2-Hydroxyisobutyrylation of SRSF9 Regulates CD44 Alternative Splicing to Promote Colorectal Cancer Liver Metastasis.

Colorectal cancer liver metastasis (CRLM) remains a major cause of cancer mortality, yet the metabolic mechanisms that remodel metastatic RNA-splicing programs are poorly defined. Here, integrated quantitative proteomics and lysine 2-hydroxyisobutyrylation (Khib) proteomics of patient-matched normal mucosa, primary CRCs, and liver metastases revealed a progressive increase in global Khib, with enrichment on RNA-splicing proteins. Serine/arginine-rich splicing factor 9 (SRSF9) emerged as a Khib-modified splicing factor whose expression and K155 Khib levels increased from normal tissue to primary tumors and liver metastases. Gain and loss of function studies in CRC cell lines, patient-derived organoids, subcutaneous xenografts, and splenic injection liver metastasis models showed that SRSF9 promotes proliferation, invasion, tumor growth, and liver colonization. Mechanistically, K155 Khib stabilized SRSF9 protein; the K155R mutation accelerated degradation and abolished SRSF9-driven malignant phenotypes. KAT7 and SIRT6 act as regulators of Khib modification, which positively and negatively modulate the K155 Khib modification of SRSF9, respectively. SRSF9 bound CD44 pre-mRNA through its RRM1 domain and promoted alternative splicing toward the oncogenic CD44V8-10 isoform, which was elevated in patient tumors and metastases and rescued the impaired proliferation and metastatic capacity caused by SRSF9 loss. Finally, a CD44V8-10-targeting antisense oligonucleotide suppressed CRC growth and liver metastasis in preclinical models. These findings define a KAT7/SIRT6-regulated Khib-SRSF9-CD44V8-10 axis that couples metabolic acylation to metastatic splicing and nominate CD44V8-10 as a therapeutic vulnerability in CRC liver metastasis.