JAK2 V617F Progenitors Utilize Adaptations to Cell-Autonomous and Microenvironment-Dependent Inflammatory Stress in Polycythemia Vera, Likely Exhibiting Barrier Against Rapid Transformation to Myelofibrosis

Abstract

It has been shown that hyperactivated JAK-STAT signaling axis in oncogenic JAK2 V617F mutation-positive polycythemia vera (PV) leads to dysregulation of cytokine receptors signaling, triggering systemic inflammatory response. However, it remains to be understood how inflammatory and oncogene-induced signaling converge in disease initiation and evolution into fibrosis or neoplastic transformation.Immunohistochemistry (IHC) staining of patients' bone marrow (BM) as well as induced pluripotent stem cell (iPSC) clones with distinct JAK2 genotypes derived from PV female patient were used for the study. The generation and characterization of the JAK2 V617F (V617F+) homozygous iPSCs was described by Ye, Stem Cells, 2014; BXS0116 iPSCs (ACS-1030, ATCC) were used as control; V617F+ iPSC lines were provided by Josef T. Prchal (University of Utah) and all patients samples were provided by Jiri Ehrmann (Palacky University, Czech Republic). We identified increased cell-autonomous expression of IFNγ and IFNγ-dependent STAT1 signaling in V617F+ cells compared to wild-type (wt) progenitors. IHC staining of PV and post-PV secondary myelofibrosis showed continuously increasing expression of TNFα and TGFβ1, confirming published data about their vital role in fibrogenesis. Staining also demonstrated that fibrotic lesions are a major site of TNFα synthesis, while TGFβ1 was highly positive in its putative storage site, in megakaryocytes. In order to mimic these conditions in vitro we tested the effect of IFNγ in combination with TNFα and/or TGFβ1 on the expression of inflammatory signature in PV progenitors. This treatment promoted upregulation of two potent pro-fibrogenic chemokines, CXCL10 and CXCL9, in PV progenitors solely, which share common regulation by STAT1 signaling and TNFα/NF-κB signaling.As pro-inflammatory milieu is known to induce DNA damage, we analyzed DNA damage response (DDR) in PV progenitors in vitro and in vivo. We show that both markers for the presence of double stranded DNA damage and DDR, γH2AX and ATM pS1981, were weakly detectable in PV patients, with increased trend of γH2AX foci accumulation in later disease stages. The same applied for the presence of oxidative-stress marker 8-oxoGuanine, suggesting presence of protection mechanisms against inflammation-evoked DNA damage in V617F+ cells. Assessment of DDR in vitro confirmed decreased levels of DNA damage, marked by lower γH2AX foci presence in V617F+ cells, compared to wt progenitors, while inducing bystander DNA damage to wt progenitors upon co-culture. As homologous recombination (HR) plays important role in repair during DNA replication and maintenance of genomic stability, altered rate of HR could form basis for disease progression. Assessment of RAD51 recruitment to DNA damage sites, marked by γ-H2AX foci as a readout of HR efficiency upon sequential staining protocol (Bennett et al, Methods, 2009) showed not significantly altered RAD51 recruitment to these sites. With RAD51 accumulation following the trends of γ-H2AX levels these data suggest intact regulation of HR in V617F+ cells. As the main source of DNA damage induced by inflammatory cytokines is mediated by reactive oxygen species (ROS), we have measured ROS kinetics followed by analysis of ROS-buffering system activity. These studies showed un-responsiveness of V617F+ progenitors to treatment with inflammatory cytokines, while wt cells reacted with increase in ROS levels. The total rates of ROS were, however, maintained at higher levels in V617F+ cells compared to wt progenitors, resembling many types of malignancies, where elevated ROS levels promote cancer development and progression. Furthermore, we observed significantly increased activity of metabolic enzymes glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) involved in ROS-buffering system in V617F+ cells, compared to wt cells. These results suggest increased ROS buffering capacity of V617F+ cells by metabolic re-wiring of G6PD and GR, which helps to keep delicate balance of elevated intracellular ROS levels.Overall, these data suggest multi-level adaptations of premalignant clone in PV, with cooperation between cell-intrinsic and microenvironment-dependent fail-safe mechanisms that serve as a barrier delaying transformation to senescence-like condition in myelofibrosis.JS and PL: first co-authors. Supported by GP14-10687P and KONTAKTII (LH15223). DisclosuresNo relevant conflicts of interest to declare.