Target In Myeloproliferative Neoplasms Research Articles

Jak2V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms.

Current JAK inhibitors to treat myeloproliferative neoplasms are ineffective at eradicating mutant cells. We developed an endogenously expressed Jak2V617F dual-recombinase knock-in/knock-out model to investigate Jak2V617F oncogenic reversion in vivo. Jak2V617F deletion abrogates MPN features and depletes disease-sustaining MPN stem cells, suggesting improved Jak2V617F targeting offers the potential for greater therapeutic efficacy. See related commentary by Celik and Challen, p. 701. This article is featured in Selected Articles from This Issue, p. 695.

Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms

The role of hematopoietic Hedgehog signaling in myeloproliferative neoplasms (MPNs) remains incompletely understood despite data suggesting that Hedgehog (Hh) pathway inhibitors have therapeutic activity in patients. We aim to systematically interrogate the role of canonical vs. non-canonical Hh signaling in MPNs. We show that Gli1 protein levels in patient peripheral blood mononuclear cells (PBMCs) mark fibrotic progression and that, in murine MPN models, absence of hematopoietic Gli1, but not Gli2 or Smo, significantly reduces MPN phenotype and fibrosis, indicating that GLI1 in the MPN clone can be activated in a non-canonical fashion. Additionally, we establish that hematopoietic Gli1 has a significant effect on stromal cells, mediated through a druggable MIF-CD74 axis. These data highlight the complex interplay between alterations in the MPN clone and activation of stromal cells and indicate that Gli1 represents a promising therapeutic target in MPNs, particularly that Hh signaling is dispensable for normal hematopoiesis.

Galectin-1 Fuels Monocyte-Driven Hyperinflammation and Represents a Novel Therapeutic Target in Myeloproliferative Neoplasms

JDP and STO co-corresponding authors Previous studies by our group and others have demonstrated that monocytes play a key role in driving hyperinflammation in myeloproliferative neoplasms (MPNs), and that aberrant inflammatory cytokine signaling contributes to disease progression and poor prognosis in MPNs. To interrogate relationships between cellular sources and targets of inflammatory cytokines, we performed single cell RNA-seq (scRNA-seq) in PBMCs and progenitor cells from MPN patients and healthy controls. Monocytes from MPN patients displayed significant enrichment of inflammation-relevant genes as expected. Cell-cell communication networks inferred from expression of ligands and receptors predicted monocytes as a pivotal mediator of cell interactions. Further analysis revealed galectin signaling as one of the most robust input/output pathways for monocytes and significant enrichment of LGALS1 (galectin-1) expression in MPN monocytes. Galectins are a class of proteins that bind specifically to β-galactoside carbohydrates, such as N-linked or O-linked glycosylated proteins. Abnormal expression of galectins in various cancers has been found to mediate broad biological functions, including cell proliferation, apoptosis, adhesion, and inflammation. However, the mechanisms by which galectins contribute to MPN pathogenesis remain incompletely understood. To corroborate our sRNA-seq findings, we performed flow cytometry analysis which demonstrated elevated expression of galectin-1 in MPN patient CD14+ monocytes. Increased plasma levels of galectin-1 were also identified in MPN patients compared to healthy individuals. Higher galectin-1 protein levels in both Kit+ progenitor cells and CD11b+ myeloid cells from Jak2 V617F knock-in mice were also observed when compared to wild type mice. Additionally, galectin-1 expression was induced by MPL W515L and JAK2 V617F and inhibited by ruxolitinib in Ba/F3 cells. Thus, our results show enrichment of galectin-1 in both MPN patients and mouse models, with evidence of direct contribution from specific MPN driver mutations. Our scRNA-seq dataset analysis showed significant correlations of galectin-1 expression with inflammatory and oxidative phosphorylation (OXPHOS) genes in MPN patient monocytes. To investigate the effects of galectin-1 in monocyte activation, we incubated CD14+ monocytes from MPN patients with recombinant galectin-1 (rGal-1) and observed markedly stimulated secretion of inflammatory cytokines, such as IL-1α, IL-1β, IL-6, IL-8 and TNF-α. Furthermore, OTX008, a galectin-1 inhibitor, inhibited the transcription and secretion of inflammatory cytokines in MPN patient CD14+ monocytes and monocytic cell lines. Notably, both genetic and pharmacologic inhibition of galectin-1 reduced cellular ATP level and oxygen consumption rate, suggesting a metabolic reprogramming in MPNs. Although galectin-1 mediates broad biological functions, the molecular mechanisms by which galectin-1 regulates signaling pathways and transcription in MPN cells had not been delineated. Our scRNA-seq heterogeneity analysis suggested that genes in the PI3K-AKT-mTOR pathway were differentially expressed between galectin-1 high- and low-expressing monocytes. We therefore incubated both monocytic cell lines and monocytes from MPN patients with rGal-1 and observed stimulation of the PI3K-AKT-mTOR pathway, represented by increased levels of phosphorylated mTOR, AKT and S6. Consistently, genetic and pharmacologic inhibition of galectin-1 inhibited activation of the PI3K-AKT-mTOR pathway. In summary, our results demonstrate upregulation of galectin-1 in MPN monocytes, potentially due to activation of JAK2-driven signaling. We further demonstrate that galectin-1 fuels inflammation potentially via metabolic reprogramming of monocytes and activation of PI3K-AKT-mTOR signaling. Our data also suggest galectin-1 as a putative therapeutic target in MPNs.

