Abstract A040: Survival mechanisms common to immunotherapy and drug tolerant persister tumor cells

Abstract Background: Persisters are a sub-population of tumor cells that survive anti-cancer therapy and are thought to be a major cause of tumor recurrence. Persisters have been identified following both drug- and immune-therapy but are generally considered to be very distinct entities. The focus of this study is to examine any similarities/differences between immunotherapy persister cells (IPCs) and drug tolerant persister cells (DTPs). Both pharmacological agents and immune cells often kill via apoptosis. Therefore, we investigated if reduced apoptotic sensitivity is a survival mechanism common to IPCs and DTPs and whether the use of BH3 mimetics to enhance apoptotic sensitivity could reduce the survival of IPCs and DTPs. Multi-therapy resistance is often observed in the clinic, therefore, we investigated if IPCs and DTPs exhibit a reciprocal multi-therapy resistant phenotype which could be conferred through a reduction in apoptotic sensitivity. Methods: IPCs were generated by co-culturing HeLa and U251 tumor cell lines with EGFR-CAR T cells for 3 weeks, replenishing CAR T cells every 3 days. Using three tumor cell line models (Hela, MC38 and B16), nine DTP cell lines were generated by chronically treating tumor cells for 7 days with an IC90 dose of drug (5-FU, oxaliplatin and etoposide), replenishing drug dose every 3 days. A CellTiter-glo or CellTox assay was used to determine the sensitivity of IPCs and DTPs to drugs and radiation. Sensitivity to T cell attack was assessed by flow cytometry (cell counting beads and zombie viability dye. Microscopy-based BH3 profiling was employed to measure the mitochondrial apoptotic sensitivity of persisters and to probe for therapeutically exploitable anti-apoptotic dependencies. Results: IPCs acquired a reduced sensitivity to multiple drug classes and radiotherapy. Likewise, DTPs also developed a reduced sensitivity to multiple drug classes and radiotherapy but also acquired a reduced sensitivity to CAR T cell killing. Depending on the cell line and therapy, some persisters developed either an increased or decreased sensitivity to mitochondrial apoptosis. Inhibition of specific anti-apoptotic dependencies using BH3 mimetics increased apoptotic sensitivity of persisters. Conclusions: Both immunotherapy and drug-tolerant persisters develop a reduced sensitivity to multiple therapeutic modalities, suggesting non-immune mechanisms are important in the survival of cancer cells after immunotherapy. These results suggest there are common mechanisms involved in producing persisters from dramatically different therapies. Our study highlights the potential of BH3 profiling to guide the selection of therapies to target persisters. Citation Format: Maria Davern, Cole J Turner, Lara Bencsics, Brenda Chan, Jasmine Yun-Tong Kung, Michael Olson, Cheyanne Walker-Williams, David Barbie, Shaili Soni, Patrick Bhola, Marcela V Maus, Anthony Letai. Survival mechanisms common to immunotherapy and drug tolerant persister tumor cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr A040.