SPHINX31 acts as a SRPK1 inhibitor targeting the ATR/DNA-PKcs/CHK1 replicative checkpoint to inhibit cell growth in non-small cell lung cancer.

Splicing targeting drugs have emerged as promising anticancer drugs. However, their mechanisms of action remain largely unknown, notably in Non-Small Cell Lung Carcinoma (NSCLC). In this study, we demonstrated that SPHINX31, which targets SRPK1, inhibits the ATR/CHK1 signaling pathway, a cornerstone of the replicative stress response, leading to decreased cell proliferation, increased DNA damage and apoptosis, notably in NSCLC cells resistant to platinum salts. Mechanistically, we found that SRPK1 is recruited at stalled replication forks upon replicative stress, co-immunoprecipitates with the ATR/ATRIP/TOPBP1 complex, directly interacts with TOPBP1 BRCT4/5 and BRCT7/8 domains, and contributes to the accumulation of TOPBP1 nuclear foci. We further showed that SRPK1 controls the splicing of genes previously related to ATR signaling, notably WIZ. All these events are prevented by SPHINX31. Lastly, we showed that the inhibitory effects of SPHINX31 on ATR are counterbalanced by the activation of DNA-PKcs. Altogether, this study uncovers SRPK1 as a new component of the ATR/DNA-PKcs/CHK1 replicative checkpoint. SRPK1 inhibitors, alone or in combination with DNA-PKcs or CHK1 inhibitors, could provide therapeutic benefit in NSCLC patients including those who relapse after platinum-based chemotherapy.