Divergent Functions of ERK2 Substrate Binding Modalities in Myeloproliferative Neoplasm

Introduction: JAK2-V617F driven myeloproliferative neoplasms (MPNs) are intractable blood disorders marked by the elevated production of erythrocytes and myeloid cells in the bone marrow. While current JAK2 inhibitors offer some respite, they fail to eradicate the mutated cell population due to acquired treatment resistance. Treatment resistance arises from compensatory activation of the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) pathway; however, even combination therapy targeting both JAK2 and the MAPK signaling has led to drug resistance. Instead of attempting to inhibit kinase activity, we propose to attenuate ERK2 substrate interactions that drive cancer. We have discovered that the two substrate binding domains of ERK2, D and DBP, play opposing roles in MPN pathogenesis. The DBP domain attenuates MPN pathogenesis and promotes oncogene-induced-senescence (OIS) through physical association with the DEF motif of Egr1, while the D-domain promotes progression, through Polq induction. Methods: To study the divergent effects of ERK2 DBP and D domain on MPN progression, we introduced single point mutations in the substrate binding domains of ERK2 using CRISPR-Cas9. The expression of mutant alleles of ERK2 is conditionally activated by excision of the floxed allele using Vav-Cre. Similarly, we constructed a CRISPR knock-in mouse model, Egr1-Y253A, to examine the role of the Egr1 DEF motif in regulating OIS. To assess the response of individual malignant clones to Polq inhibitor (Polqi), we employed single-cell NGS and the Tapestri platform to detect clinically relevant mutations in 54 genes associated with myeloid neoplasms. For the inhibitor studies, we used human MPN cell lines and independent MPN patient cohorts. Results: Ectopic expression of JAK2 V617F in DBP-inactivated progenitors enhanced their clonogenic potential and abrogated OIS in vitro. This occurs because mutating the ERK2-DBP domain prevents interaction with the senescence-inducing transcription factor, Egr1. Egr1-Y253A mutant progenitors display a similar phenotype in colony formation and OIS function supporting our previous findings that physical association with the DBP domain is required for senescence induction. Because disabling the DBP domain accelerated MPN progression (Zhang et al, Blood, 2022), it is suggested that the D domain promotes progression. Induction of Polq protein is observed with ectopic expression of oncogenic MPN drivers in a myeloid cell line, which is inhibited by blocking ERK activity. The increased expression of Polq is crucial for MPN growth and survival, as the growth of HSPCs transduced with MPN oncogenic drivers is attenuated in the absence of Polq. Notably, Polq expression is upregulated in HSPCs from ERK2-DBP mutant mice, both before and after transduction with oncogenic JAK2 and CALR, thereby suggesting that the remaining substrate interaction domain, ERK2-D, is responsible for its induction. Interestingly, the ERK2-D domain not only interacts with Polq but may also regulate its expression through ubiquitin-dependent degradation involving the E3-ubiquitin ligase, c-CBL. D-domain inhibitor (76) substantially reduced disease burden in a xenotransplantation model of SET2 MPN line. We found that targeting the ERK2-D domain substrate, Polq, was similarly effective. Lin-CD34+ MPN HSPC were more sensitive to Polq inhibitor, ART558, compared to normal implicating Polq as a therapeutic target in MPNs. Results from single cell sequencing analysis of a triple-negative MPN which transitioned to AML, indicated a high degree of clonal variation in sensitivity to Polqi, with WT1 clones being sensitive and NRAS mutant clones exhibiting resistance. Thus, a combination of Polqi + ERK2-Di might exhibit a synergistic anti-MPN effect by attenuating the resistance mechanism of malignant subclones. As an initial test of this possibility, we treated SET2 cells with Polqi + ERK2-Di combination therapy, which revealed a synergistic response. Conclusion: The findings suggest that a combination therapy, such as Polqi + ERK2-Di, may be a promising strategy to overcome treatment resistance arising from clonal variations in sensitivity to single agents. By targeting both the D domain and inhibiting the downstream substrate Polq, a synergistic anti-MPN effect may be achieved, potentially offering a more effective therapeutic approach for patients with MPNs.

