Primary Myelofibrosis CD34 Research Articles

A Calreticulin Neoepitope-Directed Monoclonal Antibody Can Overcome JAK Inhibitor Resistance and Block TPO-Independent Megakaryocyte Differentation

Introduction: Mutations within the gene encoding calreticulin (CALR) are the second most common genetic aberration associated with primary myelofibrosis (PMF), observed in 70% of non-JAK2 V617F cases. Importantly, patients with CALR mutations do not effectively respond to JAK inhibitor therapy and no mutation specific therapy is currently in use. Virtually all CALR mutations identified in PMF are small insertions or deletions clustered within exon 9 leading to a neo-epitope peptide sequence which is thought to directly or indirectly activate the thrombopoietin receptor (TpoR) by a poorly defined mechanism. Here we engineered a neo-epitope specific monoclonal antobody that has striking biological activity against ruxolitinib persistent cells.Methods TF-1 TpoR cells expressing TpoR were supplemented with 20 ng/mL of TPO. Rats were immunised with a CALR mutant peptide coupled to KLH. Serum from the immunised rats was screened by enzyme linked immunoassay, to verify a strong titre to the peptide immunogen. Primary PMF CD34+ cells were cultured in StemCell Pro with human SCF, IL-6 and IL-9. NSG mice were used to for engraftment studies after 150 cGy irradiation.Results: We engineered a panel of rat monoclonal antibodies after immunization with a 30 amino acid peptide corresponding to the C-terminal mutant CALR neoepitope sequence with an extra cysteine residue. Clone 4D7 showed superior activity of detecting mutant but not wild type CALR protein with a binding affinity of 13.5 pM and dissociation constant of 1.53 nM as measured by I 125-Scatchard. Treatment with 4D7 resulted in a significant (5-7-fold) increase in the amount of full-length mutant CALR protein in conditioned media. 4D7 inhibited Tpo-independent cell growth over 6 days in TF-1 cells expressing MPL and mutant CALR at 2, 10 and 20 µg. 4D7 blocked constitutive factor-independent phospho-STAT5 and phospho-ERK after incubation exclusively in mutant CALR cells but not in TF-1 cells expressing TpoR alone and increased the sub-G 0 fraction was observed compared to IgG control (P = 0.001, n = 3 independent experiments) consistent with induction of an apoptotic response. We tested activity in purified primary CD34+ cells obtained from patients with CALR mutant myelofibrosis using two orthogonal assays: - (i) Tpo-independent megakaryocyte differentiation in liquid culture and (ii) Tpo-independent megakaryocyte colony formation on a collagen-based medium. 4 out of 4 patient samples that displayed robust Tpo-independent growth of CD41+CD61+ megakaryocyte progenitors showed inhibition by 4D7 of at least 50%. Similarly, we saw dramatic reduction in the absolute numbers of primary Tpo-independent megakaryocyte colonies cultured on collagen (colony-forming unit-mega) treated with 4D7 in multiple patient samples (decrease of 46%, P = 0.0001, Student's t-test, n = 4 independent patient samples) Importantly, secretion of mutant CALR protein was neither upregulated nor downregulated by ruxolitinib, indicating ruxolitinib is unlikely to alter mutant CALR trafficking in patients. 4D7 had strong inhibitory activity on cells that were resistant to ruxolitinib, in both liquid culture at 96 hours or colony formation. To test whether 4D7 could block mutant CALR-dependent proliferation in vivo, we developed two distinct xenograft models, a bone marrow engraftment model, which measures mutant CALR dependent proliferation in the bone marrow microenvironment, and a chloroma model, which mimics extravascular infiltration of mutant CALR leukaemia, by injection of TPO-independent TF-1 cells in NSG mice. In the bone marrow engraftment model 4D7 treatment (12 mg/kg twice weekly via intraperitoneal injection) lowered peripheral blood engraftment of human CD33 myeloid cells at 3 weeks, bone marrow engraftment and significantly prolonged survival compared to IgG control (P=0.004, HR=0.2). In the chloroma model, 4D7 treatment resulted in significant decrease in tumour growth measured at 3 weeks (P<0.01) and improved overall survival (P=0.02, HR=0.07) compared to IgG controlConclusion: Together, these results suggest an immunotherapeutic approach may have clinical utility CALR-driven myeloproliferative neoplasms and CALR mutant acute myeloid leukaemia, as well as activity in CALR mutant patients that develop resistance/persistence to ruxolitinib. DisclosuresRoss: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

Abnormal Expression Of WT1-As, MEG3 and ANRIL Long Non-Coding RNAs In Primary Myelofibrosis and Their Clinical Correlates

A growing body of evidence suggests that long non-coding RNAs (lncRNAs) are involved in the regulation of gene expression in normal and cancer cells by recruiting chromatin remodeling complexes to specific genomic loci [Rinn JL, et al. Cell. 2007; Mattick JS, et al. Bioessays 2009]. For this reason, lncRNAs deregulation can play key roles in malignant transformation and cancer cell behavior. [Kogo R, et al. Cancer Res 2011; Yang F, et al. Hepatology 2011].Despite the increasing number of studies on lncRNAs expression and their involvement in some solid tumors, lncRNAs are far from being extensively characterized in malignant hematopoiesis [Heuston EF, Front Genet 2011]. In particular, lncRNAs expression has never been investigated in cells from primary myelofibrosis (PMF) patients.PMF is a Philadelphia negative chronic Myeloproliferative Neoplasm (MPN) that originates from deregulated clonal proliferation of hematopoietic stem cell associated with overproduction of mature blood cells. Molecular mechanisms underlying MPN pathogenesis were partially unraveled in 2005-2006 with the identification of somatic gain-of-function mutations of JAK2 [James C, et al. Nature 2005; Kralovics R, et al. N Engl J Med 2005; Levine RL, et al. Cancer Cell 2005] and MPL [Pardanani AD, et al. Blood 2006] after which many other mutations were identified. Recently, several new molecular pathogenetic mechanisms were proposed, such as the aberrant expression of coding and non-coding RNAs.After extensive screening of the long non-coding RNA database (lncRNAdb), which is a database providing comprehensive annotations of eukaryotic lncRNAs (http://www.lncrnadb.org) [Amaral PP, Nucleic Acids Res 2011], we identified some previously described lncRNAs as potentially involved in hematological malignancies, such as CDKN2B-antisense (ANRIL), MEG3 and WT1-antisense lncRNAs.With the aim to describe possible abnormalities of lncRNAs in MPN, we investigated the expression of those lncRNAs in CD34+ cells from PMF patients. Since the perturbation of the antisense RNA could alter the expression of the sense gene, we also analyzed the expression of the ANRIL and WT1-as coding genes, called CDKN2B and WT1, to evaluate the correlation of each sense/antisense couple.This study included a total of 26 PMF patients and 16 healthy donors. The diagnosis of PMF was made according to World Health Organization (WHO) criteria.The results evidenced that the majority of PMF samples (74%) displayed a co-upregulation of WT1 and its antisense RNA compared to healthy controls. We found that the large part (61%) of patients harboring concordant upregulation of WT1 sense and antisense belongs to the intermediate-2 and high risk categories of DIPSS-plus scoring system (57.4% and 43.1% in the intermediate-1 and -2, respectively versus 0% in the high-risk category; P = .006) and presents low Hb level, higher percentage of circulating CD34+ cells and a blast count > 1% .Noteworthy, almost 90% of patients with increased expression of WT1 and its antisense showed concurrent upregulation of MEG3 and shared the same clinical variables associated with poor outcome. In fact, also patients harboring higher levels of MEG3 cluster in higher DIPSS-plus scoring system categories, suffer from anemia, have high levels of circulating CD34+ cells and a blast count >1%. These data suggest that not only high levels of WT1-as we previously described [Guglielmelli P, Stem Cells 2007], but also its antisense and MEG3 could identify PMF patients with more severe disease.Finally, we could observe a correlation between CDKN2B upregulation and the grade of BM fibrosis. In fact, the majority of samples having CDKN2B upregulation clustered in categories 2 and 3 (87.3%) as compared to only 4.5% in case of samples showing CDKN2B downregulation (P = .043). Noteworthy, 82% of patients overexpressing CDKN2B were JAK2V617F positive as compared to 22.1% among those with CDKN2B downregulation (P = .004)To our knowledge, this is the first study describing the expression profiles of human lncRNAs in CD34+ cells of PMF patients. Our data demonstrate that WT1-as, MEG3 and ANRIL lncRNAs are aberrantly expressed in PMF CD34+ cells compared to healthy controls. Moreover, different patterns of expression are correlated with some patient' clinical features. Therefore, our results suggest that a deregulated expression of these lncRNAs could play a role in PMF pathogenesis and progression. Disclosures:No relevant conflicts of interest to declare.

