Dysregulation of the Notch1 receptor has been shown to facilitate the development and progression of colorectal cancer (CRC) and has been identified as an independent predictor of disease progression and worse survival. Although mutations in the NOTCH1 receptor have not been described in CRC, we have previously discovered a NOTCH1 gene copy number gain in a portion of CRC tumor samples. Here, we demonstrated that a NOTCH1 gene copy number gain is significantly associated with worse survival and a high percentage of gene duplication in a cohort of patients with advanced CRC. In our CRC patient-derived tumor xenograft (PDTX) model, tumors harboring a NOTCH1 gain exhibited significant elevation of the Notch1 receptor, JAG1 ligand and cleaved Notch1 activity. In addition, a significant association was identified between a gain in NOTCH1 gene copy number and sensitivity to a Notch1-targeting antibody. These findings suggest that patients with metastatic CRC that harbor a gain in NOTCH1 gene copy number have worse survival and that targeting this patient population with a Notch1 antibody may yield improved outcomes.
Introduction: Inhibiting the activity of the epidermal growth factor receptor (EGFR) with monoclonal antibodies has been utilized as a therapeutic strategy for patients with metastatic colorectal cancer (CRC), leading to improved clinical results alone and in combination with standard chemotherapy. Many systematic reviews and metanalyses were performed to better understand the role of EGFR inhibition in CRC, revealing that KRAS exon 2 mutations and furthermore exons 3 and 4 and NRAS exons 2, 3, and 4 were predictive of non-responsiveness to these agents. Concurrent with these results has been the development of immunotherapy targeting immune regulatory checkpoints such as CTLA-4 and PD-1 that have initiated a new era in the treatment of cancer. In order to gain a better biological understanding of the context of immune responses and facilitate preclinical evaluation of cancer immunotherapy, we developed a hematopoietic humanized mouse model utilizing patient-derived CRC xenograft tumor models to assess immune therapy for RAS mutant CRC. Not only could evaluation of humanized RAS mutant PDX models provide additional information on the potential for clinical activity of immune therapies, but could also improve the understanding of immune responses to RAS mutant cancers. We therefore hypothesize that humanized RAS mutant colorectal PDX models can be used to evaluate the preclinical activity of immune targeted agents for treatment of RAS mutant colorectal cancer. Methods: Humanized BRG mice developed from the BALB/cRag2-/-IL2Rγc-/- (BRG) strain which is known to accept human hematopoietic stem cells, have been used to enhance engraftment. BRG newborn pups were humanized through transplantation of approximately 1x105 CD34+ cells purified from umbilical cord blood. The mice were evaluated for chimerism at 8 and 12 weeks. At 14 weeks, tumor tissue from established PDX models were implanted on the right and left flank of humanized mice. The tumor was selected among a cluster within the “immune-enriched” subtype (C2) based upon the RNAseq characterization of the models. When the average tumor size reached a volume of approximately ~150-300 mm3, the mice were randomized into either vehicle or nivolumab treatment groups. Mice were monitored daily for signs of toxicity and weighed twice weekly. They were treated with nivolumab (30 mg/kg) twice a week by intraperitoneal injection for 15 days. Tumor size was evaluated twice weekly by caliper measurements using the following equation: tumor volume= (length × width2) × 0.52. At the end of the treatment, mice were euthanized while sera, lymph nodes, spleen, bone marrow and tumors were collected for further investigation. Results: Humanized RAS mutant CRC PDX models were successfully established in vivo. While no differences were observed in tumor growth among the control and treated arms, we were able to detect differences in PD1 expression among treated versus control mice, with lower expression in the nivolumab treated group. We also observed higher numbers of T cells in the lymph nodes of nivolumab treated mice, suggesting T cell expansion. Interestingly, we also observed an increase of T cells in the spleen and blood and late occupancy of T cells in the bone marrow. Two of the treated mice exhibited identifiable TILs that were comprised of a majority of CD4+ T cells with an activated phenotype (CD69+). Conclusions: Humanized KRAS mutant CRC PDX models were successfully established and tumor engraftment occurred in all humanized mice with nivolumab-treated mice demonstrating the development of lymph nodes that were populated by activated T cells. These preliminary results demonstrate that human immunity and PD-1 expressing T cells exist in these models and provide the basis for planned immunotherapy combination studies. This abstract is also being presented as Poster A23. Citation Format: Anna Capasso, Julie Lang, Todd M. Pitts, S. Lindsey Davis, Chris H. Lieu, Stacey M. Bagby, Aik Choon Tan, John J. Tentler, Jill E. Slansky, Roberta Pelanda, S. Gail Eckhardt. Characterizing the immune context of responses to immunotherapy in humanized patient derived xenograft models of CRC. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr PR03.
Pancreatic ductal adenocarcinoma (PDAC) shows little to no symptoms and is often detected at later stages which makes it difficult to treat resulting in a very low survival rate. PDAC is resistant to most chemotherapy mostly due to a high amount of stroma and low vascularity. Additionally, PDAC is resistant to the newer immunotherapies due to the lack of immune cells and/or the presence of exhausted T-cells. Expression of WNT pathway genes allows cancer to avoid the immune system by affecting immune cell activation and migration into the tumor microenvironment. In this study, we investigate Immune activation and migration by co-culturing peripheral blood mononuclear cells (PBMCs) with PDAC spheroids in the presence of SM04690 (CLK/WNTi). SM04690 is a potent CLK and downstream WNT signaling inhibitor. The migration of PBMCs into PDAC spheroids were analyzed by FLOW cell cytometry and fluorescence confocal microscopy. The total CD3+ T cell population remained unchanged in the presence of SM04690, however, T cell subpopulations were affected by WNT inhibition. The total CD4+ T cell population in the spheroid co-culture was significantly increased while the CD8+ population was significantly reduced. Among the CD4+ T cell population, there was a significant increase in CCR7+CD45RA- central memory cells and CCR7-CD45RA- effector memory cells, while there was a significant decrease in FoxP3+CD25+ regulatory T cells. Surface expression of PD-L1 and HLA-DR on PDAC cells were also significantly increased compared to the untreated co-culture control. Fluorescent labeled PDAC and PBMCs allow clear quantification of immune infiltration through spheroid and Matrigel organoid systems. Genes associated with b-catenin destruction complex (AXIN1 and 2, APC, GSK3A) and transcription factors associated with gene expression (TCF7 and TCF7L1) were also significantly reduced in the drug treatment. Additional studies are ongoing investigating the role cancer-associated fibroblast (CAFs) play in the tumor microenvironment in the presence of SM04690 (CLK/WNTi). WNT inhibition can be a potent immune modulator and influence surface marker expression on the tumor. This can lead to future drug combinations with immune checkpoint inhibitors or drug combinations taking advantage of expression changes. Future directions involve utilizing PRO-seq to identify enhancer RNAs and immediate transcriptional effects of WNT inhibition, identify novel targets for combination therapy, and in vivo PDAC humanized animal models studying the effects in the presence of an autologous immune system. Citation Format: Adrian TA Dominguez, Adreanna G. Real, Cecilia B. Levandowski, Matthew S. Lewis, Sarah Hartman, Betelehem Yacob, Stephen Smoots, Anna Schreiber, Jordi Lanis, Pragya Nepal, Jennifer Diamond, Chris Lieu, Wells Messersmith, Julie Lang, Todd Pitts. Immune modulation in immune co-cultures of pancreatic ductal adenocarcinoma by small molecule CLK/WNT pathway inhibitor SM04690 [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 3509.
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