JAK2V617F-induced Myeloproliferative Neoplasms Research Articles

PPIL2 Is a Target of the JAK2-STAT5 Pathway and Mediates p53 Polyubiquitination and Degradation

The activated JAK2-STAT pathway is characteristic of myeloproliferative neoplasms (MPNs). We previously revealed that Plek2, a downstream target of the JAK2/STAT5 pathway, functions as a scaffold protein to activate Akt and plays an important role in the pathogenesis of JAK2 V617F-induced MPNs. To further explore the mechanisms of the Plek2 signalosome, we performed a comprehensive proteomic analysis of Plek2 interacting proteins and found peptidylprolyl isomerase-like 2 (PPIL2) as a potential novel effector of the signalosome. As a U-box E3 ubiquitin ligase, PPIL2 was reported to be involved in cancer metastasis. However, its role in normal and malignant hematopoiesis remains unknown. We found that PPIL2 is required for human and murine erythropoiesis in vitro. Knockout of PPIL2 through CRISPR/Cas9 significantly reduced cell proliferation and differentiation and led to marked apoptosis. Mechanistically, PPIL2 interacts with and catalyzes p53 polyubiquitination at lysine 24 to promote proteosome-mediated p53 degradation, thereby preventing p53-dependent cell cycle inhibition and apoptosis. We further revealed that PPIL2 is a downstream target of the JAK2/STAT5 pathway and is upregulated in a JAK2 V617F-positive MPN mouse model and in patients with MPNs. Loss of Ppil2 ameliorated JAK2 V617F-induced myeloproliferative phenotypes including erythrocytosis, neutrophilia, thrombocytosis, and splenomegaly in JAK2 V617F-knockin mice. The same findings were also observed when the JAK2 V617F-knockin mice were treated with cyclosporin A, an inhibitor of PPIL2. Our findings reveal PPIL2 as an effector of the JAK2/STAT5 pathway and Plek2 signalosome. PPIL2 plays an important role in normal erythropoiesis and the pathogenesis of JAK2 V617F-induced MPNs by degrading p53, pointing to PPIL2 as a potential therapeutic target for the treatment of MPNs.

Genetic Deletion or Pharmacologic Inhibition of PTPN11 Impedes the Development and Progression of Myeloproliferative Neoplasms Induced By JAK2V617F and MPLW515L Mutants

Myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are clonal myeloid malignancies derived from mutated hematopoietic stem cells. The JAK2V617F mutation was found in ~95% cases of PV and 50-60% patients with ET and MF. Mutations in MPL and CALR were also detected in ET and MF. The JAK inhibitors, Ruxolitinib and Fedratinib, can reduce splenomegaly and alleviate constitutional symptoms but they are not sufficient to induce remission of MPN. So, there is an unmet need to identify new therapeutic targets and therapies for MPN/MF. The non-receptor protein tyrosine phosphatase 11 (PTPN11), which positively regulates hematopoietic cell signaling, is constitutively hyperphosphorylated in MPN patient cells and in hematopoietic cell lines expressing MPN driver mutants. So, we hypothesize that PTPN11 plays an important role in MPNs. To examine the role of PTPN11 in JAK2V617F-induced MPN, we generated inducible PTPN11-deficient heterozygous JAK2V617F mice by crossing PTPN11 floxed mouse with Mx1Cre and JAK2V617F knock-in mice. While expression of heterozygous JAK2V617F (JAK2 VF/+) induced a PV-like MPN characterized by increased WBC, neutrophil, RBC, hemoglobin and platelet counts in the peripheral blood and enlargement of spleen size, deletion of PTPN11 normalized the blood counts and spleen size in JAK2 VF/+ mice. PTPN11 deletion significantly reduced the expansion of hematopoietic stem/progenitors, erythroid, megakaryocytic, and granulocytic cells in the bone marrow (BM) and spleens of JAK2 VF/+ mice. Furthermore, deletion of PTPN11 abrogated Epo-independent CFU-colonies, a hallmark feature of PV disease, in the BM and spleens of JAK2 VF/+ mice. We also investigated the role of PTPN11 in the development of MF using homozygous JAK2V617F and MPLW515L mouse models. We found that deletion of PTPN11 significantly reduced WBC, neutrophil and platelet counts, normalized spleen size and abrogated BM fibrosis in JAK2V617F and MPLW515L mouse models. These results suggest that PTPN11 plays an important role in the development and progression of PV and MF. Data from our PTPN11 genetic deletion study led us to investigate the effects of PTPN11 inhibition against MPN/MF. We tested the efficacy of an allosteric PTPN11 inhibitor SHP099 in MPN hematopoietic cell lines and mouse models of MPN/MF. We observed that treatment of SHP099 significantly reduced proliferation of hematopoietic cells expressing JAK2V617F and MPLW515L. SHP099 treatment also significantly inhibited hematopoietic progenitor colony growth in PV and MF patient CD34+ cells. Next, we tested the in vivo efficacy of SHP099 against PV using heterozygous JAK2V617F mice. Treatment of SHP099 significantly inhibited the increase in WBC, neutrophil and RBC counts and markedly reduced splenomegaly in JAK2 VF/+ mice. SHP099 treatment also significantly reduced hematopoietic stem/progenitors and myeloid precursors in the BM and spleens of JAK2 VF/+ mice. Furthermore, SHP099 treatment abrogated Epo-independent CFU-E colony formation in the BM and spleens of JAK2 VF/+ mice. We also tested the efficacy of SHP099 alone or in combination with ruxolitinib against myelofibrosis using homozygous JAK2V617F (JAK2 VF/VF) and MPLW515L mouse models. SHP099 treatment alone significantly reduced WBC and neutrophil counts and markedly reduced splenomegaly in JAK2 VF/VF and MPLW515L mice. Combined treatment of SHP099 and ruxolitinib resulted in greater reduction of blood cell counts and spleen size in JAK2 VF/VF and MPLW515Lmice. Histopathologic analyses showed that SHP099 treatment significantly reduced BM fibrosis in JAK2 VF/VF and MPLW515L mice while combined treatment of SHP099and ruxolitinib resulted in greater inhibition of BM fibrosis in these mice. To gain insights into the mechanism of inhibition of MPN/MF by PTPN11 deletion or inhibition, we performed RNA-seq analysis on purified LSK cells from JAK2 VF/+ and PTPN11deleted-JAK2 VF/+ mice as well as from JAK2 VF/VF mice treated with ruxolitinib and SHP099/ruxolitinib. RNA-seq data analysis revealed that genes related to MYC targets, ribosome biogenesis and translation were significantly down-regulated by PTPN11 deletion or SHP099/ruxolitinib combination treatment. Overall, our results suggest that inhibition of PTPN11 alone or in combination with JAK2 inhibition might be useful for treatment of PV and MF.