The Role of the Axis TGF-β1/Mir-382-5p/SOD2 in the Induction of Oxidative Stress in CD34+ Hematopoietic Stem/Progenitor Cells from Primary Myelofibrosis

Philadelphia negative chronic myeloproliferative neoplasms (MPNs) are acquired clonal disorders of hematopoietic stem cell. Among MPNs, Primary Myelofibrosis (PMF) carries the worst prognosis with severe clinical hallmarks, including bone marrow failure, extramedullary hematopoiesis and splenomegaly. MNPs are intimately associated with high level of pro-inflammatory cytokines (i.e., transforming growth factor-β1, TGF-β1) and excess generation of Reactive Oxygen Species (ROS) that are, at least in part, responsible for disease progression. Recently, extensive studies have provided unequivocal evidences that differentially expressed microRNAs (miRNAs) may play a role in MNP's pathogenesis. The miRNA expression profile performed on CD34+ hematopoietic stem/progenitor cells (HSPCs) obtained from PMF patients (R. Norfo et al., Blood, 2014), allowed us to identify miR-382-5p as up-regulated miRNA. Overexpression experiments using miRNA mimic in normal CD34+ cells have demonstrated its central role in granulocyte fate decision (R. Zini et al., Stem Cell and Dev, 2016).In this study, to establish the role of miR-382-5p in PMF pathogenesis, we performed a gene expression profile (GEP) in cord blood CD34+ cells overexpressing miR-382-5p by means of Affymetrix HG-U219 microarray platform. We identified 75 downregulated genes in miR-382-5p overexpressing cells compared to controls. Then, we pointed out 6 genes downregulated both upon miRNA overexpression and in PMF CD34+ cells compared to healthy donor. Among them, we selected the anti-oxidant superoxide dismutase 2 (SOD2), depicted as the most favorable predicted target according to different prediction algorithms such as Target Scan, miRNA.org and miRDB. Luciferase reporter assay, performed in K562 cells, confirmed SOD2 as a real miR-382-5p target. Then, we showed that miR-382-5p overexpression leads to SOD2 down-regulation both at mRNA and protein level and reduced SOD activity in CB CD34+ cells. Furthermore, we demonstrated that miR-382-5p induced ROS accumulation as compared to control (42.1±1.5% vs 31.8±1.7%, p<0.05), increased DNA damages (measured by 8-OHdG level) and enhanced CD34+ cells proliferation. Afterwards, to confirm that miR-382-5p play a role in PMF pathogenesis, we performed inhibition experiments in PMF CD34+ cells using miR-382-5p antagomiR. Our data revealed that miR-382-5p silencing restored SOD2 expression and activity, induced ROS disposal (50.1±3.8% vs 62.1±3.2%, p<0,05), decreased DNA oxidation and impaired cell proliferation.Then, to verify whether miR-382-5p could be related to chronic inflammatory state typical of PMF, we treated CD34+ cells with the pro-inflammatory cytokine TGF-β1, already described up-regulated cytokine in PMF serum, and involved in PMF pathogenesis (A. Agarwal et al., Stem Cell Investig, 2016). We showed that TGF-β1 treatment induced miR-382-5p expression, which in turn suppresses SOD2 both at mRNA and protein level and impaired its activity leading to ROS over-production (54.7±1.1% vs 36.7±1.3% 24h after TGF-β1 treatment, p<0.05, 72.4±0.7% vs 51.9±2.0% 48h after TGF-β1 treatment, p<0.01). Finally, to further confirm the involvement of the axis TGF-β1/miR-382-5p/SOD2 in the induction of oxidative stress, we inhibited TGF-β1 pathway in PMF CD34+ cells using 500nM of Galunisertib, a specific TGF-β-receptor I kinase inhibitor, already demonstrated to be capable of attenuating the fibrotic phenotype in MPN mouse model (L.Yue et al., Blood, 2015). As a result, TGF-β1 inhibition reduced miR-382-5p expression, increasing SOD activity and its expression both at mRNA and protein level. Of note, the TGF-β-receptor I kinase inhibitor significantly reduced ROS accumulation 24h after Galunisertib treatment, in PMF CD34+ cells as compared to untreated sample (33.1±7.2% vs 45.3±6.3%, p< 0.05).Taken together, our data showed that miR-382-5p enhanced oxidative stress and oxidative-DNA-damages in normal and PMF CD34+ cells through the modulation of its target SOD2. We showed for the first time that the axis TGF-β1/miR-382-5p/SOD2 is responsible, at least in part, for ROS over-production that contribute to a state of chronic oxidative stress and inflammation, which are typical features of PMF. Furthermore, our results suggested that Galunisertib might be an effective therapy to reduce abnormal oxidative stress sustained by increased level of TGF-β1 in PMF CD34+ cells. [Display omitted] DisclosuresVannucchi:Novartis: Honoraria, Speakers Bureau; Shire: Speakers Bureau.

Selective Inhibition of Nucleocytoplasmic Transport Overcomes Ruxolitinib Resistance in Myelofibrosis

