Targeting CD11b+ regulatory B cells via RAS/RAF/PI3K pathway inhibition augments response to immune checkpoint blockade or CD40 agonism in metastatic melanoma

Abstract Development of effective second-line treatment options for immune checkpoint blockade (ICB) resistant patients with BRAFWTNRASWT or BRAFWTNRASmut melanoma is crucial. While systemic delivery of agonist CD40 (aCD40) plus anti-PD1 (aPD1) 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 cells. We previously reported that RAS/RAF/PI3K-inhibition elevated CD40 expression in melanoma cells and sensitized tumors to ICB. Here, we show that treatment of ICB-resistant BRAFWTNRASWT and BRAFWTNRASmut melanoma tumors with the combination of rigosertib (RGS), a RAS/PI3K/AKT-pathway inhibitor, and/or trametinib (T), a MEK1/2 inhibitor, plus aCD40 overcomes ICB resistance. Overexpression of CD40 in melanoma cells effectively reverses ICB-resistance and induces tumor regression upon aCD40+aPD1 treatment. Mechanistically, RGS+T suppressed aCD40-associated CD11b+PD-L1+ B regulatory cells (B11regs), which promoted CD8+ T-cell killing in melanoma. scRNA-Seq analyses confirmed CD40-associated B11regs across cancer types in patients. Statement of Significance Addition of aCD40 treatment to ?PD1 fails to overcome ICB resistance due to aCD40-induction of B11regs and low expression of CD40 in melanoma cells. Our data demonstrate that addition of RAS/RAF/PI3K inhibitors to aCD40 resolves these issues and provides alternative therapeutic options for ICB-resistant BRAFWTNRASWT or BRAFWTNRASmut metastatic melanoma. Overall design: CD40 overexpression (CD40-OE) and CD40-ctrl clones of 1014 NRAS-mut melanoma cells were generated and injected in C57BL/6 mice. Treatment with 200µg/mouse aPD1 (every 3 days, intraperitoneal), 30µg/mouse aCD40 (every 3 days, intraperitoneal), or 300mg/kg rigosertib (RGS, 5 days a week, oral gavage), starts at day 8 post tumor cell inoculation. Tumor samples were harvested 22 days post-treatment and proceeded with bulk RNA-Seq and TCR-Seq (n=3 per treatment group).