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Background The directed use of targeted therapy to sensitize tumors to immunotherapy remains a key therapeutic opportunity. There is significant evidence that optimal cancer cell fitness resides within a range of oncogenic pathway signaling (not too much and not too little). 1–3 Here we show that synthetically inducing a senescence-like state through oncogenic pathway hyperactivation augments immunotherapy responses.Methods BRAF inhibitors can paradoxically activate ERK 4–6 but it has been unknown whether this effect causes senescence akin to oncogene-induced senescence. We tested NRAS mutant melanoma and KRAS mutant adenocarcinoma lines to see whether paradoxical hyperactivation of ERK would lead to arrest. We termed this approach ‘pathway-synthetic senescence’ and additionally showed that this sensitizes RAS mutant tumors to immunotherapy.Results BRAF inhibitors hyperactivated ERK signaling resulting in nanomolar IC50 in long-term cultures of NRAS-mutant melanoma cell lines (figure 1A-D). The 2nd generation BRAFi dabrafenib (figure 1A) and encorafenib (figure 1B) showed nM efficacy against these lines; PLX8934, which does not cause ERK hyperactivation, had no effect figure (figure 1D). Concomitant MEK or ERK inhibition abrogated this effect proving that ERK signaling is indeed the key driver (figure 1E-F) in which cell growth could be recovered in the presence of BRAFi and slowly increasing MEKi or ERKi concentrations. We then asked whether these changes could enhance responses to immunotherapy using two novel immunocompetent models of RAS-mutant melanoma and pancreatic adenocarcinoma (PDAC). The Nras(Q61R)-mutant melanoma model is a syngeneic, transplantable tumor model on the C57BL/6 background.7 The Kras-mutant PDAC model is based upon orthotopic transplantation of Sleeping Beauty transposon-mediated mutagenesis.8 Surprisingly, both RAS-mutant murine models showed effective tumor regression and suppression of metastasis only when mice were treated with dabrafenib and anti-PD1 (figure 2A). Molecular profiling of the tumor microenvironment showed increased infiltration of activated CD8+ T-cells as well as significantly decreased myeloid populations (figure 2B). Several cytokines relevant for T-cell activation including IL2, IL15, and IFNg were increased in association with tumor cell stress responses including autophagy (figure 2C). Finally, single agent encorafenib was administered in the 3rd and 4th line in two patients with late stage refractory metastatic PDAC. One with a 66.5% VAF for KRAS(G12D) responded temporarily (figure 2D) whereas one with 13.5% VAF for KRAS(G12D) did not (figure 2E) suggesting mutant RAS-dependent effects.Conclusions Pathway-synthetic senescence drives cell cycle arrest, sensitization to immunotherapy, and is a novel conceptual analogue to synthetic lethality. A novel fragment based drug screen is planned to identify new compounds to leverage activation of additional pathways.References Chang L, et al. Systematic profiling of conditional pathway activation identifies context-dependent synthetic lethalities. Nat Genet. 2023;55:1709–1720.Unni AM, et al. Hyperactivation of ERK by multiple mechanisms is toxic to RTK-RAS mutation-driven lung adenocarcinoma cells. Elife. 2018;7.Varmus H, Unni AM, Lockwood WW. How cancer genomics drives cancer biology: does synthetic lethality explain mutually exclusive oncogenic mutations? Cold Spring Harb Symp Quant Biol. 2016;81:247–255.Hatzivassiliou G, et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature. 2010;464:431–435.Poulikakos PI, Zhang C, Bollag G, Shokat KM, Rosen N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature. 2010;464:427–430.Heidorn SJ, et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell. 2010;140:209–221.Murphy BM, et al. The OSUMMER lines: a series of ultraviolet-accelerated NRAS-mutant mouse melanoma cell lines syngeneic to C57BL/6. Pigment Cell Melanoma Res. 2023;36:365–377.Mann KM, et al. Sleeping beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma. Proc Natl Acad Sci U S A. 2012;109,5934–5941.Abstract 878 Figure 1Paradoxical activation of ERK signaling arrests NRAS-mutant melanoma cell linesAbstract 878 Figure 2Pathway-synthetic senescence augments response to immunotherapy, induces key cytokines, and induces tumor stasis in metastatic PDAC patients