Jak2V617F Knock-in Mice Research Articles

In VitroandIn VivoInteractions between Stromal and Leukemic Cells

Understanding the mechanisms underlying changes in the microenvironment caused by pathological cells is essential to develop new therapeutic strategies. Such modifications allow leukemia cells to modify their microenvironment in order to promote their growth. Here, we studiedin vivoandin vitrointeractions between Primary Myelofibrosis and the microenvironment. This clonal malignant disorder is caused by three major driver mutations: JAK2V617F, MPLW515L and CALR exon 9 mutations. We performed 72h co-cultures of the mesenchymal stromal cell line MS5 and the MO7e human hematopoietic cell line transduced with either the MPLW515L mutation (MO7eMPLW515L) or GFP as a control. As Mo7e cells are dependent on the human growth factor h-GM-CSF, co-cultures were performed with and without it Flow cytometry confirmed that leukemic cells expressing the MPLW515L mutation proliferated actively in the absence of hGM-CSF, unlike the control MO7e GFP cells. In addition, the presence of MS5 stromal cells did not have a major impact on the proliferation of MO7eMPLW515L but promoted MO7e GFP cell survival. Thus the MPLW515L mutation activates signaling pathways that make the cells independent of cytokines and stroma. On the other hand, the number of stromal cells increased upon co-culture with either MO7e GFP or MO7e W515L and in the presence of h-GM-CSF. Thus, either growth factor addition or the MPLW515L mutation allows MO7e cells to enhance the proliferation of stromal cells. To test our observations in primary cells, we also performed 72h co-cultures using isolated murine cKIT+ hematopoietic progenitor cells from JAK2V617F knock-in or wild type mice. The impact of stromal cells was evaluated in the presence or absence of thrombopoietin (TPO). Cells were analyzed by flow cytometry. The results showed that MS5 stromal cells induced proliferation of both primary wild type and JAK2V617F cKIT+ hematopoietic cells independently of TPO. Moreover, the proliferation of MS5 stromal cells was also significantly induced by both wild type and JAK2617F cKIT + primary cells. In vivointeractions were studied using transgenic mice expressing CXCL12HT-dsRED crossed with JAK2V617F mice (CXCL2-HT-JAK2V617F). We followed different parameters, namely the weight and the size of the spleen, the different blood parameters, the number of hematopoietic progenitors in bone marrow, spleen and blood, and the cellularity and the number of fibroblast colonies (CFU-F). Results showed that CXCL12 HT JAK2KI mice developed significant splenomegaly, associated with changes in blood parameters characteristic of JAK2KI mice, i.e. increases in white blood cells, red blood cells, hematocrit, and hemoglobin. There was also an increase in blood, spleenand bone marrow progenitors compared to the CXCL12HT GFP control mice. The number of fibroblast colonies (CFU-F), reflecting the activity of mesenchymal stem cells (MSC) in the microenvironment, was also greatly increased, especially in the marrow of CXCL12HT JAK2KI mice, indicating that the malignant clone modified the microenvironment of the mice. Taken together, our findings show that hematopoietic cells, either normal or leukemic, can modify their own microenvironmentin vitroandin vivo. This offers a new avenue for therapeutic targeting in the treatment of this type of hemopathologies. Disclosures No relevant conflicts of interest to declare.

N-acetylcysteine inhibits thrombosis in a murine model of myeloproliferative neoplasm

AbstractThrombosis is a major cause of mortality in patients with myeloproliferative neoplasms (MPNs), though there is currently little to offer patients with MPN beyond aspirin and cytoreductive therapies such as hydroxyurea for primary prevention. Thrombogenesis in MPN involves multiple cellular mechanisms, including platelet activation and neutrophil-extracellular trap formation; therefore, an antithrombotic agent that targets one or more of these processes would be of therapeutic benefit in MPN. Here, we treated the JAK2V617F knockin mouse model of polycythemia vera with N-acetylcysteine (NAC), a sulfhydryl-containing compound with broad effects on glutathione replenishment, free radical scavenging, and reducing disulfide bonds, to investigate its antithrombotic effects in the context of MPN. Strikingly, NAC treatment extended the lifespan of JAK2V617F mice without impacting blood counts or splenomegaly. Using an acute pulmonary thrombosis model in vivo, we found that NAC reduced thrombus formation to a similar extent as the irreversible platelet inhibitor aspirin. In vitro analysis of platelet activation revealed that NAC reduced thrombin-induced platelet-leukocyte aggregate formation in JAK2V617F mice. Furthermore, NAC reduced neutrophil extracellular trap formation in primary human neutrophils from patients with MPN as well as healthy controls. These results provide evidence that N-acetylcysteine inhibits thrombosis in JAK2V617F mice and provide a pre-clinical rationale for investigating NAC as a therapeutic to reduce thrombotic risk in MPN.

The CDK6 Inhibitor Palbociclib in Combination with Ruxolitinib Remarkably Improves Myelofibrosis in Murine Models

Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN), characterized by deposition of fibrous tissues in the bone marrow, abnormal megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. The median survival for patients with MF is ~5 years. The JAK2V617F mutation, which results in constitutive activation of the JAK2 tyrosine kinase, has been found in 50-60% patients with MF. Additional mutations in the genes encoding thrombopoietin receptor (MPL) and calreticulin (CALR) have been found at a lower frequency than JAK2V617F in MF. The JAK1/JAK2 inhibitor ruxolitinib has been approved for treatment of MF. Although ruxolitinib can reduce splenomegaly and constitutional symptoms, it does not cure or prevent fibrosis in patients with MPN/MF. Additionally, ruxolitinib treatment does not improve survival in MF patients. So, there is a critical need to identify new therapeutic targets and develop novel therapies for MF. We have found that expression of CDK6 is significantly elevated in mouse and human MPN hematopoietic cells/progenitors. We also observed that knockdown of CDK6 significantly inhibited proliferation of BA/F3-EpoR-JAK2V617F and HEL cells expressing JAK2V617F but not wild-type JAK2 expressing BA/F3-EpoR cells, suggesting that CDK6 is a bona fide target in MPN. In this study, we investigated the efficacy of CDK4/6 inhibitor Palbociclib (PD-0332991) in hematopoietic cells expressing JAK2V617F and MPLW515L and murine models of MF. Since Palbociclib is an FDA-approved drug for treatment of breast cancer, information on the dose, pharmacokinetics and toxicity of Palbociclib in human is already available. Thus, it would be easier to move this drug into clinic and repurpose it for treatment of MPN/MF. We observed that Palbociclib treatment (0.25-0.5 uM) significantly reduced the proliferation of murine BA/F3 cells expressing JAK2V617F or MPLW515L. Palbociclib treatment also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 cells. However, Palbociclibonly modestly affected proliferation of wild type JAK2-expressing BA/F3 cells at higher concentrations. Palbociclib treatment also resulted in marked apoptosis in BA/F3-EpoR cells expressing JAK2V617F. Furthermore, Palbociclib and ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Additionally, Palbociclib significantly inhibited hematopoietic progenitor colony formation in MPN/MF CD34+ cells. We previously generated conditional JAK2V617F knock-in mice. Whereas expression of heterozygous JAK2V617F induces a PV-like disease, homozygous JAK2V617F expression promotes rapid progression to myelofibrosis. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of Palbociclib alone and in combination with ruxolitinib against MF. Treatment of Palbociclib alone significantly reduced the WBC and neutrophil counts and decreased the spleen size in homozygous JAK2V617F mice. Combined treatment of Palbociclib and ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow of homozygous JAK2V617F mice treated with Palbociclib or Palbociclib and ruxolitinib combination. We also have tested the in vivo efficacy of Palbociclib and Palbociclib/ruxolitinib combination in MPL mutant (MPLW515L) mouse model of MF. We observed similar decrease in WBC and neutrophil counts and reduction in splenomegaly in MPL mutant mice upon treatment of Palbociclib alone or in combination with ruxolitinib. Combined treatment of Palbociclib and ruxolitinib remarkably reduced fibrosis in the bone marrow of MPL mutant mice. Overall, our results suggest that palbociclib in combination with ruxolitinib may have therapeutic potential against MF and support the clinical investigation of palbociclib and ruxolitinib combination in patients with MF. Disclosures No relevant conflicts of interest to declare.