PTPN1 Deficiency Accelerates the Progression to Myelofibrosis in JAK2V617F-Driven Myeloproliferative Neoplasm

The deletion of chromosome 20q (del20q) is frequently associated with myeloid malignancies including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The del20q cytogenetic abnormality was observed most frequently in patients with myelofibrosis (MF). However, the key target genes within 20q involved in the pathogenesis of myelofibrosis has remained elusive. We previously reported that PTPN1 gene encoding protein tyrosine phosphatase non-receptor type 1 is a potential tumor suppressor within 20q in MPN/MF (Jobe et al. Leukemia 2017). The del20q involving PTPN1 deletion was more commonly found in MPN/MF in comparison to other myeloid neoplasms. We also observed significant co-occurrence of del20q involving PTPN1 deletion with JAK2V617F mutation in patients with MF. So, we hypothesize that PTPN1 deficiency may cooperate with JAK2V617F in the pathogenesis of MF. In this study, we investigated the effects of heterozygous and homozygous PTPN1 deletion in JAK2V617F-induced MPN using a conditional PTPN1 knockout allele and a JAK2V617F knock-in mouse model of MPN. We previously reported the generation of a conditional JAK2V617F knock-in mouse. Mice expressing heterozygous JAK2V617F (JAK2VF/+) exhibit a PV-like MPN characterized by increase in red blood cells (RBC), white blood cells (WBC), neutrophils and platelets in the peripheral blood. Heterozygous deletion of PTPN1 significantly increased WBC, neutrophil and platelet counts but reduced RBC and hemoglobin levels in JAK2VF/+ mice. Flow cytometric analysis showed that heterozygous deletion of PTPN1 significantly increased myeloid (Gr-1+Mac-1+) and megakaryocytic (CD41+CD61+) precursors in the BM and spleens of PTPN1-deficient JAK2VF/+ mice compared with JAK2VF/+ mice. Additionally, deletion of PTPN1 significantly increased hematopoietic stem cells and myeloid progenitors in the BM and spleens of PTPN1-deficient JAK2VF/+ mice compared to JAK2VF/+ mice. Spleen weight was significantly increased in JAK2VF/+ mice, while deletion of PTPN1 further enhanced splenomegaly in PTPN1-deficient JAK2VF/+ mice. Furthermore, deletion of PTPN1 significantly enhanced bone marrow fibrosis in JAK2VF/+mice. Homozygous deletion of PTPN1 resulted in more robust and accelerated development of lethal myelofibrosis in JAK2VF/+ mice. The cooperative effects of JAK2V617F and PTPN1 deficiency in myelofibrotic transformation is associated with increased production of proinflammatory cytokines including IL-1beta, IL-6 and TGF-beta. We also observed enhanced activation of STAT1, STAT3, ERK1/2 and NF-kB in PTPN1-deficient JAK2VF/+ mice bone marrow compared with JAK2VF/+ or WT mice bone marrow. CRISPR/Cas9-mediated deletion of PTPN1 also resulted in enhanced activation of these signaling molecules and increased proliferation in JAK2V617F-positive mouse and human hematopoietic cells. Furthermore, deletion of PTPN1 significantly reduced ruxolitinib sensitivity against JAK2V617F-positive hematopoietic cells, suggesting that del20q involving PTPN1 deletion may cause ruxolitinib resistance in MPN. Overall, this study demonstrates that PTPN1 is an important target in del20q-associated MPN, and deficiency of PTPN1 collaborates with JAK2V617F in the progression to myelofibrosis.

Preclinical studies of Flonoltinib Maleate, a novel JAK2/FLT3 inhibitor, in treatment of JAK2V617F-induced myeloproliferative neoplasms

Janus kinase 2 (JAK2) hyperactivation by JAK2V617F mutation leads to myeloproliferative neoplasms (MPNs) and targeting JAK2 could serve as a promising therapeutic strategy for MPNs. Here, we report that Flonoltinib Maleate (FM), a selective JAK2/FLT3 inhibitor, shows high selectivity for JAK2 over the JAK family. Surface plasmon resonance assays verified that FM had a stronger affinity for the pseudokinase domain JH2 than JH1 of JAK2 and had an inhibitory effect on JAK2 JH2V617F. The cocrystal structure confirmed that FM could stably bind to JAK2 JH2, and FM suppressed endogenous colony formation of primary erythroid progenitor cells from patients with MPNs. In several JAK2V617F-induced MPN murine models, FM could dose-dependently reduce hepatosplenomegaly and prolong survival. Similar results were observed in JAK2V617F bone marrow transplantation mice. FM exhibited strong inhibitory effects on fibrosis of the spleen and bone marrow. Long-term FM treatment showed good pharmacokinetic/pharmacodynamic characteristics with high drug exposure in tumor-bearing tissues and low toxicity. Currently, FM has been approved by the National Medical Products Administration of China (CXHL2000628), and this study will guide clinical trials for patients with MPNs.

IL-1 Signaling Promotes Clonal Expansion and Progression of Bone Marrow Fibrosis in JAK2V617F-Induced Myeloproliferative Neoplasm

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell derived myeloid malignancies characterized by aberrant production of myeloid, erythroid or megakaryocytic lineage cells. JAK2V617F is the most common somatic driver mutation associated with MPN. Interestingly, JAK2V617F mutation can also be detected in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) who do not exhibit overt changes in blood counts. This suggests that other factors might be involved in association with JAK2 mutation in clonal expansion and initiation/progression of MPN. Chronic inflammation is frequently associated with MPN. Interleukin 1 (IL-1) is a major regulator of inflammation. IL-1 consists of two related cytokines IL-1α and IL-1β. Both IL-1α and IL-1β bind to the IL-1 receptor 1 (IL-1R1) to initiate downstream signaling. Although elevated expression of IL-1α and IL-1β has been observed in MPN, their role in the pathogenesis of MPN has remained elusive.In this study, we investigated the role of IL-1 signaling in JAK2V617F-induced MPN using a Jak2V617F knock-in mouse model. We observed elevated levels of IL-1α and IL-1β in mice expressing heterozygous (Jak2 VF/+) and homozygous Jak2V617F (Jak2 VF/VF) compared with WT control animals. Notably, IL-1α and IL-1β expression was significantly higher in Jak2 VF/VF mice exhibiting extensive bone marrow (BM) fibrosis compared with Jak2 VF/+ mice exhibiting polycythemia vera (PV), consistent with elevated levels of IL-1 in patients with myelofibrosis (MF).Since both IL-1α and IL-1β levels were elevated in Jak2 VF/VF mice exhibiting MF, we utilized conditional IL-1R1 knockout (IL-1R1cKO) and Jak2 VF/VF mice to assess the role of IL-1 signaling in the initiation/progression of MF. As expected, Jak2 VF/VF mice exhibited a significant increase in WBC, neutrophil and platelet counts compared to WT control mice. Deletion of IL-1R1in Jak2 VF/VF mice (IL-1R1cKO; Jak2 VF/VF) significantly reduced the WBC, neutrophil and platelet counts to almost control levels. Flow cytometric analysis also showed a significant reduction of myeloid (Gr-1 +) and megakaryocytic (CD41 +) precursors in the BM and spleens of IL-1R1cKO; Jak2 VF/VF mice compared to Jak2 VF/VF mice. Moreover, deletion of IL-1R1 significantly reduced hematopoietic stem and progenitor cells (HSPC) in the BM of IL-1R1cKO; Jak2 VF/VF mice compared to Jak2 VF/VF mice. Spleen weight was significantly reduced in IL-1R1cKO; Jak2 VF/VF mice compared with Jak2 VF/VF mice and they were comparable to control WT mice. More importantly, deletion of IL-1R1 markedly reduced BM fibrosis in Jak2 VF/VF mice. These data suggest an important role of IL-1 signaling in the progression of BM fibrosis in Jak2V617F-induced MPN.To test whether IL-1 signaling contributes to clonal expansion of JAK2 mutant HSPC, we performed competitive transplantation assays by mixing Mx1Cre; Jak2 VF/+ and Mx1Cre; IL-1R1 F/F; Jak2 VF/+ mice BM cells with CD45.1 + WT mice BM cells at a ratio of 1:1 and transplanted into lethally irradiated CD45.1 + recipient animals. At 4 weeks after BMT, the recipient animals were injected with pI-pC to induce Jak2V617F expression and IL-1R1 deletion. We observed significantly higher percentages of total CD45.2 + cells as well as CD45.2 + myeloid (Gr-1 +), B- and T-cells in the peripheral blood of chimeric mice receiving Jak2 VF/+ BM compared with chimeric mice receiving IL-1R1cKO; Jak2 VF/+ BM. We also observed significantly reduced percentages of CD45.2 + LSK, LK, Gr-1 + and CD41 + cells in the BM of chimeric recipient animals receiving IL-1R1cKO; Jak2 VF/+ BM compared with Jak2 VF/+ BM. These results suggest a role of IL-1 signaling in clonal expansion of Jak2V617F mutant HSPC.Additionally, we tested the effects of blocking IL-1R1 using an anti-IL-1R1 antibody in the homozygous Jak2V617F knock-in mouse model of MF. We observed that anti-IL-1R1 antibody treatment significantly reduced peripheral blood WBC and neutrophil counts and decreased HSPC and myeloid precursors in the BM of Jak2 VF/VF mice. Furthermore, anti-IL-1R1 antibody treatment significantly reduced splenomegaly and markedly reduced BM fibrosis in Jak2 VF/VF mice, suggesting that therapies targeting IL-1R1 could be useful for the treatment of myelofibrosis. Overall, our results suggest that IL-1 signaling contributes to clonal expansion of Jak2V617F mutant HSPC and progression of bone marrow fibrosis in MPN. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of SHP2 Ameliorates JAK2V617F-Induced Myeloproliferative Neoplasms

