The Hematopoietic Microenvironment in Myeloproliferative Neoplasms: The Interplay Between Nature (Stem Cells) and Nurture (the Niche)

2020 ◽  
pp. 135-145
Author(s):  
Huichun Zhan ◽  
Kenneth Kaushansky
Keyword(s):  
Stem Cells ◽  
Myeloproliferative Neoplasms ◽  
Hematopoietic Microenvironment

scholarly journals STAT5 is Expressed in CD34+/CD38− Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 1021 ◽  
Author(s):  
Emir Hadzijusufovic ◽  
Alexandra Keller ◽  
Daniela Berger ◽  
Georg Greiner ◽  
Bettina Wingelhofer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Janus Kinase ◽  
Signaling Cascades ◽  
Nuclear Compartment ◽  
Downstream Signaling ◽  
Direct Targeting ◽  
Calr Mutations

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34+/CD38− MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34+/CD38− MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

Expression of the CAMPATH-1 Antigen (CD52) on CD34+/CD38- Progenitor Cells in Patients with 5q- MDS and in a Subset of AML

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1728-1728
Author(s):  
Katharina Blatt ◽  
Harald Herrmann ◽  
Sabine Cerny-Reiterer ◽  
Susanne Herndlhofer ◽  
Wolfgang R. Sperr ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Lymphocytic Leukemia ◽  
Target Antigen ◽  
Blood Monocytes ◽  
Trisomy 8 ◽  
Monosomy 7

Abstract Abstract 1728 The target antigen CAMPATH-1 (CD52) is widely expressed in various hematopoietic lineages inlcuding lymphocytes, basophils, and blood monocytes. The anti-CD52 antibody Alemtuzumab is used successfully to treat patients with chemotherapy-refractory chronic lymphocytic leukemia. Based on its strong immunosuppressive effects, Alemtuzumab has also been considered for patients with aplastic anemia and hypoplastic myelodysplastic syndromes (MDS). Indeed, more recently, Alemtuzumab was found to induce major hematologic responses in a group of patients with MDS. Although the immunosuppressive effect was considered to play a role, the exact mechanisms underlying this drug effect remained speculative. In the current study, we asked whether CD34+ bone marrow (BM) progenitor cells in MDS and acute myeloid leukemia (AML) express the CAMPATH-1 antigen. Twelve patients with MDS (5 females, 7 males; median age: 70 years), 25 patients with AML (16 females, 9 males; median age: 62 years), and 34 control cases (normal reactive BM, n=12; idiopathic cytopenia of unknown significance, n=11; chronic myeloid leukemia, CML, n=4; chronic myelomonocytic leukemia, CMML, n=3; JAK2 V617F+ myeloproliferative neoplasms, MPN, n=4) were examined. Surface expression of CD52 on CD34+/CD38+ and CD34+/CD38- BM progenitor cells was analyzed by monoclonal antibodies and multicolor flow cytometry. In the group of MDS, CD52 was detectable on CD34+/CD38- stem cells in 3/4 patients with isolated 5q-. In most of the other MDS patients, CD52 was weakly expressed or not detectable on CD34+/CD38- cells. In AML, CD34+/CD38- cells displayed CD52 in 12/25 patients, namely 3 with complex karyotype including 5q-, 2 with inv(3), one with t(8;21), one with inv(16), one with del13q, one with trisomy 8, one with monosomy 7, and 2 with normal karyotype. Expression of CD52 mRNA in CD34+/CD38- AML stem cells was confirmed by qPCR in all patients tested (n=14). In addition, a good correlation was found between surface CD52 expression and CD52 mRNA expression in AML progenitor fractions. In patients with normal hematopoiesis (n=12) or idiopathic cytopenia (n=11), CD34+/CD38- cells stained weakly positive or negative for CD52. Almost in all cases tested, blood monocytes and blood basophils stained positive for CD52. Together, our data suggest that the target antigen CAMPATH-1 (CD52) is expressed on primitive CD34+/CD38- progenitor cells in MDS, preferentially in 5q- patients, and in a subset of patients with AML. These observations may have clinical implications and explain recently described effects of Alemtuzumab in patients with MDS. Our data also suggest that Alemtuzumab may be an interesting targeted drug in patients with refractory or relapsed AML in whom neoplastic stem cells express the target antigen CD52. Disclosures: No relevant conflicts of interest to declare.

