Targeting CLK1/SRSF7 axis-dependent alternative splicing sensitizes pancreatic ductal adenocarcinoma to chemotherapy and immunotherapy.

AIM

The persistently high mortality rate of pancreatic ductal adenocarcinoma (PDAC) is largely attributed to the acquired resistance to chemotherapy, particularly gemcitabine. This study aims to elucidate the underlying molecular mechanisms of gemcitabine resistance in PDAC, uncover additional pro-tumorigenic factors contributing to drug resistance, and develop more effective and safer targeted therapeutic strategies against this phenomenon.

METHODS

Circular RNA (circRNA) sequencing was employed to identify differentially expressed circRNAs between chemo-sensitive and resistant tumors. Liquid Chromatography-Mass Spectrometry (LC-MS) was utilized to uncover the RNA-binding proteins (RBPs) associated with circular RNA of alpha-1, 3-glucosyltransferase 8 (cALG8). Molecular biology techniques were applied to explore the biological functions and regulatory mechanisms of cALG8 in the context of gemcitabine resistance in PDAC. Single-cell sequencing was performed to reveal changes in the composition of tumor immune microenvironment of pancreatic cancer. Patient-Derived Organoid (PDO) and Patient-Derived Xenograft (PDX) were employed to further validate the molecular mechanisms. Finally, antisense oligonucleotides (ASOs) targeting cALG8 were developed for in vivo use, and their translational therapeutic potential was evaluated in mouse models.

RESULTS

This study identified that cALG8, which is associated with alternative splicing, is highly expressed in gemcitabine-resistant PDAC cells. cALG8 regulates the alternative splicing complex, thereby promoting chemoresistance and immunosuppression in PDAC. Mechanistically, high level of cALG8 functions as a protein scaffold through its 34-85 nt and 109-160 nt regions, creating spatial conditions for CDC-like kinase 1 (CLK1) to phosphorylate serine/arginine-rich splicing factor 7 (SRSF7) at site 231S. This process facilitates the formation of the SRSF7-dependent ataxia-telangiectasia mutated (ATM) kinase variant, ATM203, enhancing the translational efficiency of ATM, and consequently promoting DNA damage repair and immune microenvironment remodeling in PDAC cells to counteract the effects of chemotherapeutic drugs. A cALG8-targeting ASO that disrupts the CLK1-SRSF7 interaction, when combined with gemcitabine and anti-programmed cell death protein (PD)-1 antibody, significantly reduced tumor burden in PDX model, validating its therapeutic translational value.

CONCLUSION

We demonstrated that the cALG8/CLK1/SRSF7 axis promotes ATM expression by enhancing the splicing of ATM203, thereby facilitating gemcitabine resistance and formation of an immunosuppressive microenvironment in PDAC. This insight aids in the development of drugs targeting chemotherapy resistance induced by DNA damage repair mechanisms in PDAC.