The oncogenic JAK2V617F mutation has been found in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF and advanced PV. Although ruxolitinib treatment can reduce splenomegaly and alleviate constitutional symptoms, it does not induce disease remission or cure bone marrow (BM) fibrosis in patients with MPN/MF. Thus, there is a critical need to identify new therapeutic target and develop novel targeted therapies for MPN/MF. We found that SHP2, a protein tyrosine phosphatase that positively regulates growth factor and cytokine signaling, is constitutively hyperphosphorylated in mouse and human MPN hematopoietic cells expressing JAK2V617F. We found that genetic ablation of SHP2 markedly inhibited the development of PV disease and BM fibrosis in Jak2V617F knock-in mouse models of MPN. So, we hypothesize that targeting of SHP2 alone or in combination with JAK2 inhibition might be useful for treatment of JAK2V617F-evoked MPN. In this study, we investigated the efficacy of SHP2 inhibition using an allosteric SHP2 inhibitor, SHP099, in hematopoietic cells and progenitors expressing JAK2V617F and a Jak2V617F knock-in mouse model of MPN.Treatment of SHP099 alone significantly reduced proliferation of murine BA/F3 cells expressing JAK2V617F. SHP099 treatment also significantly inhibited human JAK2V617F-positive UKE-1 cells. However, SHP099 only modestly affected proliferation of wild type JAK2-expressing BA/F3 cells at higher concentrations. SHP099 treatment also resulted in marked apoptosis in BA/F3-EpoR-JAK2V617F and UKE-1 cells but not in wild-type JAK2 expressing BA/F3-EpoR cells. We also found that SHP099 and ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Furthermore, SHP099 significantly inhibited hematopoietic progenitor colony growth in MPN/MF patient CD34+ cells.We previously generated conditional Jak2V617F knock-in mice. Whereas expression of heterozygous Jak2V617F (Jak2 VF/+) induces a PV-like disease, homozygous Jak2V617F (Jak2 VF/VF) expression promotes rapid progression to myelofibrosis. We utilized heterozygous Jak2V617F mice to test the in vivo efficacy of SHP099 against PV. Treatment of SHP099 significantly reduced the increase in WBC, neutrophil and RBC counts and markedly reduced splenomegaly in heterozygous Jak2V617F (Jak2 VF/+) mice. SHP099 treatment also significantly reduced hematopoietic stem/progenitors (HSPC) and myeloid precursors (Gr-1 +/Mac1 +) in the BM and spleens of Jak2V617F mice. Erythropoietin (Epo)-independent erythroid colony formation in the BM and spleens, a hall-mark feature of PV disease, was observed in Jak2V617F mice. Treatment of SHP099 markedly inhibited the Epo-independent CFU-E colonies in the BM and spleens of heterozygous Jak2V617F mice.We also tested the efficacy of SHP099 alone or in combination with ruxolitinib against myelofibrosis using our homozygous Jak2V617F (Jak2 VF/VF) mice. SHP099 treatment alone or in combination with ruxolitinib significantly reduced WBC and neutrophil counts and markedly reduced splenomegaly in Jak2 VF/VF mice. Treatment of SHP099 or SHP099/ruxolitinib combination also significantly reduced the HSPC and myeloid precursor cells in the BM and spleens of Jak2 VF/VF mice. Histopathologic analyzes revealed extensive BM fibrosis in vehicle-treated Jak2 VF/VFmice. SHP099 treatment alone caused marked reduction of BM fibrosis in Jak2 VF/VF mice whereas combined treatment of SHP099 and ruxolitinib almost completely inhibited BM fibrosis in Jak2 VF/VF mice. Additionally, we performed competitive BM transplantation assays using Mx1Cre; Jak2 VF/VF GFP+ and WT mice BM cells followed by drug treatments. We observed that combined treatment of SHP099 and ruxolitinib significantly reduced the percentage of Jak2V617F mutant GFP+ LSK and myeloid cells in the BM of chimeric mice, suggesting that SHP099 and ruxolitinib combination preferentially inhibits oncogenic Jak2V617F mutant hematopoietic progenitors. Overall, our results suggest that SHP099 alone or in combination with ruxolitinib may have therapeutic potential against PV and MF and support the clinical investigation of SHP2 inhibitor in patients with PV/MF. DisclosuresNo relevant conflicts of interest to declare.

Depletion of Neoplastic CD11b Positive Cells in Jak2V617F Mutant Mice Reduced Fibrocytes in Bone Marrow and Improved Myelofibrosis

Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008). First, the distribution of neoplastic fibrocyte in the BM of Jak2V617F transgenic (TG) mice was examined. We transplanted wild-type (WT) or Jak2V617F TG cells (B6-CD45.2), together with WT BM cells (B6-CD45.1) into irradiated WT recipient mice (B6-CD45.1). Only recipient mice transplanted with a mixture of Jak2V617F cells and WT cells developed BM fibrosis. In immunofluorescent staining of fibrotic BM, cells expressing the fibrocyte marker CD45/Collagen-1(Col-1) were observed much more than cells expressing the fibroblast marker CD90(usually positive for MSCs)/Col-1. As for CD45/Col-1 positive cells, cells expressing CD45.2/Col-1 were much more than cells expressing CD45.1/Col-1, clearly indicating that these cells were derived from Jak2V617F mutant blood cells. On the other hand, in the BM of recipient mice transplanted with control WT cells, few cells expressing CD45/Col-1 or CD90/Col-1 were present. To examine the differentiation ability of Jak2V617F blood cells to fibrocytes directly, peripheral blood (PB) mononuclear cells (MNC) of Jak2V617F mice or WT mice were cultured in vitro. After 5 days of culture, PB MNCs from Jak2V617F mice differentiated into mature fibrocytes exhibiting a long spindle shape with Col-1 expression. On the other hand, there were very few fibrocytes differentiated from PB MNC from WT mice. Next, we depleted monocytes, the main source of fibrocytes, and observed its effects on BM fibrosis in vivo. Jak2V617F TG mice were mated with CD11b-diphtheria toxin receptor (DTR) TG mice (Duffield et al. JCI 2005) to obtain Jak2V617F/CD11b-DTR double TG mice. Mice transplanted with BM cells from Jak2V617F/CD11b-DTR double TG mice (hereinafter called Jak2V617F/CD11b-DTR mice) exhibit leukocytosis, thrombocytosis, anemia, splenomegaly, and BM fibrosis with increased megakaryocytes. Jak2V617F/CD11b-DTR mice was administered diphtheria toxin (DT) intraperitoneally to deplete monocytes. One day after DT administration, the number of PB monocytes (CD11b+/F4/80+) drastically decreased in Jak2V617F/CD11b-DTR mice, and the reduction of monocyte was maintained by every-other-day DT administration. After 8 weeks DT treatment, mice were sacrificed and analyzed. As a control group, Jak2V617F/CD11b-DTR mice treated with PBS were examined. DT treatment drastically decreased the number of neoplastic fibrocytes expressing CD45.2/Col-1 in BM and spleen of Jak2V617F/CD11b-DTR mice compared with control mice treated with PBS. Consistently, reticulin fibers were eliminated almost completely and collagen fibers almost fully disappeared in BM, which led to a reversal of the decrease in BM cellularity, although the number of MKs was not affected. Similar findings were observed in the spleen, although not completely. Plasma TGF-b1 level were about 2-fold higher in Jak2V617F/CD11b-DTR mice than in WT mice. Neoplastic monocyte depletion significantly decreased TGF-b1 level. Since MK numbers did not change, this indicates that fibrocytes are one of the main sources of TGF-b1. In other features of MF in Jak2V617F/CD11b-DTR mice, splenomegaly was ameliorated by DT treatment. Microscopic analysis revealed an improvement in the damaged spleen architecture and the disappearance of splenic fibrosis. In summary, most collagen-producing cells in BM were neoplastic fibrocytes in Jak2V617F-induced MPN, indicating that neoplastic fibrocytes played an essential role and mesenchymal fibroblasts had a minor contribution in fibrosis in MPN. Depletion of neoplastic monocytes also improved splenomegaly as well as BM fibrosis in mice, and this cell fraction could be a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

Metformin Suppress Cellular and Molecular Processes Related to Maintenance and Proliferation of Myeloproliferative Neoplasm Stem Cell

Background : Preclinical rationale of metformin treatment for myeloproliferative neoplasms (MPN) has recently been demonstrated (Cell Death Dis. 2018;9(3):311). Current MPN treatment options are mainly directed to symptoms and have not greatly improve clinical outcomes. It is a consensus that potential curative approaches should affects MPN stem cell function. Aims: To interrogate the metformin action in MPN stem cell function according to frequency and ex vivo differentiation capacity, as well as to underlying molecular mechanisms related to the treatment. Material and Methods: For induction of PV phenotype, 5×106 total bone marrow cells from Jak2V617F conditional mice (Jak2WT/V617F-CD45.2) were transplanted into lethally irradiated) Pep/boy mice (CD45.1-700cGy X-Ray). After 4 weeks, peripheral blood chimerism was evaluated by CD45.1 and CD45.2 markers (Becton-Dickinson) by flow cytrometry and mice were randomized for treatment by intraperitoneal injection of vehicle (PBS, n=6) or metformin (125 mg/kg/day, n=4) for 6 weeks according to chimerism and body weight. Erythroid progenitors (CD71 and Ter119) were evaluated in bone marrow (BM) and spleen. Hematopoietic stem and progenitor cells (LSK: Lin-Sca-1+C-KitHi/long-term HSC[LT-HSC]:LSKCD48-CD150+/ short-term HSC [ST-HSC]:LSKCD48+CD150+/multipotent progenitor [MPP]: LSKCD48+CD150-), myeloid progenitors (LK:Lin-Sca-1+C-KitHi/myelo-erythroid progenitor [MEP]: LKCD34-CD16/32-/common myeloid progenitors [CMP]: LKCD34+CD16/32-/granulocytic progenitors [GMP]: LKCD34+CD16/32+) were evaluated in BM by flow cytometry. BM stroma was evaluated according to frequencies of macrophages (F4/80+CD11b+), inflammatory macrophages (M1) (F4/80+CD11b+CD11c+), arterioles (Cd45/Ter119-CD31+Sca-1+), osteoblasts (Cd45/Ter119-CD51+Sca-1-) and mesenchymal stem cell (Cd45/Ter119-CD51+Sca-1+). BM c-Kit cells were enriched using AutoMacs separator with anti-CD117 magnetics beads (Miltenyi) and used for RNA extraction and PCR array for cell cycle and apoptosis (PAMM-020Z and PAMM-012Z, respectively-Qiagen). Genes with fold-change ≥2 in both directions were selected. All experiments were approved by the Animal Ethics Committee. For comparisons, unpaired T-test or Mann-Whitney test were used as appropriated. All P-values were two-sided with a significance level of 0.05. Results: At randomization, mice of both groups were similar according PB chimerism, body weight and blood counts (all P>0.05). After 6 weeks of metformin treatment mice had no evidence of hematological toxicity or weight loss. Metformin treated mice presented lower platelets counts in PB (mean for vehicle vs. metformin: 4370×103/uL vs. 3451×103/uL; P=0.04) and reduction of splenomegaly (P=0.02). Metformin reduced CD45.2 chimerism (92.7% vs. 88.9%; P=0.03), and showed a trend in reducing pro-erythroblasts (0.91% vs. 0.53%; P=0.06) and early erythroid progenitors (37.4% vs. 32.4%; P=0.11), while increased late erythroid progenitors (16.2% vs. 27.2%; P=0.008) in spleen. Metformin significantly increased ST-HSC frequency (0.06% vs. 0.29%; P=0.001) compared to vehicle. Conversely, bone marrow mononuclear cells from metformin treated mice showed decreased clonogenic capacity (2.5 colonies/1000 BMMC vs. 8.6 colonies/1000 BMMC; P=0.001). Competitive secondary transplant into lethally irradiated mice was performed and results will be show at meeting. Metformin treatment did not modulate the frequency of erythroid progenitors, LT-HSC, MPP, MEP, GMP and CMP in BM nor of the stromal cells evaluated. In c-Kit enriched cells, PCR-array identified 31 downregulated and 64 upregulated genes by metformin treatment. Metformin reduced expression of anti-apoptotic genes (Bcl2 [Fold-change: 0.33] and Xiap [0.48]), cell cycle regulators (Cdkn2a [0.21], Ccnd1 [0.27], Ccn1a [0.42]), while increased expression of TP53 family members (Trp63 [4.1] and Trp53 [2.1]), pro-apoptotic (Bid [2.5] and Bcl10 [1.9]) and cellular-stress response genes (Atm [66.9] and Cdkn2a [3.1]). Conclusions: In a conditional Jak2V617F-induced MPN murine model, daily treatment with metformin reduced tumor burden, extramedullary hematopoiesis, and clonogenic capacity of HSPC by acting on molecular processes related to maintenance and proliferation of MPN stem cell. Disclosures Figueiredo-Pontes: Novartis: Honoraria.