The histone demethylase JMJD2C constitutes a novel NFE2 target gene that is required for the survival of JAK2V617F mutated cells

The transcription factor NFE2 is overexpressed in most patients with myeloproliferative neoplasms (MPN). Moreover, mutations in NFE2, found in a subset of MPN patients, strongly predispose for transformation to acute leukemia. Transgenic mice overexpressing NFE2 as well as mice harboring NFE2 mutations display an MPN phenotype and spontaneously develop leukemia. However, the molecular mechanisms effecting NFE2-driven leukemic transformation remain incompletely understood. Here we show that the pro-leukemic histone demethylase JMJD2C constitutes a novel NFE2 target gene. JMJD2C expression is elevated in MPN patients as well as in NFE2 transgenic mice. Moreover, we show that loss of JMJD2C selectively impairs proliferation of JAK2V617F mutated cells. Our data suggest that JMJD2C represents a promising drug target in MPN and provide a rationale for further investigation in preclinical and clinical settings.

Transcriptomic Analysis Reveals JAK2/MPL-Independent Effects of Calreticulin Mutations in a C. elegans Model.

There is growing evidence that Ph-negative myeloproliferative neoplasms (MPNs) are disorders in which multiple molecular mechanisms are significantly disturbed. Since their discovery, CALR driver mutations have been demonstrated to trigger pathogenic mechanisms apart from the well-documented activation of JAK2/MPL-related pathways, but the lack of experimental models harboring CALR mutations in a JAK2/MPL knockout background has hindered the research on these non-canonical mechanisms. In this study, CRISPR/Cas9 was performed to introduce homozygous patient-like calreticulin mutations in a C. elegans model that naturally lacks JAK2 and MPL orthologs. Whole-genome transcriptomic analysis of these worms was conducted, and some of the genes identified to be associated with processes involved in the pathogenesis of MPNs were further validated by qPCR. Some of the transcriptomic alterations corresponded to typically altered genes and processes in cancer and Ph-negative MPN patients that are known to be triggered by mutant calreticulin without the intervention of JAK2/MPL. However, interestingly, we have also found altered other processes described in these diseases that had not been directly attributed to calreticulin mutations without the intervention of JAK2 or MPL. Thus, these results point to a new experimental model for the study of the JAK2/MPL-independent mechanisms of mutant calreticulin that induce these biological alterations, which could be useful to study unknown non-canonical effects of the mutant protein. The comparison with a calreticulin null strain revealed that the alteration of all of these processes seems to be a consequence of a loss of function of mutant calreticulin in the worm, except for the dysregulation of Hedgehog signaling and flh-3. Further analysis of this model could help to delineate these mechanisms, and the verification of these results in mammalian models may unravel new potential therapeutic targets in MPNs. As far as we know, this is the first time that a C. elegans strain with patient-like mutations is proposed as a potential model for leukemia research.

Mast cells as a therapeutic target in myeloproliferative neoplasms.

Mast cells have been implicated as mediators of bone marrow fibrosis and pruritus in myeloproliferative neoplasms (MPNs) with JAK2V617F or calreticulin mutations. We hypothesize that potent KIT inhibitors, already in clinical use for systemic mastocytosis, have therapeutic potential for the treatment of MPNs by directly targeting mast cells.