Myelofibrosis (MF) is a stem cell-derived hematopoietic malignancy characterized by constitutive activation of JAK/STAT signaling through mutations in JAK2, calreticulin or MPL. MF patients have reduced life expectancy due to cytopenias, progression to acute myeloid leukemia and thromboembolic events. JAK kinase inhibitors such as ruxolitinib reduce constitutive symptoms but are not curative, with persistence of malignant cells. Allogeneic stem cell transplant remains the only curative therapy for MF, but is associated with considerable morbidity and mortality, defining an urgent need for new therapies that induce more profound and durable responses.We used a barcoded lentiviral shRNA library to identify survival-critical genes for MF in HEL cells (homozygous for JAK2V617F), established from a patient with erythroleukemia. shRNAs targeting RAN and binding protein (RANBP) were significantly depleted, implicating nucleocytoplasmic transport (NCT) as one of the top survival-critical pathways. RAN is a small GTPase, whose cycling between GTP- and GDP-bound forms provides the engine for NCT. We previously presented results showing that shRNA-mediated knockdown of RAN, significantly reduced proliferation and induced apoptosis in HEL and SET-2 cells (harboring heterozygous JAK2V617F). Additionally, KPT-330 (selinexor, Karyopharm Therapeutics), an inhibitor of CRM1, the core component of NCT, also inhibited proliferation of HEL and SET-2 cells and colony formation by primary MF CD34+ cells, but not cord blood (CB). Importantly, inhibition of NCT was effective at reducing colony formation in samples from ruxolitinib-refractory MF patients, regardless of mutation status. A clinical trial to evaluate KPT-330 in ruxolitinib-refractory MF is in preparation.KPT-8602 is a second-generation inhibitor of CRM-1. Unlike KPT-330, KPT-8602 does not cross the brain-blood barrier, resulting in minimal anorexia, malaise, and weight loss [Etchin et al., Leukemia, 2017 Jan;31(1):143-150]. To determine the efficacy of KPT-8602 in MF, we tested KPT-8602 in cell lines, patient samples, and a mouse model of MF, along with ruxolitinib as comparison.In cell proliferation assays, KPT-8602 was five-fold more potent than ruxolitinib against HEL cells (IC50: 83 nM vs. 437 nM) and as potent as ruxolitinib in SET-2 cells (IC50: 95 nM vs. 85 nM). In clonogenic assays, KPT-8602 showed selectivity toward primary MF vs. CB CD34+ cells (IC50: 25 nM for MF vs. 258 nM for CB) and, in combination with ruxolitinib, resulted in a 95% reduction in colony formation. KPT-8602 inhibited colony formation by CD34+ cells from all MF patients tested, including ruxolitinib-refractory cases, irrespective of mutation status (including triple negative).To evaluate the possible use of KPT-8602 in vivo, we used a mouse transduction/transplantation model of JAK2V617F - positive myeloproliferative neoplasm (MPN). In this model, mice develop MPN resembling polycythemia vera that progresses to MF [Bumm et al., Cancer Res., 2006, 1;66(23):11156-65]. MPN was evident based on elevated blood counts and blood GFP+ cells (representing JAK2V617F allelic burden). MPN mice were randomized into four groups: vehicles, KPT-8602 (initial dose 15 mg/kg daily, orally), ruxolitinib (initial dose 50 mg/kg twice daily, orally) or combined ruxolitinib with KPT-8602 at these same concentrations, and treated for 28 days. KPT-8602 as a single agent reduced red blood cells (RBC), white blood cells (WBC), BM GFP+ cells, peripheral blood (PB) GFP+ cells and spleen size to the same extent as ruxolitinib, and the combination was no more effective than either single agent (Table 1). Spleen cytoarchitecture was fully restored by KPT-8602, but bone marrow fibrosis was not rectified in any treatment group. Mice in all groups, including the combined vehicle controls, still experienced weight loss and lethality, suggesting that toxicity may be due to vehicles, and that optimization of drug formulations and dosing is warranted.Overall, these results indicate that NCT is critical for MF survival, and that NCT inhibition may provide therapeutic benefit for MF patients. KPT-8602 is being tested in a clinical trial (NCT02649790) for refractory multiple myeloma. The results of our study provide strong rationale for a clinical trial in patients with refractory MF. [Display omitted] DisclosuresBaloglu:Karyopharm Therapeutics Inc: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Deininger:Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding; Incyte: Consultancy; Pfizer: Consultancy; Celgene: Research Funding; ARIAD: Consultancy; Gilead: Research Funding; BMS: Consultancy, Research Funding.

Impact of the rs1024611 Polymorphism of CCL2 on the Pathophysiology and Outcome of Primary Myelofibrosis.

Simple SummaryAmong myeloproliferative neoplasms, primary myelofibrosis (PMF) is considered the paradigm of inflammation-related cancer development. Host genetic variants such as single nucleotide polymorphisms (SNPs) can affect cytokine/chemokine gene expression and may therefore have a role in a disease with a strong inflammatory component such as PMF. Here we demonstrate that the homozygosity for the rs1024611 SNP of the chemokine CCL2 represents a high-risk variant and a novel host genetic determinant of reduced survival in PMF, providing opportunities for CCL2 SNP genotyping as a potential novel strategy to risk-stratify patients. The rs1024611 genotype also influences CCL2 production in PMF cells, which are electively sensitive to CCL2 effects because of their unique expression of its receptor CCR2. Finally, ruxolitinib is capable of effectively down-regulating CCR2 expression, de-sensitizing PMF cells to the IL-1β-dependent pro-inflammatory stimulus.Single nucleotide polymorphisms (SNPs) can modify the individual pro-inflammatory background and may therefore have relevant implications in the MPN setting, typified by aberrant cytokine production. In a cohort of 773 primary myelofibrosis (PMF), we determined the contribution of the rs1024611 SNP of CCL2—one of the most potent immunomodulatory chemokines—to the clinical and biological characteristics of the disease, demonstrating that male subjects carrying the homozygous genotype G/G had an increased risk of PMF and that, among PMF patients, the G/G genotype is an independent prognostic factor for reduced overall survival. Functional characterization of the SNP and the CCL2-CCR2 axis in PMF showed that i) homozygous PMF cells are the highest chemokine producers as compared to the other genotypes; ii) PMF CD34+ cells are a selective target of CCL2, since they uniquely express CCR2 (CCL2 receptor); iii) activation of the CCL2-CCR2 axis boosts pro-survival signals induced by driver mutations via Akt phosphorylation; iv) ruxolitinib effectively counteracts CCL2 production and down-regulates CCR2 expression in PMF cells. In conclusion, the identification of the role of the CCL2/CCR2 chemokine system in PMF adds a novel element to the pathophysiological picture of the disease, with clinical and therapeutic implications.

PF655 PROGNOSTIC RELEVANCE OF THE GENE EXPRESSION SIGNATURE IN PRIMARY AND SECONDARY MYELOFIBROSIS

Background: Primary myelofibrosis (PMF) together with polycythemia vera (PV) and essential thrombocythemia (ET) belongs to the classic Philadelphia-negative myeloproliferative neoplasms (MPNs). PV and ET can evolve to secondary myelofibrosis (SMF) giving rise to post-PV (PPV) and post-ET (PET) myelofibrosis (MF). PMF and SMF patients are currently managed in the same way and prediction of survival is based on the same prognostic models, even if it has been demonstrated that they can’t accurately distinguish different risk categories in SMF. In the last few years interest grew concerning the ability of gene expression profiling (GEP) to provide valuable prognostic information for clinical decision making. Several studies demonstrated that GEP can improve risk classification in hematologic malignancies such as AML, MDS and lymphoma. Aims: As a preliminary result we performed GEP in PMF and SMF CD34+ cells and identified a gene signature that was able to distinguish patients with a better prognosis from that with a worse one. To confirm these results and to ease the possible clinical use of a molecular signature based on gene expression we decided to move to a more accessible cell population. Methods: Granulocytes were isolated from 114 MF patients (35 prefibrotic/early PMF, 37 overt PMF, 26 PET and 16 PPV) and total cellular RNA was extracted. Gene expression profiling (GEP) was performed using Affymetrix platform. In order to identify genes whose expression is related to survival in MF patients, we performed a Cox regression analysis by means of Partek GS Software. Results: Cox regression analysis led to the identification of a list of 650 transcripts which discriminate high-risk (HR) MF patients from low-risk (LR) ones. According to our results, the frequency of deceased patients was increased in the HR group and survival curves demonstrated that the median overall survival was lower in the HR group compared to the LR one (3.25 y vs 6.88 y, Log-rank p-value <0.01). Moreover, we observed that 11 out of 13 AML transformed patients clustered within the HR group. Interestingly, we found that HR group was enriched in patients carrying at least one high molecular risk mutation (EZH2, ASXL1, IDH1/2, SRSF2), while there was no significant difference between the two groups regarding the disease and the driver mutation. Concerning clinical variables, we observed that median age at diagnosis was higher in the HR group (65.6 y vs 62.1 y, p-value <0.05). WBC count was increased in the HR group (12.9 x109/L vs 9.3 x109/L, p-value <0.05), while platelet count was decreased in the same group compared to LR ones (218 x109/L vs 352 x109/L, p-value <0.01). Moreover, the percentage of patients with more than 1% circulating blasts is greater in the HR group. Finally, we studied the distribution of samples classified according to DIPSS prognostic model. Strikingly, our gene signature classifies as HR several patients belonging to the DIPSS low and intermediate-1 categories. These patients are deceased or leukemia transformed into a shorter time frame than the median survival reported for the reference prognostic class. Summary/Conclusion: Our results demonstrate that GEP of primary MF cells colud be a useful tool for risk prediction in PMF and SMF since it can improve the identification of patients’ subgroups characterized by a poor prognosis. These results should be validated in independent patient cohorts in order to confirm their predictive power.

