Abstract LB075: Small molecule procaspase activating compound 1 (PAC-1) enhances CAR-T immunotherapy for solid tumors in syngeneic murine models
Authors: Ranoa Diana R. , Kang Yifei , Drnevich Jenny , Rendon Gloria , Fields Christopher J. , Bailey Keith , Roy Edward J. , Fan Timothy M. , Kranz David M. , Hergenrother Paul J. 
Published: April 2024

Abstract
Chimeric antigen receptor (CAR)-T cell therapy has been shown to effectively treat solid tumors in syngeneic murine models, but antigen loss and T cell trafficking into the tumors pose major obstacles to its long-term efficacy. Here we explored a combination therapy against advanced stages of colorectal and ovarian syngeneic murine tumor models using a tumor-specific small molecule (PAC-1) that triggers the engagement of the host immune system and synergizes with a Tn-specific adoptively transferred 237 CAR-T immunotherapy. PAC-1 has been shown to induce apoptosis in tumor cells with high expression of procaspase 3, via chelation of inhibitory zinc and release of active executioner caspase 3. PAC-1 also recently completed a phase I trial (NCT02355535) and was shown to be well-tolerated in late-stage cancer patients. RNA sequencing of CT26 colorectal cells stimulated with PAC-1 for 24 hours revealed that PAC-1 induced global changes at the transcriptome level, up-regulating genes involved in immune activation and apoptosis, and down-regulating genes involved in DNA repair, cell cycle regulation, and myc-driven proliferation. Validation of RNAseq data was performed using a combination of RT-qPCR, Western blot, flow cytometry, and co-culture studies. A combination of transcriptome and exome sequencing analyses in murine colorectal cell line CT26 revealed that the PAC-1-mediated down-regulation of DNA repair machinery led to somatic alterations in the genome and a higher mutational load in tumor cells, subsequently presented as neo-antigens by antigen-presenting cells. As a result, host CD8+ cytotoxic T cells were able to infiltrate tumors and reduce the tumor burden. Tn-dependent CARs have been used recently to demonstrate the proof of concept in the targeting of cancer-specific Tn-antigens such as the Tn-MUC1 antigen in humans, and an analogous Tn-OTS8 target in mice. In our murine ID8 ovarian tumor model, untreated mice with disseminated tumors have a median survival of 71 days. As a single agent administered intraperitoneally, PAC-1 extended the median survival to 90 days (p&lt;0.0001), while 237 CAR-T treated mice have a median survival of 103 days (p&lt;0.0001). A combination of the two treatment modalities (PAC-1 administered before 237 CAR-T) further extended the median survival to 145 days (p=0.048). Administering PAC-1 after 237 CAR-T also modestly extended the median survival to 132 days (p=0.06). Taken altogether, our results demonstrate that PAC-1 engages the immune system to treat cancer by inducing expression of pro-inflammatory cytokines in tumor cells allowing the recruitment of CD8+ effector T cells to the tumor microenvironment. Combination strategies that boost T cell trafficking shows great promise to enhance the anti-tumor efficacy and feasibility of adoptive CAR-T therapy against solid tumors.
Citation Format: Diana R. Ranoa, Yifei Kang, Jenny Drnevich, Gloria Rendon, Christopher J. Fields, Keith Bailey, Edward J. Roy, Timothy M. Fan, David M. Kranz, Paul J. Hergenrother. Small molecule procaspase activating compound 1 (PAC-1) enhances CAR-T immunotherapy for solid tumors in syngeneic murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB075.