Models Of Myeloproliferative Neoplasms Research Articles

Myeloproliferative neoplasms as a model of a stem cell disease

Myeloproliferative neoplasms (MPN) are clonal disorders of the hematopoietic stem cell. Somatic gain of function mutations in the genes for JAK2, CALR or MPL can be found in about 90% of patients with MPN and are thought to be the disease driving mutations. In addition, accumulation of mutations in genes encoding epigenetic regulators, including TET2, ASXL1, DNMT3A and EZH2, transcription factors and signaling components modify the course of the disease and can contribute to disease initiation and/or progression. We have studied the effects of the JAK2-V617F mutation on the hematopoietic stem cell (HSC) compartment in mouse models of MPN and using limiting-dilution and single-cell transplantations we could show that that MPN disease can be initiated clonally from a purified single HSC carrying JAK2-V617F as the sole genetic alteration. Expression of JAK2-V617F in HSCs promoted cell division and expanded long-term repopulating capacity to a compartment with intermediate expression levels of lineage markers. Mice double mutant for JAK2-V617F and loss of the epigenetic regulator gene Ezh2 in hematopoieisis showed acceleration of disease and a more severe MPN phenotype with early progression to myelofibrosis. RNA sequencing in Ezh2 deficient hematopoietic stem cells and megakaryocytic erythroid progenitors identified highly upregulated genes, including Lin28b, and Hmga2, and Chip-qPCR analysis of their promoters revealed decreased H3K27me3 deposition. Expression of JAK2-V617F together with homozygous loss of Ezh2 resulted in greatly increased MPN disease initiation in limiting dilution transplantation and added repopulating capacity to stem and progenitor compartments that normally do not show long-term engraftment. Our data support the proposed tumor suppressor function of EZH2 in patients with MPN and demonstrate synergistic effects with JAK2-V617F on the HSC compartment.

Lysyl oxidase is associated with increased thrombosis and platelet reactivity

AbstractLysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Loxtg/tg) were generated. Pf4-Loxtg/tg mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Loxtg/tg mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Loxtg/tg platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Loxtg/tg platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2β1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis.

Complete Inhibition of STAT5 Phosphorylation Is Achieved By Combination of JAK1/2 and PI3K/mTOR Inhibitors in in Vitro and In Vivo MPN Models

Autonomous activation of JAK/STAT pathway by JAK2 V617F (VF) and similar mutations in myeloproliferative neoplasms (MPN) is associated with enhanced signaling of other downstream pathways including the PI3K/mTOR. Targeting the PI3K pathway resulted in inhibition of JAK2 VF cells (J Cell Mol Med 2013;17:1385), and the Torc1 inhibitor Everolimus (RAD001) showed clinical benefits in a phase I/II trial (Blood 2011;118:2069). In this study we evaluated the effects of BKM120, a pan-PI3K specific inhibitor, alone and in combination with the JAK1/JAK2 inhibitor Ruxolitinib (Ruxo) and Everolimus or PP242 (Torc1/2 inhibitor), in different MPN models and correlated efficacy with the extent of STAT5 phosphorylation inhibition.In vitro, we used BaF3 and BaF3-EPOR cells expressing wild type (WT) or VF mutated JAK2, the human VF mutated HEL and SET2 cell lines, and primary CD34+ cells from MPN patients. The combination index (CI) according to Chou and Talalay was used to evaluate drug combination; a CI <1 indicates synergistic activity. For in vivo studies we used two mouse models: (1) SCID mice receiving iv BaF3-EPOR VF-luciferase (luc) cells (Mol Cancer Ther 2010;9:1945) were randomized on day 6 to different treatment groups based on baseline luminescence and animals were sacrificed when became moribund; (2) C57Bl6/J JAK2VF knock-in (KI) mice constitutively expressing JAK2VF protein in heterozygous status (Blood 2013; 122:1464) were treated for 15 days before analysis.BKM120 preferentially inhibited BaF3VF and BaF3-EpoR-VF cells compared to the WT counterpart (IC50: 364±200nM and 1100±207nM vs 5300±800nM and 3122±1000nM respectively; P<0.05) and was similarly effective against JAK2VF HEL and SET2 cells. Western blot analysis showed marked reduction of phospho (p)-mTOR and its target p-4EBP1 as well as downregulation of p-STAT5. BKM120 reduced colony formation from MF and PV CD34+ cells at concentrations 2- to 8-fold lower than controls (P<0.01). EEC colony growth was inhibited at very low nM concentrations (IC50: 9±4nM). In vivo, BKM120 alone at a dose of 60mpk increased survival of BaF3VF-luc injected mice from 21d (controls) to 28d (P<0.05). Then we evaluated triple combinations of BKM120 and Ruxo plus either Everolimus or PP242 and found strong synergism against BaF3-EpoR-VF cells (CI=0.27 and 0.52). Furthermore, triple combinations of low doses (30nM each) BKM120, Ruxo and Everolimus reduced colony formation from BM cells of JAK2VF KI mice preferentially as compared to WT mice (-93.7% vs -44.7%). In vivo, triple combination treatment of KI mice with low drug doses (30mpk BKM120 + 30mpk Ruxo + 1.5mpk Everolimus) resulted in impressive improvement of splenomegaly: median spleen reduction compared to controls was -44.5%, -36.2%, -8%, for 60mpk BKM120, 60mpk Ruxo, 60mpk Everolimus as single agents and -69.5%, for triple treatment. Similarly greater activity was seen concerning reduction of leukocytosis and reticulocyte count. Compatible with findings in vitro, the level of p-4eBP1 and p-STAT5 in the spleen were significantly reduced in mice receiving combined treatment versus single agents. We explored the mechanisms by which PI3K/mTOR inhibitors resulted in reduced p-STAT5. We found that Ruxo treatment reduced tyrosine (Tyr) phosphorylation of the STAT5a isoform while leaving serines (Ser) unaffected, while BKM120 and Everolimus specifically resulted in dephosphorylation of STAT5b serine residues, in particular Ser193 and Ser731; accordingly, combined treatment resulted in complete STAT5 inactivation by dephosphorylation of both Ser and Tyr residues. Interestingly, the mRNA and protein levels of Cip2a, Cancerous Inhibitor of Phosphatase 2a (PP2a, that is normally involved in dephosphorylation of p-STAT residues), were strongly reduced by PI3K pathway inhibitors both in cell lines and in samples from patients responsive to Everolimus treatment. PP2a inhibition by Calyculin A resulted in enhanced STAT5b Ser193 and Ser731 phosphorylation while slightly altered STAT5a phosphoresidues suggesting a role of PP2a and its repressor Cip2a in PI3K/STAT5-dependent activation of JAK/STAT signaling.Concurrent inhibition of the JAK2 and PI3K pathways by Ruxo plus BKM120 and Everolimus results in enhanced activity in preclinical models of MPN and underlines the importance of complete STAT5 inactivation in order to magnify treatment efficacy, thereby providing a rationale for clinical trials. DisclosuresVannucchi:Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees.