Abstract 1287: Targeting pleckstrin-2 for the JAK2-STAT and PI3K-Akt pathways in cancer therapy

Abstract Pleckstrin-2 (Plek2) is a widely expressed PH domain containing protein that binds to phosphoinositide with unclear functions. Our published reports reveal that Plek2 is overexpressed in JAK2 V617F mutation positive myeloproliferative neoplasms (MPNs). We identified that Plek2 is a downstream effector of the JAK2-STAT5 pathway. Furthermore, through a mouse genetic approach, we discovered that knockout of Plek2 significantly ameliorated the MPN phenotypes and reverted lethality and thrombosis in JAK2 V617F knockin mice. These studies demonstrate that Plek2 is critical for the pathogenesis of MPNs with the activated JAK2-STAT5 pathway, and form a strong foundation for the development of Plek2 inhibitors for the treatment of MPNs. Importantly, our published study shows that Plek2 knockout mice do not develop anemia or cytopenia, indicating Plek2’s oncogenic potential is only in the disease background, which makes Plek2 inhibitors less likely to cause severe side effects compared to JAK inhibitor ruxolitinib. Based on these studies, we used an in silico approach to screen for putative Plek2 binding small molecules and identified hit compounds that bind to the DEP domain of Plek2. Further medicinal chemistry studies identified lead compound NUP-17d that inhibited proliferation of the hyperproliferative hematopoietic cells with potency comparable to ruxolitinib. In addition, NUP-17d also blocked proliferation of several Plek2-overexpressing solid tumor cell lines. Our biochemical assays showed that NUP-17d inhibited Akt phosphorylation. Further mechanistic studies revealed that Plek2 functions as a central hub to mediate the JAK2-STAT and PI3K-Akt pathways to promote tumor cell proliferation. Specifically, Plek2 binds to and recruits PI3K-produced PtdIns(3,4)P2 and PI3K effector proteins including Akt, PDK1, PDK2, and mTOR, which forms a complex and significantly enhances the PI3K signaling. In addition to the biochemical assays, we also demonstrated Plek2’s function through a mouse genetic approach using a Pten hematopoietic specific knockout mouse model. Knockout of Plek2 significantly reverted the myeloproliferative phenotype in these mice and markedly extended their survival. Therefore, NUP-17d blocks tumor cell proliferation through the disruption of the Plek2 complex and inhibition of the PI3K-Akt pathway. Indeed, treatment of an erythropoietin-induced myeloproliferative mouse model with NUP-17d also demonstrated its potent inhibitory effect in myeloproliferation in vivo. These study establishes Plek2 as a oncoprotein mediating JAK2-STAT and PI3K-Akt signaling pathways. In addition to hematologic malignancies, Plek2 is also found to be highly upregulated with an associated worse prognosis in many solid tumors (oncomine, kmplot). Therefore, our Plek2 inhibitors will have a broad impact in cancer therapy, especially cancers with upregulated JAK2-STAT or PI3K-Akt pathway. Citation Format: Xu Han, Yang Mei, Gary E. Schiltz, Rama K. Mishra, Atul D. Jain, Peng Ji. Targeting pleckstrin-2 for the JAK2-STAT and PI3K-Akt pathways in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1287.

Abstract 1172: JAK2V617F-mutant megakaryocytes contribute to stem cell aging in myeloproliferative neoplasms

Abstract The myeloproliferative neoplasms (MPNs) are stem cell disorders characterized by hematopoietic stem cell (HSC) expansion and an increased propensity to develop hematologic malignancies. The acquired kinase mutation JAK2V617F plays a central role in MPNs. In this work we tested the hypothesis that the JAK2V617F-bearing megakaryocytes (MKs), an important component of the hematopoietic niche, promotes HSC dysfunction in MPNs. Methods JAK2V617F Flip-Flop (FF1) mice and Pf4-Cre mice (Radek Skoda, Switzerland) were crossed to generate MK lineage-specific human JAK2V617F knock-in mouse line (Pf4/FF1). Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline. Results We previously reported that, at 28 wks of age, the Pf4/FF1 mice developed a murine myeloproliferative syndrome with moderate thrombocytosis, splenomegaly, increased marrow MKs, and a 3-fold increase of CD45+CD201+CD150+CD48- HSCs with increased donor engraftment in a competitive repopulation assay compared to Pf4-cre control mice. (Zhan Leukemia 2016; Zhang Stem Cells 2018) Rigorous, extremely sensitive RT-PCR assays eliminated the possibility that HSCs in Pf4/FF1 mice express the mutant kinase. To study the effect of JAK2V617F-bearing MKs on HSC function during aging, we followed the Pf4/FF1 mice up to 2 years of age. At 2 yr, there were significant neutrophilia in Pf4/FF1 mice but no change was observed in hemoglobin, platelet count, or marrow HSC cell numbers compared to age-matched control mice. Indeed, while HSC numbers significantly increased in old (2-yr) control mice compared to young (6-mo) control mice (3.3-fold, p=0.0002, n=4-8), there was no significant increase of HSCs between old and young Pf4/FF1 mice (1.6-fold, p=0.158, n=6-8). A competitive marrow transplantation experiment was performed in which CD45.2 donor marrow cells from 2-yr old Pf4/FF1 or control mice were injected intravenously together with competitor CD45.1 wild-type marrow cells intro lethally irradiated wild-type recipient (CD45.1) (n=8-9 in each group). During a 4-month follow up, old HSCs from the Pf4/FF1 mice displayed hallmarks of murine hematopoietic aging including a lower peripheral blood donor chimerism, increased myeloid blood cell production, and decreased lymphoid cells compared to old HSCs from control mice. To begin to understand how JAK2V617F-bearing MKs promote the aging phenotype of HSCs in old Pf4/FF1 mice, qRT-PCR of purified HSCs identified up-regulation of p21 (2-fold, p = 0.019) in Pf4/FF1 mice compared to controls. Conclusion The Pf4/FF1 mice, in which the JAK2V617F expression is restricted to MKs, developed a myeloproliferative phenotype at young age and accelerated stem cell aging at old age. The precise mechanism by which JAK2V617F-bearing MKs affect the MPN marrow niche and stem cell dysfunction during disease progression deserve further investigation. Citation Format: Helen Wong, Melissa Castiglione, Molly Quan, Huichun Zhan. JAK2V617F-mutant megakaryocytes contribute to stem cell aging in myeloproliferative neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1172.

