis by de-repression of PAK3 via miRNA regulation." Nature Communications 12, no. 1 (August 11, 2021). http://dx.doi.org/10.1038/s41467-021-24898-9.
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AbstractSMAD4 is mutated in human lung cancer, but the underlying mechanism by which Smad4 loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated. Here, we generate a highly aggressive lung cancer mouse model bearing conditional KrasG12D, p53fl/fl LOF and Smad4fl/fl LOF mutations (SPK), showing a much higher incidence of tumor metastases than the KrasG12D, p53fl/fl (PK) mice. Molecularly, PAK3 is identified as a downstream effector of Smad4, mediating metastatic signal transduction via the PAK3-JNK-Jun pathway. Upregulation of PAK3 by Smad4 LOF in SPK mice is achieved by attenuating Smad4-dependent transcription of miR-495 and miR-543. These microRNAs (miRNAs) directly bind to the PAK3 3′UTR for blockade of PAK3 production, ultimately regulating lung cancer metastasis. An inverse correlation between Smad4 and PAK3 pathway components is observed in human lung cancer. Our study highlights the Smad4-PAK3 regulation as a point of potential therapy in metastatic lung cancer.
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Makino, Yuki, Hayato Hikita, Kenji Fukumoto, Ji Hyun Sung, Yoshihiro Sakano, Kazuhiro Murai, Sadatsugu Sakane, et al. "Constitutive activation of the tumor suppressor p53 in hepatocytes paradoxically promotes non-cell autonomous liver carcinogenesis." Cancer Research, June 13, 2022. http://dx.doi.org/10.1158/0008-5472.can-21-4390.
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Abstract In chronic liver diseases (CLDs), p53 is constitutively activated in hepatocytes due to various etiologies as viral infection, ethanol exposure, or lipid accumulation. This study was aimed to clarify the significance of p53 activation on the pathophysiology of CLDs. In Kras-mutant liver cancer model, Mdm2, a negative regulator of p53, was specifically deleted in hepatocytes (Alb-Cre KrasLSL-G12D Mdm2fl/fl) (LiKM; KrasG12D mutation and Mdm2 loss in the liver). Accumulation of p53 and up-regulation of its downstream genes were observed in hepatocytes in LiKM mice. LiKM mice showed liver inflammation accompanied by hepatocyte apoptosis, senescence-associated secretory phenotype (SASP), and the emergence of hepatic progenitor cells (HPCs). More importantly, Mdm2 deletion promoted non-cell autonomous development of liver tumors. Organoids generated from HPCs harbored tumor-formation ability, when subcutaneously inoculated into NOD/Shi-scid/IL-2Rγ (null) mice. Treatment with acyclic retinoid suppressed growth of HPCs in vitro and inhibited tumorigenesis in LiKM mice. All of the phenotypes in LiKM mice, including accelerated liver tumorigenesis, were negated by further deletion of p53 in hepatocytes (Alb-Cre KrasLSL-G12D Mdm2fl/fl p53fl/fl). Activation of hepatic p53 was noted in liver biopsy samples obtained from 182 CLD patients, in comparison to 23 normal liver samples without background liver diseases. In CLD patients, activity of hepatic p53 were positively correlated with the expression of apoptosis, SASP, HPC-associated genes and tumor incidence in the liver after biopsy. In conclusion, activation of hepatocyte p53 creates a microenvironment prone to tumor formation from HPCs. Optimization of p53 activity in hepatocytes is important to prevent CLD patients from hepatocarcinogenesis.
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Pierce, Stuart R., Ziwei Fang, Yajie Yin, Lindsay West, Majdouline Asher, Tianran Hao, Xin Zhang, et al. "Targeting dopamine receptor D2 as a novel therapeutic strategy in endometrial cancer." Journal of Experimental & Clinical Cancer Research 40, no. 1 (February 8, 2021). http://dx.doi.org/10.1186/s13046-021-01842-9.