Mesenchymal Cell Associated Fibrosis in Experimental mplW515L Mouse Model of Myelofibrosis

Background: Bone marrow fibrosis caused by excessive collagen deposition represents a significant indicator of poor prognosis in myeloproliferative neoplasms (MPNs). Genetic driver mutations have been identified as JAK2, CALR and mpl, but the precise mechanism and cell type leading to fibrosis remains unanswered. TGFβ production by mutant hematopoietic cells is the primary factor responsible for fibrosis initiation. Although effective JAK2 inhibitor therapy has been adopted to manage disease symptoms and decrease abnormal hematopoietic cells, this pharmacological inhibitor does not reverse fibrosis. Our previous observations in patients with Large granular lymphocyte (LGL) leukemia indicate that mesenchymal-derived fibroblasts, distinguished by the capacity to undergo trilineage differentiation into osteoblasts, adipocytes and chondrocytes, are responsible for the fibrosis observed in these patients. In this study, we report the identification of a similar fibrosis-initiating mesenchymal cell population in a mouse model of MPN induced by transplantation of gfp-mplW515L retrovirus-expressing hematopoietic cells and indicate a mechanism to reverse the fibrotic phenotype.Methods: Primary mesenchymal stromal cells (MSCs) were isolated from mice with MPN caused by the transplantation of gfp-mplW515L -transduced bone marrow cells (MPN mice) and from control mice that received transplants with gfp-wild-type(wt)-mpl-expressing bone marrow. MSCs were harvested 17 days post-transplant when WBC counts exceeded at least >20,000 cells/μl. Fibrillar collagen I (detected with trichrome stain in bone marrow biopsies) and collagen III (detected with reticulin stain in bone marrow biopsies) were identified as the major proteins produced in the bone marrow and by cultured MSCs in MPN mice consistent with pathologic examination. The amount of collagen was quantified in MSCs grown under reduced oxygen in undifferentiating culture conditions. To determine pluripotency, cells were grown with differentiation-specific culture media and stained with Alizarin Red S (to detect osteoblasts) and Alcian blue (chondrocytes). Collagen production was determined by qRT-PCR and polychromatic immunofluorescence staining of three-dimensional cultures and TGFβ was measured by qRT-PCR and ELISA.Results: To isolate purified populations of MSCs, gfp-positive hematopoietic cells were removed and MSCs were maintained in long-term culture in the absence of malignant cells. MSCs were isolated in micro-cultures from gfp-wt-mpl (ie, control) and mplW515L transplanted mice. Using gfp-wt-mpl-derived MSCs, we first showed that the addition of TGFβ induced collagen I and III expression as expected from previous studies. Original undifferentiated MSCs derived from control and MPN mice grew continuously in undifferentiated culture conditions with a similar life expectancy, and both populations were capable of further differentiation into osteoblasts and chondrocytes. Collagen I (p<0.0001) and III (p=0.0008) mRNA and fiber formation were increased in the MSCs from mplW515L -transplanted mice compared to control. Without exogenous cytokines or HSC supernatant, the MSCs from mutant-mpl-mice maintained greater than 2-fold increase in collagen suggesting that intrinsic reprogramming occurred in the MSC population independent of tumor cells that are normally considered the source of TGFβ and other fibrosis-initiating cytokines. TGFβ mRNA (0.9878±0.129, control vs 1.023±0.319, MPN p=ns) and total TGFβ secreted protein detected by ELISA (165.0±22.7pg/ml, control vs 144.1±21.8pg/ml, MPN p=ns) was similar in control and MPN-derived MSCs indicating that excessive TGFβ production does not maintain the fibrotic phenotype in culture. We have shown previously that collagen from fibrotic MSCs in LGL leukemia is inhibited by culturing the cells with fibroblast growth factor-b (FGFb, FGF-2). Similar to LGL leukemia-dervied MSCs, the addition of FGFb stimulated proliferation and restored normal levels of collagen I and III in the MSCs from MPN mice.Conclusions: This study indicates that expanded MPN-derived MSCs can be re-programmed to produce normal amounts of collagen, which may reverse fibrosis. This study has implications for the development of MSC-based cell therapies in MPN. DisclosuresNo relevant conflicts of interest to declare.

