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Hematopoietic stem and progenitor cells (HSPCs) sustain lifelong hematopoiesis. Mutations of pre-mRNA splicing machinery, especially splicing factor 3b, subunit 1 (SF3B1), are early lesions found in malignancies arising from HSPC dysfunction. However, why splicing factor deficits contribute to HSPC defects remains incompletely understood. Using zebrafish, we show that HSPC formation in sf3b1 homozygous mutants is dependent on STAT3 activation. Clinically, mutations in SF3B1 are heterozygous; thus, we explored if targeting STAT3 could be a vulnerability in these cells. We show that SF3B1 heterozygosity confers heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Cells carrying mutations in other splicing factors or treated with splicing modulators are also more sensitive to STAT3 inhibition. Mechanistically, we illustrate that STAT3 inhibition exacerbates aberrant splicing in SF3B1 mutant cells. Our findings reveal a conserved vulnerability of splicing factor mutant HSPCs that could allow for their selective targeting in hematologic malignancies. • Sf3b1 loss-of-function HSPCs are highly dependent on STAT3 signaling • SF3B1 heterozygosity confers heightened sensitivity to STAT3 inhibition • STAT3 inhibition aggravates splicing defects in SF3B1 mutant cells Potts et al. show that STAT3 inhibition is a conserved vulnerability for splicing factor heterozygous mutant hematopoietic cells due to its impact on splicing. These findings suggest that STAT3 inhibitors could be used alone or in combination with splicing modulators to selectively ablate splicing-defective cells in blood cancers. [ABSTRACT FROM AUTHOR]

Summary More than 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 ( SF3B1 ). We generated a conditional knockin mouse model of the most common SF3B1 mutation, Sf3b1 K700E . Sf3b1 K700E mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1 K700E myeloid progenitors and SF3B1 -mutant MDS patient samples demonstrate aberrant 3′ splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1 K700E to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1 K700E -expressing cells. Thus, SF3B1 K700E expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS. [ABSTRACT FROM AUTHOR]

Objective To characterize heparin-induced thrombocytopenia (HIT) at a single pediatric center including the prevalence and the accuracy of the 4Ts scoring system as a predictor of HIT. Study design In this retrospective cohort study, we identified 155 consecutive patients <21 years old with sufficient data for 4Ts scoring. The 4Ts scoring system is a validated pretest tool in adults that predicts the likelihood of HIT using clinical features. Hospital-wide exposure to unfractionated and low molecular weight heparin was determined by querying the hospital pharmacy database. Results The majority of patients with suspected HIT (61.2%) were on surgical services. Prediction of HIT risk using initial 4Ts scoring found 3 (2%) had high risk 4Ts scores, 114 (73%) had intermediate risk 4Ts scores, and the remaining 38 (25%) had low risk 4Ts scores. HIT was confirmed in 0/38 patients with low risk 4Ts scores, 2/114 patients with intermediate-risk 4Ts scores, and all 3 patients with high-risk 4Ts scores presented with HIT with thrombosis. Of 12 positive HIT screening tests, results were falsely positive in 66.6% of patients with intermediate risk 4Ts scores and 100% of patients with low risk 4Ts scores. The prevalence of HIT was 0.058% and HIT with thrombosis was 0.046% in pediatric patients on unfractionated heparin. Conclusions The prevalence of HIT appears significantly lower in pediatric patients compared with adults. Application of the 4Ts system as a pretest tool may reduce laboratory evaluation for HIT in heparin-exposed children with low risk 4Ts scores, decreasing unnecessary further testing, intervention, and cost. [ABSTRACT FROM AUTHOR]

Alterations in cellular homeostasis that affect protein folding in the endoplasmic reticulum (ER) trigger a signaling pathway known as the unfolded protein response (UPR). The initially cytoprotective UPR will trigger an apoptotic cascade if the cellular insult is not corrected; however, the proteins required to initiate this cell death pathway are poorly understood. In this study, we show that UPR gene expression is induced in cells treated with ER stress agents in the presence or absence of routine caspase-12 or human caspase-4 expression and in cells that overexpress Bcl-xL or a dominant negative caspase-9. We further demonstrate that ER stress-induced apoptosis is a caspase-dependent process that does not require the expression of caspase-12 or caspase-4 but can be inhibited by overexpression of Bcl-xL or a dominant negative caspase-9. Additionally, treatment of human and murine cells with ER stress agents led to the cleavage of the caspase-4 fluorogenic substrate, LEVD-7-amino-4-trifluoromethylcoumarin, in the presence or absence of caspase-12 or caspase-4 expression, whereas Bcl-xL or a dominant negative caspase-9 overexpression inhibited LEVD-7-amino-4-trifluoromethylcoumarin cleavage. These data suggest that caspase-12 and caspase-4 are not required for the induction of ER stress-induced apoptosis and that caspase-4-1ike activity is not always associated with an initiating event. [ABSTRACT FROM AUTHOR]

