Development Of Myeloproliferative Neoplasms Research Articles

SH2B3 (LNK) rs3184504 polymorphism is correlated with JAK2 V617F-positive myeloproliferative neoplasms

Abstract Background: Pathogenesis and phenotypic diversity in myeloproliferative neoplasms (MPN) cannot be fully explained by the currently known acquired mutations alone. Some susceptible germline variants of different genes have been proved to be associated with the development of these diseases. The goal of our study was to evaluate the association between the rs3184504 polymorphism of SH2B3 (LNK) gene (p.R262W, c.784T>C) and the risk of developing the four typical MPN - polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and chronic myeloid leukemia (CML). Material and methods: We investigated the SH2B3 rs3184504 T>C polymorphism by real-time PCR in 1901 MPN patients (575 with PV, 798 with ET, 251 with PMF, and 277 with CML), all of them harboring one of the specific driver mutations - JAK2 V617F or CALR in case of PV, ET and PMF, or BCR-ABL1 in case of CML, and 359 controls. Results: Overall, the TT homozygous genotype was significantly associated with BCR-ABL1-negative MPN (OR = 1.34; 95% CI = 1.03-1.74; crude p-value = 0.02; adjusted p-value = 0.04). The most significant association was seen in case of PV (OR = 1.54; 95% CI = 1.14-2.06; crude p-value = 0.004; adjusted p-value = 0.024). Also, SH2B3 rs3184504 correlated significantly with JAK2 V617F-positive MPN (OR = 1.36; 95% CI = 1.04-1.77; crude p-value = 0.02; adjusted p-value = 0.08), but not with those CALR-positive. ET (regardless of molecular subtype) and CML were not correlated with SH2B3 rs3184504. Conclusions: The SH2B3 rs3184504 polymorphism is associated with risk of MPN development, especially PV. This effect is restricted to JAK2 V617F-positive PV and PMF only.

Mechanism underlying the development of myeloproliferative neoplasms through mutant calreticulin.

Deregulation of cytokine signaling is frequently associated with various pathological conditions, including malignancies. In patients with myeloproliferative neoplasms (MPNs), recurrent somatic mutations in the calreticulin (CALR) gene, which encodes a molecular chaperone that resides in the endoplasmic reticulum, have been reported. Studies have defined mutant CALR as an oncogene promoting the development of MPN, and deciphered a novel molecular mechanism by which mutant CALR constitutively activates thrombopoietin receptor MPL and its downstream molecules to induce cellular transformation. The mechanism of interaction and activation of MPL by mutant CALR is unique, not only due to the latter forming a homomultimeric complex through a novel mutant‐specific sequence generated by frameshift mutation, but also for its ability to interact with immature asparagine‐linked glycan for eventual engagement with immature MPL in the endoplasmic reticulum. The complex formed between mutant CALR and MPL is then transported to the cell surface, where it induces constitutive activation of downstream kinase JAK2 bound to MPL. Refined structural and cell biological studies can provide an in‐depth understanding of this unusual mechanism of receptor activation by a mutant molecular chaperone. Mutant CALR is also involved in modulation of the immune response, transcription, and intracellular homeostasis, which could contribute to the development of MPN. In the present article, we comprehensively review the current understanding of the underlying molecular mechanisms for mutant molecular chaperone‐induced cellular transformation.

JAK2 inhibition in JAK2V617F-bearing leukemia cells enriches CD34+ leukemic stem cells that are abolished by the telomerase inhibitor GRN163L

The activating-mutation of JAK2V617F drives the development of myeloproliferative neoplasms (MPNs). Several JAK2 inhibitors such as ruxolitinib and gandotinib (LY2784544) currently in clinical trials and, provide improvements in MPNs including myelofibrosis. However, JAK2 inhibitors are non-curative and murine experiments show that JAK2 inhibitors don’t eradicate MPN stem cells and it is currently unclear how they escape. We thus determined the effect of the specific JAK2V617F inhibitor LY2784544 on leukemic stem (CD34+) cells (LSCs) using the JAK2V617F-bearing erythroleukemia cell line HEL. The LY2784544 treatment caused a transient proliferation inhibition and apoptosis of HEL cells, but a recovery occurred within a week. Thereafter, the continuous LY2784544 exposure induced the accumulation of CD34+ LSCs, and the CD34+ cells increased from 2% to >90% by week 9, which was accompanied by increased clonogenic potentials. LY2784544 was capable of stimulating CD34 expression even in CD34− HEL cells, which indicated cellular de-differentiation. A significantly enhanced expression of the stem cell factor KLF4 was observed in LY2784544-treated HEL cells. Inhibiting KLF4 expression attenuated LY2784544-mediated accumulation of CD34+ LSCs. Moreover, the telomerase inhibitor GRN163L abolished the LY2784544-effect. JAK2 inhibitors thus cause enrichment of LSCs and are unlikely to cure MPN as a monotherapy. Simultaneously targeting JAK2V617F and KLF4 or telomerase may be a novel strategy for MPN therapy, which should be of significance both biologically and clinically.

Mutation profiles of classic myeloproliferative neoplasms detected by a customized next-generation sequencing-based 50-gene panel

