RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b+ Bregs thereby overcoming melanoma PD1-resistance

Development of effective second-line treatment options for patients with BRAF^wtNRAS^wt or BRAF^wtNRAS^mut melanoma resistant to immune checkpoint blockade (ICB) is crucial. While systemic delivery of agonist CD40 (aCD40) plus anti-PD1 (αPD1) showed activity in patients with ICB-resistant melanoma, the objective response rate was modest (15%), in part due to induction of B regulatory cells (Bregs) which suppress CD8⁺ effector T cell responses. We previously reported that RAS/RAF/PI3K-inhibition elevates CD40 expression in melanoma cells and sensitizes tumors to ICB. Here, we show that combined treatment with a RAS/PI3K/AKT-pathway inhibitor rigosertib (RGS), and/or a MEK1/2 inhibitor trametinib (T), plus aCD40, overcomes the ICB resistance of BRAF^wtNRAS^wt and BRAF^wtNRAS^mut melanoma tumors growing in C57BL/6 mice. In addition, overexpression of CD40 in these melanoma cells effectively reverses ICB-resistance and aCD40 + αPD1 treatment induces tumor regression. Mechanistically, RGS + T suppress aCD40-associated CD11b⁺PD-L1⁺ Bregs, promoting CD8⁺ T-cell mediated killing in melanoma. scRNA-Seq analyses confirm CD40-associated CD11b⁺ Bregs across cancer types in patients. Our data demonstrate that addition of RAS/PI3K/AKT and MEK inhibitors to aCD40 resolves the issue of aCD40 induction of CD11b⁺PD-L1⁺ Bregs and provides alternative therapeutic options for ICB-resistant BRAF^wtNRAS^wt or BRAF^wtNRAS^mut metastatic melanoma.