Noncellular Niche Influences: Nervous System Mediators, Metabolic Mediators, and Hypoxia/Oxidative Stress

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-26-SCI-26
Author(s):  
Simón Méndez-Ferrer
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Nervous System ◽  
Mesenchymal Stem Cells ◽  
Myeloproliferative Neoplasms ◽  
Sympathetic Nerves ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Hsc Niche ◽  
Central Pacemaker

Hematopoietic stem cells (HSCs) traffic between bone marrow and circulation, what allows for life-saving clinical transplantation. Our previous work has shown that HSC numbers in blood follow circadian oscillations that are regulated by the central pacemaker in the brain, which reaches bone marrow nestin+ mesenchymal stem cells through peripheral sympathetic nerves. In the perinatal bone marrow, HSC-niche forming mesenchymal stem cells might be different from those that form the skeleton and some of them might be neural crest-derived, like peripheral neurons and supporting glial cells. Thus, tight regulation of the bone marrow stem-cell niche in vertebrates might build upon developmental relationships of its cellular components. We have found recently that cholinergic nerves regulate HSC maintenance, proliferation and migration in divergent niches. We will present unpublished evidence of how both branches of the autonomic nervous system cooperate to regulate HSC maintenance and function in spatially and temporally distinct niches. Moreover, we have shown recently that damage to this regulatory network is essential for the manifestation of myeloproliferative neoplasms. In these diseases, previously thought to be driven solely by mutated HSCs, protecting the HSC niche might represent a novel therapeutic strategy. Patients with myeloproliferative neoplasms have a higher risk of developing acute leukemia. However, at this stage, leukemic cells might be less sensitive to the normal control by the microenvironment and, instead, acute myelogenous leukemic cells might transform the bone marrow niches to support their own survival. We will discuss potential contributions of HSC niches to myeloproliferative neoplasms and MLL-AF9-driven acute myeloid leukemia. Disclosures Off Label Use: Potential use of selective estrogen receptor modulators and beta3-adrenergic agonists in myeloproliferative neoplasms.

scholarly journals Interrogating the Role of ASXL1 and JAK2 mutations in Myeloproliferative Neoplasms Utilizing Human Pluripotent Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2971-2971
Author(s):  
Taylor B. Collins ◽  
Stephanie A. Luff ◽  
Luis F.Z. Batista ◽  
Christopher M. Sturgeon ◽  
Stephen T. Oh
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Double Mutant ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Mutant Line ◽  
Advisory Committees ◽  
Cd34 Cells ◽  
Mutant Lines

