LAUSR

Immuno-oncologic (IO) approaches have completely transformed lung cancer treatment. Unfortunately, most patients with NSCLC develop primary resistance during IO and less than 20% achieve partial or complete response. Understanding the pathways to resistance is a significant challenge that requires a focused approach combining both clinical and basic research. Because resistance to IO is complex and likely multimodal, it is vital to find alternative strategies to overcome these challenges. IO generally employs cellular-mediated delivery of granzyme (GrB) as the final toxic payload and fusion proteins containing GrB serve as a targeted therapeutic with an identical mechanism of cytotoxic action. Subsequent development of resistance to GrB-based fusions may provide a look into the intrinsic mechanism of resistance related to cell signaling and gene expression for IO agents. The human fusion protein GrB-Fc-IT4 contains a single chain variable fragment targeting the Fn14 (fibroblast growth factor-inducible 14) receptor for TWEAK that is overexpressed in several tumor types, including NSCLC. GrB-Fc-IT4 exhibits high affinity and selective cytotoxicity within the nanomolar range when tested against a large panel of Fn14+ human cancer cell lines including NSCLC. We generated 3 resistant clones from a NSCLC GrB-sensitive cell line (A549) by exposing the cells to increasing concentrations of GrB-Fc-IT4. We then conducted a comprehensive genomic characterization of the GrB-sensitive A549 and compared to the GrB-Fc-IT4 resistant clones (A549R-F5, A549R-F7 and A549R-F8) to identify significant differentially expressed genes (DEGs) and pathways involved in GrB-resistance mechanisms. By mapping gene expression changes we have established that clones A549R-F5 and A549R-F7 have a very similar genomic profile, while the clone A549R-F8 displays a distinctive genomic profile. These results suggest that clones A549R-F5 and A549R-F7 shared a similar mechanism of resistance whereas clone A549R-F8 mechanism of resistance might be different. Comparison of the GrB-resistant clones with 7 NSCLC cell lines sensitive to GrB-Fc-IT4 revealed 43 significant DEGs (padj<0.05), suggesting these genes could be involved in the GrB response. From these DEGs, 34 genes had a higher basal expression in the GrB-resistant clonal cell lines while 9 DEGs were downregulated. Significantly, sensitivity of GrB-resistant cell lines to the chemotherapy agent cisplatin showed that the GrB-resistant clones were 6-fold more resistant than the parental cell line. We found that 13 DEGs of the 43 DEGS have also been described to be involved with cisplatin- or damage-response. These novel candidates for GrB resistance could prove to be important prognostic markers or targets for tailored combined therapy in the future. Research conducted, in part, by the Clayton Foundation for Research and Lung Spore (P50CA070907). Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Ganiraju C. Manyam, Jing Wang, John V. Heymach, Michael G. Rosenblum. Identification of genomic pathways associated with resistance of NSCLC to immunotherapies containing granzyme B. [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 3901.