Ruxolitinib Significantly Prolongs Survival in Both a Primary Mediastinal B-Cell Lymphoma (PMBL) and Hodgkin Lymphoma (HL) Xenograft NSG Mouse Model: Ruxolitinib May be a Potential Adjuvant Agent in the Treatment of PMBL and of HL

BACKGROUND: Primary Mediastinal large B-cell lymphoma (PMBL) and Hodgkin lymphoma (HL) are two of the most common malignancies among adolescents and young adults (AYA). PMBL and HL share similar molecular features by gene expression profiling and pathogenesis (Rosenwald et al., J Exp Med., 2003). HL represents only approximately 4-5% of all cancers in children younger than 15 years of age, but is the most common cancer in the 15-35 yrs AYA group. While the prognosis is excellent with AYA HL, there are significant late effects secondary to chemoradiotherapy and therefore new targeted agents are needed to avoid these morbid late effects in HL (Hochberg/Cairo et al., Br. J. Haem., 2009). Frequent gains of chromosome 9p exhibit higher Janus Kinase 2 (JAK2) transcript levels with increased JAK2 activity (Bentz et al., Genes Chromosomes Cancer, 2001), suggesting aberrant activity of JAK2 and Signal Transducer and Activator of Transcription (STAT) pathways, which may in part play an important role in the pathogenesis of HL and PMBL. Ruxolitinib is a potent and selective JAK1/JAK2 inhibitor against myeloproliferative neoplasms (MPNs) that consistently exhibits dysregulation of the JAK1/JAK2 pathway, including those MPNs with a JAK2V617F mutation. Ruxolitinb also inhibits JAK2/STAT5 signaling in vitro and in a murine model of MPN (Quintas-Cardama et al., Blood, 2010).OBJECTIVES: We hypothesize that ruxolitinib may potentially function as targeted therapeutic agent for both PMBL and HL and therefore, investigated the efficacy of ruxolitinib in PMBL and HL cells xenografted into NSG mice.METHODS: Cell proliferation and apoptosis analysis were assessed using MTS and Caspase-3/7 assay (Promega), respectively. The expression of protein was examined by immunoblotting and statistical significance was determined by Student t-test. Karpas-1106P PMBL cell line and L-428 HL cell line were stably transfected with a firefly luciferase expression plasmid (ffluc-zeo), kindly provided by Laurence Cooper MD, PhD. The six to eight weeks old Female NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were from Jackson laboratory. The ffluc-zeo NSG mice were irradiated (2.5 Gy) and then were subcutaneously injected with 1x106 ffluc-zeo Karpas-1106P or L-428 tumor cells. Tumor burden and tumor progression were monitored by bioluminescence imaging (BLI) using the Xenogen IVIS-200 (Caliper Life Sciences) for up to 60 days. Mice were orally gavaged with either vehicle or ruxolitinib (45.0mg/kg or 90.0mg/kg) (generously provided by Incyte Corporation, Wilmington, DE, USA) for 21 days. Survival rates were analyzed by the Kaplan-Meier method and differences evaluated by log-rank test using the Prism Version 6.0 software.RESULTS: We observed that ruxolitinib significantly decreased the phosphorylation of STAT3, -5 and -6 in Karpas-1106P PMBL and L-428 HL cells. The reduction in cell proliferation by 10uM ruxolitinib are 41% in Karpas-1106P (p<0.04) and 19% in L-428 (p<0.03), respectively. In addition, ruxolitinib significantly induced Caspase-3/7 activity (p<0.05) and increased cleavage of PARP and caspase-3 fragments (p<0.001). In PMBL and HL xenografted mice models, we observed a significant decrease in tumor luminescence intensity following ruxolitinib treated PMBL (45.0mg/kg and 90.0mg/kg, p<0.05) and HL xenografted NSG mice at day 21 and 28 (45.0mg/kg, p<0.05) compared to control mice (Figure 1A). Ruxolitinib (45.0mg/kg) treated xenograft NSG mice had a significantly prolonged survival in PMBL (p<0.0001) and HL (p=0.0001) compared to control mice (Figure 1B).CONCLUSIONS: Ruxolitinib significantly inhibited JAK2 enzymatic activity by the abrogation of the phosphorylation of STAT3, STAT5 and STAT6, and ruxolitinib showed significant anti-proliferative effects. Ruxolitinib (45mg/kg) significantly inhibited tumor progression and significantly prolonged survival in PMBL and HL xenografted NSG mice compared to control mice. Ruxolitinib may be a potential adjuvant agent in the treatment of PMBL and HL. Future studies will focus on whether the addition of ruxolitinib to chemoimmunotherapy improves the survival in a NSG PMBL and HL xenograft mouse models. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

HH-002 Effectively Suppresses Myeloproliferative Neoplasm Phenotypes in JAK2V617F Transgenic Mice

