Background: Lung cancer, including Non-Small Cell Lung Carcinoma (NSCLC), is the leading cause of cancer mortality worldwide. Platinum salts are the gold standard chemotherapy for NSCLC but many patients develop resistance leading to disease progression. Identifying new therapeutic strategies to counteract resistance is crucial. Pharmacological compounds targeting core components of the spliceosome machinery have emerged as promising anti-cancer agents. However, their mechanisms of action remain to be elucidated in NSCLC. Methods: Various NSCLC cell lines were used in 2D and 3D cultures or clonogenic assays. NSCLC Patient-Derived Xenografts were also used. SF3B1 was silenced by siRNA. Flow cytometry was performed to analyze cell cycle distribution and apoptosis. Western-blot, immunofluorescence, SIRF analysis and DNA repair assays were done to assess globally the DNA damage response. RNA-Seq, RT-qPCR and RT-PCR studies were performed to identify gene and splicing events impacted by SF3B1 inhibition. Publicly available transcriptomic and proteomic data were analyzed. Results: SF3B1 is a core component of the spliceosome machinery. We show that NSCLC cells with acquired resistance to platinum salts are vulnerable to pladienolide B, a SF3B1 inhibitor, or SF3B1 knock-down. Importantly, pladienolide B also slows down tumor growth of NSCLC Patient-Derived Xenografts (PDXs) poorly responsive to platinum salts. Mechanistically, we show that pladienolide B leads to genomic instability and apoptosis, that correlate with early transcription-dependent replication stress and DNA-PKcs activation, followed by the shutdown of ATR/DNA-PKcs-dependent signaling. In addition, pladienolide B profoundly regulates the expression and/or splicing, particularly exon skipping, of numerous genes i