Preclinical efficacy of a PROTAC pan-KRAS degrader in a KRAS G12D syngeneic mouse model and concurrent immune tumor microenvironment changes

Missense mutations in KRAS occur in ~20% of cancers. Although 2 KRAS inhibitors have been approved for patients with KRAS G12C-mutated disease, resistance quickly develops; moreover, no approved agents target other KRAS mutants. We developed a tool PROteolysis TArgeting Chimera (PROTAC) pan-KRAS degrader that induces the ubiquitination and subsequent proteasomal degradation of KRAS; it potently and selectively degrades active and inactive KRAS forms. Selective KRAS degradation that spares HRAS and NRAS may yield a higher therapeutic index than current investigational RAS inhibitors targeting all RAS isoforms. Additionally, the iterative activity of the PROTAC pan-KRAS degrader may overcome resistance driven by KRAS upregulation, a common RAS inhibitor resistance mechanism. Mutant KRAS is an established intrinsic driver of an immunosuppressive tumor microenvironment (TME), which can be alleviated by KRAS inhibition, suggesting potential synergy with immune checkpoint blockade (ICB). We compared the antitumor activity of PROTAC-driven KRAS degradation vs RAS inhibition (± ICB with an anti-programmed death-1 antibody) and the effects on TME in a KRAS G12D murine colorectal cancer model, CT-26. Our PROTAC potently degraded mutant KRAS and exhibited antiproliferative effects in spheroids that were comparable to a clinical RAS(ON) inhibitor. Oral administration of the PROTAC pan-KRAS degrader as a single agent in the CT-26 model led to substantial KRAS degradation (>85%), robust and durable suppression of mitogen-activated protein kinase pathway activity, and substantial tumor growth inhibition, including complete responses (CRs). The PROTAC pan-KRAS degrader combined with ICB led to deeper and more rapid tumor regressions, resulting in a higher rate of CRs and longer post-treatment survival, compared with the RAS(ON) inhibitor plus ICB. To characterize TME changes, tumors treated with the PROTAC pan-KRAS degrader or RAS(ON) inhibitor (± ICB) were compared by bulk RNA sequencing (RNAseq) and tumor-infiltration leukocyte (TIL) analysis. RNAseq revealed strong RAS pathway suppression by both agents, leading to altered gene expression in tumor-intrinsic and extrinsic molecular pathways. KRAS degradation demonstrated a differential positive enrichment for key immune-related pathways associated with antitumor immunity and immune activation, suggesting PROTAC-specific immune TME changes may contribute to its antitumor effects. TIL analysis demonstrated statistically significant changes in immune populations, including cytotoxic T cells and myeloid cells, after PROTAC treatment. The observed rapid tumor regressions, paired with deeper immune response and longer survival, suggest that the PROTAC pan-KRAS degrader is more efficacious and permissive to ICB combination than a RAS(ON) inhibitor in an immunocompetent tumor model system.