Differential cytotoxic activity of pharmacological inhibitors of IGF1R-related pathways in JAK2V617F driven cells

Myeloproliferative neoplasms (MPN) belong to a group of clonal diseases of hematopoietic stem cells characterized by aberrant proliferation of mature myeloid lineages. The constitutive activation of the JAK2/STAT signaling pathway is now well established to play a central role in MPN pathogenesis; however, accumulating evidence now indicates that the IGF1R-mediated signaling pathway contributes to the maintenance of the malignant phenotype. Studies using inhibitors of IGF1-mediated signaling have reported cytotoxic effects in cellular and murine models of MPN, but no consensus has been reached regarding the potency and efficacy of inhibitors of the IGF1R-related pathway in this context. In the present study, we compared the potency and efficacy of three inhibitors of IGF1R-related pathways in a JAK2V617F-driven cellular model. These inhibitors (NT157, OSI-906, and NVP-AEW54) present antineoplastic activity with similar efficacy in Ba/F3 JAK2V617F cells, with NT157 showing the greatest potency. Both the induction of apoptosis and reduction in cell proliferation were associated with the observed reduction in cell viability. Downregulation of JAK2/STAT signaling was an advantageous off-target effect of all three inhibitors. These preclinical studies reinforce the potential of the IGF1R-related pathway as a therapeutic target in MPN.

Targets in MPNs and potential therapeutics.

Philadelphia-negative classical Myeloproliferative Neoplasms (MPNs), including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), are clonal hemopathies that emerge in the hematopoietic stem cell (HSC) compartment. MPN driver mutations are restricted to specific exons (14 and 12) of Janus kinase 2 (JAK2), thrombopoietin receptor (MPL/TPOR) and calreticulin (CALR) genes, are involved directly in clonal myeloproliferation and generate the MPN phenotype. As a result, an increased number of fully functional erythrocytes, platelets and leukocytes is observed in the peripheral blood. Nevertheless, the complexity and heterogeneity of MPN clinical phenotypes cannot be solely explained by the type of driver mutation. Other factors, such as additional somatic mutations affecting epigenetic regulators or spliceosomes components, mutant allele burdens and modifiers of signaling by driver mutants, clonal architecture and the order of mutation acquisition, signaling events that occur downstream of a driver mutation, the presence of specific germ-line variants, the interaction of the neoplastic clone with bone marrow microenvironment and chronic inflammation, all can modulate the disease phenotype, influence the MPN clinical course and therefore, might be useful therapeutic targets.

HMGA1 Chromatin Regulators Drive Progression in Myeloproliferative Neoplasms through Epigenetic Rewiring to Induce Networks Involved in GATA2 and Proliferation