3183 – THE ROLE OF THE THROMBOPOIETIN RECEPTOR (MPL) IN JAK2V617F-POSITIVE MYELOPLROLIFERATIVE NEOPLASMS

Constitutive activation of the Jak/Stat signalling pathway is a hallmark of Myeloproliferative Neoplasms (MPN), with 60-90% of patients carrying the activating JAK2 mutation, JAK2V617F. Several studies suggest expression of a type I cytokine receptor is essential for JAK2V617F-mediated transformation, but the precise contribution of such receptors is unresolved. Here we investigate the functional role of Mpl in JAK2V617F-driven MPN in vitro and in vivo. In vitro studies confirmed that Mpl expression was required for JAK2V617F-induced transformation, but identified a Mpl truncation mutant retaining the JAK2-interaction domain, but lacking the C-terminus (Mpl-IC36), typically associated with Stat5 binding, to be sufficient to support JAK2V617F-induced transformation. Interestingly, Stat5-phosphorylation was maintained. In contrast, deletion of the entire cytoplasmic receptor domain abrogated Stat5 phosphorylation and factor-independent proliferation. In vivo Mpl-wt;JAK2V617F mice exhibited the thrombocytosis and erythrocytosis typical of MPN, while Mpl deficiency abrogated thrombocytosis but sustained high red blood cell counts. Although the Mpl-IC36 receptor was sufficient for JAK2V617F-induced factor independence in BaF/3 cells, Mpl-IC36;JAK2V617F mice did not develop thrombocytosis, indeed platelet counts were similar to those in Mpl null mice. This underlines differences that may emerge from in vitro and in vivo systems and highlights the important role of the Mpl cytoplasmic region for JAK2V617F-mediated signalling, extending the function of Mpl in JAK2V617F-induced MPN beyond a simple scaffold for active JAK2.

Loss of pleckstrin-2 reverts lethality and vascular occlusions in JAK2V617F-positive myeloproliferative neoplasms.

V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombosis is a leading cause of mortality and morbidity in MPNs, the mechanisms underlying their pathogenesis are unclear. Here, we identified pleckstrin-2 (Plek2) as a downstream target of the JAK2/STAT5 pathway in erythroid and myeloid cells, and showed that it is upregulated in a JAK2V617F-positive MPN mouse model and in patients with MPNs. Loss of Plek2 ameliorated JAK2V617F-induced myeloproliferative phenotypes including erythrocytosis, neutrophilia, thrombocytosis, and splenomegaly, thereby reverting the widespread vascular occlusions and lethality in JAK2V617F-knockin mice. Additionally, we demonstrated that a reduction in red blood cell mass was the main contributing factor in the reversion of vascular occlusions. Thus, our study identifies Plek2 as an effector of the JAK2/STAT5 pathway and a key factor in the pathogenesis of JAK2V617F-induced MPNs, pointing to Plek2 as a viable target for the treatment of MPNs.

Pleckstrin-2 Plays an Essential Role in the Pathogenesis of JAK2V617F-Induced Myeloproliferative Neoplasms

V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs). Studies on the pathogenesis of JAK2V617F positive MPNs have primarily focused on deregulation of JAK2 and STAT activities. It remains unclear exactly how JAK2-STAT downstream targets are involved in the development of the MPNs. We previously reported that pleckstrin-2 (Plek2) plays an important role in terminal erythropoiesis in vitro. Here we show that Plek2 is a downstream target of the JAK2-STAT5 pathway in erythroid, megakaryocytic, and granulocytic cells. Plek2 is significantly upregulated by JAK2V617F in both transduced primary mouse hematopoietic cells and in patients with JAK2V617F positive MPNs. Using a JAK2V617F knockin mouse model, we demonstrated that loss of Plek2 ameliorated JAK2V617F-induced myeloproliferative phenotypes including reticulocytosis, thrombocytosis, neutrophilia, and splenomegaly, thereby reverting the widespread vascular occlusions and lethality of JAK2V617F knockin mice (Figure 1A). These phenotypes were also transplantable indicating the role of Plek2 in mediating the pathogenesis of JAK2V617F-induced MPNs is cell-intrinsic (Figure 1B). Our study identifies Plek2 as a novel effector of JAK2-STAT5 pathway and a key factor in the pathogenesis of JAK2V617F-induced MPNs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Metformin inhibits JAK2V617F activity in MPN cells by activating AMPK and PP2A complexes containing the B56α subunit.

Metformin suppresses the growth of a variety of malignant hematologic cells. It is widely accepted that metformin inhibits the growth of malignant cells primarily by suppressing the mTOR pathway or regulating autophagy. In contrast, we found another possible mechanism that inhibits the growth of malignant cells, suppression of the activity of the oncogenic kinase JAK2V617F. We identified at least two distinct mechanisms involved in metformin-induced JAK2V617F inhibition. First, metformin increases reactive oxygen species levels in these cells, leading to the inhibition of SHP-2, a positive regulator of JAK2V617F. These effects of metformin require AMPK. Second, metformin activates protein tyrosine phosphatase PP2A, a negative regulator of JAK2V617F. Furthermore, we determined that among the numerous PP2A subfamily members, the PP2A complex containing the B56α subunit is responsible for the inhibition of JAK2V617F. In contrast, the B56α-containing PP2A complex functions as a positive regulator of JAK2V617F by inhibiting AMPK. Finally, we determined that metformin enhances the antileukemic action of ruxolitinib in HEL and SET-2cells. Our present observations suggest that the combination of metformin with ruxolitinib might be a new therapeutic option for treating JAK2V617F-induced myeloproliferative neoplasms. In addition, activators specific for PP2A complexes containing the B56α subunit may be usefulfor the treatment of JAK2V617F-induced myeloproliferative neoplasms.

Deletion of Stat3 enhances myeloid cell expansion and increases the severity of myeloproliferative neoplasms in Jak2V617F knock-in mice.

The JAK2V617F mutation commonly found in myeloproliferative neoplasms (MPNs) induces constitutive phosphorylation/activation of the signal transducer and activator of transcription 3 (Stat3). However, the contribution of Stat3 in MPN evoked by JAK2V617F remains unknown. To determine the role of Stat3 in JAK2V617F-induced MPN, we generated Stat3-deficient Jak2V617F-expressing mice. Whereas expression of Jak2V617F resulted in a PV-like disease characterized by increased red blood cells (RBC), hematocrit, neutrophils and platelets in the peripheral blood of Jak2V617F knock-in mice, deletion of Stat3 slightly reduced RBC, and hematocrit parameters and modestly increased platelet numbers in Jak2V617F knock-in mice. Moreover, deletion of Stat3 significantly increased the neutrophil counts/percentages and markedly reduced the survival of mice expressing Jak2V617F. These phenotypic manifestations were reproduced upon bone marrow transplantation into wild-type animals. Flow cytometric analysis showed increased hematopoietic stem cell and granulocyte-macrophage progenitor populations in the bone marrow and spleens of Stat3-deficient Jak2V617F mice. Stat3 deficiency also caused a marked expansion of Gr-1+/Mac-1+ myeloid cells in Jak2V617F knock-in mice. Histopathologic analysis revealed marked increase in granulocytes in the bone marrow, spleens and livers of Stat3-deficient Jak2V617F-expressing mice. Together, these results suggest that deletion of Stat3 increases the severity of MPN induced by Jak2V617F.

Regulation of hematopoietic progenitors by estrogens as a basis for new antileukemic strategies

We recently reported that estrogens regulate survival, proliferation, and self-renewal of hematopoietic stem cells and progenitors via estrogen receptor-α activation. Through its proapoptotic effect on malignant progenitors, tamoxifen treatment blocks the development of JAK2V617F-induced myeloproliferative neoplasms in mice and sensitizes MLL-AF9-induced leukemias to chemotherapy, without detrimental effects on normal hematopoiesis.

