Myeloproliferative Neoplasms Stem Cells Research Articles

Abstract 263: RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms for dynamic detection of clonal myeloproliferative neoplasm stem cell fitness

Abstract Introduction Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) derived disorders that can progress to acute myeloid leukemia (AML) at variable rates depending on macroenvironmental stressors and microenvironmental inflammatory cytokines. The total symptom score (TSS) and variant allele frequency (VAF) of mutations have proven valuable predictors of therapeutic response. Recently, we discovered inflammatory-cytokine-responsive ADAR1 RNA editing enzyme overexpression during MPN progression. However, dynamic prediction of clonal MPN stem cell fitness and progression to AML remains challenging. Methods We performed longitudinal 150-gene next-generation sequencing (NGS) analyses for 129 MPN patients. We developed single-cell RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms to quantify ADAR1 activity with a novel ADAR1 nanoluc-GFP reporter assay, whole transcriptome RNA editome analyses, and MPN stem cell replating assays. To identify novel RNA editing sites, we performed whole genome sequencing on CD34+ cells from 43 MPN patients and whole transcriptome sequencing on FACS-purified HSC and progenitor cells from 32 MPN patients and 24 non-MPN controls. Results Sequential NGS analysis was performed with a median follow-up of 958 days (range 0-4214; interquartile range (IQR) 314-1350). On average, 6 NGS analyses (range 1-19; IQR 3-9) were performed for each patient. In our cohort, 58.9% of patients were JAK2 V617F+, while mutations in CALR and ASXL1 were observed in 11.6% and 14.0% of cases, respectively. In total, 44.2% (N=57) of patients in our cohort received Inrebic, with 56% reaching the maximum dose of 400mg. Notably, most patients on Inrebic were JAK2 V617F+ (N=38, 67.7%). In JAK2 V617F+ patients on Inrebic, 50% (n=19) experienced a reduction in the JAK2 V617F VAF from their peak value with a median drop of 16% (range 4-57). Our analyses using READAR platforms revealed increased RNA editing levels in JAK2 V617F+ patients from whole transcriptome sequencing analyses compared with JAK2 wild-type patients. Transduction of our ADAR1 nanoluc-GFP reporter into CD34+ cells from MPN patients (N=7), enabled us to quantify ADAR1 activity in an ex vivo setting. Moreover, CD34+ cells from MPN patient samples (n=7) treated in stromal co-cultures with ADAR1 inhibitor, Rebecsinib, demonstrated inhibition of self-renewal in replating assays. A significant reduction in ADAR1-GFP reporter activity was observed in MPN CD34+ cells following Rebecsinib treatment of stromal co-cultures. Also, humanized ADAR1-GFP-luciferase reporter AML mouse models showed a significant reduction in ADAR1 activity and leukemia stem cell self-renewal following Rebecsinib treatment. Conclusion These READAR platform results suggest that ADAR1 activity can be utilized to predict clonal MPN stem cell fitness and progression. Citation Format: Inge van der Werf, Larisa Balaian, Jessica Pham, Wenxue Ma, Athena Mohebbi, Emma Klacking, Antonio Ruiz, Karla Mack, John Mascarenhas, Thomas Whisenant, Ludmil Alexandrov, Sheldon Morris, Catriona Jamieson. RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms for dynamic detection of clonal myeloproliferative neoplasm stem cell fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 263.

Evolving landscape of JAK inhibition in myelofibrosis: monotherapy and combinations.

Myeloproliferative neoplasms (MPNs) are characterized by clonal myeloproliferation in 1 or more of the hematopoietic stem cell lineages. Primary myelofibrosis (MF), post-polycythemia vera MF, and post-essential thrombocythemia MF have the worst prognosis and are characterized by the presence of cytokine-mediated symptom complex, splenomegaly, progressive marrow failure, and clonal instability, leading to leukemic transformation. The key therapeutic aims encompass the management of symptoms, splenomegaly, and anemia and the improvement of survivals. These therapeutic aims have evolved with the availability of Jak inhibitors and novel agents, making disease modification potentially achievable. Novel agents may potentially target MPN stem cells, epigenetic alterations, signaling pathways, and apoptotic pathways. In this case-based review, we outline our approach to the management of MF and discuss the therapeutic landscape of MF, highlighting the utility of Jak inhibitors and novel Jak inhibitor-based combinations.

Phenotypic characterization of disease-initiating stem cells in JAK2- or CALR-mutated myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are characterized by uncontrolled expansion of myeloid cells, disease-related mutations in certain driver-genes including JAK2, CALR, and MPL, and a substantial risk to progress to secondary acute myeloid leukemia (sAML). Although behaving as stem cell neoplasms, little is known about disease-initiating stem cells in MPN. We established the phenotype of putative CD34+ /CD38- stem cells and CD34+ /CD38+ progenitor cells in MPN. A total of 111 patients with MPN suffering from polycythemia vera, essential thrombocythemia, or primary myelofibrosis (PMF) were examined. In almost all patients tested, CD34+ /CD38- stem cells expressed CD33, CD44, CD47, CD52, CD97, CD99, CD105, CD117, CD123, CD133, CD184, CD243, and CD274 (PD-L1). In patients with PMF, MPN stem cells often expressed CD25 and sometimes also CD26 in an aberrant manner. MPN stem cells did not exhibit substantial amounts of CD90, CD273 (PD-L2), CD279 (PD-1), CD366 (TIM-3), CD371 (CLL-1), or IL-1RAP. The phenotype of CD34+ /CD38- stem cells did not change profoundly during progression to sAML. The disease-initiating capacity of putative MPN stem cells was confirmed in NSGS mice. Whereas CD34+ /CD38- MPN cells engrafted in NSGS mice, no substantial engraftment was produced by CD34+ /CD38+ or CD34- cells. The JAK2-targeting drug fedratinib and the BRD4 degrader dBET6 induced apoptosis and suppressed proliferation in MPN stem cells. Together, MPN stem cells display a unique phenotype, including cytokine receptors, immune checkpoint molecules, and other clinically relevant target antigens. Phenotypic characterization of neoplastic stem cells in MPN and sAML should facilitate their enrichment and the development of stem cell-eradicating (curative) therapies.

DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression.

Myeloproliferative neoplasms (MPNs) exhibit a propensity for transformation to secondary acute myeloid leukemia (sAML), for which the underlying mechanisms remain poorly understood, resulting in limited treatment options and dismal clinical outcomes. Here, we performed single-cell RNA sequencing on serial MPN and sAML patient stem and progenitor cells, identifying aberrantly increased expression of DUSP6 underlying disease transformation. Pharmacologic dual-specificity phosphatase (DUSP)6 targeting led to inhibition of S6 and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling while also reducing inflammatory cytokine production. DUSP6 perturbation further inhibited ribosomal S6 kinase (RSK)1, which we identified as a second indispensable candidate associated with poor clinical outcome. Ectopic expression of DUSP6 mediated JAK2-inhibitor resistance and exacerbated disease severity in patient-derived xenograft (PDX) models. Contrastingly, DUSP6 inhibition potently suppressed disease development across Jak2V617F and MPLW515L MPN mouse models and sAML PDXs without inducing toxicity in healthy controls. These findings underscore DUSP6 in driving disease transformation and highlight the DUSP6-RSK1 axis as a vulnerable, druggable pathway in myeloid malignancies.

Continuous Indexing of Fibrosis (CIF): improving the assessment and classification of MPN patients

The grading of fibrosis in myeloproliferative neoplasms (MPN) is an important component of disease classification, prognostication and monitoring. However, current fibrosis grading systems are only semi-quantitative and fail to fully capture sample heterogeneity. To improve the quantitation of reticulin fibrosis, we developed a machine learning approach using bone marrow trephine (BMT) samples (n = 107) from patients diagnosed with MPN or a reactive marrow. The resulting Continuous Indexing of Fibrosis (CIF) enhances the detection and monitoring of fibrosis within BMTs, and aids MPN subtyping. When combined with megakaryocyte feature analysis, CIF discriminates between the frequently challenging differential diagnosis of essential thrombocythemia (ET) and pre-fibrotic myelofibrosis with high predictive accuracy [area under the curve = 0.94]. CIF also shows promise in the identification of MPN patients at risk of disease progression; analysis of samples from 35 patients diagnosed with ET and enrolled in the Primary Thrombocythemia-1 trial identified features predictive of post-ET myelofibrosis (area under the curve = 0.77). In addition to these clinical applications, automated analysis of fibrosis has clear potential to further refine disease classification boundaries and inform future studies of the micro-environmental factors driving disease initiation and progression in MPN and other stem cell disorders.

UBASH3B knockout Downregulates STAT1 Mediated Interferon Signalling and Megakaryocyte Related Genes in a JAK2 V617F Positive MPN Model

