2584 Background: Mechanism-based strategies to overcome resistance to anti-PD1 therapy are urgently needed. Using CRISPR/Cas9 genome editing tools, we developed acquired resistant models through JAK1/2 and B2M loss of function (LoF) mutations in human melanoma cell lines and in the murine MC38 colon carcinoma, known for high mutational load and good response to anti-PD-1. We hypothesized that the downstream activation of the IFN-receptor pathway or the activation of natural killer (NK) cells would overcome this resistance. Methods: We studied signaling changes in four human cell lines (parental and LoFs) exposed to IFN-gamma using RNAseq. In addition, we analyzed the in-vivo antitumor activity in MC38 variants with anti-PD1 and characterized the tumor microenvironment using mass cytometry (CyTOF). Finally, we tested strategies to overcome resistance mechanisms with SD-101 (TLR-9 agonist) and bempegaldesleukin (NKTR-214, CD-122 biased agonist) with the extent of CD8 and NK1.1 depletion. Results: RNAseq differential gene expression analysis showed that the IFN-gamma induced increased expression of antigen presenting machinery, IFN-gamma signaling and chemokines (CXCL9/10) was lost in JAK1/2-LoF human melanoma cell lines. The significant antitumor activity of anti-PD-1 against MC38 parental cell line was lost in JAK1/2 and B2M LoF sublines, and CyTOF analysis revealed that anti-PD-1 therapy was unable to increase tumor CD8+ T-effectors in these LoF tumors. The intratumoral administration of SD-101 (50 μg/injection q4dx3wks) was able to overcome local resistance even in non-injected sites in JAK1/2 and IFNAR-type-I LoF tumors, and systemic administration of bempegaldesleukin (0.8 mg/kg, q9dx2, i.v.) was able to overcome resistance in B2M LoF with significantly increased survival (Table). Depletion studies showed complete abrogation of anti-tumor response with anti-NK1.1 in JAK1 LoF and B2M LoF, and partial abrogation with anti-NK1.1 or anti-CD8 in JAK2 LoF tumors. Conclusions: Even in the extreme setting of genetic resistance to PD-1 blockade by JAK1/2 LoF, resistance can be overcome by SD-101, a TLR9 agonist, while resistance of B2M LoF can be overcome by bempegaldesleukin (NKTR-214), a CD-122 biased agonist. Our findings support the testing of these rational mechanistic strategies in patients with a-PD1 resistance. [Table: see text]