Inflammatory and oncogenic signaling converge in disease evolution of BCR-ABL-negative myeloproliferative neoplasms, clonal hematopoietic stem cell disorders characterized by gain-of-function mutation in JAK2 kinase (JAK2V617F), with highest prevalence in patients with polycythemia vera (PV). Despite the high risk, DNA-damaging inflammatory microenvironment, PV progenitors tend to preserve their genomic stability over decades until their progression to post-PV myelofibrosis/acute myeloid leukemia. Using induced pluripotent stem cells-derived CD34 + progenitor-enriched cultures from JAK2V617F + PV patient and from JAK2 wild-type healthy control, CRISPR-modified HEL cells and patients' bone marrow sections from different disease stages, we demonstrate that JAK2V617F induces an intrinsic IFNγ-and NF-κBassociated inflammatory program, while suppressing inflammation-evoked DNA damage both in vitro and in vivo. We show that cells with JAK2V617F tightly regulate levels of inflammatory cytokines-induced reactive oxygen species, do not fully activate the ATM/p53/p21waf1 checkpoint and p38/JNK MAPK stress pathway signaling when exposed to inflammatory cytokines, suppress DNA single-strand break repair genes' expression yet overexpress the dual-specificity phosphatase (DUSP) 1. RNAi-mediated knock-down and pharmacological inhibition of DUSP1, involved in p38/JNK deactivation, in HEL cells reveals growth addiction to DUSP1, consistent with enhanced DNA damage response and apoptosis in DUSP1inhibited parental JAK2V617F + cells, but not in CRISPR-modified JAK2 wild-type cells. Our results indicate that the JAK2V617F + PV progenitors utilize DUSP1 activity as a protection mechanism against DNA damage accumulation, promoting their proliferation and survival in the inflammatory microenvironment, identifying DUSP1 as a potential therapeutic target in PV.

We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and show bias for differentiation towards megakaryocytes (Mk). Mouse models of myeloproliferative neoplasms (MPN) expressing JAK2-V617F (VF) or a JAK2 exon 12 mutation (E12) displayed increased frequencies and percentages of the CD41hi versusCD41lo HSCs compared to wildtype controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from MPN patients. CD41hi HSCs produced higher numbers of Mk-colonies HSC in single cell cultures in vitro, but showed reduced long-term reconstitution potential compared to CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, while CD41lo HSCs showed higher gene expression of interferon, JAK/STAT and TNFα/NFkB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in MPN patients. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the numbers of JAK2-V617F positive HSCs in mice and patients with MPN. The shift towards the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.

To better understand the molecular basis of resistance to azacitidine (AZA) therapy in myelodysplastic syndromes (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), we performed RNA sequencing on pre-treatment CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 25 MDS/AML-MRC patients of the discovery cohort (10 AZA responders (RD), six stable disease, nine progressive disease (PD) during AZA therapy) and from eight controls. Eleven MDS/AML-MRC samples were also available for analysis of selected metabolites, along with 17 additional samples from an independent validation cohort. Except for two patients, the others did not carry isocitrate dehydrogenase (IDH)1/2 mutations. Transcriptional landscapes of the patients’ HSPCs were comparable to those published previously, including decreased signatures of active cell cycling and DNA damage response in PD compared to RD and controls. In addition, PD-derived HSPCs revealed repressed markers of the tricarboxylic acid cycle, with IDH2 among the top 50 downregulated genes in PD compared to RD. Decreased citrate plasma levels, downregulated expression of the (ATP)-citrate lyase and other transcriptional/metabolic networks indicate metabolism-driven histone modifications in PD HSPCs. Observed histone deacetylation is consistent with transcription-nonpermissive chromatin configuration and quiescence of PD HSPCs. This study highlights the complexity of the molecular network underlying response/resistance to hypomethylating agents.