Abstract Objective: A sequencing panel consisting of 50 genes was customized to reveal the potential molecular land-scapes of essential thrombocytosis, polycythemia vera, and primary myelofibrosis in Chinese patients with myeloproliferative neoplasm (MPN). Methods: Sixty-five MPN patients (38 with essential thrombocytosis, 21 with polycythemia vera, and 6 with primary myelofibrosis), including 12 triple-negative patients, were recruited and were screened for their mutational spectrum using next-generation sequencing technology in this retrospective observational study. This study was approved by the Institutional Review Board of Changhai Hospital, Naval Military Medical University, China. Results: In addition to the typical driver mutations in JAK2, CALR, and MPL, pathogenic mutations in 15 other genes were frequently detected among the 65 patients with MPN. The 15 mutated genes were TET2, EZH2, ASXL1, MIR662, MLH1, MLH3, SF3B1, MSH6, BARD1, DNMT3A, KIT, MSH2, RUNX1, TP53, and NRAS in this order according to the mutational frequency detected. The average number of mutated genes was 1.2 genes per patient, while in the 12 triple-negative patients with MPN (ie, patients that lack the JAK2, CALR, or MPL mutations), at least one of the 15 pathogenic mutations was detected for each patient. Interestingly, 4 single nucleotide polymorphisms (rs4858647, rs9376092, rs58270997, rs621940) that might be correlated to individual susceptibility to myeloproliferative neoplasm were identified among the 65 patients. We also found that single nucleotide polymorphism and/or single nucleotide variation mutations occurred in multiple loci of mismatch repair-related genes, which might contribute to the development of MPN. Conclusion: Our study confirms the importance of the previously known MPN relative genes and, more importantly, provides some new and potentially valuable information about mutations associated with MPNs.

Inhibition of JAK2 Suppresses Myelopoiesis and Atherosclerosis in Apoe\u2212/\u2212 Mice

ObjectiveIncreased myelopoiesis has been linked to risk of atherosclerotic cardiovascular disease (ACD). Excessive myelopoiesis can be driven by dyslipidemia and cholesterol accumulation in hematopoietic stem and progenitor cells (HSPC) and may involve increased signaling via Janus kinase 2 (JAK2). Constitutively activating JAK2 mutants drive biased myelopoiesis and promote development of myeloproliferative neoplasms (MPN) or clonal hematopoiesis, conditions associated with increased risk of ACD. JAK2 inhibitors have been developed as a therapy for MPNs. The potential for JAK2 inhibitors to protect against atherosclerosis has not been tested. We therefore assessed the impact of JAK2 inhibition on atherogenesis.MethodsA selective JAK2 inhibitor TG101348 (fedratinib) or vehicle was given to high-fat high-cholesterol Western diet (WD)–fed wild-type (WT) or Apoe−/− mice. Hematopoietic cell profiles, cell proliferation, and atherosclerosis in WT or Apoe−/− mice were assessed.ResultsTG101348 selectively reversed neutrophilia, monocytosis, HSPC, and granulocyte-macrophage progenitor (GMP) expansion in Apoe−/− mice with decreased cellular phosphorylated STAT5 and ERK1/2 and reduced cell cycling and BrdU incorporation in HSPCs, indicating inhibition of JAK/STAT signaling and cell proliferation. Ten-week WD feeding allowed the development of marked aortic atherosclerosis in Apoe−/− mice which was substantially reduced by TG101348.ConclusionsSelective JAK2 inhibition reduces atherogenesis by suppressing excessive myelopoiesis in hypercholesterolemic Apoe−/− mice. These findings suggest selective JAK2 inhibition as a potential therapeutic approach to decrease ACD risk in patients with increased myelopoiesis and leukocytosis.

Mutant calreticulin and the molecular mechanisms in development of myeloproliferative neoplasms

This review aimed to evaluate the molecular mechanism underlying the development of myeloproliferative neoplasms (MPN) caused by mutant calreticulin (CALR). This mutation is found in a subset of patients with Philadelphia chromosome-negative MPNs, and it encodes a molecular chaperone. However, it is essentially impossible to elucidate the oncogenic property of mutant CALR from the wild-type CALR function. Studies have reported that mutant CALR forms a homomultimeric complex via intermolecular interaction between novel domains acquired due to a frameshift mutation, gains a high binding affinity for myeloproliferative leukemia protein (MPL), the thrombopoietin receptor, through a presumptive structural change, and acts as an agonist for MPL. In this review, I would like to describe the course of the discovery of this unique molecular mechanism and discuss future scope of research on mutant CALR.

Recent advances in the treatment of myelofibrosis

Primary myelofibrosis (PMF) is classified as a clonal myeloproliferative neoplasm (MPN) characterized by bone marrow fibrosis and subsequent extramedullary hematopoiesis that causes progressive anemia, symptomatic splenomegaly, and various constitutional symptoms and eventual transformation into acute leukemia. The main MPN pathophysiology is the constitutive activation of JAK2/STAT signaling. JAK2, MPL, and CALR mutations, known as phenotypic driver mutations, are directly implicated in the disease pathogenesis by the activation of JAK2/STAT signaling. Moreover, other gene mutations, including methylation-related regulators, histone modification-related factors, and RNA splicing molecules, also contribute to the pathogenesis of MPN development. Patients with PMF, unlike other MPNs, experience a significantly worse prognosis. Thus, the risk of disease should be evaluated individually, and a tailored treatment plan should be developed based on each patient's disease risk. Gene mutation information is becoming more important in evaluating the risk of disease and determining treatment options. Allogeneic hematopoietic stem cell transplantation is the only curative treatment, but its indication is limited because of the age of onset. A JAK2 inhibitor, ruxolitinib, improves splenomegaly and disease-related constitutive symptoms. To date, new JAK2 inhibitors and drugs that delay the progression of fibrosis and leukemic transformation are under development and are expected to improve the prognosis for PMF.

