Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ...ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species–induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase–mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors.
•Ruxolitinib caused DNA repair defects and sensitized MPN stem and progenitor cells to PARP inhibitors.•Quiescent and proliferating MPN cells were eliminated by ruxolitinib and olaparib plus or minus hydroxyurea.
Estrogens are potent regulators of mature hematopoietic cells; however, their effects on primitive and malignant hematopoietic cells remain unclear. Using genetic and pharmacological approaches, ...we observed differential expression and function of estrogen receptors (ERs) in hematopoietic stem cell (HSC) and progenitor subsets. ERα activation with the selective ER modulator (SERM) tamoxifen induced apoptosis in short-term HSCs and multipotent progenitors. In contrast, tamoxifen induced proliferation of quiescent long-term HSCs, altered the expression of self-renewal genes, and compromised hematopoietic reconstitution after myelotoxic stress, which was reversible. In mice, tamoxifen treatment blocked development of JAK2V617F-induced myeloproliferative neoplasm in vivo, induced apoptosis of human JAK2V617F+ HSPCs in a xenograft model, and sensitized MLL-AF9+ leukemias to chemotherapy. Apoptosis was selectively observed in mutant cells, and tamoxifen treatment only had a minor impact on steady-state hematopoiesis in disease-free animals. Together, these results uncover specific regulation of hematopoietic progenitors by estrogens and potential antileukemic properties of SERMs.
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•Hematopoietic stem and multipotent progenitor cells (MPPs) express ERα•ERα activation induces apoptosis of MPPs and proliferation of LT-HSCs•Tamoxifen blocks JAK2V617F-induced myeloproliferative neoplasia in mice•Tamoxifen enhances chemotherapy response of MLL-AF9-induced leukemias
In mice, activation of estrogen receptor (ER) signaling induces apoptosis in multipotent hematopoietic progenitors but induces proliferation and compromises function of HSCs in a reversible manner. Tamoxifen induces apoptosis of MLL-AF9+ blasts, improving conventional chemotherapy, and tamoxifen alone blocks JAK2V617F-induced myeloproliferative neoplasm by restoring normal apoptosis levels in mutant cells.
The acquired somatic JAK2-V617F mutation is present in >80% of patients with myeloproliferative neoplasms (MPNs). Stat3 plays a role in hematopoietic homeostasis and might influence the ...JAK2-V617F–driven MPN phenotype. We crossed our transgenic SclCre;V617F mice with a conditional Stat3 knockout strain and performed bone marrow transplantations into lethally irradiated recipient mice. The deletion of Stat3 increased the platelet numbers in SclCre;V617F;Stat3fl/fl mice compared with SclCre;V617F;Stat3fl/+ or SclCre;V617F;Stat3+/+ mice. Stat3 deletion also normalized JAK2-V617F–induced neutrophilia. Megakaryocyte progenitors were elevated, especially in the spleen, and a slight increase in myelofibrosis was noted. We observed increased mRNA expression levels of Stat1 and Stat1 target genes and augmented phosphorylation of Stat1 protein in bone marrow and spleen of JAK2-V617F mice after Stat3 deletion. The survival of Stat3-deficient mice expressing JAK2-V617F was reduced. Inflammatory bowel disease, previously associated with shortened survival of Stat3-deficient mice, was less prominent in the bone marrow transplantation setting, possibly by limiting deletion of Stat3 to hematopoietic tissues only. In conclusion, deletion of Stat3 in hematopoietic cells from JAK2-V617F mice did not ameliorate the course of MPN, but rather enhanced thrombocytosis and shortened the overall survival.
•Loss of Stat3 in hematopoietic cells enhances JAK2-V617F–driven thrombopoiesis and negatively impacts survival in mouse models.•The phenotypic changes of Stat3-deficient JAK2-V617F mice could in part be mediated by increased Stat1 expression and activation.
