Journal: Blood Cancer Discovery

Article Title: Mis-splicing of Mitotic Regulators Sensitizes SF3B1-Mutated Human HSCs to CHK1 Inhibition

doi: 10.1158/2643-3230.bcd-23-0230

Figure Lengend Snippet: Figure 2. Pervasive mis-splicing of cell division and genome maintenance genes in CD34+ HSPCs. A, Percent mis-splicing of TMEM14C, MAP3K7, DYNLL1, and ORAI2 mRNA in patients with SF3B1 WT or mutant (MUT) MDS, iPSC-derived HSPCs, K562 cells, and edited CD34+ CB and PB HSPCs, or normal BM. Median and range; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-sided Mann–Whitney U test. B, Gene ontology (GO) analysis of a3′ss mis-splicing targets in SF3B1 K700E edited PB CD34+ HSPCs. GO performed using Metascape showing summary terms; ≥10% mis-splicing, Bayes factor ≥5. C, Overlap between mis-spliced a3′ss events in CD34+ CB, CD34+ PB and K562 SF3B1 K700E cells, and list of recurrently mis-spliced genes in all three cell types (right); ≥10% mis-splicing, Bayes factor ≥5. D, STRING protein–protein interaction network of CB/PB a3′ss mis-spliced genes in the cell division GO category; disconnected nodes removed, line thickness proportional to interaction score >0.40. E, GO analysis of genes downregulated in SF3B1 K700E CD34+ CB/PB HSPCs (log2 fold change ≤ −0.5; P < 0.05). GO performed using Metascape showing summary terms. F, Hallmark pathways upregulated or down- regulated in SF3B1 K700E vs. control edited CD34+ CB/PB HSPCs; >25 genes, FDR < 0.01. NES, normalized enrichment score. G, Significantly down- or up-regulated genes among 186 genes with conserved a3′ss mis-splicing in both CB and PB CD34+ HSPCs from Supplementary Fig. S2B. Expression ranked by log2 fold change in SF3B1 K700E vs. control edited CD34+ HSPCs, P < 0.1. Mitosis-related terms are shown in purple color.

Article Snippet: A shRNA targeting luciferase was used as a control. shRNA sequences used are as follows: For lentivirus-mediated gene overexpression, open reading frame (ORF) for DYNLL1 was purchased from the Mission TRC3 Human LentiORF Collection and subcloned into the pSMAL overexpression vector (Addgene, Plasmid #161785).

Techniques: Mutagenesis, Derivative Assay, MANN-WHITNEY, Control, Expressing

Journal: Blood Cancer Discovery

Article Title: Mis-splicing of Mitotic Regulators Sensitizes SF3B1-Mutated Human HSCs to CHK1 Inhibition

doi: 10.1158/2643-3230.bcd-23-0230

Figure Lengend Snippet: Figure 4. SF3B1-mutant cells have delayed G2/M cell cycle progression. A, EdU cell cycle analysis of WT and SF3B1 K700E K562 cells. Representative flow plots (left) and quantitation (right) of the proportion in S or G2/M phase. Mean ± SD, n = 6 independent experiments, unpaired t test. B, EdU cell cycle analysis of control or SF3B1 K700E edited CB cells expressing HSC marker CD133. Representative flow plots (left) and quantitation (right). Mean ± SD, n = 2 inde- pendent experiments, paired t test. C, Proportion of WT and SF3B1 K700E K562 cells positive for pH3, a marker of mitosis. Representative flow plots (left) and quantitation (right). Mean ± SD, of n = 8 experiments, unpaired t test. D, Proportion of pH3-positive mitotic WT and SF3B1-mutant cells after release from G2/M block using CDK1 inhibitor RO-3306. n = 2 independent time-course experiments, unpaired t test. E, Mis-splicing of BUBR1 and CDC27 in SF3B1 WT or mutant (MUT) MDS patients, iPSC-HSPCs, K562 cells, and edited CB/PB CD34+ HSPCs, or normal bone marrow (BM); significance shown using 1-sided Mann–Whitney U test. F, Western Blot analysis of BUBR1 (top) and CDC27 (bottom) protein level in SF3B1-mutant (MUT) and WT K562 cells. Expression nor- malized to GAPDH and shown as fold change relative to WT; n = 3 experiments, mean ± SD. G, EdU cell cycle analysis of WT K562 cells transduced with control luciferase (WT), BUBR1 (B2, B3), or CDC27 (C2, C3) shRNAs, or SF3B1-mutant K562 cells transduced with control shRNA (MUT). Left: representative EdU cell cycle flow plots. Right: proportion of BUBR1 (left) or CDC27 (right) knockdown K562 cells in G2/M phase. Mean ± SD, of n = 4 experiments; unpaired t test.