Nuclear-Cytoplasmic Transport Is a Therapeutic Target in Myelofibrosis.

Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis. A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear-cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor). JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo. Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.

Role of TGF-β1/miR-382-5p/SOD2 axis in the induction of oxidative stress in CD34+ cells from primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by an excessive production of pro‐inflammatory cytokines resulting in chronic inflammation and genomic instability. Besides the driver mutations in JAK2,MPL, and CALR genes, the deregulation of miRNA expression may also contribute to the pathogenesis of PMF. To this end, we recently reported the upregulation of miR‐382‐5p in PMF CD34+ cells. In order to unveil the mechanistic details of the role of miR‐382‐5p in pathogenesis of PMF, we performed gene expression profiling of CD34+ cells overexpressing miR‐382‐5p. Among the downregulated genes, we identified superoxide dismutase 2 (SOD2), which is a predicted target of miR‐382‐5p. Subsequently, we confirmed miR‐382‐5p/SOD2 interaction by luciferase assay and we showed that miR‐382‐5p overexpression in CD34+ cells causes the decrease in SOD2 activity leading to reactive oxygen species (ROS) accumulation and oxidative DNA damage. In addition, our data indicate that inhibition of miR‐382‐5p in PMF CD34+ cells restores SOD2 function, induces ROS disposal, and reduces DNA oxidation. Since the pro‐inflammatory cytokine transforming growth factor‐β1 (TGF‐β1) is a key player in PMF pathogenesis, we further investigated the effect of TGF‐β1 on ROS and miR‐382‐5p levels. Our data showed that TGF‐β1 treatment enhances miR‐382‐5p expression and reduces SOD2 activity leading to ROS accumulation. Finally, inhibition of TGF‐β1 signaling in PMF CD34+ cells by galunisertib significantly reduced miR‐382‐5p expression and ROS accumulation and restored SOD2 activity. As a whole, this study reports that TGF‐β1/miR‐382‐5p/SOD2 axis deregulation in PMF cells is linked to ROS overproduction that may contribute to enhanced oxidative stress and inflammation. Our results suggest that galunisertib may represent an effective drug reducing abnormal oxidative stress induced by TGF‐β1 in PMF patients.Database linkingGEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103464.

Involvement of MAF/SPP1 axis in the development of bone marrow fibrosis in PMF patients

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.

MiR-494-3p overexpression promotes megakaryocytopoiesis in primary myelofibrosis hematopoietic stem/progenitor cells by targeting SOCS6.

Primary myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by hematopoietic stem cell-derived clonal myeloproliferation, involving especially the megakaryocyte lineage. To better characterize how the altered expression of microRNAs might contribute to PMF pathogenesis, we have previously performed the integrative analysis of gene and microRNA expression profiles of PMF hematopoietic stem/progenitor cells (HSPCs), which allowed us to identify miR-494-3p as the upregulated microRNA predicted to target the highest number of downregulated mRNAs.To elucidate the role of miR-494-3p in hematopoietic differentiation, in the present study we demonstrated that miR-494-3p enforced expression in normal HSPCs promotes megakaryocytopoiesis. Gene expression profiling upon miR-494-3p overexpression allowed the identification of genes commonly downregulated both after microRNA overexpression and in PMF CD34+ cells. Among them, suppressor of cytokine signaling 6 (SOCS6) was confirmed to be a miR-494-3p target by luciferase assay. Western blot analysis showed reduced level of SOCS6 protein as well as STAT3 activation in miR-494-3p overexpressing cells. Furthermore, transient inhibition of SOCS6 expression in HSPCs demonstrated that SOCS6 silencing stimulates megakaryocytopoiesis, mimicking the phenotypic effects observed upon miR-494-3p overexpression. Finally, to disclose the contribution of miR-494-3p upregulation to PMF pathogenesis, we performed inhibition experiments in PMF HSPCs, which showed that miR-494-3p silencing led to SOCS6 upregulation and impaired megakaryocyte differentiation.Taken together, our results describe for the first time the role of miR-494-3p during normal HSPC differentiation and suggest that its increased expression, and the subsequent downregulation of its target SOCS6, might contribute to the megakaryocyte hyperplasia commonly observed in PMF patients.

Role of miR-34a-5p in Hematopoietic Progenitor Cells Proliferation and Fate Decision: Novel Insights into the Pathogenesis of Primary Myelofibrosis.

Primary Myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by a skewed megakaryopoiesis and an overproduction of proinflammatory and profibrotic mediators that lead to the development of bone marrow (BM) fibrosis. Since we recently uncovered the upregulation of miR-34a-5p in PMF CD34+ hematopoietic progenitor cells (HPCs), in order to elucidate its role in PMF pathogenesis here we unravelled the effects of miR-34a-5p overexpression in HPCs. We showed that enforced expression of miR-34a-5p partially constrains proliferation and favours the megakaryocyte and monocyte/macrophage commitment of HPCs. Interestingly, we identified lymphoid enhancer-binding factor 1 (LEF1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts as miR-34a-5p-targets downregulated after miR-34a-5p overexpression in HPCs as well as in PMF CD34+ cells. Remarkably, the knockdown of NR4A2 in HPCs mimicked the antiproliferative effects of miR-34a-5p overexpression, while the silencing of LEF1 phenocopied the effects of miR-34a-5p overexpression on HPCs lineage choice, by favouring the megakaryocyte and monocyte/macrophage commitment. Collectively our data unravel the role of miR-34a-5p in HPCs fate decision and suggest that the increased expression of miR-34a-5p in PMF HPCs could be important for the skewing of megakaryopoiesis and the production of monocytes, that are key players in BM fibrosis in PMF patients.