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). However, mechanisms driving progression remain elusive and therapies are ineffective after leukemic transformation. The High Mobility Group A1 (HMGA1) gene encodes oncogenic chromatin regulators which are overexpressed in diverse tumors where they portend adverse outcomes (Resar Cancer Res 2010; Xian et al Nature Commun 2017). Hmga1 induces leukemic transformation in transgenic mice and HMGA1 is overexpressed in refractory myeloid malignancies (Resar et al Cancer Res 2018). Further, germline lesions within the HMGA1 loci increase the risk for developing MPN (Bao et al Nature 2020). We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by rewiring transcriptional networks to foster leukemogenesis, and, 2) identify mechanisms underlying HMGA1 that could be targeted with therapy.Methods: To elucidate the function of HMGA1, we disrupted HMGA1 expression via CRISPR/Cas9 or short hairpin RNA (shRNA) targeting 2 different sequences per gene and assessed proliferation, colony formation, apoptosis, and leukemogenesis. We also generated JAK2 V617F transgenic mouse models of MF with Hmga1 deficiency. To dissect molecular mechanisms underlying HMGA1, we integrated RNAseq, ATACseq, and chromatin immunoprecipitation (ChIP) from MPN-AML cell lines (DAMI, SET-2). Next, we tested whether HMGA1 depletion synergizes with ruxolitinib in preventing leukemic engraftment in mice. To identify drugs to target HMGA1 networks, we applied the Broad Institute Connectivity Map (CMAP).Results: HMGA1 is overexpressed in CD34 + cells from patients with JAK2 V617F MPN with highest levels after transformation to MF or AML in 3 independent cohorts. CRISPR/Cas9 inactivation or shRNA-mediated HMGA1 silencing disrupts proliferation, decreases the frequency of cells in S phase, increases apoptosis, and impairs clonogenicity in human MPN-AML cell lines. HMGA1 depletion also prevents leukemic engraftment in mice. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2 V617F murine models of MPN, decreasing erythrocytosis, thrombocytosis, and preventing splenomegaly and fibrosis of the spleen and bone marrow. Further, Hmga1 deficiency preferentially prevents expansion in long-term HSC, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors in JAK2 V617F mice. RNAseq revealed genes induced by HMGA1 that govern cell cycle progression (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) and cell fate decisions (GATA2 networks), including the GATA2 master regulator gene. Silencing GATA2 recapitulates anti-leukemia phenotypes observed with HMGA1 deficiency whereas restoring GATA2 in MPN-AML cells with HMGA1 silencing partially rescues leukemia phenotypes, increasing clonogenicity and leukemic engraftment. Mechanistically, HMGA1 binds directly to AT-rich sequences near the GATA2 developmental enhancer (+9.5), enhances chromatin accessibility, and recruits active histone marks (H3K4me1/3) to induce GATA2 expression. HMGA1 depletion enhances responses to the JAK/STAT Inhibitor, ruxolitinib, delaying leukemic engraftment and prolonging survival in murine models of JAK2 V617F MPN-AML. Further, epigenetic drugs predicted to target HMGA1 transcriptional networks using CMAP synergize with JAK inhibitors to disrupt proliferation in human MPN-AML cells. HMGA1 and GATA2 are co-expressed and up-regulated with progression from MF to AML in matched patient samples. Moreover, HMGA1 transcriptional networks are activated in leukemic blasts, thus underscoring the role of HMGA1 in human MPN progression.Conclusions: We uncovered a previously unknown epigenetic program whereby HMGA1 enhances chromatin accessibility and recruits activating histone marks to induce transcriptional networks required for progression in MPN, including direct transactivation of GATA2. Further, HMGA1 networks can be targeted with epigenetic therapy and synergize with ruxolitinib. Together, our studies reveal a new paradigm whereby HMGA1 up-regulates GATA2 and proliferation networks to drive disease progression and illuminate HMGA1 as a novel therapeutic target in MPN. [Display omitted] DisclosuresRampal: Jazz Pharmaceuticals: Consultancy; Incyte: Consultancy, Research Funding; Kartos: Consultancy; Constellation: Research Funding; Pharmaessentia: Consultancy; Blueprint: Consultancy; Disc Medicine: Consultancy; Stemline: Consultancy, Research Funding; BMS/Celgene: Consultancy; Novartis: Consultancy; Sierra Oncology: Consultancy; CTI: Consultancy; Abbvie: Consultancy; Memorial Sloan Kettering: Current Employment. Stubbs: Incyte Research Institute: Current Employment, Current holder of individual stocks in a privately-held company.

AXL Inhibition Represents a Novel Therapeutic Approach in BCR-ABL Negative Myeloproliferative Neoplasms.

BCR-ABL negative myeloproliferative neoplasms (MPNs) consist of essential thrombocythemia, polycythemia vera, and myelofibrosis. The majority of patients harbor the JAK2-activating mutation V617F. JAK2 inhibitors were shown to reduce symptom burden and splenomegaly in MPN patients. However, treatment options are limited after failure of JAK2 inhibitors. AXL, a member of the TAM family of receptor tyrosine kinases, mediates survival and therapy resistance of different myeloid cancers including acute myeloid leukemia and chronic myeloid leukemia. We studied the relevance of AXL as a target in MPN using primary patient cells and preclinical disease models. We found that AXL is abundantly activated in MPN cells and that its ligand growth arrest-specific gene 6 is upregulated in MPN patients. Pharmacologic and genetic blockade of AXL impaired viability, decreased proliferation and increased apoptosis of MPN cells. Interestingly, ruxolitinib treatment induced increased phosphorylation of AXL indicating that activation of AXL might mediate resistance to ruxolitinib. Consistently, the AXL inhibitor bemcentinib exerted additive effects with ruxolitinib via impaired STAT3, STAT5, and AKT signaling. Both agents had activity when employed alone and exerted an additive effect on survival and splenomegaly in vivo. Moreover, bemcentinib treatment normalized red blood cell count and hemoglobin levels in vivo. Thus, our data indicate that AXL inhibition represents a novel treatment option in MPN warranting clinical investigation.

The Tyrosine Kinase-Driven Networks of Novel Long Non-coding RNAs and Their Molecular Targets in Myeloproliferative Neoplasms.