Ph-negative myeloproliferative neoplasms (MPNs) represent a group of conditions characterized by chronic increases in some or all of the blood cells (platelets, white blood cells, and red blood cells). A major molecular defect in MPNs is JAK2V617F, a gain-of-function mutant form of tyrosine kinase JAK2 found in the majority of MPN patients. In earlier studies, we generated JAK2V617F transgenic mice by using the vav promoter which drives gene expression in all hematopoietic cells. These mice develop MPN-like phenotypes in a transgene dose-dependent manner. The objective of this study is to use this animal MPN model to test the efficacy of HH-002, a derivative of natural plant alkaloid homoharringtonine.Treatment of JAK2V617F transgenic mice with a daily dose of 1 mg/kg HH-002 through subcutaneous injection reduced blood cell counts to the normal range or slightly below the normal level. HH-002 causes a preferential reduction of myeloid cells in JAK2V617F transgenic mice since percentages of lymphocytes (CD3e+ T cells and CD19+ B cells) were increased (despite somewhat decreases in absolute numbers) while the percentage of Gr-1+/CD11b+ granulocytes sharply declined. HH-002 also effectively reduced the spleen size of JAK2V617F transgenic mice and prevented development of myelofibrosis. With a JAK2V617F bone marrow transplant mouse model, HH-002 showed a similar effect. Although it did not increase the ratio of JAK2V617F-negative cells to JAK2V617F-positive, it stopped further expansion of JAK2V617F-containing malignant cells in JAK2V617F bone marrow recipient mice. In vitro experiments with primary hematopoietic cells demonstrated that HH-002 potently inhibited formation of erythroid and myeloid colonies with an IC50 value of 1-3 nM. However, it does not show a preferential inhibition of JAK2V617F-containing cells. This is not unexpected since homoharringtonine is known to inhibit protein synthesis. Taken together, HH-002 is a promising candidate for development of therapeutic drugs to treat MPNs and related diseases. Combination drug therapies with JAK2 inhibitors need to be explored. DisclosuresHallenbeck:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Employment, Equity Ownership. Rong:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Employment, Equity Ownership. Zhao:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Research Funding.

The PIM inhibitor AZD1208 synergizes with ruxolitinib to induce apoptosis of ruxolitinib sensitive and resistant JAK2-V617F-driven cells and inhibit colony formation of primary MPN cells.

Classical myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders that exhibit excess mature myeloid cells, bone marrow fibrosis, and risk of leukemic transformation. Aberrant JAK2 signaling plays an etiological role in MPN formation. Because neoplastic cells in patients are largely insensitive to current anti-JAK2 therapies, effective therapies remain needed. Members of the PIM family of serine/threonine kinases are induced by JAK/STAT signaling, regulate hematopoietic stem cell growth, protect hematopoietic cells from apoptosis, and exhibit hematopoietic cell transforming properties. We hypothesized that PIM kinases may offer a therapeutic target for MPNs. We treated JAK2-V617F-dependent MPN model cells as well as primary MPN patient cells with the PIM kinase inhibitors SGI-1776 and AZD1208 and the JAK2 inhibitor ruxolitinib. While MPN model cells were rather insensitive to PIM inhibitors, combination of PIM inhibitors with ruxolitinib led to a synergistic effect on MPN cell growth due to enhanced apoptosis. Importantly, PIM inhibitor mono-therapy inhibited, and AZD1208/ruxolitinib combination therapy synergistically suppressed, colony formation of primary MPN cells. Enhanced apoptosis by combination therapy was associated with activation of BAD, inhibition of downstream components of the mTOR pathway, including p70S6K and S6 protein, and activation of 4EBP1. Importantly, PIM inhibitors re-sensitized ruxolitinib-resistant MPN cells to ruxolitinib by inducing apoptosis. Finally, exogenous expression of PIM1 induced ruxolitinib resistance in MPN model cells. These data indicate that PIMs may play a role in MPNs and that combining PIM and JAK2 kinase inhibitors may offer a more efficacious therapeutic approach for MPNs over JAK2 inhibitor mono-therapy.

Abstract IA41: Role of JAK-STAT pathway activation in MPN pathogenesis and therapeutic response.

Abstract The discovery of JAK2/MPL mutations in the majority of patients with Myeloproliferative Neoplasms (MPN) led to the development of JAK kinase inhibitors. JAK kinase inhibitors, including ruxolitinib, improve patient constitutional symptoms and reduce splenomegaly, but do not significantly reduce mutant allele burden. Chronic exposure to JAK kinase inhibitors results in JAK inhibitor persistence in cell lines, murine models, and patient samples, which we have shown is due to JAK2-transactivation and persistent JAK-STAT signaling. We have used murine genetic studies show that MPN cells remain dependent on JAK2 expression for proliferation and survival. These data suggest that pharmacologic approaches that better inhibit JAK2 activity in MPN cells may demonstrate increased efficacy in vivo. All JAK inhibitors in clinical development are type I inhibitors that interact with and inhibit the active confirmation of the JAK2 kinase. We hypothesized that novel, type II JAK inhibitors that interact with and inhibit JAK2 in the inactive conformation might retain activity in JAK inhibitor persistent cells and show increased efficacy in murine MPN models. Signaling studies demonstrated that CHZ868 more potently attenuated JAK-STAT signaling in JAK2/MPL-mutant cells, with suppression of JAK2 phosphorylation consistent with a type II mechanism of kinase inhibition. CHZ868 completely suppressed JAK-STAT signaling in type I JAK inhibitor-persistent cells, and prevented heterodimeric activation of JAK2 by JAK1 and TYK2. Most importantly, JAK2/MPL-mutant cells which were insensitive to type I JAK inhibitors remained highly sensitive to CHZ868, demonstrating that type I JAK inhibitor persistence does not confer resistance to type II inhibitors. CHZ868 showed significant activity in murine models of Jak2V617F-induced polycythemia vera, and MPLW515L-mutant MF, with normalization of blood counts, stem/progenitor expansion, spleen weights, and extramedullary hematopoiesis in vivo. Most importantly, CHZ868 resulted in significant reductions of mutant allele burden in these models. In addition, it has not been delineated whether the clinical benefits of JAK inhibitors are solely from target inhibition in MPN cells, or if JAK-STAT inhibition in malignant and non-malignant cells contributes to the therapeutic response. We show that the levels of circulating cytokines are elevated in MF and that cytokine production is reduced with JAK inhibitor treatment. Importantly, aberrant cytokine production in MF emanates from both malignant and non-malignant cells, and that JAK inhibition reduces cytokine production from both tumor and non-tumor populations. Our data suggest inhibition of JAK-STAT signaling in malignant and non-malignant cells is required to improve symptoms, reduce disease severity, and to reduce disease progression. Moreover, optimizing therapies to potently inhibit JAK-STAT signaling in malignant and non-malignant cells are warranted to improve clinical efficacy and outcomes in MPN patients. Citation Format: Sara Meyer, Maria Kleppe, Neha Bhagwat, Priya Koppikar, Minsuk Kwak, Thomas Radimerski, Rong Fan, Ross L. Levine. Role of JAK-STAT pathway activation in MPN pathogenesis and therapeutic response. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA41.