JAK2 V617F is the most frequent mutation found in myeloproliferative neoplasms (MPNs), with 50-60% of myelofibrosis (MF) patients harboring this mutation. Mutations in ASXL1 often co-occur with JAK2 V617F and are associated with decreased survival and increased risk of transformation to secondary acute myeloid leukemia. How mutant ASXL1 contributes to the MPN disease phenotype and confers poor prognosis is not fully understood. Controversy remains as to whether ASXL1 mutations found in patients confer a loss of function, gain of function, and/or dominant negative phenotype. Additionally, Asxl1 mutation knock in mouse models present with a relatively modest phenotype, following a very long latency period. A human model system has the potential to provide a useful tool to understand how these mutations affect ASXL1 function. This project therefore seeks to uncover how expression of mutant JAK2 in conjunction with mutant ASXL1 influences hematopoietic output and proliferation, utilizing human pluripotent stem cells. Two complementary approaches have been utilized to study how ASXL1 mutations influence MPN pathogenicity. CD34+ cells from an MF patient harboring JAK2 V617F and an ASXL1 mutation (P920Tfs*4) were FACS-sorted and reprogrammed to generate distinct iPSC clones. Because the majority of the patient-derived iPSC clones were double mutant, CRISPR gene editing was utilized to revert the JAK2 and ASXL1 mutations to generate all four possible genotypes. In parallel, two different ASXL1 mutations (the most common G646Wfs*12 mutation as well as the P920Tfs*4 mutation) were introduced into H1 human embryonic stem cells, each in isolation and in combination with JAK2 V617F, resulting in the generation of single and double mutant lines. These genetically engineered pluripotent stem cell lines were then differentiated specifically into definitive hematopoietic lineages, through a stepwise program regulated by Wnt signaling. This allowed us to analyze the role of ASXL1 and JAK2 mutations during the development of the various lineages of the hematopoietic system known to be dysregulated in MPNs, such as the myeloid and erythroid lineages. Following hematopoietic differentiation, colony forming assays were performed. The JAK2 mutant line produced four-fold more colonies than WT line, with a significant bias towards the generation of erythroid colonies. In contrast, the ASXL1 mutant line produced substantially fewer colonies than the WT line, with the majority of these colonies resembling myeloid colonies. The double mutant line (ASXL1/JAK2) generated more colonies than the WT and ASXL1 mutant lines, but fewer colonies as compared to the JAK2 mutant line. Of note, the JAK2 V617F mutation generated much larger erythroid colonies compared to the WT H1 control line or the double mutant line. Our in vitro differentiation experiments also allowed us to sort equal numbers of CD45+CD34+ cells from the different genotypes, and plate them in methylcellulose. These experiments allow the ability to assess how mutations impact differentiation on a per cell basis. Similar trends were observed (i.e. more erythroid colonies with JAK2 and fewer and mostly myeloid colonies with ASXL1) with this approach, suggesting that the phenotypes observed can be attributed at least in part to progenitor cell output on a per cell basis and not solely reflecting the total number of progenitors generated through the pluripotent stem cell differentiation protocol. In summary, with his human pluripotent stem cell model system, we have observed that JAK2 V617F induced an increase in total colony production with a marked expansion of the erythroid lineage, while mutant ASXL1 impaired colony production overall with substantial myeloid skewing. Cells expressing both mutations presented with an intermediate phenotype. Ongoing efforts include gene expression analysis to understand how JAK2 and ASXL1 mutations direct these observed functional differences. Disclosures Oh: Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Overexpression of NF-E2 In Vivo Causes Thrombocytosis and Acute Leukemia: A Murine Model of Myeloproliferative Neoplasms.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 961-961
Author(s):  
Albert Gruender ◽  
Kai Kaufmann ◽  
Tobias Hadlich ◽  
Thomas Günther ◽  
Roland Schüle ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Transgenic Mice ◽  
Acute Leukemia ◽  
Myeloproliferative Neoplasms ◽  
Transgenic Animals ◽  
Spleen Weight ◽  
Hematopoietic Stem

Abstract Abstract 961 The transcription factor nuclear factor erythroid-2 (NF-E2) is expressed in hematopoietic stem cells as well as in myeloid, erythroid and megakaryocytic precursors. NF-E2 deficient mice display marked anemia at birth and die perinatally due to thrombopenia, demonstrating an essential role for NF-E2 in both in erythropoiesis and platelet formation. We have previously shown that NF-E2 is overexpressed in the vast majority of patients with Myeloproliferative Neoplasms (MPNs). However, the effect of augmented transcription factor activity has not been studied in vivo. We therefore engineered two independent transgenic mouse lines expressing human NF-E2 under the control of the vav-Promoter, which has previously been shown to direct transgene expression in hematopoietic stem cells as well as in precursor cells of all lineages. The two founder lines differed in the degree of NF-E2 overexpression displayed. While one line showed moderate overexpression (2 – 5-fold), the other line expressed human NF-E2 between 10 and 100-fold above the murine counterpart. Both lines paralleled observations in PV patients, where a wide range of NF-E2 overexpression was noted (median overexpression, 7-fold; range 2-fold to 40-fold; n = 59). The two founder lines show overlapping but distinct phenotypes. In both strains. moderately overexpressing NF-E2 transgenic mice (2 – 10-fold) invariably develop thrombocytosis with a latency of 14 months. In addition, megakaryocyte colony formation in the bone marrow is drastically increased. In contrast, thrombocytosis is not observed in the markedly overexpressing NF-E2 transgenic mice (above 20-fold). A similar inverse correlation between the degree of NF-E2 overexpression and platelet numbers was observed in MPN patients. In both strains, Epo-independent colony formation, a pathognomonic feature of polycythemia vera, is significantly increased in NF-E2 transgenic animals. Bone marrow histopathology shows findings characteristically seen in MPNs, including the presence of increased megakaryopoiesis with cytologically abnormal forms, often in clusters. Both NF-E2 transgenic strains display significantly increased mortality. Upon autopsy, between 15 and 20% of mice in both strains present with major gastrointestinal bleeding in conjunction with splenic atrophy. Spleen weight is reduced by over 50% (Transgenic mice: 49 +/-15 mg, wild type littermates 103 +/- 30 mg; p < 0.001, n = 8 each). One third of the remaining mice show moderate to marked splenomegaly (2 – 27 fold increase in spleen weight; mean: 434 mg, range: 124 – 2700 mg; p < 0.001 vs. wt littermates, n = 12). Histopathological examination of all spleens revealed mild to moderately expanded red pulp with increased numbers of iron containing histiocytes. This observation indicates increased red cell destruction and may explain the fact that neither hematocrit nor hemoglobin are elevated in NF-E2 transgenic animals. At 18 months of age, one mouse developed acute leukemia, which is currently being phenotyped. In summary, in a murine model moderate NF-E2 overexpression causes a phenotype resembling Essential Thrombocythemia. In addition, our preliminary data indicate that NF-E2 overexpression may predispose to the development of acute leukemia. Disclosures: No relevant conflicts of interest to declare.