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Abstract Background ONC201 is a dopamine receptor D2 (DRD2) antagonist that inhibits tumor growth in preclinical models through ClpP activation to induce integrated stress response pathway and mitochondrial events related to inhibition of cell growth, which is being explored in clinical trials for solid tumors and hematological malignancies. In this study, we investigated the anti-tumorigenic effect of ONC201 in endometrial cancer cell lines and a genetically engineered mouse model of endometrial cancer. Methods Cell proliferation was assessed by MTT and colony formation assays. Cell cycle and apoptosis were evaluated by Cellometer. Invasion capacity was tested using adhesion, transwell and wound healing assays. LKB1fl/flp53fl/fl mouse model of endometrial cancer were fed a control low fat diet versus a high fat diet to mimic diet-induced obesity. Following tumor onset, mice were treated with placebo or ONC201. Metabolomics and lipidomics were used to identify the obesity-dependent effects of ONC201 in the mouse endometrial tumors. DRD2 expression was analyzed by immunohistochemistry in human endometrioid and serous carcinoma specimens. DRD2 mRNA expression from the Cancer Genome Atlas (TCGA) database was compared between the four molecular subtypes of endometrial cancer. Results Increasing DRD2 expression in endometrial cancer was significantly associated with grade, serous histology and stage, as well as worse progression free survival and overall survival. Higher expression of DRD2 mRNA was found for the Copy Number High (CNH) subtype when compared to the other subtypes. ONC201 inhibited cell proliferation, induced cell cycle G1 arrest, caused cellular stress and apoptosis and reduced invasion in endometrial cancer cells. Diet-induced obesity promoted endometrial tumor growth while ONC201 exhibited anti-tumorigenic efficacy in the obese and lean LKB1fl/fl/p53fl/fl mice. Metabolomic analysis demonstrated that ONC201 reversed the obesity-driven upregulation of lipid biosynthesis and reduced protein biosynthesis in obese and lean mice. Conclusion ONC201 has anti-tumorigenic effects in endometrial cancer cells and a transgenic mouse model of endometrial cancer, and DRD2 expression was documented in both human serous and endometrioid endometrial cancer. These studies support DRD2 antagonism via ONC201 as a promising therapeutic strategy for endometrial cancer that has already demonstrated pharmacodynamic activity and clinical benefit in both serous and endometrioid endometrial cancer patients.
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Delahoussaye, Abagail M., Joseph Abi Jaoude, Morgan Green, Tara N. Fujimoto, Jessica Molkentine, Carolina J. Garcia Garcia, Jason P. Gay, et al. "Feasibility of administering human pancreatic cancer chemotherapy in a spontaneous pancreatic cancer mouse model." BMC Cancer 22, no. 1 (February 16, 2022). http://dx.doi.org/10.1186/s12885-022-09255-3.
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Abstract Background Both modified FOLFIRINOX (mFFX) and gemcitabine/nab-paclitaxel chemotherapy regimens have been shown to improve clinical outcomes in patients with pancreatic cancer, and are often used interchangeably as the standard of care. Preclinical studies often do not use these regimens, since administering these multiagent approaches can be difficult. In this study, we assessed the feasibility of administering these two chemotherapy regimens in spontaneous pancreatic tumors using KPC mice with the ultimate goal of advancing preclinical studies. Methods KPC mice were created by breeding KrasLSL−G12D/+ to Trp53fl/fl;Ptf1αCre/+, resulting in KrasLSL−G12D/+;p53fl/+;Ptf1αCre/+ mice. At 14 weeks of age, mice were palpated for spontaneous tumor growth that was verified using ultrasounds. Mice with tumors under 15 mm in diameter were used. The mice were assigned to one of seven treatment regimens: 1 cycle of mFFX (FFX X1), 2 cycles of mFFX (FFX X2), 1 cycle of mFFXwith 40 Gy SBRT (FFX SBRT), 1 cycle of gemcitabine/nab-paclitaxel (GEM/AB X1), 2 cycles of gemcitabine/nab-paclitaxel (GEM/AB X2), 2 cycles of gemcitabine/nab-paclitaxel with 40 Gy SBRT (GEM/AB SBRT), or saline only (control). Results In total, 92 mice were included. The median OS in the FFX X2 group was slightly longer that the median OS in the FFX X1 group (15 days vs 11 days, P = 0.003). Mice in the GEM/AB X2 group had longer OS when compared to mice in the GEM/AB X1 group (33.5 vs 13 days, P = 0.001). Mice treated with chemotherapy survived longer than untreated control animals (median OS: 6.5 days, P < 0.001). Moreover, in mice treated with chemotherapy, mice that received 2 cycles of GEM/AB X2 had the longest survival, while the FFX X1 group had the poorest OS (P < 0.001). The addition of chemotherapy was associated with reduced number of myeloid and lymphoid cell types, except for CD4 + cells whose levels were largely unaltered only in tumors treated with gemcitabine/nab-paclitaxel. Lastly, chemotherapy followed by consolidative SBRT trended towards increased local control and survival. Conclusions We demonstrate the utility and feasibility of clinically relevant mFOLFIRINOX and gemcitabine/nab-paclitaxel in preclinical models of pancreatic cancer.