JAK2 inhibition has different therapeutic effects according to myeloproliferative neoplasm development in mice.

JAK2 inhibition therapy is used to treat patients suffering from myeloproliferative neoplasms (MPN). Conflicting data on this therapy are reported possibly linked to the types of inhibitors or disease type. Therefore, we decided to compare in mice the effect of a JAK2 inhibitor, Fedratinib, in MPN models of increasing severity: polycythemia vera (PV), post-PV myelofibrosis (PPMF) and rapid post-essential thrombocythemia MF (PTMF). The models were generated through JAK2 activation by the JAK2V617F mutation or MPL constant stimulation. JAK2 inhibition induced a correction of splenomegaly, leucocytosis and microcytosis in all three MPN models. However, the effects on fibrosis, osteosclerosis, granulocytosis, erythropoiesis or platelet counts varied according to the disease severity stage. Strikingly, complete blockade of fibrosis and osteosclerosis was observed in the PPMF model, linked to correction of MK hyper/dysplasia, but not in the PTMF model, suggesting that MF development may also become JAK2-independent. Interestingly, we originally found a decreased in the JAK2V617F allele burden in progenitor cells from the spleen but not in other cell types. Overall, this study shows that JAK2 inhibition has different effects according to disease phenotypes and can (i) normalize platelet counts, (ii) prevent the development of marrow fibrosis/osteosclerosis at an early stage and (iii) reduce splenomegaly through blockage of stem cell mobilization in the spleen.

IL-33 signaling contributes to the pathogenesis of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are characterized by the clonal expansion of one or more myeloid cell lineage. In most cases, proliferation of the malignant clone is ascribed to defined genetic alterations. MPNs are also associated with aberrant expression and activity of multiple cytokines; however, the mechanisms by which these cytokines contribute to disease pathogenesis are poorly understood. Here, we reveal a non-redundant role for steady-state IL-33 in supporting dysregulated myelopoiesis in a murine model of MPN. Genetic ablation of the IL-33 signaling pathway was sufficient and necessary to restore normal hematopoiesis and abrogate MPN-like disease in animals lacking the inositol phosphatase SHIP. Stromal cell-derived IL-33 stimulated the secretion of cytokines and growth factors by myeloid and non-hematopoietic cells of the BM, resulting in myeloproliferation in SHIP-deficient animals. Additionally, in the transgenic JAK2V617F model, the onset of MPN was delayed in animals lacking IL-33 in radio-resistant cells. In human BM, we detected increased numbers of IL-33-expressing cells, specifically in biopsies from MPN patients. Exogenous IL-33 promoted cytokine production and colony formation by primary CD34+ MPN stem/progenitor cells from patients. Moreover, IL-33 improved the survival of JAK2V617F-positive cell lines. Together, these data indicate a central role for IL-33 signaling in the pathogenesis of MPNs.

Deletion of Stat3 in hematopoietic cells enhances thrombocytosis and shortens survival in a JAK2-V617F mouse model of MPN

AbstractThe acquired somatic JAK2-V617F mutation is present in >80% of patients with myeloproliferative neoplasms (MPNs). Stat3 plays a role in hematopoietic homeostasis and might influence the JAK2-V617F–driven MPN phenotype. We crossed our transgenic SclCre;V617F mice with a conditional Stat3 knockout strain and performed bone marrow transplantations into lethally irradiated recipient mice. The deletion of Stat3 increased the platelet numbers in SclCre;V617F;Stat3fl/fl mice compared with SclCre;V617F;Stat3fl/+ or SclCre;V617F;Stat3+/+ mice. Stat3 deletion also normalized JAK2-V617F–induced neutrophilia. Megakaryocyte progenitors were elevated, especially in the spleen, and a slight increase in myelofibrosis was noted. We observed increased mRNA expression levels of Stat1 and Stat1 target genes and augmented phosphorylation of Stat1 protein in bone marrow and spleen of JAK2-V617F mice after Stat3 deletion. The survival of Stat3-deficient mice expressing JAK2-V617F was reduced. Inflammatory bowel disease, previously associated with shortened survival of Stat3-deficient mice, was less prominent in the bone marrow transplantation setting, possibly by limiting deletion of Stat3 to hematopoietic tissues only. In conclusion, deletion of Stat3 in hematopoietic cells from JAK2-V617F mice did not ameliorate the course of MPN, but rather enhanced thrombocytosis and shortened the overall survival.