Recent research has focused on the mechanisms by which long non-coding RNAs (lncRNAs) modulate diverse cellular processes such as tumorigenesis. However, the functional characteristics of these non-coding elements in the genome are poorly understood at present. In this study, we have explored several mechanisms that involve the novel lncRNA and microRNA (miRNA) axis participating in modulation of drug response and the tumor microenvironment of myeloproliferative neoplasms (MPNs). We identified novel lncRNAs via mRNA sequencing that was applied to leukemic cell lines derived from BCR-ABL1-positive and JAK2-mutant MPNs under treatment with therapeutic tyrosine kinase inhibitors (TKI). The expression and sequence of novel LNC000093 were further validated in both leukemic cells and normal primary and pluripotent cells isolated from human blood, including samples from patients with chronic myelogenous leukemia (CML). Downregulation of LNC000093 was validated in TKI-resistant CML while a converse expression pattern was observed in blood cells isolated from TKI-sensitive CML cases. In addition to BCR-ABL1-positive CML cells, the driver mutation JAK2-V617F-regulated lncRNA BANCR axis was further identified in BCR-ABL1-negative MPNs. Further genome-wide validation using MPN patient specimens identified 23 unique copy number variants including the 7 differentially expressed lncRNAs from our database. The newly identified LNC000093 served as a competitive endogenous RNA for miR-675-5p and reversed the imatinib resistance in CML cells through regulating RUNX1 expression. The extrinsic function of LNC000093 in exosomal H19/miR-675-induced modulation for the microenvironment was also determined with significant effect on VEGF expression.

Thrombosis in myeloproliferative neoplasms: update in pathophysiology.

This review summarizes high-impact research in myeloproliferative neoplasms (MPN) from the last 18 months, with a particular focus on basic science findings. A pseudo-hypoxia state with stabilization of hypoxia-inducible factor (HIFα exists that is central to cell growth, cell renewal, inflammation, and thrombotic potential in MPN hematopoietic cells. HIFα and inflammatory pathways are new therapeutic targets in MPN, with the potential to ameliorate thrombotic risk and perhaps eradicate mutant progenitor cells.

The Pharmacological IGF1R-IRS1/2 Inhibitor NT157 Presents Multiple Anti-Neoplastic Effects in Myeloproliferative Neoplasms