Autocrine TNF-α Signaling in Hematopoietic Stem Cells Promotes Myeloproliferative Disease Progression through Activation of TNFR2

The JAK2V617F mutation is a unifying feature in the majority of patients with myeloproliferative neoplasms (MPNs). The presence of JAK2V617 in a hematopoietic stem (HSC) or progenitor cell confers a proliferative advantage over native JAK2 counterparts. Several inflammatory cytokines are elevated in MPN patients and in murine models of JAK2V617F-driven MPN. In particular, expression of tumor necrosis factor alpha (TNF-α) is increased by constitutive JAK2 kinase activity and imparts a competitive advantage on JAK2V617Fexpressing cells.To identify cell types responsible for increased levels of TNF-α we performed intracellular cytokine staining in leukocytes from myelofibrosis (MF) patients and normal controls. Using a panel of markers to define cellular subsets we found expression of TNF-α was uniformly low in unstimulated cells. However, when cells were treated with lipopolysaccharide (LPS), TNF-α expression was 16-fold higher in HSCs of MF patients compared to normal controls (p<0.005), while expression in mature populations was not different. In a murine model of JAK2V617F driven MPN, where JAK2V617F cells are identified by GFP, TNF-α expression was 3-fold higher in JAK2V617F-expressing HSCs (GFP+) compared to GFP-controls (p<0.005), irrespective of LPS stimulation.To evaluate anti-TNF therapy in MPNs, we treated JAK2V617F mice with etanercept, a soluble TNF receptor fusion protein that binds and inactivates TNF-α. Etanercept did not significantly restrain JAK2V617F-associated WBC or hematocrit increases over a 10-week period, despite suppression of TNF-α activity in plasma. As TNF-α may activate pro-apoptotic (predominantly through TNF receptor 1, TNFR1) and pro-survival pathways (predominantly through TNF receptor 2, TNFR2), we reasoned that global blockade of TNF-α may not shift the balance in favor of normal hematopoiesis. To investigate this we sorted primitive (Lin-, cKit+) cells from mice with JAK2V617F-induced MPN by TNF receptor expression. TNFR2+ cells showed significantly increased colony formation compared to TNFR1+ cells, demonstrating that TNFR2 expression is associated with increased clonogenic potential. Additionally we treated these primitive cells with specific antibodies blocking TNFR1 or TNFR2. Colony assays performed after 3 days in liquid culture with TNFR-blocking antibodies confirmed that interrupting TNFR2-mediated survival signaling resulted in decreased colony formation (61% reduction) while blocking TNFR1 increased colony formation (5% increase). In addition we tested CD34+ cells from MF patient samples with TNFR-blocking antibodies. As seen with the murine MPN cells, colony formation was decreased with the TNFR2 blocking antibody (30% reduction) and increased with the TNFR1 blocking antibody (48% increase) consistent with a differential activation of survival signals by TNF-α in MPN cells. These studies were performed without addition of exogenous TNF-α, which confirms the ability and requirement for these primitive JAK2V617Fcells to utilize TNF-α production to actively support survival.Our data suggest that TNF-α generated by primitive MPN cells promotes their survival through activation of TNFR2, while TNFR1 activation is suppressive. If selective inhibition of TNFR2 shifts the equilibrium toward normal hematopoietic cells, this would support the use of TNFR2 blockade to treat MF and other myeloproliferative neoplasms. Genotyping of human MF and murine MPN colonies cultured with TNF receptor blocking antibodies and experiments in normal human CD34+ cells are ongoing and will be presented. DisclosuresDeininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

Design, Optimization, and Pre-Clinical Evaluation of Direct, Mechanism-Based STAT3 Inhibitors for Treating Myeloid Disorders

We have identified STAT3 as a convergence point for oncogenic signaling in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) lacking BCR-ABL1 kinase domain mutations. In addition, we found that STAT3 activity contributes to disease in other myeloid disorders, including acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs). Utilizing TKI-resistant CML as a model system, we identified BP-5-087 as a small molecule inhibitor of STAT3 that reduces STAT3 phosphorylation and nuclear transactivation (Eiring et al. Leukemia, 2014).Binding of BP-5-087 to the STAT3 SH2 domain was initially assessed using fluorescence polarization (FP) assays and high-resolution computational docking simulations. To further validate the binding motif of BP-5-087, we conducted time-resolved electrospray ionization mass spectrometry/hydrogen-deuterium exchange experiments. Fold-change in deuterium uptake was analyzed for 68 STAT3 peptides representing 71% sequence coverage, and mapped onto the crystal structure of STAT3. This analysis precisely defined the binding epitope for BP-5-087 within the STAT3 SH2 domain.We next tested the effects of BP-5-087 in several myeloid malignancies using relevant disease models. (i) CML stem and progenitor cells from TKI-resistant patients without kinase domain mutations were treated with BP-5-087 ex vivo, using short-term liquid culture, clonogenic and LTC-IC assays. BP-5-087 treatment significantly reduced colony formation by CML stem and progenitor cells (p<0.01), with no effect on normal human CD34+ cord blood (CB) cells. (ii) Similarly, BP-5-087 also increased apoptosis and reduced viability (p<0.05) of primary AML blasts treated ex vivo with BP-5-087 for 72 hours in liquid culture. (iii) CD34+ cells from patients with myelofibrosis were also treated with BP-5-087 in clonogenic assays, and similar to CML, BP-5-087 reduced myeloid colony formation, although to a lesser extent.The in vivo activity of BP-5-087 was next evaluated in a murine model of JAK2 V617F-induced MPN. Briefly, Balb/c bone marrow was transduced with JAK2 V617F-GFP, followed by injection into lethally irradiated recipients. After disease induction, mice were treated with BP-5-087 (25 mg/kg) by once-daily oral gavage. No toxicities were observed after 40 days of treatment in BP-5-087-treated mice. While BP-5-087 did not significantly reduce the percentage of GFP+ cells, there was a 41% reduction of spleen weight in BP-5-087-treated mice compared to vehicle-treated controls (p<0.05). Post study analysis revealed BP-5-087 plasma concentrations <1 μM, suggesting that insufficient bioavailability contributed to the modest in vivo effects.To advance the lead optimization of our STAT3 inhibitor series, we instituted a comprehensive screening cascade. We first developed a computational model (quantitative structure-activity relationship, QSAR) to guide and prioritize selection of new inhibitor candidates for synthesis. Compounds are initially ranked using a methanethiosulfonate (MTS)-based cell viability assay in a TKI-resistant, STAT3-dependent CML cell line (AR230R). Inhibition of STAT3 is confirmed using a cell-based STAT3 reporter assay and an in vitro FP-based binding assay. Optimization of potency is balanced by the goals of reducing molecular weight (MW) and calculated LogP (cLogP) compared to BP-5-087 (MW: 694.8; cLogP: 7.3). Compounds with improvements in these categories are then subject to toxicity testing utilizing clonogenic assays with CD34+ CB cells. Non-toxic compounds are evaluated for their pharmacokinetic profile in Balb/c mice and tested for activity in primary samples from CML, AML and MPN patients. These activities have directed us to a lead compound, AM-1-124, which displays significant improvements in potency, MW, cLogP, and in vivo half-life compared to BP-5-087. AM-1-124 had minimal effects in the CB toxicity assay and induced apoptosis in primary AML patient samples at 2-fold lower concentrations than BP-5-087. With AM-1-124 as our current lead compound, we are continuing our iterative evaluation of novel STAT3 inhibitors utilizing our screening cascade. Design and testing of optimized, orally active inhibitors will enable further evaluation of STAT3 as a target in animal models of myeloid leukemia and will justify the clinical development of these compounds for patients in need of new targeted therapies. DisclosuresDeininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

JAK2V617F and Loss of Ezh2 in Hematopoietic Cells Contribute Synergistically to Myeloproliferative Neoplasm Initiation Potential, and Accelerate Progression of Disease

