Abstract 3509: Immune modulation in immune co-cultures of pancreatic ductal adenocarcinoma by small molecule CLK/WNT pathway inhibitor SM04690
Authors: Dominguez Adrian TA , Real Adreanna G. , Levandowski Cecilia B. , Lewis Matthew S. , Hartman Sarah , Yacob Betelehem , Smoots Stephen , Schreiber Anna , Lanis Jordi , Nepal Pragya , Diamond Jennifer , Lieu Chris , Messersmith Wells , Lang Julie , Pitts Todd 
Published: June 2022

Abstract
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.