Interferon (IFN) signalling via STAT1 plays an important role in the pathogenesis and treatment of myeloproliferative neoplasms (MPN). Single cell sequencing approaches have implicated megakaryocyte (MKC) biased stem cells with enhanced intrinsic activation of IFN signalling pathways in MPN patients as critical mediators of disease initiation and maintenance. UBASH3B is a histidine phosphatase and has been identified as a potential regulator of malignant haematopoiesis and as a mediator of JAK/STAT1 IFN signalling in lymphoid cells. RNA sequencing (RNA-seq) data in JAK2 V617F positive UKE-1, HEL (both own lab) and SET2 (GSE91062) cells demonstrate that UBASH3B is significantly downregulated following administration of the JAK inhibitor ruxolitinib (RUX). We observed that UBASH3B expression in CD34+ cells in patients with primary myelofibrosis (GSE53482) is significantly enhanced but reduced in patients with essential thrombocythaemia(ET) and polycythaemia (GSE103237) and hypothesized that UBASH3B may contribute to MPN pathogenesis. UKE-1 is a JAK2 V617F positive primitive haematopoietic cell line derived from an ET patient. It shows features of intrinsic IFN signalling and expresses genes related to MKC development and differentiation providing a useful model of MPN stem cell behaviour. CRISPR-Cas9 mediated KO of UBASH3B was performed in UKE-1 cells. A ribonucleoprotein complex of guide RNA and Cas9 protein was nucleofected into cells. Individual colonies were assessed for UBASH3B status using western blotting and Sanger sequencing. Four distinct, healthy KO clones were established for the UKE-1 cell line and compared to the original wildtype cell population. RNA-seq and phospho-proteomic analysis were undertaken in two KO clones. Extracted RNA was sequenced on Illumina platforms with a minimum of 45 million individual paired reads per sample. Differential gene expression was analysed using DESEQ2. Whilst, matched cell lysates underwent tandem mass tagging, high pH fractionation, and phospho-peptide enrichment was undertaken prior to liquid chromatography-mass spectrometry for total and phospho-proteomic analysis. RNA-seq and total proteomic analysis were consistent in demonstrating a significant downregulation of STAT1 (Log2FC-0.79, FDR 9.03E-49) and STAT2 (Log2FC -0.70, FDR 9.05E-20) with over-enrichment analysis identifying downregulation of type 1 IFN signalling pathways and pathways related to MKC and platelet function as shown in Figure 1. The significant downregulation of genes associated with platelet function, MKC development and differentiation included GATA1, GP1BA, ITGA2B, SELP, VWF and genes identified as mediators of fibrosis in MPN as demonstrated in Figure 2. Targeted real time PCR for STAT1, GP1BA and LTPB1 confirmed the same transcriptional patterns were present in both additional knockout clones. Knockout clones show a diminished response to exogenous IFN alpha administration consistent with downregulation of these pathways. Consistently reduced proliferation is observed in the KO clones by a variety of methodologies. Comparison to the transcriptional response in RUX treated UKE-1 cells demonstrates that at 24 hours post RUX treatment significant STAT1 downregulation is observed but not accompanied by the same pattern of down-regulation of MKC and fibrosis associated genes. The same was seen in cells persistently treated with high dose RUX for greater than 5 weeks. These results show a significant downregulation of IFN signalling in UBASH3B KO cells linked to a significant downregulation of MKC associated genes further highlighting the link observed in MPN stem cells. They implicate UBASH3B as a potential mediator of IFN signalling in a JAK2 V617F context and support a hypothesis that downregulating IFN signalling in MPN stem cells may have disease modifying potential by altering differentiation and pro-fibrotic potential. RUX treatment downregulates STAT1 without activating the same transcriptional programme associated with MKC gene downregulation and this may help explain the limited disease modifying effect of RUX observed in patients. This work has generated an important KO cell line model which will permit the molecular characterisation of differential STAT1/IFN pathway activation and allow further study to understand the regulation of MKC differentiation and pro-fibrotic gene transcription observed in MPN stem cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Jak2V617F Reversible Activation Shows an Essential Requirement for Jak2V617F in Myeloproliferative Neoplasms (MPNs)

JAK inhibitors (JAKi) improve symptoms and outcomes in MPNs; however, mutant allele burden does not substantively change with chronic therapy. Alternative MAPK signaling and/or cooperating mutations are thought to contribute to MPN cell persistence; however, we hypothesized current JAKi fail to effectively abrogate mutant JAK2 signaling. To that end, we developed a conditionally inducible mouse model allowing for sequential activation, then deletion, of Jak2VF from its endogenous locus using a Dre-rox/Cre-lox (Jak2RL) dual recombinase system (Figure 1A). Dre mRNA electroporation of lineage-negative UbcCreER-Jak2RL bone marrow (BM) was used to induce Jak2VFex vivo, followed by transplant into lethally-irradiated recipient mice in competition with Cd45.1 BM. Transplanted mice developed a highly penetrant MPN characterized by leukocytosis, polycythemia, hepatosplenomegaly, and BM megakaryocytic hyperplasia. Tamoxifen administration 12 weeks post-transplant to delete Jak2VF resulted in abrupt normalization of blood cell parameters, spleen weights, and serum pro-inflammatory cytokines, with a marked prolongation in overall survival (OS) compared to MPN control mice. Reduced peripheral blood (PB) and BM Cd45.2 mutant fraction was also observed, including within hematopoietic stem/progenitor cell (HSPC) compartments and to a greater degree than ruxolitinib (RUX) therapy. Notably, treatment with CHZ868, a type II JAKi, showed improvements in regard to hematocrit and spleen weight reduction on par with deletion, as well as significant reductions in BM HSPC mutant fractions, albeit to a lesser degree than with Jak2VF reversion (Figure 1B). Transplant of Jak2VF deleted BM failed to form phenotypic disease in secondary recipient mice consistent with loss of disease-propagating MPN stem cells. RNA-Seq on sorted LSKs following Jak2VF deletion showed loss of STAT5, MAPK, and MTORC1 target gene expression, as well as negative enrichment in TGFb and TNFa/NFkB pathways consistent with reduced pro-inflammatory and proliferative output. The alterations in MAPK signaling were supported by Ybx1 RT-PCR of BM cKit+ cells and BM pERK IHC, both of which were increased with RUX but potently suppressed with deletion suggesting improved JAK2 targeting can overcome MAPK-mediated persistence. The initial proximity of Jak2RLlox sites grants Cre-mediated recombination prior to Dre-mediated Jak2VF knock-in allowing for assessment of Jak2VF oncogenic dependency alone or in the context of co-occurring mutation. Given the known effects of Tet2 loss on enhanced MPN HSC fitness, we evaluated Tet2 loss on sustaining MPN with Jak2VF reversion. UbcCreER-Jak2RL/Tet2f/f mice were pulsed with TAM to knock-out Tet2 followed by sacrifice and Dre Jak2RL knock-in 6 weeks later. Mice transplanted with UbcCreER-Jak2VF/Tet2-/- cells exhibited enhanced leukocytosis, spleen weights and PB/BM chimerism compared to Jak2VF mice; however, Jak2VF reversion resulted in similar reductions in MPN disease parameters, including Cd45.2 fraction, suggesting Tet2loss does not dramatically alter dependence on mutant JAK/STAT signaling. Notably, while Jak2VF/Tet2-/- mutant cells displayed increased serial replating capacity, this was abrogated with Jak2VF reversion suggesting despite the enhanced fitness of Tet2-/- HSCs, Jak2VF creates a unique dependency that renders double-mutant cells susceptible to eradication with Jak2VF reversion. In sum, Jak2VF deletion abrogates MPN features, extends OS, and reduces mutant cell fraction with depletion of disease-sustaining MPN HSCs, either alone or in the setting of cooperating Tet2 loss. These data suggest JAK2VF mutant-selective inhibition offers greater therapeutic potential than current JAKi and a potential curative option for the treatment of MPN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular Pathogenesis of Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs) are chronic hematological malignancies characterized by increased proliferation of MPN stem and myeloid progenitor cells with or without bone marrow fibrosis that typically lead to increased peripheral blood cell counts. The genetic and cytogenetic alterations that initiate and drive the development of MPNs have largely been defined, and we summarize these here. In recent years, advances in understanding the pathogenesis of MPNs have defined a long-preclinical phase in JAK2-mutant MPN, identified genetic loci associated with MPN predisposition and uncovered mechanistic insights in CALR-mutant MPN. The integration of molecular genetics into prognostic risk models is well-established in myelofibrosis and ongoing studies are interrogating the prognostic implications of concomitant mutations in ET and PV. Despite all these advances, the field is deficient in clonally selective therapies to effectively target the MPN clone at any stage of disease, from pre-clinical to advanced. Although the biological understanding of the pathogenesis of MPNs has progressed quickly, substantial knowledge gaps remain, including in the molecular mechanisms underlying MPN progression and myelofibrotic transformation. An ongoing goal for the MPN field is to translate advances in biological understanding to improved treatments for patients.