PS1450 THE PHARMACOLOGICAL IGF1R‐IRS1/2 INHIBITOR OSI‐906 REDUCES ERYTHROID PROGENITORS AND INHIBITS STAT3 IN JAK2V617F KNOCKIN‐MYELOPROLIFERATIVE NEOPLASM MICE

Background:The recurrent V617F mutation in JAK2 confers growth factor‐independent proliferation for hematopoietic cells and plays an important role in the pathogenesis of myeloproliferative neoplasms (MPN). Absence of complete clinical remission in most MPN patients treated with the JAK1/2 inhibitor ruxolitinib highlights the need of new therapeutic strategies. Recent evidence indicates that IGF1R/IRS pathway is a potential target in MPN. In mice, AIRAPL down‐regulation induces MPN transformation by upregulation of IGF1R signaling. Linsitinib (OSI‐906) is a potent highly selective IGF1R/IR inhibitor, with anti‐neoplastic effects in solid tumors, but underexplored in hematological neoplasms. We have previously noticed that OSI‐906 reduces cell viability and inhibited IRS1/2, IGF1R, AKT1/2/3 and P70S6K in Ba/F3 JAK2V617F cells (EHA 2018 ‐ Abstract #3288).Aims:To investigate the in vivo and ex vivo effects of OSI‐906 treatment in MPN phenotype and progenitors cells using JAK2V617F knockin‐MPN mice.Methods:For MPN phenotype induction, 5 × 106 bone marrow cells from JAK2V617F knockin mice were transplanted into lethally irradiated Pep boy mice. After 4 weeks, chimerism was evaluated by CD45.1 and CD45.2 markers (Becton‐Dickinson) by flow cytrometry in peripheral blood. Mice with >70% CD45.2 cells were randomized and treated with vehicle (25 mM tartaric acid solution, n = 5) or OSI‐906 (50 mg/kg, n = 4), both were administered oral by gavage, five days per week, from days 1 to 36. In day 37, animals were harvested and subjected to analysis of spleen and hematological parameters. Erythroid progenitors in the spleen and bone marrow were evaluated by CD71 and Ter119 markers. The immature subpopulation LSK (Sca1+/c‐Kit+), the myeloid progenitor (Sca1−/c‐Kit+), multipotent progenitors (MPP) (CD48+/C150−), long‐term (LT‐HSC) (CD48−/CD150+) and short‐term (ST‐HSC) (CD48−/150−) hematopoietic stem cells were evaluated by flow cytometry. All experiments were approved by the Animal Ethics Committee. Mann‐Whitney test or ANOVA test and Bonferroni post‐test was used. Alternatively, JAK2V617F knockin mice were euthanized between 10–16 weeks of age and the bone marrow was isolated, treated ex vivo with vehicle or OSI‐906 (≥20 μM); and submitted to clonogenicity and to protein expression/activation studies.Results:OSI‐906 treatment in vivo was well‐tolerated by JAK2V617F‐kockin MPN mice, but did not reduce the spleen volume. OSI‐906 significantly reduced erythroid progenitors (p = 0.03) and increased myeloid progenitors (p = 0.01) in bone marrow when compared with vehicle. Hemoglobin, hematocrit and the number of MPP, ST‐HSC, or LT‐HSC were not modulated by OSI‐906 treatment. Bone marrow cells from JAK2V617F knockin‐MPN mice treated ex vivo with OSI‐906 presented absence of colony formation, and decreased of protein expression/activation of IGF1R, AKT1/2/3, ERK1/2, and STAT3.Summary/Conclusion:OSI‐906 exhibited promising results ex vivo, including inhibition of IGF1R signaling pathways and progenitor cells proliferation. OSI‐906 treatment in vivo in the current protocol reduced erythroid progenitors in bone marrow from JAK2V617F knockin‐MPN mice. The lack of better results in vivo reinforces the need to adjust treatment regimen to this disease model.

N-Acetylcysteine As a Therapeutic for JAK2V617F Myeloproliferative Neoplasms

Chronic inflammation is common in MPN and drives disease progression and worsens symptom burden. It has been reported that JAK2V617F hematopoietic stem and progenitor cells (HSPCs) exhibit elevated basal oxidative stress compared to wild-type cells. However, JAK2V617F HSCs have a differential response than normal HSPCs upon stimulation with pro-inflammatory cytokines or lipopolysaccharide (LPS) invivo and in vitro. Specifically, JAK2V617F HSPCs do not significantly increase reactive oxygen species, exit quiescence, or increase DNA damage following LPS exposure (Blood 2017 130:4211). Therefore, we hypothesized that oxidative stress is important for differential responses of wild-type and JAK2V617F HSCs to inflammation.To compare cell responses to oxidative stress, we treated bone marrow cells from JAK2V617F knock-in and wild-type mice ex vivo with L-buthionine-S,R-sulfoximine (BSO), which reduces glutathione levels, then measured the impact on myeloid colony formation in methylcellulose. JAK2V617F knock-in bone marrow cells are resistant to oxidative stress-induced reduction in colony formation compared to wild-type bone marrow cells. This data suggests that JAK2V617F myeloid progenitors produce less reactive oxygen species in response to BSO or that these cells are resistant to oxidative stress-induced cell death.Next, we tested the effect of the anti-oxidant n-acetylcysteine (NAC) in a JAK2V617F knock-in mouse model. All hematopoietic cells in this model express JAK2V617F, these mice develop elevated blood counts, splenomegaly, and die suddenly at approximately 2-3 months of age. Surprisingly, addition of NAC (2g/L) in the drinking water extended the lifespan of JAK2V617F-knock in mice (p<0.02, n=6-12). However, aspirin (16mg/L) in drinking water did not extend the lifespan of JAK2V617F-knock in mice. NAC did not alter peripheral blood counts in either JAK2V617F-knock in or wild-type mice, which suggests that NAC's utility in JAK2V617F is not through cytoreduction but is due to its ability to reduce oxidative stress or thrombosis. Additionally, NAC had no effect on thrombin-induced platelet activation, which was assayed by P-selectin expression, phosphatidylserine exposure, and platelet-leukocyte aggregation via flow cytometry.There is a positive correlation between leukocytosis and thrombotic risk in MPN patients. Additionally, MPN patients and mice exhibit elevated neutrophil extracellular trap (NET) formation compared to healthy controls, which contributes to the increased thrombosis in MPN. To test the effect of antioxidants on NET formation in vitro, we treated neutrophils from MPN patients or healthy donors with phorbol myristate acetate concurrently with antioxidants (NAC or ferulic acid) and assayed the presence of extracellular DNA using a SYTOX Green nucleic acid stain. Unstimulated neutrophils from MPN patients exhibited sporadic NET formation while neutrophils from healthy donors did not. Additionally, NAC and ferulic acid reduced DNA release, which is indicative of NET formation.Taken together, these data demonstrate that JAK2V617F progenitors are resistant to oxidative stress-induced cell death. Furthermore, reduction of oxidative stress with n-acetylcysteine in vivo prevented NET-induced thrombosis in JAK2V617F knock-in mice as well as in vitro in normal and MPN neutrophils. These data provide a rationale for investigating the utility of n-acetylcysteine as a therapeutic in myeloproliferative neoplasms. DisclosuresNo relevant conflicts of interest to declare.