Cell Competition and Microenvironment in the Development of Myeloproliferative Neoplasms

Introduction The myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion. The acquired kinase mutation JAK2V617F plays a central role in MPNs. Both JAK2 wild-type (WT) clones and JAK2V617F-mutant clones co-exist in many MPN patients. Little is known about earliest steps of MPN disease initiation and how the mutant cells expand over WT cells. In this work, we tested the hypothesis that competition between WT and JAK2V617F-mutant cells acts to prevent mutant cell expansion in MPNs, but that a JAK2V617F-bearing microenvironment can overcome this competition, leading to the development of a MPN. Methods JAK2V617F Flip-Flop (FF1) mice and Tie2-Cre mice were crossed to generate a strain in which human JAK2V617F is expressed specifically in hematopoietic cells and vascular endothelial cells (Tie2FF1). Results First, we isolated JAK2WT and JAK2V617F-mutant CD45+CD201+CD150+CD48- (here termed HSPCs) from either WT mice or Tie2FF1 mice and performed a series of in vitroco-culture assays. We found that JAK2V617F HSPCs displayed a higher proliferation rate than JAK2WT HSPCs when cultured separately. In contrast,when placed in direct co-culture, proliferation of JAK2WT HSPCs was greatly increased that no significant difference between WT and mutant HSPCs was detected. Similar results were also obtained when using a transwell co-culture system, suggesting that cell-cell contact was not required for the competitive co-existence we observed in mixed JAK2WT / JAK2V617F HSPC cultures in vitro. (Figure 1) Next, we performed marrow transplantation assays to assess the competition between JAK2WT and JAK2V617F HSPCs in vivo. We found that, when 50% JAK2V617F marrow cells and 50% JAK2WT marrow cells were transplanted together into lethally irradiated WT mice, the JAK2V617F donor cells had no engraftment advantage over JAK2WT donor cells and the recipient mice had normal blood cell counts during more than 4-month follow up. This result contrasts our and other reports that a JAK2V617F-positive MPN develops following transplantation of 100% JAK2V617F-mutant donor cells into WT recipients. These observations suggest that the presence of WT cells can alterthe behavior of JAK2V617F-mutant stem cells in vivo. This conclusion was further supported by in vivoBrdU labeling experiments, which demonstrated that the proliferation rate of JAK2V617F-mutant Lin-cKit+Sca1+CD150+CD48-(here termed HSCs)was 2-fold higher than JAK2WT HSCs when transplanted separatelyinto lethally irradiated WT recipients; however, when transplanted together, WT HSCs displayed a significantly higher proliferation rate (3-fold) than mutant HSCs. (Figure 2) Endothelial cells (ECs) are an essential component of the hematopoietic niche and many studies have established that a significant number of ECs in MPN patients carry the JAK2V617F mutation. Using the same Tie2FF1 murine model, we previously reported that a JAK2V617F-bearing vascular niche promotes the expansion of JAK2V617F HSPCs over JAK2WT HSPCs, and the level of Chemokine (C-X-C motif) ligand 12 (CXCL12) was upregulated in JAK2V617F-mutant ECs compared to WT ECs. Cell cycle analysis using Hoechst33342 and Pyronin Y staining demonstrated that, while there were no differences in WT and mutant HSPC cell cycle status when transplanted into a WT vascular niche, JAK2V617F-mutant HSPCs were less quiescent (more cycling) than WT HSPCs when transplanted into a JAK2V617F-mutant vascular niche. (Figure 3) To test the hypothesis that JAK2V617F-mutant vascular niche overcomes the competition between WT and mutant cells to promote mutant HSPC expansion through increased CXCL12 level, we used the same competitive repopulation assay as in Figure 2 where both JAK2WT and JAK2V617F marrow cells were injected together into lethally irradiated WT recipient. At 12wks post transplantation, the recipient mice were treated with a single dose of CXCL12 (400ng, intravenously) before BrdU labeling. We found that CXCL12 treatment stimulated JAK2V617F-mutant HSC proliferation to a greater extent than JAK2WT HSPCs. (Figure 4) Conclusions Cell competition between WT and JAK2V617F-mutant cells prevents mutant stem cells from expansion, but a mutant microenvironment (e.g. JAK2V617F-mutant vascular niche) can overcome this competition to promote mutant stem cell expansion and the development of a murine MPN. Disclosures No relevant conflicts of interest to declare.

Transcriptional Landscape of the Microenvironment in Bone Marrow Fibrosis at Single Cell Level

Although the molecular alterations in hematopoietic cells which drive the development of myeloproliferative neoplasms (MPN) have been largely defined, reactive cellular alterations in the non-hematopoietic compartment remain rather obscure and have not been studied at single cell level. We therefore profiled enriched non-hematopoietic bone marrow cells by scRNAseq in bone marrow (BM) fibrosis compared to healthy marrow. BM fibrosis was induced by transplantation of hematopoietic stem and progenitor cells (HSPCs) with overexpression of Thrombopoietin (ThPO) into lethally irradiated mice. As ThPO-overexpression robustly leads to reticulin fibrosis in all mice (100%), we were able to study 1) pre-fibrosis (5 weeks after transplantation; reticulin fibrosis grade 0) and 2) manifest bone marrow fibrosis (10 weeks after transplantation, reticulin grade 2-3). The analysis revealed a total of 8 distinct clusters: 1-4) subpopulations of mesenchymal stromal cells (MSC-1: adipogenic, MSC-2: osteogenic, MSC-3: transition, MSC-4: interferonhigh), 5) osteoblastic lineage cells (OLCs), 6) arterial endothelial cells (ECs) and 7-8) Schwann cell precursors (SCP-1: non-myelinating SCPs; SCP-2: myelinating SCPs). Exposure to ThPO overexpressing HSPCs resulted in an overrepresentation of adipogenic MSCs at the expense of all other MSC subclusters. Differential gene expression analysis revealed a functional reprogramming of the "adipogenic" expanding MSCs with down-regulation of hematopoiesis-support and induction of a secretory phenotype including upregulation of various extracellular matrix (ECM) proteins driving fibrosis. Interestingly, only two MSC subclusters gained significant ECM expression indicating myofibroblast differentiation. Expansion of OLCs in BM fibrosis suggested a differentiation of the underrepresented MSC subpopulations into osteolineage cells which was confirmed by pseudotime analysis. Myelinating SCPs, highly expressing interleukin-33 (IL-33), showed the largest expansion in fibrosis. IL-33 is described to play a significant role in solid organ fibrosis by having both pro- and anti-fibrotic effects. Nerve injury triggers the expansion of myelinating and non-myelinating Schwann cells to promote repair, suggesting that mSCPs increase as compensatory and regenerative mechanism for the previously described MPN-induced sympathetic neuropathy. Dissection of cellular and molecular alterations in pre-fibrosis and manifest fibrosis demonstrated that only one MSC subpopulation was already significantly expanded in the pre-fibrotic phase, but only showed minor transcriptional changes. The upregulation of ECM proteins, osteogenesis as well as proinflammatory genes were hallmark features of manifest fibrosis. Interestingly, the overrepresentation of IL-33 expressing mSCPs was more pronounced in the pre-fibrotic phase, indicating that the expansion is a regenerative phenomenon failing in the stages of manifest fibrosis. Our findings were validated in the clinically relevant JAK2(V617F)-induced model of myelofibrosis. In conclusion, we here identified two distinct MSC subsets that are pro-fibrotic and contribute to osteosclerosis in PMF. The functional reprogramming of these MSCs in the bone marrow niche was accompanied by expansion of mSCPs with regenerative capacities, most likely caused by neural damage and Schwann cell death triggered by mutant HSCs. Disclosures Crysandt: Amgem: Other: travel grant; Pfizer: Other: travel grant; Gilead: Other: travel grant; Incyte: Membership on an entity's Board of Directors or advisory committees; celgene: Other: travel grant. Koschmieder:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers-Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AOP Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis Foundation: Research Funding.