The interferon-γ (IFNγ)/signal transducer and activator of transcription 1 (Stat1) pathway shows higher activity in patients with essential thrombocythemia (ET) than in polycythemia vera (PV) and was ...proposed to be promoting the ET phenotype. We explored the phenotypic consequences of Stat1 deficiency on the effects of Janus kinase 2 (JAK2)–V617F in vivo by crossing mice expressing JAK2-V617F with Stat1 knockout mice. JAK2-V617F;Stat1−/− double transgenic mice showed higher red cell parameters and lower platelet counts compared with JAK2-V617F;Stat1+/+ mice. Bone marrow transplantation reproduced these phenotypic changes in wild-type recipients, demonstrating that the effect of Stat1 is cell-intrinsic and does not require a Stat1-deficient microenvironment. Deletion of Stat1 increased burst-forming unit–erythroid and reduced colony-forming unit–megakaryocyte colony formation driven by JAK2-V617F, but was not sufficient to completely normalize the platelet count. Gata1, a key regulator of megakaryopoiesis and erythropoiesis, was decreased in Stat1-deficient platelets. V617F transgenic mice with thrombocytosis had higher serum levels of IFNγ than normal controls and patients with ET showed higher IFNγ serum levels than patients with PV. Together, these results support the concept that activating Stat1 in the presence of JAK2-V617F, for example, through IFNγ, constrains erythroid differentiation and promotes megakaryocytic development, resulting in ET phenotype.
•Stat1 deletion in the presence of JAK2-V617F alters phenotypic manifestations by reducing megakaryopoiesis and favoring erythropoiesis.•IFNγ is elevated in serum of mice with thrombocytosis and in patients with essential thrombocythemia and may drive Stat1 activation.
Heat shock protein 27 (HSP27/HSPB1) is a stress-inducible chaperone that facilitates cancer development by its proliferative and anti-apoptotic functions. The OGX-427 antisense oligonucleotide ...against HSP27 has been reported to be beneficial against idiopathic pulmonary fibrosis. Here we show that OGX-427 is effective in two murine models of thrombopoietin- and JAKV617F-induced myelofibrosis. OGX-427 limits disease progression and is associated with a reduction in spleen weight, in megakaryocyte expansion and, for the JAKV617F model, in fibrosis. HSP27 regulates the proliferation of JAK2V617F-positive cells and interacts directly with JAK2/STAT5. We also show that its expression is increased in both CD34
circulating progenitors and in the serum of patients with JAK2-dependent myeloproliferative neoplasms with fibrosis. Our data suggest that HSP27 plays a key role in the pathophysiology of myelofibrosis and represents a new potential therapeutic target for patients with myeloproliferative neoplasms.
Norepinephrine‐deficient mice harbor a disruption of the gene for dopamine‐β‐hydroxylase (DBH‐KO). Corticotropin‐releasing hormone knockout mice (CRH‐KO) have markedly reduced HPA activity. The aim ...of the present work was to study how deficiency of DBH and CRH would affect tyrosine hydroxylase (TH), DBH, and phenylethanolamine N‐methyltransferase (PNMT) gene expression and protein levels in the adrenal medulla (AM) and stellate ganglia (SG) of control and stressed mice. Both in AM and SG, single immobilization significantly increased TH and DBH mRNA and protein levels both in wild‐type (WT) and CRH‐KO mice. On the other hand, the stress‐triggered increase in PNMT mRNA and protein levels seen in WT mice was absent in CRH‐KO mice. DBH‐KO mice are more sensitive to stress but survive a single 2 h restraint stress in a tube. The increase in TH mRNA levels induced by restraint stress in WT was not observed in DBH‐KO mice. PNMT mRNA and especially PNMT protein levels were significantly elevated in AM of DBH‐KO mice. In SG of DBH‐KO mice, TH mRNA levels were not affected; however, PNMT gene expression was highly elevated. Thus, disruption of the DBH gene surprisingly blocks the stress‐induced elevation of TH mRNA levels in AM but increases PNMT gene expression in both AM and SG. Our data indicate that adrenergic signaling is required for stress‐induced increase in TH mRNA and that this signaling restrains stress‐induced increase in PNMT mRNA. They also confirm that the HPA system plays a crucial role in the stress‐induced regulation of PNMT gene expression.