The PIM Kinase Inhibitor TP-3654 in Combination with Ruxolitinib Exhibits Marked Improvement of Myelofibrosis in Murine Models

Myelofibrosis (MF) is the most severe form of myeloproliferative neoplasms (MPN). The median survival for MF patients is ~5 years. A somatic JAK2V617F mutation, which results in constitutive activation of the JAK2 tyrosine kinase, has been found in 50-60% patients with MF. Additional mutations in the genes encoding thrombopoietin receptor (MPL) and calreticulin (CALR) have been found in MF but at a lower frequency than JAK2V617F. Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF. Although ruxolitinib treatment can reduce splenomegaly and constitutional symptoms in patients with MF, it failed to induce disease remission or cure fibrosis. Therefore, there is a critical need to identify new therapeutic target(s) and develop novel targeted therapies for MF. We found that PIM1 expression is significantly increased in mouse and human MPN/MF hematopoietic progenitors. Moreover, PIM1 knockdown markedly inhibited proliferation in JAK2V617F-expressing cells but not in wild type JAK2-expressing cells. In this study, we investigated the efficacy of the second-generation pan-PIM kinase inhibitor, TP-3654, in hematopoietic cells expressing JAK2V617F and MPLW515L and murine models of MF.TP-3654 (0.25-1.0 uM) treatment significantly reduced proliferation of murine Ba/F3 cells expressing JAK2V617F or MPLW515L. TP-3654 (0.5-1.0 uM) treatment also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 cells. However, TP-3654 only modestly affected proliferation of wild type JAK2-expressing Ba/F3 cells at higher concentrations. TP-3654 treatment also resulted in marked apoptosis in Ba/F3-EpoR-JAK2V617F, HEL and UKE-1 cells but not in wild-type JAK2 expressing Ba/F3-EpoR cells. Additionally, TP-3654 and ruxolitinib synergistically induced apoptosis in JAK2V617F- expressing hematopoietic cells. Moreover, TP-3654 treatment can overcome resistance to JAK2 inhibition in ruxolitinib-resistant Ba/F3-EpoR-JAK2V617F cells. Furthermore, TP-3654 significantly inhibited MPN/MF CD34+ hematopoietic progenitor colony growth.We previously reported the generation of JAK2V617F knock-in mice. Whereas heterozygous JAK2V617F knock-in mice exhibit a polycythemia vera phenotype, mice expressing homozygous JAK2V617F rapidly develop high-grade MF. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of TP-3654 alone or in combination with ruxolitinib against MF. Treatment of TP-3654 alone significantly reduced the increase in white blood cell (WBC) and neutrophil counts as well as spleen size in mice expressing homozygous JAK2V617F compared with vehicle treatment. Combined treatment of TP-3654 and ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow and spleens of TP-3654-treated mice whereas ruxolitinib treatment did not significantly alter fibrosis. Combined TP-3654 and ruxolitinib treatment almost completely eliminated fibrosis in the bone marrow and spleens of homozygous JAK2V617F mice.Additionally, we have tested the in vivo efficacy of TP-3654 and TP-3654/ruxolitinib combination using bone marrow transplantation mouse model of MPL mutant (MPLW515L). We observed similar decrease in white blood cell and neutrophil counts in MPL mutant mice upon treatment of TP-3654 alone or in combination with ruxolitinib. We also observed significant decrease in splenomegaly in MPL mutant mice treated with TP-3654 alone and in combination with ruxolitinib. Combined treatment of TP-3654 plus ruxolitinib almost completely ablated the fibrosis in the bone marrow and spleens of MPL mutant mice.To gain insights into the mechanism of inhibition of myelofibrosis by TP-3654 or TP-3654/ ruxolitinib drug combination, we performed RNA-sequencing on purified LSK (Lin-Sca-1+c-kit+) cells from these drug-treated homozygous JAK2V617F mice. Analysis of RNA-sequencing data revealed that the genes related to HSC maintenance and TNF signaling pathways were significantly down-regulated by TP-3654 alone or TP-3654/ruxolitinib combination compared with vehicle. Overall, our results suggest that TP-3654 alone or in combination with ruxolitinib may have therapeutic potential against MF and support the clinical investigation of TP-3654 and ruxolitinib combination in patients with MF. DisclosuresWhatcott:Tolero Pharmaceuticals, Inc: Employment. Warner:Tolero Pharmaceuticals: Employment.