Germline Mutations Predispose to Familial Myeloproliferative Neoplasms

Background: Myeloproliferative neoplasms (MPNs), commonly polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET), are a group of malignant hematologic disorders characterized by proliferation and accumulation of terminally differentiated blood cells of erythroid, myeloid and megakaryocytic origin (Cazzola, Blood 2014).Somatic driver mutations, frequently JAK2V617F, MPLW515L/K or mutant CALR in sporadic MPNs affect signaling by the thrombopoietin (THPO) and/or the erythropoietin (EPO) receptor (Pickman, PLoS 2016). MPL is the receptor of THPO and JAK2 is the key downstream mediator of THPO/MPL signaling (Nangalia NEJM 2013). In familial MPNs (families with more than 1 member developing a MPN) a germline mutation predisposes to disease development. These mutations can reveal pathways or processes involved in the pathogenesis of the disease, independent of, or cooperating with, the somatic driver mutations (Rumi, JCO 2007). We identified 3 putative predisposing germline mutations in 1 family, 3 affected generations developed MPN (PMF, ET and PV): RBSN frameshift,JAK2R340Q & PRKAA2Q425P missense variants. RBSN encodes Rabenosyn-5 protein (RBSN) which plays a role in early growth-factor receptor clathrin-dependent endocytosis (Nielsen, J Cell Biol 2000) (Fig.1).We discovered that wild type (WT) Rabenosyn-5 regulates cell surface MPL levels in hematopoietic progenitor cells and predict that MPL levels on the cell surface are negatively associated with WT Rabenosyn-5 levels. We hypothesize that the RBSN mutation predisposes to MPN development in this family, leading to abnormal activation of THPO signaling by reducing MPL turnover. Methods: Peripheral blood DNA from patient samples were analyzed for potential putative mutations using Agilent SureSelect Human ALL Exon V5+UTRs exome capture kit followed by parallel sequencing with Illumina HiSeq 2000. In vitro studies involved using the BaF3 system and CRISPR-Cas9 gene editing technique. We generated BaF3-MPL (BaF3 cells transduced with human CD4-MPL fusion) cell lines that rely on IL3 or human THPO cytokines for growth & proliferation (Fig.2&3). We transduced RBSN into the BaF3-MPL cell lines and produced RBSN heterozygous (HT) and homozygous (HM) mutants. We first generated LentiCRISPRV2GFP-mRBSN vector by inserting RBSN specific guide RNA into the LentiCRISPRV2GFP vector and generated lentivirus expressing both Cas9 and guide RNA by co-transfecting 293T cells with LentiCRISPRV2GFP vector and viral packaging vectors. The final virus was used to infect BaF3-MPL cells. Transduced cells were purified by FACS to select GFP+ cells and individual cells were grown in culture. RBSN gene mutations were examined using PCR and sequenced accordingly. To study whether the RBSN mutation alters the surface MPL levels, we stained BaF3-MPL, HT-RBSN-BaF3 and HM-RBSN-BaF3 cells with APC-CD4 antibody and the surface levels of MPL were detected by FACS by comparing the mean fluorescence intensity (MFI) of APC. Results: We identified several colonies with either HT-RBSN-BaF3 or HM-RBSN-BaF3. The guide RNA we designed specifically targeted the 12th exon of the RBSN gene which best recapitulates the mutations observed in our patients, thus, the frameshift mutations in HT-RBSN-BaF3 and HM-RBSN-BaF3 cell lines we predict will make truncated forms of RBSN which will be verified by Western Blotting for future experiments. Surface levels of MPL in the HT-RBSN-BaF3 and HM-RBSN-BaF3 cells are greater than that in BaF3-MPL cells, suggesting RBSN negatively regulates THPO-MPL signaling by regulating the cell surface MPL levels (Fig. 4). Conclusions: RBSN is a regulator of receptor trafficking, which mediates the fusion of the endocytosed receptor to the early endosome and then lysosome for degradation. We propose that genetic inactivation of RBSN might prevent the endocytosed MPL from endosome-degradation and enhance the reuse of MPL by recycling. Future experiments are planned to include in vivo studies to study whether inactivation of RBSN promoted MPN-like disease development into NSG mice and transducing our established cell lines to label individual cell compartments to study THPO-stimulated signaling. Our findings, if confirmed, will further clarify aspects of familial and somatic MPN pathogenesis and may inform efforts to devise new therapeutic and diagnostic strategies for this disease complex. Disclosures No relevant conflicts of interest to declare.

