SRP312766 PRJNA718591 GSE171173 A novel regulatory mechanism of U2 spliceosome proteostasis that controls the transcriptome and therapy response in cancer [RNAseq_CUTLL1_shSF3B1.1] Aberrant splicing is an important cause of cancer, and the production of noncanonical, cancer-specific, mRNA transcripts associate with transformation. Studies have focused on characterizing the effect of spliceosome mutations on disease progression. Here, we have uncovered a novel mode of regulation of the U2 complex component SF3B1 in leukemia via lysosomal degradation. Elevated levels of SF3B1 protein control splicing and active transcription via direct interaction with the general transcriptional machinery and are critical for leukemia growth due to the control of cell cycle and DNA damage response-related transcripts, such as the serine/threonine kinase CHEK2. We further exploited these findings to show that clinically-used SF3B1 inhibitors synergize with transcriptional inhibitors, CHEK2 inhibitors and chemotherapy drugs to block leukemia growth. Our study provides the proof-of-principle for post-translational regulation of the splicing machinery via the lysosome and associated splicing-dependent and -independent roles of the U2 complex in cancer, that can be exploited for therapeutic purposes. Overall design: Whole RNA was extracted from 1-5 million T-ALL (lines) cells using the Total RNA Mini Kit (Bio-Rad) according to the manufacturer's protocol. Poly-A+ (magnetic oligodT-containing beads (Illumina)) or Ribominus RNA was used for library preparation. cDNA preparation and unstranded library construction was performed using the TruSeq RNA Sample Preparation Kit. Libraries were sequenced on the NextSeq 500/550 using 76bp paired-end read method. Differential gene expression analysis was performed for each matched treatment vs control pairs, separately in each biological or technical replicate in CUTLL1 cell line. Five types of comparisons were tested: (a) Control vs SF3B1 knockdown CUTLL1 cells with shSF3B1.1, (b) Control vs SF3B1 knockdown CUTLL1 cells with shSF3B1.2, (c) Control vs E7107 treated CUTLL1 cells (24hr), (d) Control vs E7107 treated CUTLL1 cells (15 and 30 min), (e) Control vs E7107 and/or NMDi treated CUTLL1 cells. Analysis was performed using edgeR package. GSE171173 pubmed 35061527 parent_bioproject PRJNA718587