Myeloproliferative neoplasm (MPN) patients frequently show co-occurrence of JAK2-V617F and mutations in epigenetic regulator genes, including EZH2 In this study, we show that JAK2-V617F and loss of ...Ezh2 in hematopoietic cells contribute synergistically to the development of MPN. The MPN phenotype induced by JAK2-V617F was accentuated in JAK2-V617F;Ezh2(-/-) mice, resulting in very high platelet and neutrophil counts, more advanced myelofibrosis, and reduced survival. These mice also displayed expansion of the stem cell and progenitor cell compartments and a shift of differentiation toward megakaryopoiesis at the expense of erythropoiesis. Single cell limiting dilution transplantation with bone marrow from JAK2-V617F;Ezh2(+/-) mice showed increased reconstitution and MPN disease initiation potential compared with JAK2-V617F alone. RNA sequencing in Ezh2-deficient hematopoietic stem cells (HSCs) and megakaryocytic erythroid progenitors identified highly up-regulated genes, including Lin28b and Hmga2, and chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) analysis of their promoters revealed decreased H3K27me3 deposition. Forced expression of Hmga2 resulted in increased chimerism and platelet counts in recipients of retrovirally transduced HSCs. JAK2-V617F-expressing mice treated with an Ezh2 inhibitor showed higher platelet counts than vehicle controls. Our data support the proposed tumor suppressor function of EZH2 in patients with MPN and call for caution when considering using Ezh2 inhibitors in MPN.
The majority of patients with myeloproliferative neoplasms (MPNs) carry a somatic JAK2-V617F mutation. Because additional mutations can precede JAK2-V617F, it is questioned whether JAK2-V617F alone ...can initiate MPN. Several mouse models have demonstrated that JAK2-V617F can cause MPN; however, in all these models disease was polyclonal. Conversely, cancer initiates at the single cell level, but attempts to recapitulate single-cell disease initiation in mice have thus far failed. We demonstrate by limiting dilution and single-cell transplantations that MPN disease, manifesting either as erythrocytosis or thrombocytosis, can be initiated clonally from a single cell carrying JAK2-V617F. However, only a subset of mice reconstituted from single hematopoietic stem cells (HSCs) displayed MPN phenotype. Expression of JAK2-V617F in HSCs promoted cell division and increased DNA damage. Higher JAK2-V617F expression correlated with a short-term HSC signature and increased myeloid bias in single-cell gene expression analyses. Lower JAK2-V617F expression in progenitor and stem cells was associated with the capacity to stably engraft in secondary recipients. Furthermore, long-term repopulating capacity was also present in a compartment with intermediate expression levels of lineage markers. Our studies demonstrate that MPN can be initiated from a single HSC and illustrate that JAK2-V617F has complex effects on HSC biology.
: Recently we have described the existence of phenylethanolamine N‐methyltransferase (PNMT) mRNA in the heart of adult rats. In this study, we report the first data on distribution of the PNMT ...protein in rat hearts, which follows the distribution of PNMT mRNA (high levels in the atria and low levels in ventricles). The main aim of this study was to determine the localization of the PNMT mRNA in the heart and to examine whether gene expression of this enzyme is affected by immobilization (IMO) stress in a time‐dependent manner. PNMT mRNA levels were detected in all seven studied parts of the heart (atria without and with intramural ganglion cells, ventricles, and septum), with the highest levels in the left atrium and its ganglionic part. Both Southern blot and sequencing verified the specificity of PNMT detected by RT‐PCR. Single IMO for 2‐h increased gene expression of PNMT, as determined by both RT‐PCR and Real‐Time PCR in the right and left atria. Surprisingly, the ganglionic parts of the atria did not respond to stress stimulation. Peak levels of PNMT mRNA were found in the 3‐h interval after the IMO terminated, and also 24 h after the first or sixth IMO. Expression of aromatic L‐amino acids decarboxylase and dopamine‐beta‐hydroxylase has also been detected in the heart of control and stressed rats. In the atria, the effect of stress is clearly modulated by glucocorticoids, since in mice with corticotrophin‐releasing hormone knocked out gene the immobilization‐induced increase in the PNMT mRNA levels seen in wild‐type animals was abolished. Thus, our data have shown that gene expression of the PNMT is localized, not predominantly in cardiac ganglion cells, but in a wide range in atrial cardiomyocytes. Mechanism responsible for the regulation of stress‐induced increase of PNMT gene expression in cardiac atria is clearly dependent on the presence of glucocorticoids.
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