Connexin-43 Expression Regulates the Migration of Hematopoietic Stem Cells and Progenitors towards and From Bone Marrow.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 562-562
Author(s):  
Daniel Gonzalez-Nieto ◽  
Kyung-Hee Chang ◽  
Anja Koehler ◽  
Jorden Arnett ◽  
Susan Dunn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Connexin 43 ◽  
Cell Types ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Hematopoietic Microenvironment ◽  
Deficient Mice

Abstract Abstract 562 In the bone marrow (BM) cavity, the migratory traffic of hematopoietic stem cells and progenitors (HSC/P) from the endosteal niches to circulation and viceversa depends on their response to chemokine gradients and interaction with endothelial and mesenchymal pre-osteoblastic cells located at the endosteal niches, forming the hematopoietic microenvironment (HM). Several lines of evidence have pointed out the possible role of the gap junction-forming protein connexin-43 (Cx43) in the control of stem cell and progenitor migration. Our group previously demonstrated that Cx43 expression in the hematopoietic microenvironment (HM) is critical in the fetal liver and BM hematopoietic regeneration after administration of 5-fluorouracil (5-FU) and other investigators have shown that Cx43 is crucial controlling the migration of neural progenitors along radial glial during brain development. We hypothesized that Cx43 could regulate the bidirectional migration of HSC/P in the BM stroma. Since Cx43 is expressed by mesenchymal cells, endothelial cells and hematopoietic stem cells and progenitors, we decided to analyze the Cx43 contribution in the control of HSC/P migration in cell-specific conditional knock-out mice. To achieve this objective, we have used mice that were selectively deficient for Cx43 in the osteoblast/stromal cells (Collagen 1a-Creflox/flox; O/S-Cx43-deficient), in endothelial cells (Tek-Creflox/flox; E-Cx43-deficient) or in hematopoietic cells (Vav1-Creflox/flox; H-Cx43-deficient). O/S-Cx43-deficient mice have been shown to be a model of osteoblast loss of function (Chung DJ et al., J. Cell. Sci., 2006) and E-Cx43-deficient mice have been shown to be a model of arterial hypotension induced by both increase nitric oxide and angiotensin levels (Liao Y et al, PNAS 2001). Analysis with reporter crossings with Rosa-loxP-Stop-LoxP-LacZ mice showed anatomical specificity of the Cre recombinase expression in different cell types of BM, and western-blot and RT-PCR expression indicated practical abolishment of the expression of Cx43 in each of the specific cell types. First, we analyzed whether there were changes in the levels of circulating progenitors in O/S-, E- or H-Cx43-deficient mice. While H-Cx43-deficient mice did not show any change in the levels of circulating HSC/P, E-Cx43-deficient mice showed a 3.5-fold and 4.7-fold, respectively, increase of circulating CFU-C and competitive repopulating units while maintaining normal repopulation ability of BM HSC. O/S-Cx43-deficient mice showed a 30% reduction in basal conditions which was more accentuated when administered G-CSF (50% reduction on day +6), compared with their WT counterparts. Interestingly, while osteoblast loss-of-function was induced in O/S Cx43-deficient mice, the intramarrow expression levels of CXCL12a/b and mesenchymal progenitor content (CFU-F) were increased (4- and 2-fold, respectively). In correlation with the increased levels of CXCL12, the distance to endosteum of transplanted CFSE+/lin-/c-kit+ BM cells into non-myeloablated O/S-Cx43-deficient mice was dramatically decreased (36.1±4.3 vs 23.2±2.1 mm, p<0.01), suggesting a major change in the cellular composition and chemokinesis within the hematopoietic microenvironment “in vivo”. Interestingly, the 16-hour homing of HSC/P transplanted into lethally irradiated O/S-Cx43KO recipient mice showed a ∼60% reduction and a significantly decreased survival in a limiting-dose transplantation radioprotection assay (50% survival in WT mice vs 0% survival in O/S Cx43-deficient recipients). The homing/engraftment defect of these mice correlated with a reversal of the increased levels of CXCL12 in irradiated BM and a 50% reduction of the migration of WT HSC/P through O/S-Cx43-deficient stroma in response to CXCL12. Altogether, these data indicate that intercellular communication through Cx43 shares distinct functions between the different cell components of the hematopoietic microenvironment, and mediates CXCL12-dependent and CXCL12-independent mechanisms in control of the BM homing and retention of HSC/P. Disclosures: No relevant conflicts of interest to declare.