Highly Deregulated Fibulins in Patients with Philadelphia-Negative Chronic Myeloproliferative Neoplasms

Introduction The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) develop in a biological continuum from the early cancer stages (ET/PV) to the advanced myelofibrosis stage characterized by huge splenomegaly, bone marrow failure and -fibrosis. Importantly, bone marrow fibrosis also increases from the early stages with reticulin fibrosis only and later deposition of mature collagen as well. Fibulins are glycoproteins that are important constituents of the extracellular matrix (ECM). Thus, fibulins have been shown to modulate cell morphology, growth, adhesion and motility. Dysregulation of fibulins has been reported in several cancers. In addition, upregulation of fibulins and elevated circulating fibulins have been reported in diseases - other than cancers -, in which chronic inflammation is an important pathogenetic factor, such as cardiovascular diseases. Thus, deregulated fibulins have been described in patients with type 2 diabetes mellitus. Herein, using whole blood gene expression profiling, we for the first time report deregulated fibulins in patients with MPNs. Aim To detect if genes associated with pre-atherosclerotic changes in type II diabetes are deregulated in patients with MPNs. Material and methods Gene expression microarrays were applied to generate gene expression profiles of whole blood from control subjects (n=21) and patients with ET (n=19), PV (n=41), and PMF (n=9). Total RNA was purified, amplified to biotin-labeled aRNA and hybridized to microarray chips. The statistical software R was applied to perform initial data processing and statistical analysis of gene expression changes between patients and control subjects. An FDR <0.05 was considered significant. Results We identified 23,657, 25,567, and 17,417 probe sets which were significantly differentially expressed between controls and patients with ET, PV, and PMF, respectively (FDR < 0.05). We focused upon the top 15 upregulated genes from a previous gene expression microarray study performed on arterial tissue from patients with type 2 diabetes compared to non-diabetic patients undergoing artery bypass graft surgery. Several of these genes were significantly deregulated in patients with MPNs (Table 1). In patients with ET, FBLN1, FBLN2,FAM107A, IGF2, MEG3, and ELN were significantly upregulated and ZFP36L2 were significantly downregulated. In patients with PV, FBLN1, FBLN2, ELN, LEPR, FAM107A, IGF2, CRISPLD2, and MEG3 were significantly upregulated and ZFP36L2 and SERPINF1 were significantly downregulated. In patients with PMF, MEG3, LEPR, FBLN1, FAM107A, ELN, IGF2, and VWF were significantly upregulated and ZFP36L2 and SERPINF1 were significantly downregulated. Discussion and conclusions Fibulins regulate several cellular functions including tissue homeostasis and remodeling after injury, angiogenesis, and tumorigenesis. Thus, fibulins have been reported to be upregulated in several cancer types, in which deregulated fibulins have been associated with cancer invasiveness and disease progression. We have for the first time shown that fibulins are also highly deregulated in patients with MPNs. The significance of our findings is presently unknown but since interactions between fibulins and transforming growth factor (TGFbeta) have been demonstrated, upregulated fibulins may enhance the capacities of TGFbeta, which shares several of the regulatory functions excerted by fibulins. In the context of chronic inflammation being a driving force for MPN development during the biological continuum from early cancer stages to the advanced myelofibrosis stage and chronic inflammation likely also accelerates the development of atherosclerosis in MPNs, it is highly intriguing to note that fibulins are also elevated in arteries from patients with type 2 diabetes mellitus. Thus, upregulated fibulins in blood cells may actually indirectly reflect ongoing matrix modelling during atherosclerosis development, including remodeling and turnover of basement membranes in the inflamed endothelium. In conclusion, highly deregulated fibulins have been demonstrated in circulating blood cells by whole blood gene expression profiling. Further studies are needed to assess the significance of our findings in terms of potential associations to modelling of ECM in the bone marrow and during development of atherosclerosis in MPNs. Disclosures Hasselbalch: Novartis: Research Funding; AOP Orphan Pharmaceuticals: Other: Data monitoring board.

Gene Polymorphism of Xenobiotic Biotransformation Enzymes in Patients with Classical Ph-Negative Myeloproliferative Neoplasms.

The correlation of gene polymorphisms rs4025935 (large deletion), rs1695 (313A>G), rs71748309 (large deletion), and rs1800566 (609C>T) of GSTM1, GSTT1, and NQO1 genes encoding glutathione-S-transferases (GST) M1, P1, and T1 and NADPH-quinone oxidoreductase with the risk of development of classical Ph-negative myeloproliferative neoplasms (polycythemia vera, essential thrombocythemia, and primary myelofibrosis) was studied in the Caucasian ethnicity population of Vyatka region of the Russian Federation. It was found that NQO1*609T allele, NQO1*609T genotypes, and homozygous carriage of a deletion (null) allele of GSTT1 gene are associated with the risk of development of myeloproliferative neoplasms (OR=1.29, 95%CI=1.02-1.64, p=0.04; OR=1.39, 95%CI=1.04-1.85, p=0.03; and OR=1.48, 95%CI=1.03-2.12, p=0.03, respectively). However, no influence of GSTM1 and GSTP1 gene polymorphisms on the risk of development of myeloproliferative disorders was registered.

Mutant calreticulin interacts with MPL in the secretion pathway for activation on the cell surface

Studies have shown that mutant calreticulin (CALR) constitutively activates the thrombopoietin (TPO) receptor MPL and thus plays a causal role in the development of myeloproliferative neoplasms (MPNs). To further elucidate the molecular mechanism by which mutant CALR promotes MPN development, we studied the subcellular localization of mutant CALR and its importance for the oncogenic properties of mutant CALR. Here, mutant CALR accumulated in the Golgi apparatus, and its entrance into the secretion pathway and capacity to interact with N-glycan were required for its oncogenic capacity via the constitutive activation of MPL. Mutant CALR-dependent MPL activation was resistant to blockade of intracellular protein trafficking, suggesting that MPL is activated before reaching the cell surface. However, removal of MPL from the cell surface with trypsin shut down downstream activation, implying that the surface localization of MPL is required for mutant CALR-dependent activation. Furthermore, we found that mutant CALR and MPL interact on the cell surface. Based on these findings, we propose a model in which mutant CALR induces MPL activation on the cell surface to promote MPN development.

