Rigosertib, a potential RAS-targeted agent, induces CD40 expression on melanoma cells to enhance response to immune checkpoint inhibitors

Immune checkpoint blockade (ICB) is the current first-line treatment for metastatic melanoma. However, ICB fails in many patients and has dangerous side effects. Rigosertib (RGS), a non-ATP-competitive small molecule RAS mimetic, has the potential to block oncogenic RAS-RAF-MEK-ERK and/or PI3K-AKT-mTOR signaling pathways. RGS treatment (300mg/kg) of melanoma tumor-bearing mice is well tolerated and results in ~50% inhibition of tumor growth as monotherapy and ~70% inhibition in combination with ICB. RGS-induced tumor inhibition depends on the induction of CD40 expression on melanoma cells, followed by immunogenic cell death, leading to an inflamed tumor microenvironment with enrichment of dendritic cells and activated CD8+ T cells. Highlights • RGS impairs melanoma tumor growth via direct killing and immunogenic cell death that promotes an inflamed tumor microenvironment. • RGS-induced tumor inhibition depends on the induction of CD40 expression on melanoma cells. • Combining RGS with αPD1+αCTLA4 improves tumor response. • Tumor CD40 levels are prognostic for therapeutic response to RGS and BRAF inhibitor. Significance: A high CD40 expression level correlates with CD80, ICOS-L, beneficial type I T cell responses, and better survival in melanoma patients (cBioPortal database). Tumor CD40 levels are prognostic for therapeutic response to RGS and BRAF inhibitor (DepMap and Oncomine databases), and RGS induces CD40 expression in melanoma tumor cells. Our preclinical data support the therapeutic use of RGS plus αPD1+αCTLA4 in RAS/RAF/MEK and/or PI3K pathway-activated melanoma tumors and point to the need for clinical trials to determine the clinical benefit of RGS plus ICB for metastatic melanoma patients who do not respond to ICB alone. The goal of this study is to identify Rigosertib-induced gene expression in YUMM3.3 melanoma tumors by RNA-Seq. Tumors were collected into RNAlater. RNA was extracted using Qiagen RNAeasy plus mini Kit for three replicates from H20 and Rigosertib treated groups. Samples were submitted to Vanderbilt Technologies for Advanced Genomics (VANTAGE) for RNA QC, library prep and sequencing with a mean of ~30M reads/sample. Samples were sequenced on the Illumina NovaSeq 6000 using 150bp paired-end reads. Reads were then aligned to the STAR aligner for reads.