Erythroid Lineage-Restricted Expression of Jak2V617F Is Sufficient to Induce a Myeloproliferative Disease in Mice,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3861-3861
Author(s):  
Hajime Akada ◽  
Saeko Hamada ◽  
Golam Mohi
Keyword(s):  
Stem Cells ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Myeloproliferative Neoplasms ◽  
Jak2v617f Mutation ◽  
Target Cells ◽  
Dependent Manner ◽  
Myeloproliferative Disease ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem

Abstract Abstract 3861 A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in most cases of Ph-negative myeloproliferative neoplasms (MPNs) including ∼95% patients with polycythemia vera (PV) and 50–60% patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). To investigate the contribution of JAK2V617F in MPNs, we generated a conditional Jak2V617F knock-in mouse (Akada et al., Blood 2010; 115: 3589–3597). Expression of Jak2V617F in all hematopoietic compartments including the hematopoietic stem cells (HSC) resulted in a PV-like disease associated with a marked expansion of erythroid progenitors in the bone marrow and spleen. Since Jak2 is essential for normal erythropoiesis and expression of Jak2V617F mutant enhances erythropoiesis, so we asked if erythroid progenitors are actual target cells for Jak2V617F mutation. To address this question, we have specifically expressed Jak2V617F in erythroid progenitors using the EpoR-Cre mice. Expression of heterozygous Jak2V617F in erythroid progenitors resulted in a polycythemia-like phenotype characterized by increase in hematocrit and hemoglobin, increased red blood cells, Epo-independent erythroid colonies, and splenomegaly. Erythroid lineage-specific expression of homozygous Jak2V617F resulted in significantly greater increase in hematocrit, hemoglobin, red blood cells, Epo-independent erythroid colonies, and splenomegaly compared to heterozygous Jak2V617F expression. These results suggest that erythroid lineage-restricted expression of Jak2V617F is sufficient to induce a polycythemia-like disease in a gene-dose dependent manner. However, transplantation of Jak2V617F-expressing erythroid progenitors (c-kithighTer119lowCD71high or c-kitlowTer119highCD71high) from the diseased mice into lethally irradiated recipients could not transfer the disease suggesting that Jak2V617F mutation does not confer self-renewal capacity to erythroid progenitors. We also observed that only Jak2V617F-expressing HSC has the unique capacity to serially transplant the myeloproliferative disease in mice. Taken together, our results suggest that HSCs are the disease-initiating cancer stem cells and erythroid progenitors are the target cells in Jak2V617F-evoked MPN. Disclosures: No relevant conflicts of interest to declare.