DLGAP1 directs megakaryocytic growth and differentiation in an MPL dependent manner in hematopoietic cells

BackgroundThe MPL protein is a major regulator of megakaryopoiesis and platelet formation as well as stem cell regulation. Aberrant MPL and downstream Jak/STAT signaling results in the development of the Myeloproliferative Neoplasms (MPN). The pathogenetic and phenotypic features of the classical MPNs cannot be explained by the known mutations and genetic variants associated with the disease.MethodsIn order to identify potential pathways involved in MPN development, we have performed a functional screen using retroviral insertional mutagenesis in cells dependent on MPL activation. We have used viral transduction and plasmid transfections to test the effects of candidate gene overexpression on growth and differentiation of megakaryocytic cells. The shRNA approach was used to test for the effects of candidate gene downregulation in cells. All effects were tested with candidate gene alone or in presence of hematopoietic relevant kinases in the growth medium. We assayed the candidate gene cellular localization in varying growth conditions by immunofluorescence. Flow Cytometry was used for testing of transduction efficiency and for sorting of positive cells.ResultsWe have identified the DLGAP1 gene, a member of the Scribble cell polarity complex, as one of the most prominent positive candidates. Analyses in hematopoietic cell lines revealed DLGAP1 centrosomal and cytoplasmic localization. The centrosomal localization of DLGAP1 was cell cycle dependent and hematopoietic relevant tyrosine kinases: Jak2, SRC and MAPK as well as the CDK1 kinase promoted DLGAP1 dissociation from centrosomes. DLGAP1 negatively affected the growth rate of MPL dependent hematopoietic cells and supported megakaryocytic cells polyploidization, which was correlated with its dissociation from centrosomes.ConclusionsOur data support the conclusion that DLGAP1 is a novel, potent factor in MPL signaling, affecting megakaryocytic growth and differentiation, relevant to be investigated further as a prominent candidate in MPN development.

PS1442 PROTEASOME INHIBITOR CARFILZOMIB HAS SYNTHETIC LETHALITY WITH RUXOLITINIB IN MYELOPROLIFERATIVE NEOPLASMS

Background:Ruxolitinib, like other clinically tested JAK2 inhibitors, is effective in reducing spleen size and symptom burden in patients with myelofibrosis (MF), but in most patients does not significantly reduce the mutant clone or bone marrow fibrosis. Additionally most patients develop resistance after an initial response or require dose reductions due to toxicity. Lastly, given compelling evidence that the microenvironment plays a crucial role in the development of myeloproliferative neoplasms (MPN), targeting the MPN cell clone in isolation may have limited efficacy.Aims:Therefore, we aimed to identify the signalling pathways promoting the survival of MPN cells in the presence of Ruxolitinib and the microenvironment.Methods:We applied a pooled shRNA library screen of ∼5000 genes (Cellecta, Inc.) to HEL cells, a JAK2 V617F‐mutated MPN cell line model, and we used conditioned medium (CM) from human‐derived stromal cells (HS‐5) to recapitulate in vitro the microenvironment component. Two culture conditions were tested, each run in duplicate: CM + ruxolitinib and RPMI + ruxolitinib. We also performed shRNA library screen for CD34+ cells derived from peripheral blood from a patient with primary MF treated with JAK2‐inhibitor. Primary cells were cultured in CM and the identified candidate shRNAs were compared with those resulting from the cell line screen. Chemical validation of the top‐depleted shRNAs was carried out in vitro in both the HEL cell line and primary CD34+ cells from MF patients using proliferation (MTS) and Trypan‐blue viability assays following culture with the inhibitors.Results:shRNAs were considered significant for the cell survival if their depletion was ≥ 2‐fold compared to baseline sample and was observed in at least 2 shRNAs targeting the same gene.Our screening of the ruxolitinib‐treated HEL cells cultured in CM identified 13 candidate genes with high fold depletion only in presence of the microenvironment, which will be part of future analysis. We decided in first instance to focus our attention on those genes whose depletion was maintained in the presence of the microenvironment. Among these, nucleocytoplasmic transport (confirming the recently published evidence: D Yan et al., 2018) and ubiquitin‐proteasome pathway genes were the most depleted in both settings (with and without CM). Interestingly, some of the proteasome machinery genes were highly depleted also in the primary cell shRNA screen.The in vitro proliferation and viability assays showed that proteasomal inhibition reduces the viability of both HEL cells and MF CD34+ cells and that the combination of carfilzomib with ruxolitinib is additive or synergistic.Summary/Conclusion:The proteasome is crucial to cell response to oxidative stress, which may be induced by JAK/STAT pathway. The latter represents the main pathogenic driver of MPNs. Furthermore, proteasome inhibitors are known to negatively affect the NF‐Kb pathway, which has been shown to be hyperactive in MPN, especially in MF. In view of the above evidence, it is possible to speculate that proteasome inhibition may have a significant role in the lethality of MPN cells, although potential alternative mechanisms responsible for the efficacy of carfilzomib cannot be ruled out.Although our findings still require a mechanistic explanation, they show for the first time that the combination of JAK2 and proteasome inhibitors increases the lethality of MPN cells and deserves further investigation as a potential therapeutic strategy for MPN patients.

Research progress on function of regulatory T cell in myeloproliferative neoplasms

Myeloproliferative neoplasms (MPN) are a group of abnormal clonal diseases of hematopoietic stem cell, which characterized by sustained clonal proliferation of relatively mature differentiated one or more myeloid cells. MPN mainly include chronic myeloid leukemia (CML) and Ph-/MPN. In recent years, studies have reported that abnormal number and function of regulatory T cell (Treg) play an important role in the occurrence and development of MPN. Therefore, this article reviews recent research advances in the biological characteristics of Treg, relationship between number of Treg in CML patients and CML clinical phase, Sokal score, efficacy and transcription level of BCR-ABL fusion gene, as well as the role of Treg in the pathogenesis and progression of Ph- MPN. Key words: T-lymphocytes; T-lymphocytes, regulatory; Myeloproliferative disorders; Leukemia, myelogenous, chronic, BCR-ABL positive; Treatment outcome; Neoplasm staging

GATA2 hypomorphism induces chronic myelomonocytic leukemia in mice.

