Supplementary Figures from Mis-splicing of Mitotic Regulators Sensitizes SF3B1-Mutated Human HSCs to CHK1 Inhibition

<jats:p>&lt;p&gt;Supplementary Figure S1: Multiplex gene editing of SF3B1 K700E in CD34+ cells. Supplementary Figure S2: Splicing and single cell transcriptome analysis of SF3B1-mutant HSPCs. Supplementary Figure S3: Mis-splicing and expression of BUBR1 and CDC27. Supplementary Figure S4: Drug sensitivity of SF3B1-mutant cells. Supplementary Figure S5: The role of candidate mis-spliced genes in CHK1i sensitivity. Supplementary Figure S6: Prexasertib testing in CB HSPCs, MDS samples, and in vivo.&lt;/p&gt;</jats:p>

section_content: constitutively spliced junctions (cj), alternative retention of normally constitutively spliced introns (ci), alternative 5'ss (a5ss), or alternative 3'ss (a3'ss). Events were restricted to ≥ 10% mis-splicing, Bayes factor ≥ 5. (B) Overlap between mis-spliced a3'ss events in CB and PB SF3B1 K700E cells (left), and GO annotation using Metascape showing a single summary term per group. (C) GO analysis of a3'ss mis-splicing targets in SF3B1 K700E K562 cells. GO was performed using Metascape showing summary terms. (D) Progenitor cell types annotated using lineage marker genes on the combined UMAP: HSCs (AVP, HLF, PROM1), immature HSPCs (CD34, PROM1), erythroid (GATA1, TFRC), myeloid (MPO, ELANE), megakaryocyte (PF4, ITGA2B), and mast cell (KIT, KRT1) precursors.

section_name: SUPPLEMENTAL FILES

section_content: and peripheral blood (PB) CD34 + cells. BFP + CD34 + cells were sequenced with regular (TruSeq) or low-input (SMART-Seq) RNA sequencing kits as described in Methods. Table includes Ensembl, geneID, description, and genomic coordinates for each gene identified as mis-spliced. For each splicing event, genomic coordinates, splicing type, isoform, spliceosome information and nonsensemediated RNA decay (NMD) prediction is annotated. NMD prediction: NA = the splicing event does not affect the NMD status of the transcript; TRUE = the isoform listed in the table is an NMD substrate, while the isoform not listed is not; FALSE = the isoform listed in the table is not an NMD substrate, while the isoform not listed is a substrate. The degree to mis-splicing (dpsi) and total splicing (psi) are listed for SF3B1 WT and mutant cells. Log fold change (lfc) of splicing events and respective Bayes factor are also indicated. 2. Splicing analysis of WT and SF3B1 K700E mutant K562 cells. Table includes Ensembl, geneID, description, and genomic coordinates for each gene identified as misspliced. For each splicing event, genomic coordinates, splicing type, isoform, spliceosome information and nonsense-mediated RNA decay prediction (NMD) is annotated. NMD prediction: NA = the splicing event does not affect the NMD status of the transcript; TRUE = the isoform listed in the table is an NMD substrate, while the isoform not listed is not; FALSE = the isoform listed in the table is not an NMD substrate, while the isoform not listed is a substrate. The degree to mis-splicing (dpsi) and total splicing (psi) are listed for SF3B1 WT and mutant cells. Log fold change (lfc) of splicing events and respective Bayes factor are also indicated. 3
section_name: Supplemental Table 1. Splicing analysis of control and SF3B1 K700E edited cord blood (CB)

section_name: Supplemental Table