Background;The gain-of-function JAK2 mutation, JAK2V617F, is the most common molecular abnormality in myeloproliferative neoplasms (MPNs) and appears in patients with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Ezh2 is a component of PRC2, catalyzing methylation of H3K27, and frequently mutated in MPN patients. Loss-of-function mutation of Ezh2 was reported as a poor prognostic marker in myelofibrosis patients. Many JAK2V617F-positive MPN patients harbor other mutations, but combination effects of JAK2V617F and Ezh2 mutation have not been analyzed.Methods & Results; To investigate the interaction between the two kinds of mutation in hematopoiesis, we studied transgenic mice with conditional expression of JAK2V617F, and inducible loss-of-function of Ezh2, both singly and in combination. Conditional expression of Cre was achieved using the SclCreER system with four weeks of tamoxifen injection, leading to excision of loxP-flanked alleles of Ezh2, and simultaneously induced expression of JAK2V617F, via the Flip-Flop recombination system (FF1). Mice with heterozygous deletion of Ezh2 showed no changes in peripheral blood, but homozygous deleted mice displayed slightly increased platelet counts compared to control mice. JAK2V617F-expressing (FF1) mice showed a typical PV phenotype, including erythrocytosis, thrombocytosis and neutrophilia. Mice expressing JAK2V617F with heterozygous deletion of Ezh2 (FF1;Ezh2+/-) also showed typical PV, but with more profound thrombocytosis and neutrophilia, and faster progression to fibrosis, than FF1 mice. Mice expressing JAK2V617F with homozygous deletion of Ezh2 (FF1;Ezh2-/-) had even shorter survival and showed more profound thrombocytosis and myelofibrosis than FF1;Ezh2+/- mice, but without erythrocytosis. These mice had dacryocytes and mobilized c-kit positive progenitor cells in peripheral blood. Bone marrow histology revealed mild fibrosis (grade: 1) in FF1 mice; more excessive fibrosis (grade: 1-2) in FF1;Ezh2+/- mice; and obvious fibrosis associated with collagen fiber formation and osteosclerosis (grade: 2) in FF1;Ezh2-/- mice. Cell compartment analysis in FF1 mice revealed expansion of hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MegP) in bone marrow and spleen, while common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP) and megakaryocyte erythrocyte progenitor (MEP) were expanded principally in spleen. The expansion of HSCs and all progenitors were accelerated in FF1;Ezh2+/- mice and FF1;Ezh2-/- mice. Especially MegP and mature megakaryocytes were further expanded in FF1;Ezh2-/- mice compared to FF1;Ezh2+/-. To clearly estimate disease progression, transgenic bone marrow cells were transplanted to lethally irradiated recipient mice with WT competitor bone marrow cells and tamoxifen was administrated after transplantation. FF1 bone marrow cells clearly showed outcompeting potential to WT bone marrow cells. FF1;Ezh2+/- and FF1;Ezh2-/- bone marrow cells showed more stronger outcompeting potential than FF1 bone marrow cells. Finally, disease initiative potential was evaluated by limiting dilution transplantation. Total bone marrow cells, with heterozygous deletion of Ezh2 (Ezh2+/-), or expressing JAK2V617F (FF1), or the combination of the two mutations were transplanted (following Cre induction and recombination) to lethally irradiated recipient mice, mixed with competitor cells (control or Ezh2+/- bone marrow cells were transplanted by 1:100 dilution and FF1 or FF1;Ezh2+/- bone marrow cells were transplanted by 1:250 dilution against WT competitor cells). Bone marrow cells with Ezh2+/- showed higher reconstitution capacity than control (35% vs 10%; Ezh2+/- vs control) without MPN phenotype. Bone marrow cells with FF1;Ezh2+/- showed stronger reconstitution capacity (39% vs 15%; FF1;Ezh2+/- vs FF1), and greater disease initiative potential (50% vs 33%; FF1;Ezh2+/-vs FF1) than FF1.Summary; We clarified that loss-of-function of Ezh2 accelerated JAK2V617F-induced MPNs. Especially JAK2V617F with Ezh2 homozygous loss-of-function induced excessive megakaryopoiesis and resulted in PMF. JAK2V617F with heterozygous deletion of Ezh2 synergistically enhanced bone marrow reconstitution and disease initiative potential. DisclosuresSkoda:Novartis: Consultancy; Sanofi: Consultancy.

IL3-Receptor Signaling Is Dispensable For The Generation and Maintenance Of Jak2V617F-Induced Myeloproliferative Neoplasm (MPN

Myeloproliferative neoplasms (MPN) such as polycythemia vera, essential thrombocythemia and primary myelofibrosis are clonal diseases driven by acquired somatic mutations in hematopoietic stem cells (HSCs), the most common of which is JAK2V617F. JAK2V617F leads to cytokine hypersensitivity and activation of JAK-STAT signaling in the presence of an erythropoietin (EPO), thrombopoietin (TPO) or interleukin-3 (IL3) cytokine receptor scaffold (Nature 2005; 434:1144-8, Leukemia 2008; 22:1828-40). Physiological Jak2V617F expression in long-term hematopoietic stem cells (LT-HSCs; lineagelowcKit+Sca1+CD150+CD48-) is necessary and sufficient to generate MPN, and these LT-HSCs contain the sole reservoir of Jak2V617F MPN-initiating stem cells (Cancer Cell 2010; 17:584-96; Blood 2012; 120:166-72). Although Jak2V617F-mediated EPO hypersensitivity drives erythroid expansion and in vitro EPO-independent colony formation, the EPO receptor is not expressed on Jak2V617F HSC populations (Blood 2012; 120:166-72). This suggests that hypersensitivity to IL3 and/or TPO signaling is responsible for driving LT-HSC proliferation and survival in vivo.To determine the respective contributions of IL3 and TPO signaling to Jak2V617F-induced MPN, pStat5 was measured in HSC and progenitor populations from E2ACre+Jak2V617F+/- knockin mice (hereafter Jak2VF) after stimulation with rmIL3 or rmTPO. Committed myeloid progenitors showed robust pStat5 stimulation with rmIL3, but only low level stimulation with rmTPO (TPO 460.25 ± 25.02 MFI vs. IL3 598.25 ± 69.05 MFI, p<0.05, Vehicle 362.25 ± 76.66 MFI). In contrast, LT-HSCs showed strong induction of pStat5 signaling with rmTPO stimulation but less so with rmIL3 stimulation (TPO 571 ± 47.36 MFI vs. IL3 487.5 ± 53.69 MFI, p=0.05, Vehicle 249.5 ± 47.63 MFI). Concordant with these results, TPO signaling appears essential for the generation of Jak2V617F-induced MPN (ASH 2012 Abstract 427). To determine the contribution of IL3 receptor signaling in Jak2V617F-induced MPN, we crossed Jak2VF knockin mice with mice lacking the common beta subunit of the IL3 receptor that is responsible for signal transduction of IL3, IL5 and GM-CSF (Jak2VFIL3Rb-/-). Jak2VFIL3Rb-/- mice developed MPN with similar latency and mortality to Jak2VF controls. There were no differences in peripheral blood white cell count (16.51 ± 1.5×109/L vs. 14.89 ± 2.4×109/L, p=0.39, n=3), haematocrit (67.97 ± 7.85×109/L vs. 61.97 ± 5.73×109/L, p=0.35, n=3) or extramedullary erythropoiesis (spleen weight, 665 ± 107mg vs. 541 ± 106mg, p=0.22, n=3) between Jak2VFIL3Rb-/- and Jak2VF mice respectively. In competitive bone marrow transplantation assays, all recipients of Jak2VFIL3Rb-/- or Jak2VF bone marrow developed MPN with similar diagnostic parameters such as elevated white cell count (14.29 ± 3.41×109/L vs. 18.38 ± 5.78×109/L, p=0.11, n=8), hematocrit (63.4 ± 16.5% vs. 50.5 ± 13%, p=0.16, n=8) and splenomegaly (529 ± 86mg vs. 465 ± 119mg, p=0.23, n=8) in Jak2VFIL3Rb-/- and Jak2VF mice respectively. Jak2VFIL3Rb-/- bone marrow cells initially showed reduced short-term (4 weeks) engraftment and white cell count in recipients compared to the Jak2V617F group (p<0.0005). However after 16 weeks post-transplant there was no difference in chimerism between recipients of Jak2VFIL3Rb-/- or JakVF cells. IL3 was unable to stimulate pStat5 in Jak2VFIL3Rb-/- LT-HSCs or progenitors, but was preserved in Jak2VF LT-HSCs and progenitors.These data show that IL3Rb signaling is dispensable for Jak2V617F-induced MPN and LT-HSC function, however may regulate short-term myeloid progenitor cell expansion. TPO signaling appears preferentially important for Jak2VF LT-HSC pStat5 induction, whereas IL3 is more important for pStat5 induction in progenitor cells. These findings will help to inform strategies aimed at targeting long term Jak2V617F-initiating HSC populations. Disclosures:No relevant conflicts of interest to declare.

