Abstract 5511: Frontline therapy with anti-PD1 enhances the durability of combination targeted therapy in NRAS-mutant melanoma
Authors: Phadke Manali S. , Chen Zhihua , Mohamed Eslam , Davies Michael , Chen Ann , Rix Uwe , Rodriguez Paulo , Smalley Keiran 
Published: September 2020

Abstract
Melanoma is one of the deadliest form of skin cancer. Although the activating NRAS mutations are present in 20% of melanoma patients, currently, there are no FDA-approved therapies for treating NRAS-mutant melanoma. In this project, our goal is to prime the immune system by immunotherapy and then improve the targeted therapy responses by suppressing recruitment of inhibitory immune cell populations and by increasing T cell infiltration, which would lead to enhanced responses in NRAS-mutant melanoma. We used a syngeneic C3H/HeNCrl mouse model of NRAS-mutant melanoma. The mice were treated with two doses of either anti-PD1 or IgG every 5 days followed by everyday treatment with either single agent or combination of ceritinib and trametinib for a total period of 40 days. Single cell RNA-seq platform was used to determine the impact of immunotherapy and targeted therapy upon each individual cell type. Flow cytometry experiments were carried out to assess the percentage of T cells and MDSCs recruitment. Altered signaling responses and chemokines/ cytokines responsible for MDSCs recruitment were determined from RPPA and RNA-seq data respectively. The in vivo studies demonstrated that the tumors in the mice treated with anti-PD1 followed by combination shrunk significantly over a period of 40 days without any relapse. Single cell RNA seq analysis and flow cytometry experiments revealed a 15- and 20-fold increase in T cell infiltration in these tumors. Immune cells depletion experiments showed that the cytotoxic effects of combination therapy was dependent upon CD8+ T cells only. Single cell RNA seq analysis also demonstrated that priming with immunotherapy increased IFN γ expression on T cells and subsequent treatment with ceritinib+trametinib increased melanoma antigen presentation by increased MHC class I expression. The RPPA data from tumors treated with anti-PD-1 followed by combination demonstrated inhibition of RTKs implicated in therapy escape such as c-MET, EGFR, IGF1R, HER2/3. Flow cytometric analysis also revealed around 60% decrease in Tregs and 80% decrease in MDSCs recruitment in the tumors treated with anti-PD1 followed by combination. RNA seq analysis revealed that ceritinib decreased mRNA expression of cytokines/ chemokines such as Cxcl5, Cxcl3, Cxcl2, Csf1, Cx3cl1, Cklf, that have been implicated in the recruitment and differentiation of MDSCs. Hence, we demonstrate that frontline immunotherapy primes the immune system, making the tumors more immunogenic with an increased accumulation of activated CD8+ T cells. Successive treatment with combination targeted therapy not only increases antigen presentation to T cells but also decreases recruitment of immunosuppressive MDSCs thus preventing immune escape and tumor progression. In summary, we have developed novel targeted therapy combinations that can be sequentially used with immunotherapy to maximize the durations of response in treatment of NRAS-mutant melanoma.
Citation Format: Manali S. Phadke, Zhihua Chen, Eslam Mohamed, Michael Davies, Ann Chen, Uwe Rix, Paulo Rodriguez, Keiran Smalley. Frontline therapy with anti-PD1 enhances the durability of combination targeted therapy in NRAS-mutant melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5511.