Selective Inhibition of Nuclear Cytoplasmic Transport As a New Treatment Paradigm in Myelofibrosis

▪Myelofibrosis (MF) is a hematopoietic stem cell neoplasm characterized by constitutive activation of JAK/STAT signaling due to mutations in JAK2, calreticulin or MPL. Many MF patients suffer from severe constitutional symptoms and have reduced life expectancy due to cytopenias, progression to acute myeloid leukemia and thromboembolic events. JAK kinase inhibitors such as ruxolitinib (RUX) reduce MF symptoms, but like all other drugs used in MF, are not curative, with persistence of mutant cells and prompt symptom rebound upon discontinuation. This defines a clinical need to identify strategies capable of inducing more profound and durable responses in MF.To identify previously unrecognized molecular vulnerabilities in MF, we infected HEL cells (homozygous for JAK2V617F) with a barcoded lentiviral shRNA library targeting ~5,000 human signal transduction genes, with 5-6 shRNAs/gene (Cellecta Human Module 1). Conditions were optimized to achieve a multitude of infection (MOI) of ~1. Barcode abundance was compared between days 0 and 9 after infection by next generation sequencing. Candidates were selected based on ≥ 15-fold reduction of abundance by ≥ 2 shRNAs targeting the same gene, similar to Khorashad et al. [Blood. 2015;125(11):1772-81]. Amongst the genes meeting these criteria, nuclear cytoplasmic transport (NCT) was significantly enriched, with RAN and RANBP2 amongst the top genes, suggesting that HEL cells may be highly dependent on NCT. For confirmation, HEL cells were stably transduced with doxycycline (DOX)-inducible shRAN. After 72 hours DOX-induced knockdown of RAN reduced viable cells by 77.3±5.5% and colony formation by 82.8±1.3% and dramatically increased apoptosis (uninduced: ~10% vs. induced: ~50%). Similar results were observed in SET-2 cells (heterozygous for JAK2V617F). We next cultured HEL and SET-2 cells with graded concentrations of the KPT-330 (selinexor, Karyopharm), an inhibitor of CRM-1, the core component of NCT, or RUX as a comparison. Selinexor was five-fold more potent than RUX against HEL cells (IC50: 98nM for KPT vs. 536 nM for RUX) and as potent as RUX in SET-2 cells (IC50:~100 nM). Importantly, RUX-resistant HEL cells (IC50:24µM) were highly sensitive to inhibition of NCT by knockdown of RAN or selinexor (IC50:160nM). Selinexor also selectively inhibited colony formation by primary MF vs. cord blood (CB) CD34+ cells (IC50:93nM for MF vs. 203nM for CB). Lastly, selinexor enhanced RUX-induced growth inhibition and apoptosis in primary MF CD34+cells cultured ex vivo for 72h (including both JAK2 mutation positive and negative MF samples, n=3 for each, and RUX resistant patient samples, n=6). Nuclear:cytoplasmic fractionation of HEL cells revealed that the expression and nuclear localization of the tumor suppressors FoxO3A and APC, but not of PP2A and nucleophosmin (NPM) were significantly increased upon knockdown of RAN, which may contribute to the increased apoptosis following NCT inhibition.To determine the in vivo effects of selinexor in MF, we induced MPN in Balb/c mice by transplanting donor marrow infected with JAK2V617F for three weeks, and then treated mice (n=13/group) with vehicle, selinexor (initial dose 20 mg/kg, 3x weekly, orally) or RUX (initial dose 50 mg/kg twice daily, orally) or the combination of RUX plus selinexor for up to 4 weeks. Combination treatment significantly reduced white blood cell counts and normalized spleen size. Compared to vehicle, selinexor alone significantly reduced GFP+cells in the spleen, and this effect was further enhanced with the combination treatment. Histopathology revealed that combination treatment restored splenic architecture, while bone marrow fibrosis was not significantly altered by selinexor or the combination. Mice in all groups, including the combined vehicle controls, experienced considerable weight loss, suggesting that toxicity may be partially due to high dose and frequent drug administration. Experiments with the next generation NCT inhibitor KPT-8602 [Etchin et al., Leukemia, 2016 Jun 24] are underway.In summary, our results suggest that MF cells are exquisitely dependent on NCT, and that NCT inhibition alone or in combination with RUX may reduce JAK2V617F allelic burden. This identifies NCT as a prime therapeutic target in MF. A phase I clinical trial of selinexor in refractory MF is in preparation. DisclosuresBaloglu:Karyopharm Therapeutics: Employment, Equity Ownership. Deininger:BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

MiR-494-3p Overexpression Leads to SOCS6 Downregulation and Supports Megakaryocytopoiesis in Primary Myelofibrosis CD34+ Hematopoietic Stem/Progenitor Cells

Primary Myelofibrosis (PMF) belongs to the Philadelphia negative Myeloproliferative Neoplasms (MPNs) and is characterized by hematopoietic stem-cell derived clonal myeloproliferation, involving especially megakaryocyte (MK) lineage, bone marrow fibrosis and extramedullary hematopoiesis. Recent studies have suggested that alterations in miRNAs expression could play a critical role in MPN's pathogenesis. In order to shed some light on this issue, we have previously performed the integrative analysis (IA) of gene and miRNA expression profiles of PMF CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 42 PMF patients compared with 31 healthy donors (R. Norfo et al., Blood, 2014). IA identified miR-494-3p as one of the most upregulated miRNAs in PMF CD34+ cells associated to the highest number of downregulated predicted targets (eighty-six, Fig. 1).In order to study the role of miR-494-3p in hematopoietic commitment and differentiation, and to elucidate its possible involvement in PMF pathogenesis, we performed miRNA overexpression experiments in cord blood (CB) CD34+ cells through miRNA mimic electroporation. The data showed that miR-494-3p promotes MK differentiation of HSPCs. Indeed, the fraction of cells expressing MK surface antigen CD41 was steadily increased in miR-494-3p overexpressing samples compared with controls (75.4±0.3% vs 53.2±3.5% at day 8, 82.6±1.3% vs 60.4±4.3% at day 10 of culture, p<0.05), as well as the percentage of cells expressing the late MK antigen CD42b was similarly increased. Furthermore, the percentage of MK colonies was increased in collagen-based clonogenic assay upon miR-494-3p overexpression compared to control (44.8±4.1% vs 24.1±2.1%, p<0.05). Next, to better characterize the molecular mechanisms underlying megakaryocytopoiesis stimulation by miR-494-3p, we profiled CB CD34+ cells overexpressing this miRNA using the Affymetrix HG-U219 microarray platform. Gene Expression profile analysis allowed the identification of 134 differentially expressed genes between cells overexpressing miR-494-3p and controls. In particular, we highlighted the presence of 13 genes downregulated both upon miRNA overexpression and in PMF CD34+ cells. Among them, Suppressor of Cytokine Signaling 6 (SOCS6) turned out to be the miR-494-3p predicted target associated to the most favorable prediction scores according to TargetScan, microRNA.org and miRDB prediction algorithms. Furthermore, 3’ UTR luciferase reporter assays, performed in K562 cell line, proved the predicted interaction between miR-494-3p and SOCS6 3’UTR. Subsequently, we studied the role of SOCS6 in HSPCs differentiation by inhibiting its expression in CB CD34+ cells through small interfering RNAs. SOCS6 silencing stimulated megakaryocytopoiesis in CB CD34+ cells, as demonstrated by the expansion of CD41+ and CD42b+ cell fractions in SOCS6 silenced samples compared with controls (52.8±7.0% vs 37.7±4.5% at day 8, 66.9±7.2% vs 50.7±7.2% at day 10 for CD41+ cells, p<0.05). Moreover, MK colonies were increased upon SOCS6 silencing in collagen-based clonogenic assays (62.4±7.7% vs 51.3±6.5%, p<0.05) and morphological analysis further supported these results. Finally, in order to study the possible contribution of miR-494-3p overexpression to PMF pathogenesis, we performed inhibition experiments in PMF CD34+ cells by means of miR-494-3p antagomiR. As a result, miR-494-3p silencing led to SOCS6 upregulation and impaired MK differentiation in PMF HSPCs as demonstrated by the decrease in CD41+ cell fraction in silenced samples compared with controls (28.6±7.1% vs 39.2±7.7% at day 12 of culture, p<0.05) and by the reduction of MK colonies in collagen-based clonogenic assay (44.4±3.6% vs 54.7±2.5%, p<0.05). The values are reported as mean±S.E.M from 3 independent experiments.Taken together, our results showed that miR-494-3p overexpression promotes megakaryocytopoiesis in HSPCs. Moreover, we demonstrated for the first time that SOCS6 is a direct target of miR-494-3p. Since SOCS6 downregulation promotes MK differentiation of HSPCs, SOCS6 could be considered, at least in part, responsible for the biological effects observed after miR-494-3p overexpression. As miR-494-3p and SOCS6 showed the same expression trend in PMF CD34+ cells, our results could suggest that miR-494-3p/SOCS6 axis is involved in the induction of MK hyperplasia typically observed in PMF patients. [Display omitted] DisclosuresVannucchi:Novartis: Consultancy, Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Shire: Speakers Bureau.