The NF-KB Inhibitor DMAPT in Combination with Ruxolitinib Displays Efficacy in Jak2V617F Knock-in Mouse Model of Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are a group of hematologic malignancies characterized by excessive proliferation of myeloid/erythroid lineage cells. The JAK2V617F mutation has been found in a majority of patients with MPN including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF and PV. Although ruxolitinib treatment provides some benefits, it does not produce disease remission or reverse bone marrow (BM) fibrosis in patients with MPN/MF. This underscores the need to identify new therapeutic targets and develop novel therapies for treatment of MPN.In order to identify new therapeutic targets in MPN, we performed microarray gene expression analysis on sorted hematopoietic stem cells (HSC) from wild type and JAK2V617F mice. We found significant enrichment of the NF-κB signaling pathway in JAK2V617F HSC compared with wild type HSC. We also observed increased phosphorylation/activation of NF-κB (p65 RelA) in the BM of JAK2V617F mice. Hyper activation of NF-κB also has been observed in patients with MPN. Lentiviral shRNA-mediated knockdown of RelA significantly reduced the proliferation of murine Ba/F3-EpoR cells expressing JAK2V617F. RelA knockdown also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 leukemia cells. Furthermore, forced expression of a dominant negative IκBα mutant markedly reduced proliferation in Ba/F3-EpoR-JAK2V617F cells. These data suggest a role for the NF-κB signaling pathway in MPN pathogenesis.In this study, we investigated the efficacy of the NF-κB inhibitor, dimethylaminoparthenolide (DMAPT), in hematopoietic cells expressing JAK2V617F and in JAK2V617F knock-in mouse model of MPN. DMAPT is a water soluble, orally bioavailable NF-κB inhibitor that has shown promise against several solid tumors in pre-clinical models. Treatment of DMAPT (1-2.5 uM) significantly reduced the proliferation of Ba/F3-EpoR-JAK2V617Fcells. DMAPT (2.5-7.5 uM) treatment also significantly inhibited the human JAK2V617F-positive HEL, SET-2 and UKE-1 leukemia cells. Furthermore, DMAPT treatment significantly inhibited the hematopoietic progenitor colony outgrowth in JAK2V617F mice BM and MPN patient peripheral blood CD34+ cells.We previously generated conditional Jak2V617F knock-in mice. Whereas expression of heterozygous Jak2V617F induces a PV-like disease, homozygous Jak2V617F expression promotes rapid progression to myelofibrosis. We have utilized the homozygous Jak2V617F mice to test the in vivo efficacy of DMAPT alone and in combination with ruxolitinib against MPN. We observed that combined treatment of DMAPT and ruxolitinib significantly reduced the WBC and neutrophil counts and spleen size in Jak2V617F mice compared with vehicle or single drug treatment. Combined treatment of DMAPT and ruxolitinib also significantly reduced hematopoietic stem/progenitors and myeloid precursors in the BM and spleens of Jak2V617F mice. Furthermore, combined treatment of DMAPT and ruxolitinib significantly decreased the Jak2V617F mutant-expressing myeloid precursors in the BM and spleens of transplanted mice. Histopathologic analysis revealed significant reduction of fibrosis in the bone marrow of Jak2V617F mice treated with the combination of DMAPT and ruxolitinib. Overall, our results suggest that simultaneous inhibition of JAK2 and NF-κB pathways might be useful for treatment of MPN/MF. DisclosuresCrooks:University of Arkansas for Medical Sciences: Patents & Royalties: Inventor on the DMAPT patents.

Differential Impact of Interferon Alpha on JAK2V617F and Calr Mutated Hematopoietic Stem and Progenitor Cells in Classical MPN

Introduction:Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are acquired clonal disorders of hematopoietic stem cells (HSC) leading to the hyperplasia of one or several myeloid lineages. They are due to three main recurrent mutations affecting the JAK/STAT signaling pathway: JAK2V617F and mutations in the calreticulin (CALR) and thrombopoietin receptor (MPL). Interferon alpha (IFNα) is the only drug that not only induces a hematological response in ET, PV and early MF, but also a significant molecular response on both JAK2V617F or CALR-mutated cells.Our broad aim was to understand the mechanism of action of IFNα. Previously, our group and others have shown that IFNα specifically targets JAK2V617F HSC in a chimeric JAK2V617F knock-in mouse model. In this study, we wanted to know how and how fast IFNα impacts the different mutated human hematopoietic compartments.Methods:A prospective study was performed with a cohort of 47 patients treated by IFNα for 3-5 years. The MPN disease distribution was 40% ET, 49% PV and 11% MF. This cohort included 33 JAK2V617F-mutated patients, 11 CALR-mutated patients (7 type 1/type 1-like and 4 type 2/type 2-like), 2 both JAK2V617F- and CALR-mutated patients and 1 MPLW515K-mutated patient. At 4-month intervals, the JAK2V617F or/and CALR mutation allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, the clonal architecture was also determined by studying the presence of the JAK2V617F or CALR mutations in colonies derived from the different hematopoietic stem and progenitor cell (HSPC) populations (CD90+CD34+CD38- HSC-enriched progenitors, CD90-CD34+CD38- immature progenitors and CD90- CD34+CD38+ committed progenitors).Results:After a median follow-up of 33 months, IFNα targets more efficiently and rapidly the HSPC particularly in HSC-enriched progenitors, than the mature blood cells in JAK2V617F patients (p<.05). Moreover, homozygous JAK2V617F clones responded more rapidly than heterozygous clones in all hematopoietic cell compartments showing that the intensity of JAK2V617F signaling is correlated with the efficacy of IFNα. This efficacy was slightly increased after a median follow-up of 51 months. In contrast, during a median follow-up of 33 months for CALR-mutated patients, IFNα targeted similarly the HSPC and the mature cells. Moreover, IFNα induced a slower response in targeting CALR-mutated HSPC than the JAK2V617F HSPC (p<.05) (see Figure). The role of associated mutations at diagnosis was also investigated in the IFNα-mediated HSPC molecular responses using a NGS targeted myeloid panel. In JAK2V617F-mutated patients, the number of associated mutations did not impact the HSPC molecular response. In contrast, in CALR-mutated patients, the only molecular responders were not associated with other mutations, although the lower number of cases should be expanded.Using Ba/F3-MPL cellular models and primary cells, we observed that JAK2V617F was more prone to sensitize to IFNα signaling (increased Phospho-STAT1 and IFN-stimulating genes (ISGs)) compared to controls or CALRdel52 mutated cells.Conclusion:Altogether, our results show that IFNα targets more efficiently the human JAK2V617F-HSPCthan the mature cells. Moreover, IFNα has a greater efficacy on JAK2V617F HSPC thanCALR-mutated HSPC. This former result was associated with a greater priming of the IFNα signaling by JAK2V617F than by CALRdel52. The molecular response was dependent not only on mutational status, but also on the presence of other associated mutations for the CALR-mutated HSPC. Patient data are currently incorporated into a mathematical model taking into account clonal architecture and associated mutations to develop an algorythm able to predict patient response. [Display omitted] DisclosuresKiladjian:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.

In Vivo Blockade of Beta-1 and Beta-2 Integrin Activity Inhibits Splenomegaly in JAK2-V617F Positive Myeloproliferative Disease