Introduction: The recurrent V617F mutation in JAK2 plays an important role in the pathogenesis of BCR-ABL1 negative myeloproliferative neoplasms (MPN). Absence of complete clinical response to ruxolitinib, a JAK1/2 inhibitor, highlights the need to target other signaling pathways that contribute to JAK2. Recent evidence indicates that the IGF1R/IRS pathway is a potential target in MPN: (i) insulin receptor substrate 2 (IRS2) cooperates with malignant transformation induced by JAK2V617F, and (ii) increased IGF1 signaling induced by AIRAPL down-regulation causes MPN in mice. NT157 is a synthetic compound designed as an IRS1/2 inhibitor, which presented anti-neoplastic effects in solid tumors. Aims: The objective herein was tocharacterize the molecular and cellular effects of NT157 treatment, combined or not with ruxolitinib, in MPN JAK2V617F. Methods: HEL and SET2 cell lines (both carrying JAK2V617F mutation) were treated or not with vehicle, NT157 and/or ruxolitinib and submitted to evaluation of cell viability (MTT assay), apoptosis (annexin V/PI and caspase cleavage), proliferation (Ki-67), clonogenicity (colony forming assay), cell cycle (PI), gene expression (PCR array and qPCR) and protein expression/activation (Western blot). Primary cells from polycythemia vera (PV) patients (n=3) were exposed to NT157 (3.2 µM) treatment and evaluated for erythropoietin-independent colony formation. Statistical analysis was performed using ANOVA or Student's t test. P -value <0.05 was considered statistically significant. Results: NT157 treatment significantly decreased cell viability, clonogenicity and cell proliferation (all p <0.05). NT157 induced apoptosis at doses equal or higher than 0.8 μM as verified by flow cytometry analysis of annexin V/PI (p ˂0.001) and confirmed by increased levels of cleaved caspase 3, 8 and 9. The percentage of apoptotic cells (annexin V positive) identified for untreated cells and cells exposed to NT157 0.8 μM was respectively, 14 and 56% after 24 hours of treatment; 11 and 60% after 48 hours, 6 and 40% after 72 hours (all p ˂0.001). Cell cycle analysis was performed 24 hours after NT157 exposition and revealed cell cycle arrest in G2/M, associated with decrease in G0/G1 population (p ˂0.001). In a molecular scenario, NT157 treatment strongly reduced IRS1 and IRS2 expression, and STAT3, STAT5 and ERK1/2 phosphorylation in a dose dependent manner. PCR array analyzes revealed that NT157 treatment modulated 23 oncogenes and tumor suppressor genes. Based on the cellular effects observed after NT157 treatment, we selected six genes, three repressed oncogenes and three upregulated apoptotic-related genes, to be validated in a larger number of experiments. Upon NT157 exposition, CCND1 (cyclin D1), MYB and WT1 mRNA levels were reduced, while CDKN1A (p21), FOS and JUN mRNA levels increased (all p ˂0.05). The combined treatment of NT157 plus ruxolitinib did not present potentializing effects when compared to the monotherapies. In contrast, the combined treatment prevented, at least in part, the cytotoxic effects of NT157. In HEL cells, NT157 0.8 µM monotherapy promoted enhanced apoptosis rate compared to the combined treatment, using NT157 0.8 plus ruxolitinib 0.3 µM (52% vs. 38%, respectively [ p ˂0.05]). Ruxolitinib also prevented NT157 effect on cell cycle progression, causing a reduction in G2/M arrest when compared to NT157 monotherapy (p <0.05). In primary hematopoietic cells from PV patients, NT157 exposition reduced erythropoietin-independent colony formation by 49 % (patient #1), 12% (patient #2) and 23% (patient #3); the mean reduction of erythroid colony number for all PV samples was 29% (p <0.05). Conclusions: IGF1R-IRS1/2 pharmacological inhibitor NT157 displayed remarkable anti-neoplastic effects in JAK2V617F cells lines and MPN primary cells. Our exploratory approaches provided new insights about molecular mechanisms of NT157. Our findings indicate that inhibition of IGF1R/IRS signaling, using NT157 monotherapy, emerges as a novel therapeutic opportunity for MPN JAK2V617F. DisclosuresNo relevant conflicts of interest to declare.

Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis

SummaryFunctional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs.

Heat Shock Proteins and PD-1/PD-L1 as Potential Therapeutic Targets in Myeloproliferative Neoplasms.

Simple SummaryMyeloproliferative neoplasms (MPN), which are a heterogeneous group of rare disorders that affect blood cell production in bone marrow, present many significant challenges for clinicians. Though considerable progress has been made, in particular with the JAK1/2 inhibitor ruxolitinib, more effective alternative therapeutic approaches are needed. In the search for new and more efficient therapies, heat shock proteins, also known as stress proteins, and the programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immune checkpoint axis have been found to be of great interest in hematologic malignancies. Here, we review the therapeutic potential of stress protein inhibitors in the management of patients diagnosed with MPN and summarize the accumulating evidence of the role of the PD-1/PD-L1 axis in MPN in order to provide perspectives on future therapeutic opportunities relative to the inhibition of these targets.Myeloproliferative neoplasms (MPN) are a group of clonal disorders that affect hematopoietic stem/progenitor cells. These disorders are often caused by oncogenic driver mutations associated with persistent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. While JAK inhibitors, such as ruxolitinib, reduce MPN-related symptoms in myelofibrosis, they do not influence the underlying cause of the disease and are not curative. Due to these limitations, there is a need for alternative therapeutic strategies and targets. Heat shock proteins (HSPs) are cytoprotective stress-response chaperones involved in protein homeostasis and in many critical pathways, including inflammation. Over the last decade, several research teams have unraveled the mechanistic connection between STAT signaling and several HSPs, showing that HSPs are potential therapeutic targets for MPN. These HSPs include HSP70, HSP90 (chaperoning JAK2) and both HSP110 and HSP27, which are key factors modulating STAT3 phosphorylation status. Like the HSPs, the PD-1/PD-L1 signaling pathway has been widely studied in cancer, but the importance of PD-L1-mediated immune escape in MPN was only recently reported. In this review, we summarize the role of HSPs and PD-1/PD-L1 signaling, the modalities of their experimental blockade, and the effect in MPN. Finally, we discuss the potential of these emerging targeted approaches in MPN therapy.

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.

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 &gt;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.