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Rodriguez, B. Leticia, Limo Chen, Yanli Li, Shucheng Miao, David H. Peng, Jared J. Fradette, Lixia Diao, et al. "Targeting immunosuppressive Ly6C+ classical monocytes reverses anti-PD-1/CTLA-4 immunotherapy resistance." Frontiers in Immunology 14 (June 28, 2023). http://dx.doi.org/10.3389/fimmu.2023.1161869.
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IntroductionDespite significant clinical advancement with the use of immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) there are still a major subset of patients that develop adaptive/acquired resistance. Understanding resistance mechanisms to ICB is critical to developing new therapeutic strategies and improving patient survival. The dynamic nature of the tumor microenvironment and the mutational load driving tumor immunogenicity limit the efficacy to ICB. Recent studies indicate that myeloid cells are drivers of ICB resistance. In this study we sought to understand which immune cells were contributing to resistance and if we could modify them in a way to improve response to ICB therapy.ResultsOur results show that combination anti-PD-1/CTLA-4 produces an initial antitumor effect with evidence of an activated immune response. Upon extended treatment with anti-PD-1/CTLA-4 acquired resistance developed with an increase of the immunosuppressive populations, including T-regulatory cells, neutrophils and monocytes. Addition of anti-Ly6C blocking antibody to anti-PD-1/CTLA-4 was capable of completely reversing treatment resistance and restoring CD8 T cell activity in multiple KP lung cancer models and in the autochthonous lung cancer KrasLSL-G12D/p53fl/fl model. We found that there were higher classical Ly6C+ monocytes in anti-PD-1/CTLA-4 combination resistant tumors. B7 blockade illustrated the importance of dendritic cells for treatment efficacy of anti-Ly6C/PD-1/CTLA-4. We further determined that classical Ly6C+ monocytes in anti-PD-1/CTLA-4 resistant tumors are trafficked into the tumor via IFN-γ and the CCL2-CCR2 axis. Mechanistically we found that classical monocytes from ICB resistant tumors were unable to differentiate into antigen presenting cells and instead differentiated into immunosuppressive M2 macrophages or myeloid-derived suppressor cells (MDSC). Classical Ly6C+ monocytes from ICB resistant tumors had a decrease in both Flt3 and PU.1 expression that prevented differentiation into dendritic cells/macrophages.ConclusionsTherapeutically we found that addition of anti-Ly6C to the combination of anti-PD-1/CTLA-4 was capable of complete tumor eradication. Classical Ly6C+ monocytes differentiate into immunosuppressive cells, while blockade of classical monocytes drives dendritic cell differentiation/maturation to reinvigorate the anti-tumor T cell response. These findings support that immunotherapy resistance is associated with infiltrating monocytes and that controlling the differentiation process of monocytes can enhance the therapeutic potential of ICB.
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Zhang, Hui, Tiantian Wu, Chao Ren, Ning Dong, Yao Wu, and Yongming Yao. "p53 promotes the expansion of regulatory T cells via DNMT3a- and TET2- mediated Foxp3 expression in sepsis." Burns & Trauma 11 (January 1, 2023). http://dx.doi.org/10.1093/burnst/tkad021.