The Hen or the Egg: Inflammatory Aspects of Murine MPN Models.

It has been known for some time that solid tumors, especially gastrointestinal tumors, can arise on the basis of chronic inflammation. However, the role of inflammation in the genesis of hematological malignancies has not been extensively studied. Recent evidence clearly shows that changes in the bone marrow niche can suffice to induce myeloid diseases. Nonetheless, while it has been demonstrated that myeloproliferative neoplasms (MPN) are associated with a proinflammatory state, it is not clear whether inflammatory processes contribute to the induction or maintenance of MPN. More provocatively stated: which comes first, the hen or the egg, inflammation or MPN? In other words, can chronic inflammation itself trigger an MPN? In this review, we will describe the evidence supporting a role for inflammation in initiating and promoting MPN development. Furthermore, we will compare and contrast the data obtained in gastrointestinal tumors with observations in MPN patients and models, pointing out the opportunities provided by novel murine MPN models to address fundamental questions regarding the role of inflammatory stimuli in the molecular pathogenesis of MPN.

Type II Inhibition of JAK2 with NVP-CHZ868 Reverses Type I JAK Inhibitor Persistence and Demonstrates Increased Efficacy in MPN Models

The identification of JAK2 mutations in patients with myeloproliferative neoplasms (MPN) led to the clinical development of JAK2 inhibitors, and the JAK1/2 inhibitor ruxolitinib has been approved for the treatment of myelofibrosis (MF). Although clinically tested JAK inhibitors improve MPN-associated splenomegaly and systemic symptoms, they do not significantly reduce the MPN clone in most MPN patients.We previously demonstrated that MPN cells can acquire persistence to ruxolitinib and other type I JAK inhibitors which bind the active conformation of JAK2, and that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. We have now extended our studies to other type I JAK inhibitors in clinical development, including CYT387, BMS911543 and SAR302503. In each case we see the same mechanism of persistence as observed with ruxolitinib, with transactivation of JAK2 by other JAK kinases. Most importantly, we found that MPN cells which were persistent to one JAK inhibitor were insensitive to the other JAK inhibitors, suggesting that the mechanisms which limit overall efficacy of ruxolitinib will limit the efficacy of other JAK inhibitors in clinical development.All JAK inhibitors in clinical development are type I inhibitors that interact with and inhibit the active confirmation of the JAK2 kinase. We hypothesized that novel, type II JAK inhibitors that interact with and inhibit JAK2 in the inactive conformation might retain activity in JAK inhibitor persistent cells and show increased efficacy in murine MPN models. We therefore characterized the efficacy of NVP-CHZ868, a novel type II JAK inhibitor, in MPN cells and in murine MPN models. CHZ868 potently inhibited proliferation of cells expressing the JAK2V617F mutation or the TEL-JAK2 fusion. We found that JAK2/MPL-mutant cell lines were universally sensitive to NVP-CHZ868. CHZ868 treatment of JAK2-mutant SET2 cells induced a higher degree of apoptosis compared to ruxolitinib. Signaling studies demonstrated that CHZ868 more potently attenuated JAK-STAT signaling in JAK2/MPL-mutant cells, with suppression of JAK2 phosphorylation consistent with a type II mechanism of kinase inhibition.We next investigated the ability of CHZ868 to inhibit the proliferation and signaling of MPN cells that had acquired persistence to type I JAK inhibitors. Type II inhibition with CHZ868 completely suppressed JAK-STAT signaling in type I JAK inhibitor-persistent cells, and prevented heterodimeric activation of JAK2 by JAK1 and TYK2. Most importantly, JAK2/MPL-mutant cells which were insensitive to type I JAK inhibitors remained highly sensitive to CHZ868, demonstrating that type I JAK inhibitor persistence does not confer resistance to type II inhibitors.We next evaluated the efficacy of CHZ868 in murine models of JAK2/MPL-mutant MPN. CHZ868 showed significant activity in conditional knock-in and bone marrow transplant (BMT) models of Jak2V617F-induced polycythemia vera, with normalization of hematocrit, reversal of stem/progenitor expansion, normalization of splenomegaly/splenic architecture, and reversal of bone marrow fibrosis. CHZ868 demonstrated similar activity in the MPLW515L BMT model of MF, with normalization of blood counts, stem/progenitor expansion, spleen weights, and extramedullary hematopoiesis in vivo. Most importantly, CHZ868 resulted in significant reductions of mutant allele burden (mean allele burden reduction 49%) in the Jak2V617F model. We observed analogous reductions in allele burden in the Jak2V617F and MPLW515L BMT models, consistent with disease modifying activity.Taken together, our data demonstrate that a spectrum of type I JAK inhibitors induce JAK inhibitor persistence, by a similar mechanism of JAK2 transactivation as observed with ruxolitinib. By contrast, type II JAK inhibition with CHZ868 remains highly active in JAK inhibitor persistent cells, and shows increased activity in murine MPN models. These data demonstrate that novel JAK inhibitors can increase target inhibition and therapeutic efficacy and should be pursued as an approach to improve outcomes for MPN patients. [Display omitted] [Display omitted] DisclosuresKoppikar:Amgen: Employment. Sellers:Novartis: Employment. Hofmann:Novartis: Employment. Baffert:Novartis: Employment. Gaul:Novartis: Employment. Radimerski:Novartis: Employment. Levine:Novartis: Consultancy, Grant support Other.