The Persistent Survival of MPN Cells to JAK2 Inhibition Is Dependent on SHP2 Activity, Which May Provide a Therapeutic Target to Enhance Current Anti-MPN Therapies

Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis, are hematopoietic stem cell disorders that affect about 300,000 people in the U.S. PV and ET can progress to myelofibrosis where patients are susceptible to bone marrow failure, increased incidence of acute leukemia, and poor survival. MPNs are driven by aberrant JAK2 activity that is a consequence of mutations in JAK2, Mpl, or CalR. The JAK1/2 inhibitor ruxolitinib, which is FDA-approved for a subset of MPN patients, is effective in reducing patient symptomology but fails to induce remission, as it has little effect on the neoplastic cell burden. However, a modest increase in survival and decrease of allele burden has been suggested following long-term ruxolitinib treatment. The quality of life benefit afforded by ruxolitinib suggests it will likely remain a mainstay of future MPN therapies. Identifying the mechanism(s) by which cells persistently survive in the presence of ruxolitinib could prove pivotal to improving JAK2 inhibitor therapies in MPN patients.In order to identify mechanisms by which JAK2-driven MPN cells survive ruxolitinib treatment, we utilized global phospho-tyrosine proteomics to compare ruxolitinib-sensitive and persistent cells, the latter of which are drug persistent by nature due to their reacquisition of JAK2 inhibitor sensitivity upon removal of ruxolitinib. We utilized the JAK2-V617F-driven MPN model cell lines UKE1 and SET2 for this analysis. Pathway analysis from the datasets obtained indicated changes in phosphorylation of proteins involved in growth factor and cytokine signaling. Notably, we identified changes in phosphorylation of several tyrosine phosphatases, including SHP1 and SHP2. The SH2 domain-containing tyrosine phosphatase SHP2 (encoded by PTPN11) plays important roles in relaying signals from cell surface receptors, notably mediating activation of ERKs, and was the first phosphatase identified as leukemogenic. SHP2 regulates JAK2 signaling and is found mutated in ~8% of post-MPN leukemia, suggesting it may play a role in the biology of MPNs. In addition, SHP2 has been identified as a potential mediator of targeted therapy resistance in cancer. Therefore, we hypothesized that SHP2 activity may contribute to the persistent survival of MPN cells in ruxolitinib and provide a therapeutic target in MPNs. To test our hypothesis, we treated ruxolitinib-sensitive (drug naïve) and persistent cells with a specific allosteric inhibitor of SHP2, SHP099. Ruxolitinib-persistent cell growth was more sensitive to SHP2 inhibition, and SHP099 enhanced the growth inhibitory effects of ruxolitinib on ruxolitinib-sensitive cells. Treatment of UKE1 cells with a high concentration (~10X GI50) of ruxolitinib leads to the persistent survival of a small population of cells and this was prevented by SHP099. SHP2 inhibition was also deleterious to the maintenance of an existing drug persistent state in a high concentration of ruxolitinib. Interestingly, signaling pathways in cells persistently growing in ruxolitinib differentially respond to SHP2 inhibition. In both ruxolitinib-sensitive and persistent cells activation of ERK was inhibited by SHP099. However, in ruxolitinib-persistent UKE1 cells, this inhibition was more rapid, suggesting the kinetic regulation of ERK activation by SHP2 is altered in these cells. Also, SHP099 led to an increase in pSTAT3 in both ruxolitinib-sensitive and persistent cells. However, SHP099 led to an increase in pSTAT5 in ruxolitinib sensitive UKE1 cells, but not in cells persistently growing in ruxolitinib. Re-activation of signaling pathways following ruxolitinib treatment likely contributes to cell survival. We observed that ERK becomes re-activated shortly after ruxolitinib treatment, and this re-activation depends on SHP2 activity. Finally, we observed dose-dependent inhibition of the neoplastic growth of primary MPN patient cells by SHP099 and synergistic inhibition with ruxolitinib, suggesting co-targeting SHP2 may improve JAK2-targeted MPN therapies.In conclusion, our data suggest that re-wiring of signaling in cells challenged with ruxolitinib may create greater dependence on SHP2 activity that contributes to cell survival to JAK2 inhibition. Thus, targeting SHP2 may improve the efficacy of ruxolitinib as an anti-MPN therapy. DisclosuresHaura:Incyte Corporation: Research Funding. Reuther:Incyte Corporation: Research Funding.

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.