Depletion of Jak2V617F MPN Stem Cells by IFNα in a Murine Model of Polycythemia Vera

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 806-806
Author(s):  
Ann Mullally ◽  
Claudia Bruedigam ◽  
Dirk Heckl ◽  
Luke Poveromo ◽  
Florian H. Heidel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Murine Model ◽  
Myeloproliferative Neoplasms ◽  
Spleen Weight ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Type 1 Interferon

Abstract Abstract 806 Interferon alpha (IFNα) is an effective treatment for patients with myeloproliferative neoplasms (MPN). In addition to inducing hematological responses in most MPN patients, IFNα reduces the JAK2V617F allelic burden and can render the JAK2V617F mutant clone undetectable in some patients. The mechanism underlying these responses is incompletely understood and whether the molecular responses that are seen occur due to the effects of IFNα on JAK2V617F mutant stem cells is debated. Using a murine model of Jak2V617F MPN, we investigated the effects of IFNα on Jak2V617F MPN stem cells in vivo. Chimeric transplant recipients were generated with purified stem cell enriched populations (lin−Kit+Sca1+) and these were treated for 4 weeks with either IFNα or vehicle control. IFNα treatment caused a reduction in extramedullary hematopoiesis (spleen weight, vehicle 262mg vs IFNα 192mg, p<0.01), hematocrit (vehicle 76.0% vs IFNα 65.5%, p<0.05) and white blood cell count (vehicle 13.9×109/L vs IFNα 7.5×109/L, p<0.01) in this disease model. IFNα treatment caused a reduction in early (CD71+Ter119+) erythroid progenitors that had accumulated in the spleen of Jak2V617F mice. IFNα treatment caused selective depletion of Jak2V617F MPN hematopoietic stem cells (HSC, lin−kit+Sca1+CD150+CD48−) over time and this was associated with reduced Jak2V617F chimerism in the long-term HSC compartment (Jak2V617F chimerism Vehicle 41.4% vs. IFNα 23.9%, p<0.05). IFNα treatment impaired the transmission of Jak2V617F-MPN and reduced Jak2V617F chimerism in transplanted recipient mice, demonstrating functional depletion of disease-specific stem cells. Mechanistically, IFNα treatment preferentially induced cell-cycle activation of Jak2V617F mutant long-term HSCs. Gene expression profiling revealed relative enrichment of cell cycle genes and depletion of quiescence related genes in IFNα treated Jak2V617F HSC compared to IFNα treated WT HSC. IFNα treatment promoted a predetermined terminal erythroid-lineage differentiation program within myeloid progenitor cells. The effects on Jak2V617F long-term HSC were absent in Jak2V617F+/−IFNAR1−/− (lacking the type 1 interferon receptor) chimeric mice demonstrating that the effects of IFNα treatment were cell autonomous and specific for type 1 interferon signalling. These findings provide insights into the differential effects of IFNα on Jak2V617F mutant and normal hematopoiesis and suggest that IFNα achieves molecular remissions in MPN patients through its effects on MPN stem cells. Furthermore, these results support combinatorial therapeutic approaches in MPN, by concurrently depleting dormant JAK2V617F MPN-propagating stem cells with IFNα and targeting the proliferating downstream progeny with JAK2-inhibitors or cytotoxic chemotherapy. Disclosures: Heidel: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ebert:Celgene: Consultancy; Genoptix: Consultancy.

RUNX1 and NF-E2 Transcriptional Upregulation Is Not Specific For Myeloproliferative Neoplasms But Is Seen In Those Polycythemic Disorders With Augmented HIFs Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2177-2177
Author(s):  
Katarina Kapralova ◽  
Lucie Lanikova ◽  
Felipe R Lorenzo V ◽  
Monika Horvathova ◽  
Vladimir Divoky ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloproliferative Neoplasms ◽  
Cell Mass ◽  
Homozygous Mutation ◽  
Erythroid Progenitors ◽  
Gain Of Function ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Exon 2 ◽  
Secondary Polycythemia

