SIPA1L3缺失通过Rap1-MAPK激活在ALK-/RET-融合阳性NSCLC中诱导TKI耐药

ALK-/RET-fusion-positive non-small cell lung cancer (NSCLC) accounts for 3-5% and 1-2% of total NSCLC cases, respectively. Selective tyrosine kinase inhibitors (TKIs) against ALK or RET have demonstrated remarkable efficacy, and six ALK-TKIs and two RET-TKIs have been approved for the treatment of ALK- or RET-rearranged NSCLC. However, resistance can occur even in patients who initially achieve marked tumor shrinkage. To date, many investigators have reported mechanisms of ALK- or RET-TKI resistance, focusing on secondary mutations, activation of bypass signaling pathways, or EMT transition. Still, in a significant number of cases, the mechanism of resistance remains unknown.To discover additional potential resistance mechanisms, we conducted a genome-wide CRISPR/Cas9 screening under floating culture condition culture conditions in CCDC6-RET fusion positive LC2/ad cells treated with selpercatinib or pralsetinib for nine days. sgRNAs in surviving drug-tolerant cells were then analyzed by NGS. We identified multiple candidate genes, including MED12 and SIPA1L3, that contribute to drug-tolerant persister (DTP) cell survival during RET-TKI treatment. SIPA1L3 encodes a Rap1 GTPase-activating protein (GAP) that promotes the conversion of the active GTP-bound form of Rap1 to its inactive GDP-bound form. Knockout of SIPA1L3 induced significant increase of Rap1-GTP, leading to resistance through activation of the MAPK and PI3K/Akt/mTOR pathways. Phosphoproteomic analysis confirmed upregulation of phospho-Raf, -ERK, and -S6, indicating enhanced MAPK signaling in SIPA1L3-deficient LC2/ad cells after selpercatinib treatment as compared to control cells after the same treatment.These findings highlight the critical regulatory role of SIPA1L3 on Rap1 activation in bypassing RET inhibition through MAPK activation. Notably, combining RET-TKIs with MAPK pathway inhibitors-particularly the Raf-MEK dual inhibitor avutometinib-markedly reduced DTP cell survival in vitro, and induced tumor shrinkage in SIPA1L3-knocked-out tumors that relapsed on selpercatinib single treatment in vivo , suggesting a promising strategy to overcome such resistance. Furthermore, loss of SIPA1L3 also conferred ALK-TKI resistance in the ALK-fusion H3122 cell line, accompanied by increased Rap1-GTP expression. Co-treatment with alectinib and avutometinib effectively suppressed DTP formation.In conclusion, this study identifies SIPA1L3 as a critical mediator of ALK- or RET-TKI resistance in NSCLC from genome-wide CRISPR screening and underscores the therapeutic potential of avutometinib in combination with ALK-/RET-TKIs to overcome drug-tolerant persistence and improve treatment outcomes in ALK-/RET-rearranged NSCLC.