Limited Efficacy of BMS-911543 in a Murine Model of JAK2V617F Myeloproliferative Neoplasm

Activation of JAK2, frequently as a result of the JAK2V617F mutation, is a characteristic feature of the classical myeloproliferative neoplasms (MPN) polycythemia vera, essential thrombocythemia and myelofibrosis and thought to be responsible for the constitutional symptoms associated with these diseases. BMS-911543 is a JAK2 selective inhibitor that induces apoptosis in JAK2-dependent cell lines and inhibits the growth of CD34+ progenitor cells from patients with V617F+ MPN. To explore the clinical potential of this inhibitor, we tested BMS-911543 in a murine retroviral transduction – transplantation model of JAK2V617F MPN. Treatment was initiated at two dose levels (3 mg/kg and 10 mg/kg) when the hematocrit exceeded 70%. Following the first week, white blood cell counts were reduced to normal in the high dose group and were maintained well below the vehicle-treated mice throughout the study. However, BMS-911543 had no effect on red cell parameters. After 42 days of treatment, the proportion of JAK2V617F - positive cells in hematopoietic tissues was identical to controls or slightly increased compared to controls. Plasma concentrations of IL-6, IL-15, and TNFα were elevated in MPN mice and reduced in the high dose treatment group, while other cytokines were unchanged. Collectively, these results show that BMS-911543 has limited activity in this murine model of JAK2V617F – driven MPN and suggest that targeting JAK2 alone may be insufficient to achieve effective disease control. DisclosuresDeininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

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.

Mathematical Optimization of JAK Inhibitor Dose and Scheduling for MPN Patients

The identification of JAK-STAT pathway mutations in the majority of patients with the myeloproliferative neoplasms (MPN) polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF) led the to clinical development of JAK inhibitors, and the resultant approval of ruxolitinib for the treatment of PMF. However, despite this important therapeutic advance, there are significant limitations to JAK inhibitor therapy both with respect to efficacy and toxicity. First, although JAK inhibitors reduce splenomegaly, ameliorate symptoms, and improve long-term outcome, they do not achieve molecular or pathologic remission at currently utilized dosing strategies. Second, JAK2 has a role in hematopoiesis and other biological processes, and JAK inhibition leads to significant hematologic toxicities including anemia and thrombocytopenia. We recently used genetic and pharmacologic studies to demonstrate that JAK inhibitor persistent cells which survive JAK inhibitor therapy in vitro and in vivo remain JAK2 dependent, consistent with incomplete target inhibition. As such, we hypothesized that alternate dosing regimens which allow for intermittent, maximal target inhibition might increase efficacy and reduce toxicity. We therefore used experimental and modeling approaches to investigate the potential efficacy of alternate dosing regimens.We first explored the effects of chronic vs intermittent dosing in vitro by altering the treatment regimen in cell lines. To this end, we treated the JAK2 V617F mutant cell line, SET-2, and JAK2-wild-type (control) cell lines with ruxolitinib (1µM vs 0.5 µM) on a chronic or intermittent (alternating 1 week on and 1 week off the drug) basis. We then performed cell viability assays using flow cytometry to estimate the effect of the drug on the cell division and death rates of each cell population. Using this data, we developed a mathematical model to predict responses to varying dose therapy. Cell proliferation was described using an exponential growth model (pt2 = pt1e(birth rate-death rate)Dt, p=population size). Birth and death rates as a function of the drug concentration was fitted using a simple iterative least squares estimation from the in vitro collected data, where death(c) = 0.0046log(1.5014 + 30.4910c) and birth(c) = 0.0098 + 0.0051e-1.2946c. Treatment cycles were modeled by ton + toff for pulsitile versus chronic (toff = 0) regimens for time on and off drug. We also added a toxicity constraint based on preclinical testing and the mathematical model T(c) = (α/c) –β, where α = 539 and β=5.2, which will inform our in vivo studies. Inputting these rates into a mathematical model to predict optimal treatment schedule, our in silico analysis suggest that high dose pulse treatment of INCB18424 has the same efficacy as chronic dosing and is associated with reduced toxicity. We are currently testing our dosing and administration schedules using in vivo models of MPN, and we will present these data at the meeting. Preliminary studies suggest intermittent JAK inhibition shows similar efficacy as chronic JAK inhibition, with reduced toxicity, suggesting our in silico models inform the development of more optimal dosing regimens. We are now testing higher doses of JAK inhibitors in an intermittent administration regimen in order to maximize efficacy and mitigate hematologic and non-hematologic toxicity.In conclusion, our proof-of-principle studies show that intermittent treatment with JAK kinase inhibitors demonstrates equivalent efficacy in vitro and our in silico data suggests that we will see reduced toxicity with intermittent dosing in the mouse models. Our in vivo data will inform further clinical optimization of treatment regiments for patients with myeloproliferative neoplasms DisclosuresKoppikar:Amgen: Employment. Levine:Novartis: Consultancy, Grant support Other.