Introduction:Recently, we showed that in chronic myeloproliferative neoplasia (CMN), β1 and β2 integrins are overactivated on leukocytes in patients and in a JAK2-V617F knock-in mouse model (Gupta et al., Leukemia, 2017; Edelmann-Stephan et al., JCI, In-Press Preview) resulting in increased adhesion of leukocytes to vascular cell adhesion molecule 1 (VCAM-1) and to intercellular cell adhesion molecule 1 (ICAM-1). VCAM-1 and ICAM-1 are abundantly expressed on endothelial cells and in spleen tissue. In JAK2-V617F induced CMN, current therapeutic strategies for symptomatic splenomegaly as hydroxyurea, JAK inhibitors or allogeneic stem cell transplantation (SCT) are effective, however, often limited in duration of efficacy or in eligibility for SCT. Therefore, there is a need to deeper understand the pathophysiology of splenomegaly in CMN in order to develop novel therapeutic approaches. We sought to investigate the effects of in vivo blockade of β1 and β2 integrin overactivation on spleen size, cellular composition of spleen and peripheral blood counts.Material and Methods:11 weeks old Vav1-Cre x JAK2+/V617F mice were treated with a combination of 200µg neutralizing anti-α4β1-integrin (VLA4) antibody (BioXCell, USA) plus 200µg neutralizing anti-β2 integrin antibody (BD Pharmingen, USA) (N=6) and with isotype controls (each 200µg) (N=3), respectively. Animals were injected i.p. on day 1 and day 8. Small animal MRI was used to serially assess the development of spleen volume on days 1, 8, and 15. Abdominal MRI-images employing a slice thickness of 1-2 mm were obtained. The volume of spleens were computed according to radiology standards. Peripheral blood was collected via retro-orbital bleeding and blood counts were measured on days 1, 8, and 15. Thereafter, mice were sacrificed by cervical dislocation and spleens were removed for further analysis by FACS.Results and Discussion:The applied antibody injections were well tolerated and all animals survived the planned observation period. At the start of the experiments (day 1), splenomegaly was confirmed by MRI. As expected, the spleen volumes were similar in the cohorts of isotype and β1/β2 integrin antibody treated JAK2+/V617F mice: 550±76 mm3 (mean±SD) and 518±96 mm3, respectively (Fig. 1). At day 8, upon a single injection of β1/β2 integrin antibodies, a significant decrease in mean spleen volume from baseline 518±96 mm3 to 414±32 mm3 (p=0.0302) was observed (Fig. 1). The correspondent mean±SD for the isotype antibody treated animals was 594±63 mm3. Thus, on day 8, a 30.3% reduction in spleen volume was demonstrated upon β1/β2 integrin antibody treatment in comparison to isotype treatment (p=0.0006). On day 15, the mean spleen volumes of isotype-treated and β1/β2 integrin antibody-treated mice were 665±187 mm3 and 460±69 mm3 (p=0.0410). These results demonstrate that in JAK2-V617F induced disease, blocking of overactivated β1/β2 integrins reduces splenomegaly within a short period of time (8 days). Longer application schedules may build up even stronger and stable decrease in spleen size.In addition to MRI imaging, we also analyzed the composition of peripheral blood cells. At the start of the experiments on day 1, blood parameters showed the typical abnormalities of myeloproliferative disease in both cohorts. Mean counts in isotype treated animals were: WBC = 14,300/µL, hematocrit = 80.3 %, PLT = 1283/nL. Mean counts in β1/β2 integrin antibody treated animals were: WBC = 13,850/µL, hematocrit = 76.0 %, PLT = 1348/nL. This confirms that the JAK2-V617F mouse model used indeed resembles polycythemia vera. In general, the changes observed upon β1/β2 integrin antibody treatment in blood were subtle: the WBC slightly decreased from 13,850/µL to 12,520/µL on day 15, whereas RBC, HCT and PLT remained nearly unchanged. This indicates tolerability of β1/β2 integrin antibody treatment at the level of the hematopoietic system. In isotype treated mice, peripheral blood counts also stayed stable. Data obtained by FACS analysis of peripheral blood and of spleen tissue (composition in granulocytes, B-cells and T-cells) will be presented.Overall, our results indicate a novel role of JAK2-V617F induced overactivation of β1/β2 integrins in development of splenomegaly. Thus, modulation of integrin activity may have therapeutic potential to reduce the spleen volume in CMN patients insufficiently responding to standard treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)

Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro.We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. DisclosuresBrümmendorf:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

Abstract 1874: The PIM kinase inhibitor TP-3654 demonstrates efficacy in a murine model of myelofibrosis

Abstract Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN). The JAK2V617F mutation has been found in ~50% patients with MF. Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF. However, Ruxolitinib treatment has failed to induce disease remission or cure fibrosis. Therefore, there is a need to develop novel targeted therapies for MF. We found that PIM1 expression is significantly increased in mouse and human MPN/MF hematopoietic progenitor cells. Moreover, PIM1 knockdown markedly inhibited proliferation in JAK2V617F-expressing cells but not in wild-type JAK2-expressing cells. In this study, we investigated the efficacy of the second-generation pan-PIM kinase inhibitor, TP-3654, in hematopoietic cells expressing JAK2V617F and in a murine model of MF. We found that TP-3654 (0.5-1.0 μM) treatment significantly reduced proliferation of murine Ba/F3-EpoR cells expressing JAK2V617F or human JAK2V617F-positive HEL and UKE-1 cells. TP-3654 Treatment also resulted in marked apoptosis in Ba/F3-EpoR-JAK2V617F, HEL and UKE-1 cells but not in wild-type JAK2 expressing Ba/F3-EpoR cells. Additionally, TP-3654 and Ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Furthermore, TP-3654 significantly inhibited MPN/MF CD34+ hematopoietic progenitor colony growth. We previously reported the generation of JAK2V617F knock-in mice. Whereas heterozygous JAK2V617F knock-in mice exhibit a polycythemia vera phenotype, mice expressing homozygous JAK2V617F rapidly develop high-grade MF. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of TP-3654 alone or in combination with Ruxolitinib against MF. Treatment of TP-3654 alone significantly reduced the increase in white blood cell (WBC) and neutrophil counts as well as spleen size in mice expressing homozygous JAK2V617F compared with vehicle treatment. Combined treatment of TP-3654 and Ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow and spleens of TP-3654-treated mice whereas Ruxolitinib treatment did not significantly reduce fibrosis. Combined TP-3654 and Ruxolitinib treatment almost completely eliminated fibrosis in the bone marrow and spleens of homozygous JAK2V617F mice. TP-3654 treatment was well tolerated and did not cause any significant toxicity in wild type mice. RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells from these drug-treated mice revealed that the genes related to TNFα and WNT signaling pathways were significantly down-regulated by treatment of TP-3654 alone or TP-3654 and Ruxolitinib combination compared with vehicle. Overall, our results suggest that TP-3654 alone or in combination with Ruxolitinib may have therapeutic potential for treatment of MF. Citation Format: Avik Dutta, Dipmoy Nath, Yue Yang, Golam Mohi. The PIM kinase inhibitor TP-3654 demonstrates efficacy in a murine model of myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1874.

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.