Phenformin increases early hematopoietic progenitors in the Jak2V617F murine model.

Myeloproliferative neoplasms (MPN) are disorders characterized by an alteration at the hematopoietic stem cell (HSC) level, where the JAK2 mutation is the most common genetic alteration found in classic MPN (polycythemia vera, essential thrombocythemia, and primary myelofibrosis). We and others previously demonstrated that metformin reduced splenomegaly and platelets counts in peripheral blood in JAK2V617F pre-clinical MPN models, which highlighted the antineoplastic potential of biguanides for MPN treatment. Phenformin is a biguanide that has been used to treat diabetes, but was withdrawn due to its potential to cause lactic acidosis in patients. We herein aimed to investigate the effects of phenformin in MPN disease burden and stem cell function in Jak2V617F-knockin MPN mice. In vitro phenformin treatment reduced cell viability and increased apoptosis in SET2 JAK2V67F cells. Long-term treatment with 40mg/kg phenformin in Jak2V617F knockin mice increased the frequency of LSK, myeloid progenitors (MP), and multipotent progenitors (MPP) in the bone marrow. Phenformin treatment did not affect peripheral blood counts, spleen weight, megakaryocyte count, erythroid precursors frequency, or ex vivo clonogenic capacity. Ex vivo treatment of bone marrow cells from Jak2V617F knockin mice with phenformin did not affect hematologic parameters or engraftment in recipient mice. Phenformin increased the percentages of LSK, MP, and MPP populations, but did not reduce disease burden in Jak2V617F-knockin mice. Additional studies are necessary to further understand the effects of phenformin on early hematopoietic progenitors.

Ruxolitinib-Induced Defects in DNA Repair Cause Sensitivity to PARP Inhibitors in Myeloproliferative Neoplasms

Philadelphia chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), which are associated with mutations in JAK2, CALR and MPL genes. Current treatment options for Ph- MPNs include cytoreductive therapy with hydroxyurea, and the JAK2 kinase inhibitors (e.g., ruxolitinib), which produce durable reductions in splenomegaly and improvement of symptoms and probably of survival, but do not eliminate the disease-initiating cell population. All three disease “driver” mutations, [JAK2(V617F), CALR(del52), and MPL(W515L)] have been detected not only in mature MPN cells, but also in MPN stem cells, and therefore these cells must be eliminated to eradicate the disease. Since JAK2 kinase inhibitors did not eliminate the disease-initiating population novel therapeutic approaches are needed. We show here that cell lines expressing JAK2(V617F), MPL(W515L) or CALR(del52) accumulated reactive oxygen species-induced lethal DNA double-strand breaks (DSBs), which were efficiently repaired thus preventing MPN cell death. Inhibition of JAK2 kinase by ruxolitinib caused downregulation of BRCA1, RAD51 and LIG4, the key proteins in two major DSB repair mechanisms, BRCA-mediated homologous recombination (HR) and DNA-PK -mediated non-homologous end-joining (NHEJ). We recently reported (Nieborowska-Skorska et al., J.Clin.Invest., 2017) that NHEJ-deficient acute myeloid leukemia (AML) quiescent stem cells and HR/NHEJ-deficient AML proliefrating stem and progenitor cells were extremely sensitive to synthetic lethality triggered by poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673 (talazoparib). Therefore, we hypothesized that PARP inhibitors should be very effective against ruxolitinib-treated HR/NHEJ-deficient MPN quiescent and proliferating cells. Olaparib, when combined with ruxolitinib caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of samples. Importantly, combination of BMN673, ruxolitinib and hydroxyurea was highly effective in vivo against JAK2(V617F)-positive murine MPN-like disease and also against pre-selected JAK2(V617F), CALR(del52), and MPL(W515L)-positive primary MPN xenografts. However, MPN samples from individual patients displayed high degree of variability in the sensitivity to JAK2 inhibitor combined with PARP inhibitor and/or hydroxyurea. The differences between susceptibility of individual MPNs to these drugs may depend on additional mutations accompanying the “driver” mutations (e.g., TET2 mutation) and on homo/heterozygosity of the “driver” mutation allele. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized pre-selected MPN cells to synthetic lethality triggered by PARP inhibitors. DisclosuresKoschmieder:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche: Other: Clinical Trial participation. Skoda:Novartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Consultancy, Speakers Bureau.