The transcription factor GATA2 regulates normal hematopoiesis, particularly in‐ stem cell maintenance and myeloid differentiation. Various heteroallelic GATA2 gene mutations are associated with a variety of hematological neoplasms, including myelodysplastic syndromes and leukemias. Here, we report that impaired GATA2 expression induces myelodysplastic and myeloproliferative neoplasm development in elderly animals, and this neoplasm resembles chronic myelomonocytic leukemia in humans. GATA2 hypomorphic mutant (G2 f GN / fGN) mice that were generated by the germline insertion of a neocassette into the Gata2 gene locus avoided the early embryonic lethality observed in Gata2‐null mice. However, adult G2 f GN / fGN mice suffered from exacerbated leukocytosis concomitant with progressive anemia and thrombocytopenia and eventually developed massive granulomonocytosis accompanied by trilineage dysplasia. The reconstitution activity of G2 f GN / fGN mouse stem cells was impaired. Furthermore, G2 f GN / fGN progenitors showed myeloid lineage‐biased proliferation and differentiation. Myeloid progenitor accumulation started at a younger age in G2 f GN / fGN mice and appeared to worsen with age. G2 f GN / fGN mice showed increased expression of transcripts encoding cytokine receptors, such as macrophage colony‐stimulating factor receptor and interleukin‐6 receptor, in granulocyte‐monocyte progenitors. This increased expression could be correlated with the hypersensitive granulomonocytic proliferation reaction when the mice were exposed to lipopolysaccharide. Taken together, these observations indicate that GATA2 hypomorphism leads to a hyperreactive defense response to infections, and this reaction is attributed to a unique intrinsic cell defect in the regulation of myeloid expansion that increases the risk of hematological neoplasm transformation.

Copy number alterations and copy-neutral loss of heterozygosity in Ukrainian patients with primary myelofibrosis

To examine frequencies and spectrum of genomic alterations in Ukrainian patients diagnosed with primary myelofibrosis(PMF). We enrolled 30 Ukrainian patients diagnosed with PMF who were previously tested for usual mutations in mye-loproliferative neoplasms driver genes (JAK2, MPL and CALR). Genomic DNA samples were obtained from peripheral blood leukocytes of these patients. Copy number alterations and copy-neutral loss of heterozygosity (cnLOH) were assessed using a high-density CytoScanHD microarray platform. Statistical significance was evaluated by the Fisher exact test. We identified frequent genomic alterations, but no significant difference in the rates of copy-number loss, copy-number gain, cnLOH, or multiple genomic alterations were found in the groups of PMF patients that were positive for one of the usual mutations in driver genes or negative for such mutations (33.3% and 55.6%, p = 0.4181, 19.0% and 11.1%, p = 1.0000, 61.9% and 44.4%, p = 0.4434, 33.3% and 55.6%, p=0.4181, respectively). The most frequent alterations were cnLOH at 1p36-1p22, 9p24.3-9p13.3 and 11q12.3-11q25; copy number loss at 7q21-7q36.3 and 13q12.3-13q14.3. Copy number alterations and cnLOH commonly affected the EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53 genes, in addition to JAK2, MPL and CALR. We demonstrated the spectrum of genomic alterations in the groups of the Ukrainian PMF patients with or without the usual mutations in the specific driver genes. We identified several potential genes, which may be involved in the myeloproliferative neoplasms development and their phenotype modification (EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53).

Genetic Evidence for Involvement of Human Endogenous Retrovirus Herv-Fc1 in the Pathogenesis of MPNs

Introduction: Human endogenous retroviruses (HERVs) are genomic sequences that result from ancestral germ-line infections by exogenous RVs and therefore are transmitted in a Mendelian fashion. Several lines of genetic evidence suggest the involvement of HERVs in the etiology of autoimmune diseases and cancer. Here, by means of genetic epidemiology, we describe a case-control association study of SNPs located in the proximity of HERVs elements in patients with myeloproliferative neoplasms (MPNs).Methods: Genomic DNA from two cohorts recruited from 4 university hospitals, in total 376 patients with MPN and 527 control subjects, was isolated from whole blood. 148 SNPs related to 49 HERV loci were tested using the Sequenom platform. To quantify genomic DNA copy number variation, real-time PCR was performed using the LightCycler system. Data was analyzed using the χ2-test. For synergy, we recoded controls and cases as 0 and 1, respectively, and used ANOVA with type III sum of squares. Adjustment for multiple hypothesis testing was done using the Bonferroni correction (pB <0.05).Results: In cohort 1, analysis of 148 SNPs in 188 MPN patients and 453 controls demonstrated a significant association in MPNs of the two SNPs rs219077 and rs5993426 belonging to HERV-K and HERV-9, respectively. The two SNPs were validated in an additional cohort of 188 MPNs and were again highly significant, rs219077 (OR 1.97 (1.43 - 2.7), combined pB = 0.005) and rs5993426 (OR 3.94 (2.72 - 5.71), combined pB = 9x10-11). Additionally, we report that in patients carrying the SNP risk allele (G), rs219077, qPCR analysis results in significantly higher copy numbers compared to patients not carrying the risk allele.Discussion and conclusion: About 30 years ago HERV-K particles were reported in megakaryocytes cultured from patients with ET. Surprisingly, since then no studies have focused upon a potential link between virus infection and the development of MPNs. In the present study, we revive the HERV-story in MPNs. Thus, we show that two SNPs, rs5993426 and rs219077, close to HERVs correlate with risk of MPNs supporting a role of these genetics elements in MPN pathogenesis. In our study, rs219077, located on the chromosome 2, associates with the disease and maps in the proximity of the HERV-9 sequence. Additionally, the SNP risk allele (G) in rs219077 results in significantly higher copy numbers. There are two possible explanations for the amplification of rs219077 in MPNs. First, it might be an amplification of a part of chromosome 2, as genomic features of MPNs often include clonal, non-random chromosomal amplifications. Second, it might be due to the formation of a transducing virus from the right LTR of HERV-9, possibly now (after a few rounds of replication) with HERV-K LTRs. The observed amplification of risk allele (G) in MPN may indicate that the recombinant virus pathogenic copy can retrotranspose in PBMCs with an active viral reverse transcriptase and, eventually, integrate through recombination/integration events. rs219077 is also located in genetic proximity to MIR3 SINE elements. Thus, one fascinating possibility is that the putative transducing ERV-9 virus could encode or regulate microRNA. The second marker showing significant association with MPN risk was rs5993426. This marker in the proximity of HERV-K encoding Gag, Pol has been reported to be associated with rheumatoid arthritis. Our genetic data are currently exclusively based on genetic association and cannot as such predict any functional relationship beyond presence or absence of SNPs. Nevertheless, statistical associations are most easily explained by functional relations and it is therefore pertinent to pursue the potential molecular mechanisms mediated by HERVs in MPNs. As an example, the induction and progression of rodent erythroleukemias by Friend spleen-forming virus are mainly due to the ability of proviruses to activate cellular oncogenes or inactivate tumor suppressor genes. In this regard, we have most recently shown downregulation of TP53 in MPNs to be significantly upregulated during treatment with interferon-alpha2. In conclusion, we have for the first time demonstrated a genetic association of MPNs with HERV elements. The association supports a pathogenetic role of these loci in MPNs. Our observations stimulate to further studies on HERVs in the pathogenesis of MPNs and our results need to be confirmed in larger cohorts of patients. DisclosuresHasselbalch:Novartis: Research Funding.

