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Background Acquired resistance in cancer is a major obstacle to durable response after immune checkpoint blockade (ICB). While interferon (IFN) signaling is frequently linked with effective ICB response, prolonged interferon signaling specifically within cancer cells paradoxically drives immune suppression and acquired resistance to therapy. 1–3 In preclinical mouse models, in vivo relapsed tumors as well as tumors treated chronically ex vivo with IFNy display resistance to ICB, increased expression of immune evasive molecules, and an altered epigenetic landscape characterized by inflammatory memory domain accessibility that is maintained by STAT1 and IRF3.4 Here, we further characterize the function of cancer IFN signaling in treatment failure, and we restore ICB response by targeting pathways that maintain IFN-associated resistance.Methods Res 499 tumor cells, derived from a late-relapse B16 melanoma, 5 were manipulated as indicated and flank-injected into C57BL/6 mice, followed by ICB (aCTLA4 plus aPDL1). Tumors were harvested on day 16 post-implantation for RNA-seq, ATAC-seq, and flow cytometry.Results We find that prolonged exposure of cancer cells to IFN selectively increases the expression level and duration of a subset of IFNy stimulated genes (ISGs), which we term ‘memory ISGs’, that are enriched for genes with known immune evasive properties. Moreover, persistent cancer IFN signaling induces elevated expression of not only dsRNA-forming endogenous retroelements (EREs), but also of IFN-inducing dsRNA sensors such as MDA5, establishing a cell intrinsic feed-forward loop. Consequently, CRISPR mediated knock out of dsRNA pattern recognition receptors (PRRs) disrupts this loop - lowering ERE RNA expression, decreasing chromatin accessibility at inflammatory memory loci, and increasing tumor sensitivity to ICB ( figure 1). We observe similar epigenetic effects and ICB responsiveness when we pharmacologically block IFN and PRR signaling through 3-week ex vivo treatment with Ruxolitinib and Cmpd1,6 inhibitors of JAK1/2 (downstream of IFN) and TBK1(downstream of dsRNA PRRs) respectively. Via flow cytometry, we note that ex vivo JAKi and TBK1i treatment, in a cancer cell intrinsic manner, induces a less exhausted and more stem-like CD8 T cell response following ICB, possibly through improved interactions with dendritic cells. Notably, short term in vivo co-administration of JAKi and TBK1i following ICB further improves therapeutic efficacy beyond either agent alone (figure 2). Given emerging clinical data supporting JAK inhibition in ICB-refractory tumors,7 8 these findings may suggest additional benefits from targeting TBK1 as well.Conclusions Targeting IFN and PRR-driven feedback with combined JAK and TBK1 inhibition may overcome IFN-associated resistance and enhance ICB efficacy.References Benci JL, Xu B, Qiu Y, Wu TJ, Dada H, Twyman-Saint Victor C, Cucolo L, et al. Tumor interferon signaling regulates a multigenic resistance program to immune checkpoint blockade. Cell. 2016;167(6):1540–1554.e12.Benci JL, Johnson LR, Choa R, Xu Y, Qiu J, Zhou Z, Xu B, et al. Opposing functions of interferon coordinate adaptive and innate immune responses to cancer immune checkpoint blockade. Cell. 2019;178(4):933–948.e14.Wong CW, Evangelou C, Sefton KN, Leshem R, Zhang W, Gopalan V, Chattrakarn S, et al. PARP14 inhibition restores PD-1 immune checkpoint inhibitor response following IFNγ-driven acquired resistance in preclinical cancer models. Nat Commun. 2023;14(1):5983.Qiu J, Xu B, Ye D, Ren D, Wang S, Benci JL, Xu Y, et al. Cancer cells resistant to immune checkpoint blockade acquire interferon-associated epigenetic memory to sustain T cell dysfunction. Nat Cancer. 2023 Jan. https://doi.org/10.1038/s43018-022-00490-y.Twyman-Saint Victor C, Rech AJ, Maity A, Rengan R, Pauken KE, Stelekati E, Benci JL, et al. Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature. 2015;520(7547):373–377.Jenkins RW, Aref AR, Lizotte PH, Ivanova E, Stinson S, Zhou CW, Bowden M, et al. Ex vivo profiling of PD-1 blockade using organotypic tumor spheroids. Cancer Discov. 2018;8(2):196–215.Mathew D, Marmarelis ME, Foley C, Bauml JM, Ye D, Ghinnagow R, Ngiow SF, et al. Combined JAK inhibition and PD-1 immunotherapy for non-small cell lung cancer patients. Science. 2024;384(6702):eadf1329.Zak J, Pratumchai I, Marro BS, Marquardt KL, Zavareh RB, Lairson LL, Oldstone MBA, et al. JAK inhibition enhances checkpoint blockade immunotherapy in patients with hodgkin lymphoma. Science. 2024;384(6702):eade8520.Abstract 451 Figure 1(A) Summary GSVA enrichment scores and heatmap for expression of endogenous retroelements in Res 499 cells sorted from in vivo tumors. (B) Volumes of either WT or MDA5 and RIG-I knockout (PRR_DKO) Res 499 tumors treated with ICB (aCTLA4 and aPDL1)Abstract 451 Figure 2Volumes of tumors with indicated treatment. After standard ICB regimen, JAK inhibitor (60 mg/kg), TBK1 inhibitor (40 mg/kg), or both were dosed daily via oral gavage beginning on day 11 and continuing for 5 days (denoted in bisque)