Pims: Potential Therapeutic Targets for Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders characterized by the abnormal production of various myeloid cells. Aberrant JAK2 signaling (e.g. induced by JAK2-V617F) plays an etiological role in MPN formation. While JAK2 inhibitors improve patient symptoms, they do not induce cell death as neoplastic cells appear to be rather insensitive to JAK2 inhibition and effectively rapidly become resistant to treatment. Therefore, the development of additional therapeutic approaches for MPNs is needed. Pim kinases are serine/threonine kinases that protect hematopoietic cells from apoptosis and also play a role in regulating hematopoietic stem cell growth. In mouse models, elevated Pim expression contributes to the development of lymphoma. Pims are constitutively active and thus regulated by protein expression, which is controlled by Pim gene expression and Pim protein stability. Pim1 gene expression is normally induced by JAK2/STAT5 signaling in response to extracellular growth factor stimulation, as Pim1 is a direct transcriptional target of STAT5. The deregulated JAK2 signaling in MPNs also induces Pim expression. STAT5 is required for MPN disease in mouse models, suggesting genes transcriptionally regulated by STAT5 are required for MPN disease formation. Together with the anti-apoptotic signaling and transforming properties of Pims, this suggests Pims may play a role in MPNs. We hypothesized that Pim kinases may offer a therapeutic target for MPNs and that Pim kinase inhibitors in combination with JAK inhibitors may cause neoplastic cytotoxicity, improving on current JAK2-inhibitor mono-therapy for MPNs. JAK2-V617F-dependentMPN model cells (HEL, SET2, Uke1, and BaF3/JAK2-V617F, including cells that are resistant to the JAK2 inhibitor ruxolitinib) as well as MPN patient cells, were treated with Pim kinase inhibitors, SGI-1776 and AZD1208, and the JAK2 inhibitor, ruxolitinib. The effects on cell growth, cell cycle, viability, and cell signaling were studied. High concentration SGI-1776 (10 μM) inhibited cell growth and viability of MPN model cells while lower doses (1 and 3 μM) had little effect on the growth and viability of these cells. Combination of 3 μM SGI-1776 with low dose ruxolitinib significantly enhanced growth inhibition and cell death of HEL and SET2 cells. Similar results were obtained with the much more effective and selective Pim inhibitor, AZD1208. We show that ruxolitinib inhibits Pim expression in MPN cells, and Pim expression is restored in ruxolitinib-resistant cells. Importantly, low dose SGI-1776 or AZD1208 (100 nM) re-sensitized ruxolitinib-resistant MPN cells to ruxolitinib treatment. Significantly, as single agents, both SGI-1776 and AZD1208 inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but not erythroid colonies of normal controls. The combination of AZD1208 and ruxolitinib exhibited enhanced inhibition of colony formation of primary cells from MPN patients compared to treatment with either drug alone. These data indicate that Pim kinase inhibitors in combination with a JAK2 inhibitor may offer a more efficacious therapeutic approach over JAK2 inhibitor mono-therapy for MPNs. DisclosuresNo relevant conflicts of interest to declare.

Improved targeting of JAK2 leads to increased therapeutic efficacy in myeloproliferative neoplasms

AbstractThe discovery of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) led to clinical development of Janus kinase (JAK) inhibitors for treatment of MPN. These inhibitors improve constitutional symptoms and splenomegaly but do not significantly reduce mutant allele burden in patients. We recently showed that chronic exposure to JAK inhibitors results in inhibitor persistence via JAK2 transactivation and persistent JAK–signal transducer and activator of transcription signaling. We performed genetic and pharmacologic studies to determine whether improved JAK2 inhibition would show increased efficacy in MPN models and primary samples. Jak2 deletion in vivo led to profound reduction in disease burden not seen with JAK inhibitors, and deletion of Jak2 following chronic ruxolitinib therapy markedly reduced mutant allele burden. This demonstrates that JAK2 remains an essential target in MPN cells that survive in the setting of chronic JAK inhibition. Combination therapy with the heat shock protein 90 (HSP90) inhibitor PU-H71 and ruxolitinib reduced total and phospho-JAK2 and achieved more potent inhibition of downstream signaling than ruxolitinib monotherapy. Combination treatment improved blood counts, spleen weights, and reduced bone marrow fibrosis compared with ruxolitinib alone. These data suggest alternate approaches that increase JAK2 targeting, including combination JAK/HSP90 inhibitor therapy, are warranted in the clinical setting.

JAK2V 617 F-positive endothelial cells contribute to clotting abnormalities in myeloproliferative neoplasms

The Janus kinase 2 (JAK2) V617F mutation is the primary pathogenic mutation in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombohemorrhagic incidents are the most common causes of morbidity and mortality in patients with MPNs, the events causing these clotting abnormalities remain unclear. To identify the cells responsible for the dysfunctional hemostasis, we used transgenic mice expressing JAK2V617F in specific lineages involved in thrombosis and hemostasis. When JAK2V617F was expressed in both hematopoietic and endothelial cells (ECs), the mice developed a significant MPN, characterized by thrombocytosis, neutrophilia, and splenomegaly. However, despite having significantly higher platelet counts than controls, these mice showed severely attenuated thrombosis following injury. Interestingly, platelet activation and aggregation in response to agonists was unaltered by JAK2V617F expression. Subsequent bone marrow transplants revealed the contribution of both endothelial and hematopoietic compartments to the attenuated thrombosis. Furthermore, we identified a potential mechanism for this phenotype through JAK2V617F-regulated inhibition of von Willebrand factor (VWF) function and/or secretion. JAK2V617F(+) mice display a condition similar to acquired von Willebrand syndrome, exhibiting significantly less high molecular weight VWF and reduced agglutination to ristocetin. These findings greatly advance our understanding of thrombohemorrhagic events in MPNs and highlight the critical role of ECs in the pathology of hematopoietic malignancies.