Distinct small molecule inhibitors of Kras specifically prime CTLA4 blockade therapy to transcriptionally reprogram Tregs and overcome resistance to suppress pancreas cancer

Lack of sustained response to oncogenic Kras (Kras*) inhibition in preclinical models and patients with pancreatic ductal adenocarcinoma (PDAC) emphasizes the need to identify impactful synergistic combination therapies to achieve robust clinical benefit. Kras* targeting results in an influx of T cell infiltrates including Tregs, effector CD8 ⁺ T cells and exhausted CD8 ⁺ T cells expressing several immune checkpoint molecules in PDAC. Here, we probe whether the T cell influx induced by different Kras* inhibitors enable a therapeutic window to prime adaptive immune response in PDAC. Here we report a specific synergy between Kras ^G12D allele specific inhibitor, MRTX1133 or multi-selective pan-RAS inhibitor, RMC-6236 and anti-CTLA4 immune checkpoint blockade. In contrast, attempted therapeutic combination with multiple other immune checkpoint inhibitors, including anti-PD1, anti-Tim3, anti-Lag3, anti-Vista and anti-4-1BB agonist antibody failed due to compensatory mechanisms mediated by other checkpoints on exhausted CD8 ⁺ T cells. Specifically, anti-CTLA4 therapy in Kras* targeted PDAC transcriptionally reprograms effector T regs to a naïve phenotype, reverses CD8 ⁺ T cell exhaustion and is associated with recruitment of tertiary lymphoid structures (TLS) containing follicular B cells, interferon (IFN)- stimulated/ activated B cells, plasma cells and germinal center B cells to functionally enable efficacy of immunotherapy with long-term survival. In this regard, inhibition of the TLS with lymphotoxin-β inhibitor (LTBi) or direct B cell depletion reversed the survival benefit conferred by the combination therapy and highlights the function of TLS in generating productive anti-tumor immune responses. Further, single cell ATAC sequencing analysis revealed that transcriptional reprogramming of Tregs is epigenetically regulated by downregulation of AP-1 family of transcription factors including Fos, Fos-b, Jun-b, Jun-d in the IL-35 promoter region. This study reveals an actionable vulnerability in the adaptive immune response in Kras* targeted PDAC with relevant clinical implications.