Targeting the RIG-I-like receptor signaling pathway to improve the efficacy of immunotherapy in mucosal and uveal melanoma.
Authors: MacBeth Morgan L. , Van Gulick Robert J. , Tobin Richard P. , Cogswell Dasha T. , Medina Theresa Michelle , McCarter Martin D. , Robinson William , Couts Kasey L. 
Published: June 2021

e21593 Background: Patients with cutaneous (CM) or acral (AM) melanoma have high response rates to immune checkpoint blockade (ICB) therapies including anti-CTLA4, anti-PD1, and their combination. In contrast, patients with mucosal (MM) or uveal (UM) melanoma have low ICB response rates and a poorer overall prognosis. In this study, we analyzed melanoma transcriptomes to identify potential mechanisms underlying ICB resistance in MM and UM. We also evaluated epigenetic modifying drugs as potential agents to improve ICB responses in these patients. Methods: RNA sequencing was performed on 13 MM, 8 AM, and 24 CM patient-derived xenograft tumors. These data were analyzed along with published RNA sequencing data for 80 UM and 443 CM tumors. MM and UM cells were treated with several different classes of epigenetic modifying drugs, and the ICB-resistant YUMM1.7 mouse melanoma syngeneic model was used to evaluate the tumor growth effect of one particular drug, decitabine, at low (2.5 ug) and intermediate (10 ug) doses in combination with anti-PD1 (200 ug). Results: We identified 2513 differentially expressed genes (DEG) between MM and CM tumors (ANODEV adjusted p-value < 0.05, FDR < 0.1) compared to only 495 DEG between AM and CM. Ingenuity Pathway Analysis (IPA) analysis of the 2513 DEG in MM identified the RIG-I-like receptor (RLR) pathway as inhibited in MM versus CM ( p < 1x10-5). The RLR pathway was also inhibited in ICB-resistant CM versus ICI-sensitive (anti-CTLA4, p < 0.006 and anti-PD1, p < 0.0002) and in UM versus CM ( p < 1x10-8), but was not inhibited in AM. The RLR pathway is an innate immune pathogen sensing pathway that is critical for ICB responses in CM; therefore, we sought to identify FDA-approved drugs which activate the RLR pathway in MM and UM. Since RLR signaling is frequently repressed by epigenetic silencing, we screened direct DNMT1 inhibitors 5’-azacitidine (AZA) and 5’-aza-deoxycitidine (decitabine, DEC), several indirect DNMT1 inhibitors, and two HDAC inhibitors. We found that only DEC strongly induced the expression of RLR pathway genes in MM, UM, and ICB-resistant CM cells. Compared to vehicle, treatment of YUMM1.7 tumors with single agent anti-PD1 ( p > 0.87) and low dose ( p > 0.90) or intermediate dose ( p > 0.80) DEC had no effect on tumor growth. However, the addition of either low or intermediate dose DEC to anti-PD1 showed a trend of decreased tumor growth (low dose, 31% decrease, p = 0.22 and intermediate dose, 41% decrease, p = 0.55). Conclusions: We identified low RLR pathway signaling as one possible mechanism underlying ICB resistance in MM and UM. We show that an FDA-approved DNMT1 inhibitor, decitabine, strongly induces RLR pathway genes in MM and UM cells in vitro and leads to tumor growth suppression in combination with anti-PD1 in vivo. Therefore, co-administration of decitabine and anti-PD1 agents is a rational and novel strategy for improving outcomes for MM and UM patients.