MAF Induces Inflammatory Mediators Involved in the Pathogenesis of Primary Myelofibrosis

Primary Myelofibrosis (PMF) is a Philadelphia-negative myeloproliferative neoplasm characterized by the presence of hyperplastic/dysplastic megakaryocytes and the development of myelofibrosis and osteosclerosis. This disruption of the bone marrow (BM) structure results from a deregulated release of growth factors by malignant hematopoietic cells, particularly monocytes and megakaryocytes, that activate BM stromal cells (Desterke-C, Mediators Inflamm, 2015). Therefore, it remains of primary importance to identify novel therapeutic strategies to restore BM microenvironment homeostasis in PMF patients.In order to better characterize the molecular mechanisms underlying PMF pathogenesis, we have recently compared the gene expression profile (GEP) of CD34+ hematopoietic progenitor cells (HPCs) from PMF patients and healthy donors, and we observed the upregulation of the transcription factor MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) (Norfo-R, Blood, 2014).With the aim to investigate the role of MAF in PMF pathogenesis and to reproduce the condition observed in PMF CD34+ cells, we studied the effects of MAF overexpression on HPCs differentiation. Human cord blood-derived CD34+ cells were transduced with a retroviral vector carrying the coding sequences for the two MAF transcript variants (LMAFVAR1IDN and LMAFVAR2IDN). The megakaryocyte lineage-committed CD41+ population was increased in LMAFVAR1IDN (38.2±2.3%) and LMAFVAR2IDN (39.4±2.3%) samples compared with the empty vector LXIDN (16.1±2.7%) at day 3 after NGFR+ cells purification. Values are reported as mean ± SEM from 3 independent experiments, p<.05. Moreover, MAF overexpression determined a remarkable increase of the CD14+ monocyte population in LMAFVAR1IDN (45.5±5.7%) and LMAFVAR2IDN (43.1±8.7%) compared to LXIDN control (8.0±0.4%) at t0 post-purification. Data represent mean ± SEM from 3 independent experiments, p<.05.Next, we analyzed the GEP of MAF-overexpressing CD34+ cells and observed the upregulation of genes involved in megakaryopoiesis, such as MERTK and RCAN1, and in monocyte/macrophage differentiation (e.g. EGR2 and KLF4). In particular, GEP revealed the upregulation of genes related to processes deregulated in PMF, such as fibrosis (i.e. ENPP2, ADAM9) and inflammation (i.e. TNF, CCL2 and CCL3). Noteworthy, several upregulated transcripts encoded for secreted proteins.Based on their role in inflammation and fibrosis, we selected a set of secreted molecules (IL8, MMP9, CCL2, SPP1, LGALS3 and PLAUR) and assessed them by quantitative enzyme-linked immunoassay (ELISA) in the culture supernatants from LMAFVAR1IDN-, LMAFVAR2IDN- and LXIDN-transduced cells. The levels in culture supernatants of all these molecules were increased in MAF-overexpressing samples compared to control. In order to correlate the expression of these molecules with MAF upregulation in PMF, we assessed them in plasma samples of PMF patients (n=30) and healthy donors (n=10). All the selected molecules displayed higher plasma levels in PMF patients than in healthy controls (Figure 1).Since the increase of SPP1 and LGALS3 has never been described before in PMF, we investigated which cell type could be responsible for SPP1 and LGALS3 production. Real Time qRTPCR performed on healthy donors peripheral blood-derived cells showed that the highest SPP1 expression levels were observed in CD34+ cells-derived megakaryocytes and granulocytes, whereas LGALS3 was expressed at higher levels in monocytes compared to the other cell types.Because the increased proliferation of fibroblasts is involved in BM fibrosis and is a PMF feature, we investigated the effects of LGALS3 and SPP1 on normal human primary dermal fibroblasts. The treatment with recombinant human LGALS3 (1000 ng/ml) or SPP1 (500 ng/ml) caused an increase in fibroblasts cell count of 20.9±2.7% (p<.001) and 14.9±4.1% (p<.01) compared to the untreated control, respectively. Values are reported as mean ± SEM from 7 independent experiments.Finally, we observed that increased SPP1 plasma levels in PMF patients correlate with a more severe fibrosis and lower overall survival (p<.05).In conclusion, our data unveil the involvement of MAF in PMF pathogenesis through the induction of a pro-fibrotic and pro-inflammatory phenotype in the malignant myeloid differentiated progeny. [Display omitted] DisclosuresVannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

The Cytotoxic Effect of the PARP Inhibitor ABT-888 on Myeloproliferative Neoplasms

▪DNA instability, including increased DNA double strand breaks, has recently been demonstrated in myeloproliferative neoplasms (MPN). Because PARP1 has a central role in DNA repair and maintaining genomic integrity, we tested whether DNA repair inhibition with the PARP inhibitor ABT-888 (Abbvie, Chicago, IL) could affect the growth of myeloid neoplasms in-vitro and in-vivo, and/or enhance the cytotoxic effect of a standard alkylating agent commonly used in preparative regimens for stem cell transplants.Since the JAK2V617Fmutation is common in chronic MPNs or acute myeloid leukemia (AML) secondary to MPNs, we initially utilized JAK2 mutated AML cell lines (SET2 and HEL) in-vitro and in-vivo. We then tested the effect of PARP inhibition on CD34+ cells obtained from patients with primary myelofibrosis.JAK2 mutated SET2 and HEL cells were treated with ABT-888 in liquid cultures. Both cell lines were relatively sensitive with IC50 of 11.3μM and 74.2μM respectively. When the cells were treated with a combination of increasing doses of busulfan and 8μM ABT-888, significant synergy was observed, with busulfan IC50 decreasing from 27μM to 4μM in SET2 cells and from 45.1μM to 28.1μM in HEL cells. Treatment of SET2 cells with 8μM ABT-888, or with 8μM ABT888 and 10μM busulfan, was also accompanied by marked increase in γH2AX foci compared to control at 24 hours. We subsequently showed that ABT-888, busulfan and combination treatments resulted in increasing numbers of cells in G2/M arrest (30% vs. 35% vs 53% respectively, p=0.002). Western blot analysis of cells in G/2M arrest after combination treatment demonstrated a strongly increased phosphorylation of checkpoint kinase 1 (Chk1). In an immunofluorescence assay on treated cells we then showed that activated Chk1 translocated to the nucleus, suggesting that the Chk1-ATR pathway may be responsible for cell cycle arrest. To demonstrate the in-vivo effect of PARP inhibition, we transplanted 5x106 SET2 cells into a NOD/SCID/ gamma null mice to create a xenograft model. Mice developed AML with splenomegaly and bone marrow engraftment resulting in a median survival of 36 days. In mice that were treated with daily intraperitoneal injections of low dose ABT-888 (1.5mg/kg), starting 14 days after leukemic cell injection, survival increased to a median of 40 days (p=0.002).We subsequently tested the effect of PARP inhibition on primary myelofibrosis CD34+ cells obtained from the bone marrow or peripheral blood of 3 patients with JAK2V617Fmutation and 2 with CALR mutation. Control experiments were performed with normal CD34+ progenitors. In a standard clonogenic assay in methylcellulose, cells were plated with or without 4μM ABT-888, 5μM busulfan or a combination of both. Compared to untreated cells, ABT-888, busulfan and the combination of the two drugs reduced colony formation by 39%, 70% and 89%, respectively (p=0.01). Both CALR and JAK2 mutated cells were sensitive to ABT-888. On the contrary, treatment of normal CD34+ cells with ABT-888 did not affect colony growth.Here we describe the potential for PARP inhibition to exploit the genomic instability of MPNs. Potential clinical applications include the addition of PARP inhibition to busulfan in pretransplant conditioning or novel drug combinations including PARP inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Integrative Analysis of Copy Number and Gene Expression Data Suggests Novel Pathogenetic Mechanisms in Primary Myelofibrosis

Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, we integrated gene expression and copy number (CN) signals and identified several genomic abnormalities leading to a concordant alteration in gene expression levels. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus is accompanied by a coordinated transcriptional up-regulation in PMF patients. To assess the impact of PAOX deregulation on the survival of PMF and normal primary hematopoietic cells, PMF and normal donors cells were incubated with increasing doses of the PAOX inhibitor MDL-72,572. PAOX inhibition resulted in rapid cell death of PMF progenitor cells (5,7±0,9% of late apoptotic cells in NT sample vs 20,1±3,2% in 150μM treated-sample, p<0.05), while survival of normal CD34+ cells was not affected (0,97±0,09 of late apoptotic cells in NT sample vs 2,7±0,4 in 150μM treated-sample, p<0.05), as monitored by Annexin V/PI staining. These data suggest that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival.Moreover, our integrative analysis of gene expression and CN data pointed out the concomitant copy number loss and transcriptional down-regulation in PMF patients of the chromatin modifier HMGXB4. Interestingly, silencing of HMGXB4 in CD34+ stem/progenitor cells induced megakaryocyte differentiation, as demonstrated by increased expression of CD41 (36±1,9% vs 26,1±2,9% at day 12, 52,5±4,9% vs 33±1% at day 14 of serum free liquid culture, p<0.05) and increased number of CFU-MK colonies (27,7±2,5 vs 18,1±1,3% in small CFU-MK colonies, p<0.05). On the other hand, HMGXB4 silencing repressed the erythroid differentiation, as indicated by a decrease in the percentage of cells positive for the erythroid marker GPA (10.8%±5.6 vs 24.8%±5, p<0.05) and significant decrease in Burst Forming Unit-Erythroid (BFU-E) and Colony-Forming Unit-Erythroid (CFU-E) (47,9±6 vs 60,5±7,6, p<0.05). Taken together, these data suggest that HMGXB4 silencing in human HSPCs favors megakaryocyte differentiation while restraining the erythroid lineage. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterize PMF patients.In conclusion, our work sheds light on the influence of genomic abnormalities on gene expression regulation in PMF CD34+ cells and on their impact to features typical of PMF, such as a hyperplastic megakaryopoiesis and resistance to apoptosis, and therefore potentially contributing to the development of the myeloproliferative disease. DisclosuresVannucchi:Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Small RNA Sequencing Uncovers New miRNAs and moRNAs Differentially Expressed in Normal and Primary Myelofibrosis CD34+ Cells

Myeloproliferative neoplasms (MPN) are chronic myeloid cancers thought to arise at the level of CD34+ hematopoietic stem/progenitor cells. They include essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). All can progress to acute leukemia, but PMF carries the worst prognosis. Increasing evidences indicate that deregulation of microRNAs (miRNAs) might plays an important role in hematologic malignancies, including MPN. To attain deeper knowledge of short RNAs (sRNAs) expression pattern in CD34+ cells and of their possible role in mediating post-transcriptional regulation in PMF, we sequenced with Illumina HiSeq2000 technology CD34+ cells from healthy subjects and PMF patients. We detected the expression of 784 known miRNAs, with a prevalence of miRNA up-regulation in PMF samples, and discovered 34 new miRNAs and 99 new miRNA-offset RNAs (moRNAs), in CD34+ cells. Thirty-seven small RNAs were differentially expressed in PMF patients compared with healthy subjects, according to microRNA sequencing data. Five miRNAs (miR-10b-5p, miR-19b-3p, miR-29a-3p, miR-379-5p, and miR-543) were deregulated also in PMF granulocytes. Moreover, 3’-moR-128-2 resulted consistently downregulated in PMF according to RNA-seq and qRT-PCR data both in CD34+ cells and granulocytes. Target predictions of these validated small RNAs de-regulated in PMF and functional enrichment analyses highlighted many interesting pathways involved in tumor development and progression, such as signaling by FGFR and DAP12 and Oncogene Induced Senescence. As a whole, data obtained in this study deepened the knowledge of miRNAs and moRNAs altered expression in PMF CD34+ cells and allowed to identify and validate a specific small RNA profile that distinguishes PMF granulocytes from those of normal subjects. We thus provided new information regarding the possible role of miRNAs and, specifically, of new moRNAs in this disease.