The JAK2V617F-Bearing Megakaryocytes Promote Hematopoietic Stem/Progenitor Cell Expansion through the Thrombopoietin/MPL Signaling

IntroductionThe myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The acquired mutation JAK2V617F plays a central role in these disorders. The mechanisms responsible for MPN HSPC expansion are not fully understood. One hallmark of MPNs is megakaryocytic (MK) hyperplasia. Very recent evidence has implicated MKs in regulating HSPC activity. Since thrombopoietin (TPO) acting through its receptor MPL are key regulators of both megakaryopoiesis and HSPC activity, we hypothesize that JAK2V617F-bearing MKs promote MPN stem cell function via altered TPO/MPL signaling.MethodsJAK2V617F Flip-Flop (FF1) mice and Pf4-Cre mice were crossed to generate a MK cell lineage-specific human JAK2V617F knock-in mouse strain (Pf4/FF1). TPO or MPL was ablated in Pf4/FF1 by crossing Pf4/FF1 mice with TPO knock-out mice (TPOnull) or MPL knockout mice (MPLnull) to generate TPOnullPf4+FF1+ mice and MPLnullPf4+FF1+ mice. Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline.ResultsPreviously, we have demonstrated that JAK2V617F-bearing MKs cause a murine myeloproliferative syndrome with HSPC expansion in a Pf4/FF1 murine model. Here, we studied the effects of the JAK2V617F-bearing MK niche on MPN stem cell function. First, we isolated LineagenegcKit+ (Lin-cKit+) HSPCs from 28wk old Pf4/FF1 mice and their age-matched littermate controls (Pf4-Cre) (n=4 in each group) and analyzed their cell cycle status using propidium iodide staining. We found that the percentage of Lin-cKit+ cells in the G0/G1 phase was moderately increased in the Pf4/FF1 mice compared to control mice (63% vs 56%, P=0.012), suggesting that HSPCs in the JAK2V617F-bearing MK niche are more quiescent than in the WT MK niche. (Figure 1)Next, we performed a competitive repopulation assay in which both CD45.2 marrow cells from Pf4/FF1 mice or control mice were injected intravenously together with competitor CD45.1 WT marrow cells into lethally irradiated (950 cGy) CD45.1 recipients. During 16 weeks follow up post transplantation, recipients of Pf4/FF1 marrow cells displayed a higher peripheral blood donor (CD45.2) chimerism than the control mice. Marrow cell colony assays demonstrated a moderate increase of CD45.2+ hematopoietic progenitors in the Pf4/FF1 marrow recipients compared to control mice (1.5-fold, p = 0.0025). Marrow cell flow cytometry analysis revealed that, although there was no significant difference in the total CD41+ MK cell numbers between the two groups, CD45.2+ MKs were significantly increased (3.3-fold, P=0.012) while CD45.1+ MKs were significantly decreased (3.4-fold, P=0.024) in the Pf4/FF1 marrow recipient compared to the controls. Similarly, although the total numbers of marrow CD45+EPCR+CD48-CD150+ (E-SLAM) cells, which is a highly purified long-term repopulating HSPC population, were not changed between the two groups, CD45.2+ E-SLAM cells were significantly increased (3.5-fold, P=0.012) while CD45.1+ E-SLAM cells were significantly decreased (5.4-fold, P=0.024) in the Pf4/FF1 marrow recipient compared to the controls. Therefore, the JAK2V617F-bearing MK niche promoted the expansion of JAK2V617F HSPCs, probably at the expense of suppressing WT hematopoiesis. No significant difference in blood cell count, spleen weight, or total femoral cell count was observed between the Pf4/FF1 marrow recipients and control marrow recipients. (Figure 2)To determine the role(s) of TPO/MPL signaling in the development of JAK2V617F MK-induced myeloproliferation, we ablated TPO or MPL in the Pf4/FF1 mice. At 28 weeks of age, in contrast to the Pf4/FF1 mice which developed thrombocytosis, increased numbers of hematopoietic progenitors, and a substantial expansion of MKs and HSPCs compared to age-matched WT controls, TPOnullPf4+FF1+ mice and MPLnullPf4+FF1+ mice did not display any of these features. (Figure 3) These data suggest that TPO and MPL are required for the development of JAK2V617F MK-induced myeloproliferation.ConclusionsOur study establishes that JAK2V617F-bearing MKs form an important part of the MPN HSPC niche and TPO/MPL signaling is critical for the MK niche function. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting Abnormal Hematopoietic Stem Cells in Chronic Myeloid Leukemia and Philadelphia Chromosome-Negative Classical Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs) are unique hematopoietic stem cell disorders sharing mutations that constitutively activate the signal-transduction pathways involved in haematopoiesis. They are characterized by stem cell-derived clonal myeloproliferation. The key MPNs comprise chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). CML is defined by the presence of the Philadelphia (Ph) chromosome and BCR-ABL1 fusion gene. Despite effective cytoreductive agents and targeted therapy, complete CML/MPN stem cell eradication is rarely achieved. In this review article, we discuss the novel agents and combination therapy that can potentially abnormal hematopoietic stem cells in CML and MPNs and the CML/MPN stem cell-sustaining bone marrow microenvironment.