Tobacco Use in the Myeloproliferative Neoplasms: Patient Behavior, Opinions, and Care

Background:Smoking increases the risk of Philadelphia-chromosome negative myeloproliferative neoplasm (MPN) development as well as increases the risk of thrombotic complications, the major cause of mortality in this population (Blood 2017 130:4199). Guidelines suggest that management of more indolent forms of MPNs include the assessment and mitigation of cardiovascular risk, which includes the management of smoking-related risks (NCCN Guidelines. 2017). In this analysis we sought to examine smoking outcomes and opinions from the perspective of the MPN patient, with the goal of helping to guide MPN patient care.Methods:The internet-based survey was developed by a team of MPN investigators and administered via Mayo Clinic's REDCap. Online recruitment was facilitated via multiple MPN-related webpages including the MPN Forum, MPN Net, MPN Research Foundation, and MPN Voice during June of 2018. Surveyed data included disease demographics such as thrombosis, medications, and smoking history. MPN-specific symptoms were assessed utilizing the MPN-10 (Blood. 2011 Jul 14;118(2):401-8).Data:Participant Demographics: Of the 607 patients who clicked the survey link, 435 patients were eligible for the survey and completed the questions regarding smoking history. Of these, 254 (58%) reported no history of tobacco use, 161 (37%) reported being ex-tobacco users, and 20 (5%) reported being current users. Among those who reported current tobacco use, 50% used premade cigarettes, 27% used self-rolled cigarettes, 20% reported vaping, and 3% reported chewing tobacco or snuff use. Respondents were most often from the US (67%), Australia (12%), the UK (6%) or Canada (6%). The distribution of respondents included 28% with myelofibrosis, 44% with polycythemia vera and 27% with essential thrombocythemia. Over one quarter (26%) of patients had experienced a prior thrombosis, with the most frequent thrombosis types being DVT (9%), stroke or transient ischemic attack (8%), abdominal vein thrombosis (5%), myocardial infarction (2%) and cerebral vein thrombosis (2%). When comparing current/former smokers to never smokers, no significant difference in thrombotic frequency was observed.Smoking Correlates: When evaluating MPN symptoms, current/former smokers reported higher severity of fatigue (mean 5.6 vs 5.0, p=0.02) and inactivity (mean 4.0 vs 3.1, p=0.03) compared to nonsmokers. Additionally, current/former smokers were more likely to experience early satiety (69% vs 58%, p=0.03), inactivity (80% vs 71%, p=0.04), concentration difficulties (82% vs 73%, p=0.04), and reduced quality of life (mean 6.1 vs 6.5, p=0.03) compared to nonsmokers. Although not significant, a trend of higher symptomatology was observed for former/current smokers compared to nonsmokers (MPN-10 total symptom score [TSS] mean 30.4 vs 27.0, p=0.07). Former/current smokers were also more likely than nonsmokers to report heavy alcohol consumption (>7 drinks per week in 9.6% vs 4.0%, p=0.03) and using opioids for pain management (24% vs 11%, p=0.001).Smoking Opinions and Care: As shown in Table 1, less than half of current/former smokers (43%) reported having their physician discuss tobacco use with them. Less than 40% of MPN current or previous smokers were aware of the risk of MPN development due to smoking, although the majority (83%) were aware of the increased thrombotic risk. Less than one quarter (43/181) of current/former smokers reported having utilized pharmacologic therapy to aid cessation.ConclusionsMPN patients with current or previous tobacco use demonstrate significantly higher symptom burden than non-smoking counterparts. In terms of patient care, less than half of patients who are current or previous smokers recall having a physician discuss their smoking habits with them. These results highlight the need for enhanced MPN patient counseling by health care providers, both regarding the risks of smoking and available methods to aid cessation. DisclosuresScherber:Orphan Pharmaceuticals: Honoraria; Incyte: Consultancy. Dueck:Phytogine: Employment; Pfizer: Honoraria; Bayer: Employment. Palmer:Novartis: Research Funding. Mesa:Incyte: Research Funding; Galena: Consultancy; Gilead: Research Funding; Ariad: Consultancy; Celgene: Research Funding; Novartis: Consultancy; CTI: Research Funding; Promedior: Research Funding.