Mutations in components of the 3′ mRNA splicing machinery are found in almost 50% of myelodysplastic syndrome (MDS) cases. In this issue of Cancer Cell , Kim and colleagues, Colla and colleagues, and Shirai and colleagues report on the impact of mutated or dysregulated splicing factors to hematopoiesis, mRNA splicing, and MDS pathogenesis. [ABSTRACT FROM AUTHOR]

• The clonal complexity of the hematopoietic system increases during early ontogeny but declines with age. • Novel non-invasive methods are shedding new light on our understanding of the clonal complexity of the blood throughout development • Clonal hematopoiesis is common in an aging hematopoietic system and associated with an increased risk of hematologic cancer and cardiovascular disease. • Bone marrow stress from cytotoxic chemotherapy or chronic inflammation can cause clonal hematopoiesis in younger individuals. Hematopoietic stem and progenitor cells (HSPCs) govern the daily expansion and turnover of billions of specialized blood cells. Given their clinical utility, much effort has been made toward understanding the dynamics of hematopoietic production from this pool of stem cells. An understanding of hematopoietic stem cell clonal dynamics during blood ontogeny could yield important insights into hematopoietic regulation, especially during aging and repeated exposure to hematopoietic stress—insults that may predispose individuals to the development of hematopoietic disease. Here, we review the current state of research regarding the clonal complexity of the hematopoietic system during embryogenesis, adulthood, and hematologic disease. [ABSTRACT FROM AUTHOR]

Hematopoietic stem and progenitor cells (HSPCs) maintain hematopoiesis throughout the lifetime of an organism. HSPC dysfunction can lead to disorders such as myelodysplastic syndrome (MDS). Spliceosome mutations are prevalent in MDS, but how splicing regulates HSPCs remains poorly understood. Using a zebrafish loss-of-function mutant for the top mutated splicing factor in MDS, splicing factor 3b, subunit 1 (sf3b1), we found that impaired splicing hindered HSPC specification. To identify Sf3b1-regulated transcripts, we performed RNA-sequencing on kdrl:gfp+ endothelial cells from sf3b1 mutant and unaffected siblings at 24hpf. ∼900 genes were mis-spliced with significant enrichment for Signaling Transducer and Activator of Transcription 3 (Stat3) pathway components. Mis-spliced Stat3 signaling components observed in sf3b1 mutants were predicted to dampen protein levels or inhibit functionality, and Stat3 target genes were significantly downregulated in sf3b1 mutants. Constitutively-active Stat3 overexpression in sf3b1 mutants partially suppressed the HSPC defect. Induced mis-splicing of stat3 using morpholinos caused a significant HSPC decrease in sf3b1 heterozygotes at 28hpf, but elicited no effect in wildtype embryos, demonstrating a synthetic lethal interaction. Inhibiting Stat3 with small molecule STATTIC similarly reduced HSPCs in heterozygous but not wildtype embryos. We next tested if STAT3 inhibition selectively affected SF3B1-mutated human cells. K562 cells expressing the MDS-associated SF3B1-K666N mutation were more sensitive to STAT3 inhibition with STATTIC than wildtype cells. Similarly, primary SF3B1-mutant MDS patient cells produced fewer hematopoietic colonies when treated with STATTIC than controls. Together, STAT3 inhibition is a conserved synthetic lethal vulnerability for SF3B1-mutated cells with potential to treat splicing factor-mutated MDS. [ABSTRACT FROM AUTHOR]

Summary SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remains elusive. Here, we show that conditional expression of Sf3b1- K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment. Graphical Abstract Highlights • Sf3b1-K700E mutation impairs 3′ splice site selection and induces cellular senescence • Sf3b1-K700E mutation in combination with Atm deletion causes CLL • CLL cells show genome instability and dysregulated BCR signaling • Human CLL cells with SF3B1 mutations show dysregulation of BCR signaling Yin et al. show that Sf3b1 -K700E mutation in mouse B cells induces cellular senescence, but deletion of Atm overcomes senescence and leads to the development of CLL-like disease. Both human and mouse CLL harboring mutant SF3B1 exhibit deregulated B cell receptor signaling and increased sensitivity to ibrutinib. [ABSTRACT FROM AUTHOR]