GPATCH3, a splicing regulator that facilitates tumor immune evasion via the modulation of ATPase activity of DHX15

Tingrong RenGaigai WeiHaiping ZhaoHuiling ZhangYuqi ZhangJingjing YiZhihan GuoYihan WangJiating KuangZhaoying ShengDuanwu Zhang^*

• Fudan University, Shanghai, China

Introduction: Aberrant pre-mRNA splicing is increasingly recognized as a key contributor to tumorigenesis and immune evasion. However, the regulatory factors orchestrating splicing dynamics within the tumor microenvironment remain incompletely understood. Here, we identify GPATCH3, a previously uncharacterized G-patch domain-containing protein, as a critical modulator of alternative splicing and immune regulation in cancer. Methods: We employed biochemical studies, splicing reporter assays, and transcriptomic analyses to elucidate the function of GPATCH3. In vitro and in vivo models, including GPATCH3depleted cell lines and mouse xenografts, were used to assess its roles in tumor progression.Immune infiltration patterns were analyzed using TIMER2.0 and validated by immunological profiling. Results: GPATCH3 interacts with the RNA helicase DHX15 and enhances its ATPase activity, promoting proper spliceosome disassembly. Loss of GPATCH3 led to splicing alterations, including in immunoregulatory genes such as CXCR3, FOXP3, and CD44. Functional studies demonstrated that GPATCH3 deficiency reduced tumor growth in vivo and was associated with enhanced infiltration of cytotoxic T cells and NK cells. Conversely, high GPATCH3 expression correlated with immunosuppressive cell populations, including MDSCs and CAFs, across multiple cancer types. Transcriptomic analysis further revealed that GPATCH3 deficiency upregulates immunomodulatory genes such as CXCL8 and LAG3, suggesting a role in shaping the TME via splicing regulation. Discussion: Our findings establish GPATCH3 as a critical regulator that governs alternative splicing and immunosuppressive microenvironment remodeling. By modulating the splicing 3 fidelity of key immune genes and altering their expression, GPATCH3 may facilitate immune escape and tumor progression. These results provide mechanistic insights into how RNA splicing factors interface with immune regulation and highlight GPATCH3 as a potential therapeutic target for immunomodulatory cancer therapy.