Stat3 Negatively Regulates Myeloproliferative Neoplasm Induced By Jak2V617F

The JAK2V617F mutation has been found in most patients with Ph-negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Expression of JAK2V617F results in constitutive activation of several signaling molecules/pathways, such as Stat5, Stat3, Akt and Erk. Unraveling the contribution of these signaling pathways in MPNs will improve our understanding of the pathogenesis of MPNs and allow us to develop more effective targeted therapies.We have previously reported the generation of a conditional Jak2V617F knock-in mouse, which exhibits all the clinical features of human PV. Using this mouse model, we have demonstrated that Stat5 is absolutely required for the pathogenesis of PV induced by Jak2V617F. However, the contribution of other signaling molecules activated by Jak2V617F in the development and progression of MPNs still remains elusive. Stat3, a member of the family of signal transducer and activator of transcription (Stat), is often found activated in solid tumors and hematologic malignancies including MPNs. Although Stat3 is known to play a tumor-promoting function in various human malignancies, recent studies also have found a tumor suppressive function of Stat3 in certain malignancies. For instance, Stat3 negatively regulates BRAFV600E-induced thyroid tumorigenesis (Couto et al., Pro Natl Acad Sci USA 2012) or suppresses PTEN loss-induced malignant transformation of astrocytes (Iglesia et al., Genes Dev 2008). Thus, Stat3 can positively or negatively regulate cell growth and tumor progression. Here, we sought to determine the role of Stat3 in Jak2V617F-evoked MPN using conditional Stat3 knock-out (Stat3 floxed) and Jak2V617F knock-in mice.Whereas expression of Jak2V617F resulted an increase in red blood cells (RBC), hemoglobin, hematocrit, white blood cells (WBC), neutrophils and platelets in the peripheral blood of the Jak2V617F knock-in mice, deletion of Stat3 did not cause any significant change in RBC, hemoglobin, hematocrit and platelet numbers in Jak2V617F knock-in mice. Strikingly, Stat3 deficiency significantly increased nertrophil counts in mice expressing Jak2V617F. Flow cytometric analysis showed that deletion of Stat3 increased the hematopoietic stem cell (HSC) compartments (LSK, LT-HSC, ST-HSC) and GMP populations in the bone marrow (BM) and spleens of mice expressing Jak2V617F. However, MEP population was unaffected by Stat3 deletion. Cell cycle analysis using Hoechst/Pyronin Y staining revealed that Jak2V617F expression alone resulted in increased cycling of HSC-enriched LSK cells, and Stat3-deficiency further enhanced the cycling of Jak2V617F-expressing LSK cells. Stat3-deficiency also caused a marked expansion of Gr-1+/Mac-1+ population in the BM and spleens of mice expressing Jak2V617F. As a consequence, CD71+/Ter119+ population was proportionally reduced in Stat3-deficient Jak2V617F-expressing mice BM. Histopathologic analysis showed marked increase in granulocytes in the BM and spleens of Stat3-deficient Jak2V617F-expressing mice compared with mice expressing Jak2V617F. Stat3-deficient Jak2V617F-expressing mice also exhibited marked infiltration of neutrophils in their livers. Furthermore, deletion of Stat3 significantly reduced the survival of Jak2V617F knock-in mice. Together, these results suggest a negative role for Stat3 in Jak2V617F-induced MPN. Thus, Stat3 may not be a suitable therapeutic target for treatment of PV and other JAK2V617F-positive MPNs. Disclosures:No relevant conflicts of interest to declare.

The Role Of p53 In JAK2V617F-Induced Myeloproliferative Neoplasms

Ph- myeloproliferative neoplasms (MPNs) are hematopoietic malignancies in which one or more myeloid lineages are abnormally amplified. These diseases represent a group of chronic conditions including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. MPNs mainly affect older people and have an average onset age of 55 years. Complications associated with MPNs include development of acute leukemia as well as thrombosis, hemorrhage, and myeloid metaplasia. JAK2V617F is found in the majority of patients with MPNs, and an overwhelming number of studies have demonstrated its pathogenicity. However, the precise action of JAK2V617F is not well defined, and other molecular defects involved in MPNs remain elusive. In earlier studies, we have generated transgenic mice expressing JAK2V617F in the hematopoietic system and demonstrated that JAK2V617F can cause MPN-like phenotypes in an age- and transgene dose-dependent manner. In this study, we address the involvement of tumor suppressor p53 in the progression of JAK2V617F-induced MPN phenotypes. We crossed our JAK2V617F transgenic mice with p53 knockout mice. Under the p53+/+ background, our JAK2V617F transgenic mice developed a mild MPN-like phenotypes in 15 weeks. However, under the heterozygous knockout p53-/+ condition, the mice developed a strong MPN-like phenotype in 8 weeks, manifested in higher blood cell counts, more severe splenomegaly, and earlier onset of myelofibrosis. This suggests that p53 affects the progression of MPNs, and reduced levels of p53 activity may be responsible for the heterogeneous phenotypes observed in MPN patients. Furthermore, when p53 was deleted from both chromosomes, JAK2V617F mice developed acute erythroleukemia, megakaryoblastic leukemia, or myeloid leukemia and died in 20 weeks with greatly enlarged spleen (>10 times the normal size) and liver (twice the normal size) infiltrated with leukemic cells. This indicates that loss of p53 in addition to JAK2V617F causes leukemic transformation. This is consistent with the earlier findings that p53 mutations exist in JAK2V617F-positive leukemia cell lines and JAK2V617F-positive MPNs patients who developed acute myeloid leukemia. Our study also suggests that targeting JAK2V617F and p53 simultaneously may provide effective treatment for MPNs. We employed nutlin-3 that disrupts the MDM2-p53 interaction thereby reducing degradation of p53. In vitro cell culture studies demonstrated that JAK2 inhibitors and nutlin-3 synergistically inhibited the growth of hematopoietic progenitor cells from JAK2V617F transgenic mice and MPN patients. Finally, from one of the p53-/- JAK2V617F transgenic mice, we derived an erythroleukemia cell line designated J53Z1. J53Z1 cells are CD71high and Ter119low and are dependent on erythropoietin for survival. They were effectively inhibited by known JAK2 inhibitors and should be useful in cell-based assays for screening of JAK2 inhibitors. Altogether, our study demonstrates that the level of p53 dictates the progression of JAK2V617F-induced MPNs and targeting p53 and JAK2V617F simultaneously may provide effective treatment for JAK2V617F-positive MNPs. Disclosures:No relevant conflicts of interest to declare.