Article Snippet: A shRNA targeting luciferase was used as a control. shRNA sequences used are as follows: For lentivirus-mediated gene overexpression, open reading frame (ORF) for DYNLL1 was purchased from the Mission TRC3 Human LentiORF Collection and subcloned into the pSMAL overexpression vector (Addgene, Plasmid #161785).

Techniques: Mutagenesis, Cell Cycle Assay, Quantitation Assay, Control, Expressing, Marker, Blocking Assay, MANN-WHITNEY, Western Blot, Transduction, Luciferase, shRNA, Knockdown

Journal: Blood Cancer Discovery

Article Title: Mis-splicing of Mitotic Regulators Sensitizes SF3B1-Mutated Human HSCs to CHK1 Inhibition

doi: 10.1158/2643-3230.bcd-23-0230

Figure Lengend Snippet: Figure 5. SF3B1-mutant cells are selectively sensitized to CHK1 inhibition. A–C, IC50 value of CHK1 inhibitor AZD-7762 (A), CHK1 inhibitor prexa- sertib LY2606368 (B), or SF3b inhibitor pladienolide B (C) in WT (Ctrl) or SF3B1-mutant (S-A, S-R, S-S) K562 cells. Cells were treated with drugs in a dose-response format for 5 days. Mean ± SD, of four independent experiments, one-way ANOVA with Dunnett’s correction for multiple comparisons. D, CHK1 phosphorylation in WT (Ctrl) or SF3B1-mutant (S-A, S-R, S-S) K562 cells. Left: Western blot analysis of pCHK1 (S345) and total CHK1 treated for 5 hours with DMSO or 10 nmol/L CHK1i prexasertib. Right: Ratio of pCHK1 to total CHK1 in SF3B1-mutant normalized to WT cells. n = 2 indepen- dent experiments, two technical replicates. E, Cell growth of WT and SF3B1-mutant K562 cells after CRISPR-mediated ablation of CHK1. Left: CHK1 protein level after control AAVS1 (sgCtrl) or CHK1 (sgCHK1) knockout. Right: Cell growth of WT and SF3B1-mutant cells with sgCHK1 or sgCtrl at 3–6 days of culture, relative to day 0 (72 hours after editing). n = 3 independent experiments, ratio paired t test. F, Number of CB-derived control (Ctrl) or SF3B1 K700E-edited (S-A, S-R, S-S) CD34+ HSPCs after treatment with 2.5 nmol/L pladienolide B (right) or 2.5 nmol/L CHK1i prexasertib (left) for 7 days. Fold change of CD34+ cell number after drug treatment was calculated relative to vehicle (DMSO). Mean ± SD, of n = 3 independent experi- ments; paired t test. G, IC50 value of CHK1i prexasertib in WT K562 cells transduced with control luciferase (WT), BUBR1 (B2, B3), or CDC27 (C2, C3) shRNAs, or SF3B1-mutant K562 cells transduced with control shRNA (MUT). Cells were treated with prexasertib in a dose response format for 5 days; two independent shRNAs, two sets of lines independently generated for each condition. Mean ± SD, of three independent experiments, one-way ANOVA with Dunnett’s correction for multiple comparisons.

Article Snippet: A shRNA targeting luciferase was used as a control. shRNA sequences used are as follows: For lentivirus-mediated gene overexpression, open reading frame (ORF) for DYNLL1 was purchased from the Mission TRC3 Human LentiORF Collection and subcloned into the pSMAL overexpression vector (Addgene, Plasmid #161785).

Techniques: Mutagenesis, Inhibition, Phospho-proteomics, Western Blot, CRISPR, Control, Knock-Out, Derivative Assay, Transduction, Luciferase, shRNA, Generated