Abstract 4124: Targeting the clock pathway to modulate immune response in MSI-high colorectal cancer (CRC): evidence from a preclinical in vivo model
Authors: Soni Shivani , Battaglin Francesca , Castanon Sofi , Lo Jae Ho , Smbatyan Goar , Chan Priscilla , Qu Meng , Jayachandran Priya , Arai Hiroyuki , Kawanishi Natsuko , Zhang Wu , Kay Steve A. , Lenz Heinz Josef , Torres Evanthia T. 
Published: April 2023

Abstract
Background. Immune checkpoint inhibitors have substantial clinical success in MSI-H/dMMR metastatic CRC. However, only a subset of patients exhibits durable responses, emphasizing the importance of identifying mechanism of innate and acquired resistance. The relation between the circadian clock and the immune system is well established. We used a syngeneic mouse model of MSI-H CRC to investigate whether a targeted enhancement of clock repressor proteins cryptochrome 1/2 (CRY 1/2) may affect tumor growth and response to anti-PD1 (aPD1).
Methods. 1 × 106 MC38 cells were subcutaneously implanted into the right flank of 8 weeks old C57BL/6 mice. Mice were randomized into 1 control and 3 treatment arms (10 mice/group): A. CRY stabilizer SHP656(10 mg/kg PO, 5 days/week); B. aPD1 (100 μg/mouse IP, twice/week administered for 2 weeks); C. combination SHP656 + aPD1. Tumors from 4 mice/group were processed for tumor infiltration studies. Remaining 6 mice/group were kept for 3 weeks on maintenance aPD1 (twice/week) for survival studies. Treatment response was measured by tumor growth and survival. Treatment effects on immune cell infiltration in the tumor microenvironment (TME), functional myeloid derived suppressor cells (MDSCs) and T cells markers were investigated via flow cytometry.
Results. Single agent SHP656 significantly improved survival when compared with vehicle treated mice. Similarly, combination treatment with SHP656 + aPD1 improved survival, however no additional benefit was observed over treatment with aPD1 alone. All treatment groups had a response rate of 67% (4/6 mice/treatment group showed 2-fold decrease in tumor volume 24 days after tumor challenge vs vehicle). In responders, SHP656 as a single agent or in combination with aPD1 were more effective in suppressing tumor growth as compared to aPD1 alone. Flow cytometry of 14 tumor samples demonstrated that monocytic-MDSC infiltration was significantly decreased in the combination therapy and aPD1 alone treatment arms (p = .016) as compared to vehicle suggestive of a less suppressive TME. T cell analysis suggested a decrease in intratumoral exhausted CD8+ T cells (TIM3−PD-1+, p = .02) in SHP656 + aPD1 vs vehicle, while infiltration of CD4+ T cells and T regulatory cells were not significantly affected by any treatment. Nanostring profiling on treated tumors also confirmed immune activation in responders.
Conclusions. Our results show for the first time that targeting the clock pathway through modulation of CRY affects TME and tumor progression in in vivo models of MSI-H CRC. A decrease in infiltration of mMDSCs and exhausted CD8+ T cells seems to play a critical role in combination treatment efficacy but needs to be studied further. These results, suggests that pharmacological targeting of the circadian machinery holds great potential and could significantly impact the efficacy of immunotherapy in metastatic CRC.
Citation Format: Shivani Soni, Francesca Battaglin, Sofi Castanon, Jae Ho Lo, Goar Smbatyan, Priscilla Chan, Meng Qu, Priya Jayachandran, Hiroyuki Arai, Natsuko Kawanishi, Wu Zhang, Steve A. Kay, Heinz Josef Lenz, Evanthia T. Torres. Targeting the clock pathway to modulate immune response in MSI-high colorectal cancer (CRC): evidence from a preclinical in vivo model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4124.