Dysregulation of the Let-7/HMGA2 Axis with Methylation of p16 Promoter As a Possible Target of Histone Deacetylase Inhibitor in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are clonal hematological disorders characterized by proliferation of mature blood cells. Recently, several agents that influence epigenetic modifications, such as histone deacetylase inhibitors (HDACi), as well as JAK2 inhibitors, have been investigated for high-risk MPNs. For example, an HDACi, panobinostat has shown significant efficacy including nearly complete response in PMF (Mascarenhas et al, BJH, 2013), but molecular targets of HDACi remain largely unknown. The High Mobility Group AT-hook 2 (HMGA2) is a non-histone chromatin protein that modulates transcriptions of various genes and contributes to chromatin modification and epigenetic regulation including DNA methylation (Fusco et al, Nat Rev Cancer, 2007; Sun et al, PNAS 2013). Let-7 micro RNAs (miRNAs) negatively regulate expression of HMGA2 through 3’UTR of HMGA2 mRNA, although HMGA2 mRNA consists of both the major variant containing 3’UTR with let-7-specific sites (variant 1) and some minor variants without 3’UTR. We previously reported that overexpression of HMGA2 due to transgenic expression of Hmga2 cDNA without 3’UTR caused proliferative hematopoiesis with providing a clonal advantage to hematopoietic stem cells in mice (Ikeda et al, Blood, 2011). We also showed a deregulation of HMGA2 mRNA expression due to reduced let-7 miRNA level in granulocytes from patients with almost all of PMF and over 20% of PV and ET (Harada-Shirado et al, Blood [Abst], 2013), being associated with splenomegaly, elevated serum LDH values, and methylation of p16 promoter. Thus, we hypothesized that HMGA2 may be a candidate gene as a therapeutic target in MPNs. Since association of HDAC with HMGA2 has been reported in cord blood-derived cells (Lee et al, Cell Mol Life Sci, 2011), we here studied effects of the panobinostat on expressions of HMGA2 and let-7 in HMGA2-expressing myeloid cells including PMF-derived CD34+ cells. First, we found significantly higher HMGA2 mRNA levels in CD34+ cells from 2 PMF patients compared with CD34+ cells from 2 healthy individuals (P<0.001), as well as U937 cells compared with HL60 cells (P<0.001). Thus, we used CD34+ cells from one of these 2 PMF patients and U937 cells for further experiments. Interestingly, treatment with panobinostat at the concentration of 40 nM for 8 hours significantly increased expressions of let-7a (P<0.001 and P=0.003, respectively), -7b (P<0.001 and P<0.001), and -7c (P<0.001 and P=0.06) in U937 cells and PMF CD34+ cells, compared with samples without the treatment. In contrast, Western blotting showed clearly reduced expression of HMGA2 protein in U937 cells after the treatment with panobinostat. Moreover, we found that variant 1 of HMGA2 mRNA with 3’UTR was significantly reduced by the treatment with panobinostat, compared with samples without the treatment in both U937 cells and PMF CD34+ cells (P<0.001 and P<0.001, respectively), while expression levels of variant 2 lacking let-7-specific sites were not changed by the treatment. These findings strongly suggested that panobinostat decreased expression of HMGA2 through 3’UTR of HMGA2 mRNA by increasing expressions of let-7 miRNAs. Of note, we next found much higher expression of variant 1 of HMGA2 mRNA than variant 2 in granulocytes from 15 of 17 (88%) MPN patients whose HMGA2 mRNA levels were higher than controls in our previous study (Blood [Abst], 2013). We also assessed if treatment by panobinostat for the dysregulated let-7/HMGA2 axis may be a therapeutic option for MPNs with respect to DNA methylation. Panobinostat treatment substantially reduced the expressions of DNMT1 and DNMT3a as well as HMGA2 proteins, significantly demethylated the p16 promoter (P<0.001), and decreased survival (P<0.001) in U937 cells. Moreover, knocking-down of HMGA2 with small interfering RNA in U937 cells significantly increased expression of TET3 mRNA and demethylated the p16 promoter, suggesting that HMGA2 expression may contribute to methylation of the p16 promoter. In conclusion, deregulated expression of HMGA2 due to downregulation of let-7 miRNAs, which may lead to some epigenetic modifications such as methylation of the p16 promoter, is a possible therapeutic target of HDACi in MPNs. DisclosuresIkeda:Novartis Pharmaceuticals: Other.

Defect in Ribosome Biogenesis Contributes to Impaired Megakaryopoiesis in Primary Myelofibrosis

Primary myelofibrosis (PMF) is a clonal hematologic malignancy resulting from the transformation of a pluripotent hematopoietic progenitor cell leading to increased hematopoiesis and overproduction of abnormal blood cells. PMF is characterized by bone marrow (BM) fibrosis, extramedullary hematopoiesis, increased numbers of circulating CD34+ cells, splenomegaly, and a propensity to evolve to AML. Patients also display anemia and thrombocytopenia and harbor abnormal, immature megakaryocytes (Mks) in their BM and spleen. Numerous mutations are associated with PMF including JAK2, CALR, MPL, TET2, CBL, ASXL1, and IDH.The presence of abnormal Mks in the BM of PMF patients is an important characteristic of the disease, as these cells are believed to contribute to the fibrosis.Here, we performed an analysis of Mk differentiation from individuals with PMF compared to healthy individuals as a way to better understand the mechanism leading to their defects. CD34+ cells obtained from PMF and healthy donors were cultivated in presence of thrombopoietin to induce Mks differentiation. Under these conditions, we observed that the proliferative capacity of CD34+ cells from PMF patients was increased as compared to normal CD34+ cells. At the same time, the percentage of mature Mks obtained was reduced. Semi-solid culture conditions confirmed the proliferative advantage of the PMF CD34+ cells. To investigate the mechanism responsible for this phenotype, we performed genome wide expression analysis of sorted Mks from patients versus controls. Nine PMF patients with different mutations (5 JAK2V617F, 1 MPLW515L, 2 CALR and 1 triple negative) and 4 healthy donors were included in the study. Gene Set Enrichment Analysis (GSEA) analysis revealed that the top scoring pathways included the RPS14 signature, ribosome biogenesis, rRNA metabolic process, and rRNA processing. We first studied the RPS14 geneset, which consists of a group of genes that were up-regulated in CD34+ cells after knock-down of RPS14, a component of the small ribosome subunit. Using shRNA against RPS14, we discovered that its knock down leads to a significant reduction in the development of mature Mks from normal CD34+ cells.The microarray data also revealed changes in the expression of several myeloid genes. CEBPa and GFI-1 were over-expressed in PMF Mks, whereas GFI-1b and PF4, which are associated with Mk differentiation, were down-regulated. Surprisingly the expression of GATA1, a master regulator of megakaryopoiesis, was not changed. However, consistent with previous report, we discovered that GATA1 protein level was reduced in PMF Mks. GATA1 deficient Mks are impaired in their maturation, so we reasoned that the reduction in GATA1 contributes to the defects in PMF. Indeed, we found that overexpression of GATA1 in PMF CD34+ cells rescued Mk differentiation. To understand how GATA1 protein but not mRNA, is reduced, we knocked down RPS14 in the JAK2 mutant SET2 megakaryoblastic cell line. As predicted, we observed a reduction in GATA1 protein, but not mRNA. By ribosomal profiling, we saw that the shRPS14 led to an abnormal ribosomal profile with a reduction of the 40s peak. Finally, by RT-PCR analysis of the ribosomal and polysomal fractions, we found that GATA1 mRNA was less abundant in the polysomal fraction in cells expressing the shRPS14. This finding is consistent with recent data showing that alterations in ribosomal genes cause a decrease in GATA1 protein in Diamond-Blackfan Anemia.Although we identified the RPS14 gene signature, we did not observe changes in RPS14 expression or in any of the ribosomal subunits. Since our GSEA analysis also identified deficiencies in three ribosomal biogenesis pathways, we looked for genes present in all three signatures. NOP58, a ribonucleoprotein required for ribosome biogenesis, was significantly reduced in all samples of PMF Mks. To link the reduction in NOP58 expression with PMF associated mutations, we expressed MPLW515L or the CALR type II mutant in hematopoietic progenitor cells and detected a reduction in NOP58 expression. Our findings suggest that the activation of JAK/STAT signaling leads to a reduction in NOP58, which contributes to defects in ribosome assembly, decreased GATA1 expression and subsequent impairment in Mk differentiation. This work reveals, for the first time, that defective ribosomes contribute to the pathogenesis of PMF and suggests new avenues for therapeutic intervention. DisclosuresStein:Incyte Corporation: Honoraria, Speakers Bureau; Sanofi Oncology: Honoraria.

MiRNA-mRNA integrative analysis in primary myelofibrosis CD34+ cells: role of miR-155/JARID2 axis in abnormal megakaryopoiesis

AbstractPrimary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte (MK) hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with somatic mutations in JAK2, MPL, or CALR. Previous studies have shown that abnormal MKs play a central role in the pathophysiology of PMF. In this work, we studied both gene and microRNA (miRNA) expression profiles in CD34+ cells from PMF patients. We identified several biomarkers and putative molecular targets such as FGR, LCN2, and OLFM4. By means of miRNA-gene expression integrative analysis, we found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, we identified a network gathering several miRNAs with oncogenic potential (eg, miR-155-5p) and targeted genes whose abnormal function has been previously associated with myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Because the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, we studied the function of this axis in normal and PMF CD34+ cells. We showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41+ MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF.