Summary Over 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 knock-in mouse model of the most common SF3B1 mutation, Sf3b1K700E. Sf3b1K700E mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1K700E myeloid progenitors and SF3B1-mutant MDS patient samples demonstrate aberrant 3’ splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1K700E to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1K700E-expressing cells. Thus, SF3B1K700E expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS.

Thrombosis is a major cause of morbidity and mortality in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), clonal disorders of hematopoiesis characterized by activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. Neutrophil extracellular trap (NET) formation, a component of innate immunity, has been linked to thrombosis. We demonstrate that neutrophils from patients with MPNs are primed for NET formation, an effect blunted by pharmacological inhibition of JAK signaling. Mice with conditional knock-in of , the most common molecular driver of MPN, have an increased propensity for NET formation and thrombosis. Inhibition of JAK-STAT signaling with the clinically available JAK2 inhibitor ruxolitinib abrogated NET formation and reduced thrombosis in a deep vein stenosis murine model. We further show that expression of PAD4, a protein required for NET formation, is increased in-expressing neutrophils and that PAD4 is required for -driven NET formation and thrombosis in vivo. Finally, in a population study of more than 10,000 individuals without a known myeloid disorder,-positive clonal hematopoiesis was associated with an increased incidence of thrombosis. In aggregate, our results link expression to NET formation and thrombosis and suggest that JAK2 inhibition may reduce thrombosis in MPNs through cell-intrinsic effects on neutrophil function.

Mutations in encoding the histone 3 lysine 36 trimethyltransferase, are enriched in relapsed acute lymphoblastic leukemia and MLL-rearranged acute leukemia. We investigated the impact of mutations on chemotherapy sensitivity in isogenic leukemia cell lines and in murine leukemia generated from a conditional knockout of mutations led to resistance to DNA-damaging agents, cytarabine, 6-thioguanine, doxorubicin, and etoposide, but not to a non-DNA damaging agent, l-asparaginase. H3K36me3 localizes components of the DNA damage response (DDR) pathway and mutation impaired DDR, blunting apoptosis induced by cytotoxic chemotherapy. Consistent with local recruitment of DDR, genomic regions with higher H3K36me3 had a lower mutation rate, which was increased with SETD2 mutation. Heterozygous conditional inactivation of in a murine model decreased the latency of MLL-AF9-induced leukemia and caused resistance to cytarabine treatment in vivo, whereas homozygous loss delayed leukemia formation. Treatment with JIB-04, an inhibitor of the H3K9/36me3 demethylase KDM4A, restored H3K36me3 levels and sensitivity to cytarabine. These findings establish alteration as a mechanism of resistance to DNA-damaging chemotherapy, consistent with a local loss of DDR, and identify a potential therapeutic strategy to target -mutant leukemias.

Background: Philadelphia-negative myeloproliferative neoplasms (MPNs) are a group of clonal stem-cell disorders that are associated with an elevated risk for thrombosis. The reason for this association is incompletely understood. While elevated white blood cell (WBC) count is a risk factor for thrombosis in some models, the pathogenic pathways linking WBC abnormalities to thrombosis in MPNs are not clear. Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA, histones and serine proteases that contain and kill pathogens. There is a growing body of evidence linking NETs to thrombosis in various disease states, including cancer (Demers et al, PNAS 2012). We explored the contribution of NETs to the pro-thrombotic phenotype of MPNs. Methods: NET formation was assessed in neutrophils isolated from patients with MPNs and age-matched controls by using previously published immunofluorescence (IF) methods. c-KIT enriched marrow cells from Vav-Cre/Jak2V617F Knock-In (KI) mice were grafted into lethally irradiated wild-type (WT) recipients leading to constitutive heterozygous expression of the mutation in hematopoietic cells. Inferior vena cava (IVC) stenosis model was used to assess thrombotic tendency in mice. Mice were treated with ruxolitinib by oral gavage at a dose of 90 mg/kg BID for 3 days prior to thrombosis and NETosis experiments. NET formation was stimulated by ionomycin (4 µM). Results: Neutrophils from patients with MPNs (n=14) demonstrated significantly higher NET formation as compared to those from age-matched controls (n=10; p=0.003). This was true across specific MPN diagnoses and driver mutations (JAK2, CALR, MPL). Ex-vivo treatment of neutrophils with ruxolitinib (at 300nM for 2.5 hours) resulted in abrogation of NETosis as assessed by IF studies and was comparable to the effect achieved with the addition of compounds known to inhibit NET formation such as specific PAD4 and NADPH inhibitors; no difference was observed in Annexin V-mediated apoptosis between ruxolitinib treated and untreated neutrophils suggesting that ruxolitinib effect was not confounded by alternative apoptosis pathways. We next evaluated NETosis and its effect on thrombosis in MPN mouse models driven by Jak2 V617F. Neutrophils isolated from Vav-Cre/Jak2V617F demonstrated increased NET production ex-vivo as compared to WT littermate controls. Vav-Cre/Jak2V617F KI mice were grafted into lethally irradiated WT and assessed for their prothrombotic phenotype. VavCre/Jak2V617F KI demonstrated significantly higher thrombus formation at 2 hours using the IVC stenosis model (45% versus 0% for Jak2V617F KI (n=11) and Jak2WT (n=8), respectively; p=0.04 by Fisher's exact test). This was associated with significantly higher levels of free dsDNA in the plasma of KI stenosis mice as compared to WT stenosis controls (p=0.03 by Mann-Whitney). Furthermore, IHC and IF studies of lung tissue demonstrated that Vav-Cre/Jak2V617F KI have widespread thrombosis as compared to controls and IF studies demonstrated higher content of citrullinated histone H3 in the lung suggesting increased NETosis at that site. Treatment of Vav-Cre/Jak2V617F KI mice with ruxolitinib significantly abrogated thrombus formation (45% versus 0% for untreated (n=11) and treated (n=8) mice, respectively; p=0.04 by Fisher's exact test). We noted that expression of PAD4, an essential driver of NETosis, is elevated in MPN patients (Rampal Blood 2014). We found that PAD4 content was higher in protein lysates of neutrophils isolated from MPN patients as compared to controls. Moreover, abnormal nuclear localization was observed in patients. These observations may support a role for PAD4 in the excess NETosis observed in MPNs. Conclusion: We found that neutrophils from MPN patients and Jak2 V617F KI mice are sensitized to NET formation that may be mediated by increased PAD4 expression and nuclear localization. In mice, the increased ex-vivo NET formation is associated with an increased thrombosis rate and an abundance of NET related products in thrombus and tissue. Treatment with ruxolitinib abrogates NET formation in humans and significantly reduces thrombus formation in MPN mice models. These findings provide a pathogenic link between the genetic drivers of MPNs and thrombosis in these diseases. Pharmacologic inhibition of JAK-STAT pathway to reduce NETosis and thrombosis should be further investigated. Disclosures DeAngelo: Novartis: Consultancy; Ariad: Consultancy; Celgene: Consultancy; Baxter: Consultancy; Amgen: Consultancy; Incyte: Consultancy; Pfizer: Consultancy.