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Abstract Background Immunosuppression is an important characteristic of sepsis and is closely related to poor outcomes. Regulatory T cells (Tregs) contribute to immune suppression by inhibiting effector T cell (Teff) proliferation and differentiation. We aimed to investigate the role of p53 in Treg expansion after sepsis. Methods We constructed a sepsis model in wild-type (WT) and p53f/f/CD4-Cre+ mice by cecal ligation and puncture (CLP) and evaluated the proportions of CD4+CD25+ Foxp3+ Tregs by flow cytometry. The expression levels of forkhead/winged helix transcription factor p3 (Foxp3), DNA methyltransferase enzyme (DMNT)3a and ten–eleven translocation (TET)2 were examined using quantitative real-time PCR and Western blot analysis. Treg-specific demethylation region (TSDR) methylation sites in cells were analyzed by bisulfite-sequencing PCR. Furthermore, the direct binding of p53 to the Dnmt3a and TET2 promoters was illustrated using a luciferase assay. The suppressive ability of Tregs was indicated by enzyme-linked immunosorbent assay analysis of cytokine levels and the proliferation of cocultured Teffs. Finally, mortality rates after CLP were compared among WT and p53f/f/CD4-Cre+ mice. Results The proportion of CD4+CD25+ Foxp3+ Tregs was significantly reduced in p53f/f/CD4-Cre+ mice compared to WT mice after CLP. The enhanced expression of Foxp3 in WT mice was downregulated in the p53f/f/CD4-Cre+ group. We found decreased DMNT3a and increased TET2 levels after CLP. However, the dysregulation of DNMT3a and TET2 was significantly reversed in p53f/f/CD4-Cre+ mice. TSDR underwent increased demethylation in p53f/f/CD4-Cre+ mice. Luciferase activity indicated direct binding of p53 to the promoter regions of DNMT3a and TET2 to regulate their transcription. Consequently, Tregs from p53f/f/CD4-Cre+ CLP mice exhibited limited suppressive ability, as indicated by the reduced production of transforming growth factor-β and interleukin 10 (IL-10). In the coculture system, Teffs showed preserved production of IL-2, differentiation into Th1 cells and proliferation in the presence of Tregs isolated from p53f/f/CD4-Cre+ CLP mice. Finally, the mortality rate of the p53f/f/CD4-Cre+ group after CLP was significantly reduced in comparison to that of the WT group. Conclusion p53 appears to be critical for Foxp3 expression and consequent Treg expansion by regulating the induction of DNMT3a and TET2, thereby resulting in Foxp3-TSDR demethylation in the context of sepsis.
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Ma, Alvin Chun Hang, Christopher Chun Yu Mak, Kit San Yeung, Steven Lim Cho Pei, Dingge Ying, Mullin Ho Chung Yu, Kazi Md Mahmudul Hasan, et al. "Monoallelic Mutations in CC2D1A Suggest a Novel Role in Human Heterotaxy and Ciliary Dysfunction." Circulation: Genomic and Precision Medicine 13, no. 6 (December 2020). http://dx.doi.org/10.1161/circgen.120.003000.
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Background: Human heterotaxy is a group of congenital disorders characterized by misplacement of one or more organs according to the left-right axis. The genetic causes of human heterotaxy are highly heterogeneous. Methods: We performed exome sequencing in a cohort of 26 probands with heterotaxy followed by gene burden analysis for the enrichment of novel rare damaging mutations. Transcription activator-like effector nuclease was used to generate somatic loss-of-function mutants in a zebrafish model. Ciliary defects were examined by whole-mount immunostaining of acetylated α-tubulin. Results: We identified a significant enrichment of novel rare damaging mutations in the CC2D1A gene. Seven occurrences of CC2D1A mutations were found to affect 4 highly conserved amino acid residues of the protein. Functional analyses in the transcription activator-like effector nuclease–mediated zebrafish knockout models were performed, and heterotaxy phenotypes of the cardiovascular and gastrointestinal systems in both somatic and germline mutants were observed. Defective cilia were demonstrated by whole-mount immunostaining of acetylated α-tubulin. These abnormalities were rescued by wild-type cc2d1a mRNA but not cc2d1a mutant mRNA, strongly suggesting a loss-of-function mechanism. On the other hand, overexpression of cc2d1a orthologous mutations cc2d1a P559L and cc2d1a G808V (orthologous to human CC2D1A P532L and CC2D1A G781V) did not affect embryonic development. Conclusions: Using a zebrafish model, we were able to establish a novel association of CC2D1A with heterotaxy and ciliary dysfunction in the F2 generation via a loss-of-function mechanism. Future mechanistic studies are needed for a better understanding of the role of CC2D1A in left-right patterning and ciliary dysfunction.