Abstract 4285: Modulation of spliceosomal protein hnRNPH1 in BRAF, NRAS, and triple-wild type melanoma induces immunogenicity and in vivo immune response

Abstract Background: We previously reported that treating WM266-4 with H1 siRNA, followed by RNAseq, resulting in increased immune related pathways. Additionally, we reported novel melanoma leads, 2155-14 and 2155-18, which act via downregulation of spliceosomal heterogenous nuclear ribonuclear proteins hnRNPH1/H2 (H1/H2) and demonstrated efficacy in an in vivo model of A375 BRAF mutation. This study aimed to determine the effect of H1 modulation on immune-related genes in vitro across melanoma cell lines with diverse mutational backgrounds and in vivo using a syngeneic mouse model. Methods: To assess in vivo immune response, 2155-14 and 2155-18 were tested in the B16F10 C57BL/6J syngeneic mouse model (n=5). In vitro, we knocked down H1/H2 in BRAF-mutant, NRAS-mutant, and triple-wild type melanoma cell lines and treated BRAF and NRAS cell lines with both compounds to evaluate immune responses to spliceosomal modulation. Data from in vivo experiments were analyzed using one-way ANOVA to compare means, followed by Sidak’s multiple comparisons for significant differences. Statistical significance was set at p < 0.05. For NanoString experiments, a 1.4-fold change and p-value <0.05 (n=3) were used as thresholds. Results: Treatment of B16F10 syngeneic melanoma mouse model with 2155-14 and 2155-18 resulted in increased tumor infiltration of macrophages (p-value <0.01), dendritic cells (p-value <0.05), B cells (p-value <0.05), and CD4+ T cells (p-value <0.05) as determined by flow cytometry analysis. Moreover, NanoString gene expression analysis of both transfected and treated samples confirmed upregulation of interferon signaling and cellular immune responses, processes known to enhance cellular immunogenicity. Conclusion: These findings highlight the therapeutic promise of targeting spliceosomal proteins H1 and H2, and they underscore the potential of 2155-14 and 2155-18 as promising drug candidates for melanoma treatment. Furthermore, the results suggest that spliceosomal inhibition can enhance immune system activation, presenting an opportunity to combine these therapies with FDA-approved immunotherapies to achieve improved treatment outcomes. Citation Format: Maab Sultan, Sadeeshkumar Velayutham, Vladimir Beljanski, Keiran S.M. Smalley, Nhut Minh Nguyen, Shweta Shah, Tulsi Desai, Aveta Singh, Jun Yong Choi, Dmitriy Minond. Modulation of spliceosomal protein hnRNPH1 in BRAF, NRAS, and triple-wild type melanoma induces immunogenicity and in vivo immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4285.