A knockout combination for MPN stem cells.

Myeloproliferative neoplasms (MPNs) are a group of blood cancers that are maintained by stem cell populations. In this issue of JEM, Dagher et al. (https://doi.org/10.1084/jem.20201268) combine arsenic and interferon α to deliver a knockout punch to MPN stem cells and provide new hope to cure patients with MPNs.

Overexpression of PD-L1 Correlates with JAK2-V617F Mutational Burden and Is Associated with Chromosome 9p Uniparental Disomy in MPN

Myeloproliferative neoplasms (MPN) are characterized by clonal hematopoiesis, hyperproliferation of myeloid cells, hyperinflammation and immune deregulation. The three classical BCR-ABL1-negative MPN are essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The disease is driven by JAK2, CALR or MPL somatic mutations in most patients. Drug resistance is a major problem in MPN. Recent data suggest that MPN cells display certain immune checkpoint molecules that may contribute to resistance, including PD-L1. Antibodies targeting the PD1/PD-L1 axis are highly promising anti-cancer drugs. Their potential use in MPN is being explored but it is unclear which MPN subtypes are most suitable for testing in clinical trials. The aim of our project was to assess PD-L1 expression in disease-initiating neoplastic stem cells (SC) and differentiated cells of MPN patients and to develop therapeutic approaches capable of blocking PD-L1 expression in MPN SC. In a first step, PD-L1 expression was assessed by RNA-sequencing of granulocytes of 106 MPN patients and 15 healthy donors (HD). The cohort included 56 PMF, 33 ET and 17 PV patients. For 102 patients data from Human Genome-wide Affymetrix 6.0 SNP arrays were available. We observed a ~5-fold higher expression of PD-L1 mRNA in patients with PV compared to other MPN (P<.01) or HD (P<.01). JAK2-V617F positive ET patients had higher expression of PD-L1 compared to CALR-mutated ET (p<.005) and the same was observed in PMF (p<.01). Other mutations (TET2, DNMT3A) detected by NGS did not affect PD-L1 expression. Since PD-L1 and JAK2 are located on chromosome 9p24, we looked into our previously published dataset of 400 MPN patients analyzed by SNP arrays and found that in all 195 patients with 9p uniparental disomy (UPD) the aberrations covered both genes. As PD-L1 is more centromeric it could represent the second target of 9pUPD which can precede the acquisition of JAK2-V617F in MPN. Granulocytes in JAK2-V617F positive patients with 9pUPD expressed significantly higher levels of PD-L1 compared to patients without 9pUPD (P<.0001; Figure 1A). Moreover, the JAK2-V617F mutational burden significantly correlated with PD-L1 expression (R=.52, P<.0001; Figure 1B). This correlation was lost when cases with 9pUPD were excluded from the analysis (R=.03, P=.9), indicating that the UPD is relevant for PD-L1 upregulation. To investigate PD-L1 surface expression on MPN SC we analyzed CD34+CD45dimCD38- cells isolated from fresh bone marrow (BM) samples of another 51 MPN patients and 7 HD by flow cytometry (FC). MPN patients showed a significantly higher surface expression of PD-L1 on CD34+CD45dimCD38- cells compared to HD (p<.001; Figure 1C). PD-L1 levels on the SC surface were elevated in both JAK2- and CALR-mutated MPN patients compared to HD (p<.001 and P<.005, respectively). PD-L2 was neither expressed in MPN granulocytes nor on MPN SC. CD4+ and CD8+ T-cells from BM samples of 17 MPN patients expressed the PD-L1 receptor PD-1 as assessed by FC. We cultured ex vivo primary MPN cells from 7 JAK2-V617F positive patients and showed that PD-L1 expression on MPN SC spontaneously decreases in culture, that interferon-gamma (IFN-γ) can promote expression of PD-L1 on these cells, and that ruxolitinib and the BRD4-degrader dBET6 block IFN-γ-induced PD-L1 expression in CD34+CD45dimCD38- MPN SC (P<.05). Together, we show that PD-L1 is overexpressed on the surface of disease-initiating MPN SC, that PD-L1 mRNA is overexpressed in granulocytes of MPN patients and that PD-L1 overexpression in granulocytes correlates with the JAK2-V617F mutational burden. In patients with JAK2-V617F positive MPN, 9pUPD leads to further PD-L1 upregulation either through increasing the mutant JAK2 gene dosage, loss of wt-JAK2,or amplification of PD-L1 allele with higher expression. Our data suggest the possibility that 9pUPD and the subsequent elevation of PD-L1 expression may provide an immune escape mechanism and may contribute to positive selection of JAK2-V617F homozygous SC. Ruxolitinib and dBET6 downregulate PD-L1 expression on MPN SC suggesting a role for the JAK2 and BRD4-MYC pathway. As recent studies revealed an immunogenic potential of JAK2 and CALR mutants, overcoming the disease-mediated immune escape may be of particular importance. Further preclinical and clinical studies are now required to examine the value of PD1/PD-L1 inhibitors in patients with MPN. Disclosures Gisslinger: Celgene: Honoraria; MyeloPro Diagnostics and Research: Honoraria; AOP Orphan Pharmaceuticals AG: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; PharmaEssentia: Honoraria; Janssen-Cilag: Honoraria; Roche: Honoraria. Kralovics:AOP Orphan Pharmaceuticals AG: Honoraria; PharmaEssentia: Honoraria; Qiagen: Honoraria; Novartis: Honoraria; MyeloPro Diagnostics and Research: Current equity holder in private company. Valent:Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.