Abstract RUNX1 and NF-E2 are transcription factors that regulate hematopoietic stem cell homeostasis. It has been reported that increased RUNX1 expression in the granulocytes is present in all three classical myeloproliferative neoplasms (MPN): polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis (Wang et al, Blood 2010), and that elevated NF-E2 promotes erythropoietin (EPO)-independent erythroid maturation of hematopoietic stem cells in vitro (Bogeska et al, Stem Cells Transl Med 2013). A mouse model overexpressing the NF-E2 transgene in hematopoietic cells was reported to be a new model of myeloproliferative neoplasms (Kaufmann et al, J Exp Med 2012). Polycythemic states can be divided into primary polycythemias, characterized by intrinsically hyperproliferative erythroid progenitors that are hypersensitive to EPO, and secondary polycythemias, wherein erythroid progenitors respond normally to EPO but circulating EPO is elevated or inappropriately normal for the level of increased red cell mass. Some congenital disorders including those with mutations in the hypoxia sensing pathway may share features of both primary and secondary polycythemias. We considered the possibility that increased transcripts of RUNX1 and NF-E2 might be the feature of other primary polycythemic states as well. We report a study of 19 polycythemic patients with primary or secondary polycythemia with diverse etiologies including mutations in positive and negative regulators of hypoxia sensing pathway. RUNX1 and NF-E2 transcripts were quantitated in granulocytes and BFU-E colonies by qPCR. All primary polycythemic patients had erythroid progenitors hypersensitive to or independent to EPO; all secondary polycythemic subjects had normal erythroid progenitor response to EPO. RUNX1 and NF-E2 gene transcripts were increased in granulocytes and BFU-E colonies in all PV patients, two unrelated subjects with the VHLR200W homozygous mutation (Chuvash polycythemia), one polycythemic patient homozygous for the VHLP138L exon 2 mutation, and a patient with the HIF2αM535V gain-of-function mutation. We also found upregulated expression of RUNX1 and NF-E2 in granulocytes and BFU-Es from a polycythemic patient (with no detectable EPOR, JAK2V617F or JAK2 exon 12 mutations and low level of EPO < 1 mU/mL) who was heterozygous for a SNP in exon 3 (rs147341899) in the LNK gene. We examined transcripts of RUNX1 and NF-E2 genes in granulocytes from two Croatian polycythemic patients with a homozygous VHLH191D exon 3 mutation whose erythroid progenitors were not hypersensitive to EPO and whose RUNX1, but not NF-E2, transcript was increased. We found similar results in two compound heterozygotes for VHLT124A exon 2 and VHLL188V exon 3 mutations. These two polycythemic siblings had hypersensitive erythroid colonies, increased RUNX1 transcripts and decreased NF-E2 transcripts in granulocytes. RUNX1 and NF-E2 transcripts were normal in two subjects with primary polycythemia due to the EPOR gain-of-function EPORQ434Xmutation, and in five unrelated subjects with secondary polycythemia. We next examined granulocyte transcripts of HIF-regulated genes: TFRC, SLC2A1, VEGF, BNIP3 and HK1, and found them to be increased in all PV patients and all studied polycythemic patients with VHL, HIF2α or LNK mutations, but not in polycythemic EPORQ434Xpatients or five patients with secondary polycythemia. Increased transcripts of HIF regulated genes are compatible with the previously unappreciated Warburg effect in PV (see S. Sana's Abstract at this ASH meeting). We propose that increased expression of RUNX1 and NF-E2 is not specific for myeloproliferative neoplasms but also is not universal for primary polycythemic disorders. Therefore, increased expression of RUNX1 and NF-E2 do not seem to be underlying mechanism for MPNs development but rather represent factors associated with diverse primary polycythemia states with augmented HIF signaling. (Note: KK and LL contributed equally to this work.) This work was supported by 1P01CA108671-O1A2 (NCI) Myeloproliferative Disorders (MPD) Consortium (PI Ron Hoffman) project#1 (PI Prchal) and the Leukemia & Lymphoma Society. Work by KK, LL, MH and VD was in part supported by the European Structural Funds (project CZ.1.07/2.3.00/20.0164 and CZ.1.07/2.3.00/30.0041), grant LF_2013_010 and by Czech Science Foundation (Project-P301/12/1503). Disclosures: No relevant conflicts of interest to declare.

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