Therapeutic Targeting of Spliceosomal Proteins hnRNPH1/H2 Demonstrates Potent Anti-Melanoma Activity In BRAF Inhibitor Resistant Melanoma, and Enhances Tumor Immune Response

Background: Melanoma remains one of the most aggressive malignancies, and resistance to targeted therapies such as BRAF inhibitors continues to limit long-term patient outcomes. We previously identified small molecule compounds, 2155-14 and 2155-18, that inhibit melanoma growth by downregulating spliceosomal proteins hnRNPH1 and H2. Methods: Acute toxicity was evaluated in male and female Balb/C mice treated subcutaneously with 50 mg/kg/day of each compound for 21 days. Clinical behavior, body weight, hematology, blood chemistry, and organ morphology were assessed. Antitumor efficacy was examined in both BRAF inhibitor-sensitive and BRAF/NRAS inhibitor-resistant A375 melanoma xenografts in nude athymic mice treated with 25 mg/kg of 2155-14 or 2155-18 three times weekly, compared with vemurafenib monotherapy and vemurafenib/cobimetinib combination. Immunomodulatory effects were evaluated in an immunocompetent B16F10 melanoma-bearing syngeneic mouse model. Results: No significant toxicities were detected in treated mice, demonstrating a favorable short-term safety profile. In A375 xenografts, including the BRAF inhibitor-sensitive and BRAF/NRAS inhibitor-resistant models, both compounds significantly reduced tumor growth, with efficacy comparable to monotherapy and the vemurafenib/cobimetinib combination. Tumor volume, tumor weight, and histopathology confirmed robust inhibition of tumor progression. In the B16F10 model, treatment with either compound increased infiltration of multiple immune cell populations within the tumor microenvironment, indicating enhanced antitumor immune activation. Conclusions: Compounds 2155-14 and 2155-18 exhibit strong preclinical anti-tumor activity, minimal toxicity, and evidence of immune engagement. These findings support hnRNPH1/H2 spliceosomal protein modulation as a promising therapeutic approach for advanced or drug-resistant melanoma and provide a rationale for future development, such as monotherapy or in combination with immunotherapy. Keywords: Melanoma, Spliceosomal proteins, BRAF and NRAS inhibitor resistance, Xenograft models, cell line xenograft, immunotherapy