Addiction to DUSP1 protects JAK2V617F-driven polycythemia vera progenitors against inflammatory stress and DNA damage, allowing chronic proliferation

Abstract

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.

Role of DNA Damage Response in Suppressing Malignant Progression of Chronic Myeloid Leukemia and Polycythemia Vera: Impact of Different Oncogenes

“…In addition, the aforementioned extent of Chk2 activation in CML (high) and PV (low) likely reflects subcellular localization of P-ATM [90,91]. Our further (published in [82]) IHC staining of patients' bone marrow sections from PV revealed low nuclear staining for activated ATR (P-ATR at T1989), and barely detectable expression of a marker for oxidative DNA lesions 8-oxoguanine (8-oxoG), as well as very low staining for a marker of global nuclear DDR activation, Ser 139-phosphorylated histone H2AX (γH2AX) [82]. These data suggested that despite inflammatory microenvironment and JAK2 V617F oncogene-driven myeloproliferation, certain mechanisms must mitigate the potential genotoxic impact of the overall oncogenic program controlled by JAK2 V617F, thereby allowing for PV chronic proliferation with relatively stable genome.…”

“…Our own early (unpublished) analysis depicted in Figure 1b revealed differences between Chk2 activation in CML and PV, suggesting differences in functions of its upstream regulator ATM kinase. Although the activated, auto-phosphorylated form of ATM (P-ATM) localizes to the nucleus in bone marrow progenitors of myeloid malignancies including MDS [87,88] and CML ( [45] and Figure 2a), our immunohistochemistry (IHC) staining against P-ATM at S1981 revealed predominantly cytoplasmic P-ATM immunoreactivity in PV progenitors and distinct nuclear staining (and lack of cytoplasmic staining) only in post-PV MF ( [82] and Figure 2b). These data indicate that although ATM in CML (and MDS and post-PV MF) progenitors regulate its downstream substrates and cell cycle checkpoints mainly from the nucleus, this is different in PV progenitors, where ATM exerts its actions mainly from the cytoplasm.…”

“…These data suggested that despite inflammatory microenvironment and JAK2 V617F oncogene-driven myeloproliferation, certain mechanisms must mitigate the potential genotoxic impact of the overall oncogenic program controlled by JAK2 V617F, thereby allowing for PV chronic proliferation with relatively stable genome. Importantly, a gradual increase in the nuclear P-ATM ( Figure 2b) and γ-H2AX levels [82] following the progression of PV to post-PV MF implied that such buildup of the DDR threshold signaling is linked to suppression of proliferation and fibrogenesis. Supplementary Table S1 for patients' numbering and details.…”

“…These adaptive intrinsic mechanisms are capable of "buffering" the potential genotoxic impact of the cell autonomous and microenvironment-dependent inflammatory stress in PV. However, such a delicate balance can be experimentally altered by enhanced DNA damage and robust activation of the DDR checkpoint response in JAK2 V617F + cells, higher than that in the JAK2 wild-type (wt) cells [82].…”

“…In our recent publication, we further elaborated on the concept of protection mechanisms that guard myeloproliferative progenitors from cell-intrinsic and cell-extrinsic DNA damage and thus DDR, facilitating creation of a barrier preventing cell cycle arrest, myelofibrosis, and rapid malignant transformation in JAK2 V617F-positive PV. We used induced pluripotent stem cell (iPSC)-derived CD34 + progenitor-enriched cultures (CD34 + P-ECs) from a JAK2 V617F-positive PV patient and from a JAK2 wild-type healthy control [82]. The CD34 + P-ECs were cultured in the absence or presence of IFNγ, TNFα, and TGFβ1, in order to mimic the PV patients' microenvironment.…”