Imetelstat Inhibits Telomerase and Prevents Propagation of ADAR1-Activated Myeloproliferative Neoplasm and Leukemia Stem Cells

BACKGROUND Clonal stem cell derived myeloproliferative neoplasms (MPNs) have a propensity to evolve to acute myeloid leukemia (AML). Deregulation of the innate immune deaminase associated with RNA1 (ADAR1) has been linked to malignant progression and therapeutic resistance. Increased expression of the stem cell gene, human telomerase reverse transcriptase (hTERT), has also been linked with malignant transformation. However, the combinatorial role of ADAR1 and hTERT in the evolution of MPN stem cells to therapy resistant acute myeloid leukemia stem cells (LSCs) and the capacity of a telomerase inhibitor, imetelstat, to prevent survival and self-renewal of pre-LSC and LSC had not been established. Recent clinical trials show early signs of efficacy of imetelstat in treatment of myelofibrosis (MF). However, its role in selectively inhibiting pre-LSC transformation to self-renewing LSC has not been elucidated. Here we show that targeting telomerase activity prevents pre-LSC and LSC maintenance both in vitro and in vivo, suggesting telomerase inhibition as an effective strategy for preventing MPN progression. METHODS To quantify hTERT level and ADAR1 activity in the setting of normal HSPC and MPN stem cell evolution, whole genome sequencing (WGS) analysis was performed on 76 normal and MPN blood CD34+ cells and matching saliva samples. Results were compared with RNA-seq of 100 FACS purified young, aged, MPN and AML CD34+CD38- stem cells and CD34+CD38+ progenitor cells. Confocal fluorescence microscopic evaluation of stem cell ADAR1 and hTERT localization, telomere length by Flow-FISH and telomerase activity by TRAP assays, lentiviral ADAR1 overexpression and shRNA knockdown were performed. In vitro stromal co-cultures, and humanized immunocompromised mouse models were established to determine the impact of imetelstat (a oligonucleotide inhibitor of telomerase) on normal, MPN stem cell and LSC maintenance. RESULTS Combined hTERT overexpression, ADAR1 activation and a significant reduction in telomere length correlated with accelerated stem cell aging during MPN progression to AML. Increased ADAR1 mediated adenosine to inosine (A-to-I) transcript editing coincided with accelerated telomere shortening in high risk MPN stem cells. Moreover, lentiviral ADAR1 overexpression enhanced pre-LSC engraftment. Treatment with imetelstat reduced MPN stem cell and LSC propagation in stromal co-cultures as well as in humanized mouse models commensurate with reduced hTERT expression levels and telomerase activity and decreased ADAR1 editing activity. Specifically, stromal co-culture assays revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 µM or 5 µM significantly inhibited survival and replating of blast crisis (BC) CML progenitors compared with aged bone marrow progenitors (p < 0.001, ANOVA). As a single agent, imetelstat (5 µM) inhibited survival and replating of pre-LSC derived from myelofibrosis compared with normal bone marrow progenitor samples (p < 0.001, ANOVA). In pre-LSC MPN NSG-SGM mouse models established from 4 different MF samples, a significant reduction in proliferation of human CD45+ cells (p < 0.01, t test) was observed in bone marrow and spleen, when compared with vehicle control. Treatment of humanized LSC mouse models, established with 5 different BC CML, with 30 mg/kg of imetelstat, 3 times a week for 4 weeks resulted in a significant reduction in proliferation of malignant progenitors and human CD45+ cells (p < 0.001, ANOVA). As measured by a Flow-FISH assay, abnormal telomere length was reversed by imetelstat treatment compared with mismatch control (p < 0.05, ANOVA). In addition, FACS analysis revealed a significant reduction in activated beta-catenin expression after imetelstat treatment of LSC engrafted mice compared with vehicle control (p < 0.01, ANOVA). Finally, RNA-seq analysis performed on human CD34+ cells from imetelstat treated LSC mouse models revealed a significant reduction in LSC harboring malignant ADAR1-mediated A-to-I editing at doses that spared normal hematopoietic stem cells. CONCLUSIONS Combined WGS and RNA-Seq analyses, lentiviral ADAR1 overexpression, stromal co-culture assays and humanized pre-LSC and LSC mouse model studies reveal that pre-LSC evolution into LSC coincides with both ADAR1 and hTERT activation, which can be prevented with imetelstat. Disclosures Rizo: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Huang:Geron Corp: Current Employment, Current equity holder in publicly-traded company. Jamieson:Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.