Hmga2 promotes the development of myelofibrosis in Jak2V617F knockin mice by enhancing TGF-β1 and Cxcl12 pathways

AbstractMyelofibrosis (MF) is a devastating blood disorder. The JAK2V617F mutation has been detected in ∼50% cases of MF. Elevated expression of high-mobility group AT hook 2 (HMGA2) has also been frequently observed in patients with MF. Interestingly, upregulation of HMGA2 expression has been found in association with the JAK2V617F mutation in significant cases of MF. However, the contribution of HMGA2 in the pathogenesis of MF remains elusive. To determine the effects of concurrent expression of HMGA2 and JAK2V617F mutation in hematopoiesis, we transduced bone marrow cells from Jak2V617F knockin mice with lentivirus expressing Hmga2 and performed bone marrow transplantation. Expression of Hmga2 enhanced megakaryopoiesis, increased extramedullary hematopoiesis, and accelerated the development of MF in mice expressing Jak2V617F. Mechanistically, the data show that expression of Hmga2 enhances the activation of transforming growth factor-β1 (TGF-β1) and Cxcl12 pathways in mice expressing Jak2V617F. In addition, expression of Hmga2 causes upregulation of Fzd2, Ifi27l2a, and TGF-β receptor 2. Forced expression of Cxcl12, Fzd2, or Ifi27l2a increases megakaryocytic differentiation and proliferation in the bone marrow of Jak2V617F mice, whereas TGF-β1 or Cxcl12 stimulation induces collagen deposition in the bone marrow mesenchymal stromal cells. Together, these findings demonstrate that expression of Hmga2 cooperates with Jak2V617F in the pathogenesis of MF.

Endothelial-to-Mesenchymal Transition in Bone Marrow and Spleen of Primary Myelofibrosis

Primary myelofibrosis is characterized by the development of fibrosis in the bone marrow that contributes to ineffective hematopoiesis. Bone marrow fibrosis is the result of a complex and not yet fully understood interaction among megakaryocytes, myeloid cells, fibroblasts, and endothelial cells. Here, we report that >30% of the endothelial cells in the small vessels of the bone marrow and spleen of patients with primary myelofibrosis have a mesenchymal phenotype, which is suggestive of the process known as endothelial-to-mesenchymal transition (EndMT). EndMT can be reproduced invitro by incubation of cultured endothelial progenitor cells or spleen-derived endothelial cells with inflammatory cytokines. Megakaryocytes appear to be implicated in this process, because EndMT mainly occurs in the microvessels close to these cells, and because megakaryocyte-derived supernatant fluid can reproduce the EndMT switch invitro. Furthermore, EndMT is an early event in a JAK2-V617F knock-in mouse model of primary myelofibrosis. Overall, these data show for the first time that microvascular endothelial cells in the bone marrow and spleen of patients with primary myelofibrosis show functional and morphologic changes that are associated to the mesenchymal phenotype.

Inhibitors of the PI3K/mTOR pathway prevent STAT5 phosphorylation in JAK2V617F mutated cells through PP2A/CIP2A axis.

Inhibition of the constitutively activated JAK/STAT pathway in JAK2V617F mutated cells by the JAK1/JAK2 inhibitor ruxolitinib resulted in clinical benefits in patients with myeloproliferative neoplasms. However, evidence of disease-modifying effects remains scanty; furthermore, some patients do not respond adequately to ruxolitinib, or have transient responses, thus novel treatment strategies are needed. Here we demonstrate that ruxolitinib causes incomplete inhibition of STAT5 in JAK2V617F mutated cells due to persistence of phosphorylated serine residues of STAT5b, that conversely are targeted by PI3K and mTORC1 inhibitors. We found that PI3K/mTOR-dependent phosphorylation of STAT5b serine residues involves Protein Phosphatase 2A and its repressor CIP2A. The levels of CIP2A were found increased in cells harboring the JAK2V617F mutation, and we provide evidence of a correlation between clinical responses and the extent of CIP2A downregulation in myelofibrosis patients receiving the mTOR inhibitor RAD001 in a phase II clinical trial. To achieve maximal inhibition of STAT5 phosphorylation, we combined ruxolitinib with BKM120, a PI3K inhibitor, and RAD001, an mTOR inhibitor, obtaining improved efficacy in JAK2V617F mutated cell lines, primary patients’ cells, and JAK2V617F knock-in mice. These findings contribute to understanding the effectiveness of PI3K/mTOR inhibitors in MPN and argue for the rationale to develop combination clinical trials.

A Phenotypic Screen for Small-Molecule Inhibitors of Constitutively Active Mutant Thrombopoietin Receptor Implicated in Myeloproliferative Neoplasms.

Rather than a Janus Kinase 2 inhibitor (ruxolitinib), a specific thrombopoietin receptor (TpoR) inhibitor would be more specific for the treatment of myeloproliferative neoplasms due to TpoR mutations. A cell-based phenotypic approach to identify specific TpoR inhibitors was implemented and a library of 505,483 small molecules was screened for inhibitory effects on cells transformed by TpoR mutants. Among the identified hits are two analogs of 3-(4-piperidinyl) indole. The analogs showed about five-fold preferential inhibition of cell viability towards Ba/F3 cells expressing the TpoR W515L mutation compared to the parental cells. There was no significant difference in inhibition of cell viability between the TpoR wild type and the TpoR W515L mutant cells. Preferential inhibition of viability was observed in Ba/F3 cells expressing erythropoietin receptor (EpoR) when stimulated with Epo compared to stimulation with interleukin-3 (IL3). The indole analog inhibited ex vivo colony formations of primary bone marrow cells from heterozygous JAK2 V617F knock-in mice. Drug combination treatment study was performed using ruxolitinib and the indole analog. Drug synergistic effects were observed when cells were stimulated to proliferate through both the IL3 and TpoR pathways. Our compound specifically targets monoamine receptors in the rhodopsin-like receptor family of G protein-coupled receptor. This screen has identified a monoamine receptor inhibitor that can inhibit viability of cells with active TpoR or EpoR signalings. Drug synergism with ruxolitib is demonstrated.

Expression of HMGA2 Cooperates with Jak2V617F in the Development of Myelofibrosis

High Mobility Group AT Hook 2 (HMGA2) is a non-histone chromatin protein that regulates gene transcription and controls cell proliferation, survival and self-renewal of stem cells. HMGA2 is expressed at a low level in normal adult hematopoietic progenitors but is highly expressed in hematopoietic progenitors of patients with Myelofibrosis (MF). However, the contribution of HMGA2 to the pathogenesis of MF remains unknown.MF is the deadliest form of myeloprolifearative neoplasm (MPN) characterized by deposition of fibrous tissues in the bone marrow, increased megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. Median survival of patients with MF is less than 6 years. The JAK2V617F mutation has been found in 50-60% patients with MF. However, it is not clear whether JAK2V617F mutation alone is sufficient to cause MF. Interestingly, up-regulation of HMGA2 expression has been found in association with the JAK2V617F mutation in a significant percentage of patients with MF. To understand the role of JAK2V617F mutation in the pathogenesis of MPN, we previously generated a conditional Jak2V617F knock-in mouse. We observed that expression of heterozygous Jak2V617F in mouse hematopoietic compartments is sufficient to induce a polycythemia vera (PV)-like MPN. Recently, we have shown that deletion of EZH2 promotes the development of MF in Jak2V617F knock-in mice and EZH2 deletion increases the expression of HMGA2 in hematopoietic progenitors of EZH2-deleted Jak2V617F mice. To directly assess the effects of concomitant expression of HMGA2 and heterozygous Jak2V617F in mice hematopoietic compartments, we expressed control vector or HMGA2 in wild type and heterozygous Jak2V617F knock-in mice BM by lentiviral transduction and performed bone marrow transplantation into lethally irradiated C57BL/6 recipient mice.Whereas recipients of vector-transduced Jak2V617F knock-in BM cells exhibited a PV-like MPN characterized by increased red blood cells (RBC), hemoglobin, hematocrit and platelets in their peripheral blood, recipients of HMGA2-transduced Jak2V617F knock-in BM showed reduced hemoglobin and hematocrit parameters compared with recipients of vector-expressing Jak2V617F BM cells. Interestingly, peripheral blood neutrophil and platelet counts were further increased in transplanted animals receiving HMGA2-transduced Jak2V617F BM cells. Expression of HMGA2 also resulted in significantly larger spleen size in the transplanted animals receiving HMGA2-expressing Jak2V617F BM cells. Flow cytometric analysis showed significant increase in megakaryocytic precursors (CD41+) but decrease in erythroid precursors (CD71+/Ter119+) in the BM and spleens of transplanted animals receiving HMGA2-expressing Jak2V617F BM compared with control vector-expressing Jak2V617F BM. Furthermore, the frequency of hematopoietic stem/progenitor cells (LSK; Lin-Sca-1+c-kit+) was significantly increased in recipients of HMGA2-transduced Jak2V617F knock-in BM compared with control vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM. Histopathologic analysis revealed extensive fibrosis in the BM and spleens from recipients of HMGA2-expressing Jak2V617F mice at 32 weeks after transplantation while BM and spleens from recipients of vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM showed very little or no fibrosis at this age. Together, these data suggest that expression of HMGA2 promotes megakaryopoiesis and accelerates the development of MF in mice expressing Jak2V617F.To gain insights into the mechanisms by which expression of HMGA2 accelerates the development of MF in Jak2V617F mice, we performed RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells. Gene set enrichment and pathway analyses revealed that the genes related to chemokine, TGF-β, MAP Kinase, PI3 kinase-Akt, mTOR and WNT signaling pathways were up-regulated in HMGA2-expressing Jak2V617F mice LSK compared with vector-expressing Jak2V617F LSK cells. We also found that HMGA2 directly binds to the promoter regions of some of these target genes and regulate their expression. Further studies will validate the targets of HMGA2 and determine their contribution in MF mediated by Jak2V617F. In conclusion, our studies show that expression of HMGA2 cooperates with Jak2V617F in the development of MF. DisclosuresNo relevant conflicts of interest to declare.

Thrombosis in Myeloproliferative Neoplasms Is Linked to Increased Neutrophil Extracellular Trap (NET) Formation

▪Background: Philadelphia-negative myeloproliferative neoplasms (MPNs) are a group of clonal stem-cell disorders that are associated with an elevated risk for thrombosis. The reason for this association is incompletely understood. While elevated white blood cell (WBC) count is a risk factor for thrombosis in some models, the pathogenic pathways linking WBC abnormalities to thrombosis in MPNs are not clear.Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA, histones and serine proteases that contain and kill pathogens. There is a growing body of evidence linking NETs to thrombosis in various disease states, including cancer (Demers et al, PNAS 2012). We explored the contribution of NETs to the pro-thrombotic phenotype of MPNs.Methods: NET formation was assessed in neutrophils isolated from patients with MPNs and age-matched controls by using previously published immunofluorescence (IF) methods. c-KIT enriched marrow cells from Vav-Cre/Jak2V617F Knock-In (KI) mice were grafted into lethally irradiated wild-type (WT) recipients leading to constitutive heterozygous expression of the mutation in hematopoietic cells. Inferior vena cava (IVC) stenosis model was used to assess thrombotic tendency in mice. Mice were treated with ruxolitinib by oral gavage at a dose of 90 mg/kg BID for 3 days prior to thrombosis and NETosis experiments. NET formation was stimulated by ionomycin (4 µM).Results: Neutrophils from patients with MPNs (n=14) demonstrated significantly higher NET formation as compared to those from age-matched controls (n=10; p=0.003). This was true across specific MPN diagnoses and driver mutations (JAK2, CALR, MPL). Ex-vivo treatment of neutrophils with ruxolitinib (at 300nM for 2.5 hours) resulted in abrogation of NETosis as assessed by IF studies and was comparable to the effect achieved with the addition of compounds known to inhibit NET formation such as specific PAD4 and NADPH inhibitors; no difference was observed in Annexin V-mediated apoptosis between ruxolitinib treated and untreated neutrophils suggesting that ruxolitinib effect was not confounded by alternative apoptosis pathways.We next evaluated NETosis and its effect on thrombosis in MPN mouse models driven by Jak2 V617F. Neutrophils isolated from Vav-Cre/Jak2V617F demonstrated increased NET production ex-vivo as compared to WT littermate controls. Vav-Cre/Jak2V617F KI mice were grafted into lethally irradiated WT and assessed for their prothrombotic phenotype. VavCre/Jak2V617F KI demonstrated significantly higher thrombus formation at 2 hours using the IVC stenosis model (45% versus 0% for Jak2V617F KI (n=11) and Jak2WT (n=8), respectively; p=0.04 by Fisher's exact test). This was associated with significantly higher levels of free dsDNA in the plasma of KI stenosis mice as compared to WT stenosis controls (p=0.03 by Mann-Whitney). Furthermore, IHC and IF studies of lung tissue demonstrated that Vav-Cre/Jak2V617F KI have widespread thrombosis as compared to controls and IF studies demonstrated higher content of citrullinated histone H3 in the lung suggesting increased NETosis at that site. Treatment of Vav-Cre/Jak2V617F KI mice with ruxolitinib significantly abrogated thrombus formation (45% versus 0% for untreated (n=11) and treated (n=8) mice, respectively; p=0.04 by Fisher's exact test).We noted that expression of PAD4, an essential driver of NETosis, is elevated in MPN patients (Rampal Blood 2014). We found that PAD4 content was higher in protein lysates of neutrophils isolated from MPN patients as compared to controls. Moreover, abnormal nuclear localization was observed in patients. These observations may support a role for PAD4 in the excess NETosis observed in MPNs.Conclusion: We found that neutrophils from MPN patients and Jak2 V617F KI mice are sensitized to NET formation that may be mediated by increased PAD4 expression and nuclear localization. In mice, the increased ex-vivo NET formation is associated with an increased thrombosis rate and an abundance of NET related products in thrombus and tissue. Treatment with ruxolitinib abrogates NET formation in humans and significantly reduces thrombus formation in MPN mice models.These findings provide a pathogenic link between the genetic drivers of MPNs and thrombosis in these diseases. Pharmacologic inhibition of JAK-STAT pathway to reduce NETosis and thrombosis should be further investigated. DisclosuresDeAngelo:Novartis: Consultancy; Ariad: Consultancy; Celgene: Consultancy; Baxter: Consultancy; Amgen: Consultancy; Incyte: Consultancy; Pfizer: Consultancy.