Phenotyping of Disease-Initiating CD34+/CD38─ Stem Cells in BCR-ABL1─ MPN Reveals Expression of Multiple Cytokine Receptors and Resistance-Related Antigens

The classical BCR-ABL1-negative myeloproliferative neoplasms (MPN) are characterized by over-production of myeloid cells, disease-related mutations in certain driver-genes (JAK2, CALR, MPL) and an increased risk to transform to secondary acute myeloid leukemia (sAML). Although considered stem cell-derived neoplasms, little is known about the phenotype and functional properties of disease-initiating neoplastic stem cells (NSC) in MPN and sAML. Recent data suggest that MPN NSC reside in a CD34+ fraction of the malignant clone. Therefore, these cells are considered most critical target populations to be examined for expression of molecular and immunological targets with the aim to develop improved or even curative NSC-eliminating therapies, such as antibody-based or CAR-T cell approaches. Using a panel of monoclonal antibodies (n=40) and multicolor flow cytometry, we established the immunological phenotype and target expression profiles of putative CD34+/CD38─ NSC and CD34+/CD38+ progenitor cells in patients with polycythemia vera (PV, n=18), essential thrombocythemia (ET, n=29), primary myelofibrosis (PMF, n=38) and post-MPN sAML (n=11). In almost all patients, the putative MPN stem cells expressed the stem cell invasion receptors Hermes (CD44) and ADGRE5 (CD97), C1qR1 (CD93), the migration/adhesion receptor MIC2 (CD99), and the stem cell antigen AC133 (CD133). Contrasting normal stem cells, MPN NCS and sAML stem cells failed to express Thy-1 (CD90). Among the cytokine receptors tested, MPN NSC invariably displayed the TGFßR-related antigen endoglin (CD105), TPOR (CD110), SCFR KIT (CD117), IL-3RA (CD123), CXCR4 (CD184) and IGF-1R (CD221). NSC expressed particularly high levels of KIT and low levels of TPOR and IGF-1R. The IL-2RA (CD25) was identified on NSC in most patients with PMF and sAML, and in a few with ET, but not in patients with PV. Similarly, the GM-CSFR (CD116) was found to be expressed on NSC in most patients with PMF, a few with ET and no with PV. MPN NSC did not exhibit substantial amounts of M-CSFR (CD115), IL-3RB (CD131), FLT3 (CD135), NGFR (CD271) VEGFR-2 KDR (CD309), EPOR, MET or OSMRB. The CD34+/CD38+ MPN progenitor cells displayed a similar profile of cytokine receptors. In addition, MPN and sAML progenitor cells expressed IL-1RAP and CLL-1 in most donors examined. We next examined the expression of various immunological targets and resistance-mediating immune checkpoint antigens on NSC and MPN progenitor cells. In all MPN patients and all sAML patients tested, NSC were found to express substantial amounts of Siglec-3 (CD33) and low levels of Campath-1 (CD52) and MDR-1 (CD243). In addition, MPN NSC and sAML stem cells invariably displayed the "don't eat" me checkpoint IAP (CD47) and the classical checkpoint PD-L1 (CD274). Exposure to interferon-gamma (200 U/ml, 24 hours) resulted in an upregulation of PD-L1 on NSC. In a subset of patients, MPN NSC expressed low levels of HB15 (CD83). In contrast, MPN NSC and sAML stem cells failed to express B7-1 (CD80), B7-2 (CD86), PD-L2 (CD273) and PD1 (CD279). MPN progenitor cells and sAML progenitors expressed an identical profile of cell surface targets and checkpoint antigens. Finally, we confirmed the disease-initiating capacity of MPN stem- and progenitor cells (CD34+ cells) using primary PMF cells in xenotransplantation experiments employing NSGS mice expressing human interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF). After 28 weeks post injection, engraftment of human CD45+ cells in the bone marrow of NSGS mice was found in 15/15 mice injected with bulk mononuclear cells (MNC) containing CD34+ cells and in 0/15 NSGS mice injected with MNC depleted of CD34+ cells. Together, MPN NSC reside in a CD34+ fraction of the malignant clone and display a unique phenotype, including cytokine receptors, immune checkpoint molecules and other target antigens. The phenotypic characterization of neoplastic stem cells should facilitate their enrichment and the development of NSC-eradicating treatment concepts in MPN. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.

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.

STAT5 is Expressed in CD34+/CD38- Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms.

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34+/CD38− MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34+/CD38− MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

Metformin Suppress Cellular and Molecular Processes Related to Maintenance and Proliferation of Myeloproliferative Neoplasm Stem Cell

Background : Preclinical rationale of metformin treatment for myeloproliferative neoplasms (MPN) has recently been demonstrated (Cell Death Dis. 2018;9(3):311). Current MPN treatment options are mainly directed to symptoms and have not greatly improve clinical outcomes. It is a consensus that potential curative approaches should affects MPN stem cell function. Aims: To interrogate the metformin action in MPN stem cell function according to frequency and ex vivo differentiation capacity, as well as to underlying molecular mechanisms related to the treatment. Material and Methods: For induction of PV phenotype, 5×106 total bone marrow cells from Jak2V617F conditional mice (Jak2WT/V617F-CD45.2) were transplanted into lethally irradiated) Pep/boy mice (CD45.1-700cGy X-Ray). After 4 weeks, peripheral blood chimerism was evaluated by CD45.1 and CD45.2 markers (Becton-Dickinson) by flow cytrometry and mice were randomized for treatment by intraperitoneal injection of vehicle (PBS, n=6) or metformin (125 mg/kg/day, n=4) for 6 weeks according to chimerism and body weight. Erythroid progenitors (CD71 and Ter119) were evaluated in bone marrow (BM) and spleen. Hematopoietic stem and progenitor cells (LSK: Lin-Sca-1+C-KitHi/long-term HSC[LT-HSC]:LSKCD48-CD150+/ short-term HSC [ST-HSC]:LSKCD48+CD150+/multipotent progenitor [MPP]: LSKCD48+CD150-), myeloid progenitors (LK:Lin-Sca-1+C-KitHi/myelo-erythroid progenitor [MEP]: LKCD34-CD16/32-/common myeloid progenitors [CMP]: LKCD34+CD16/32-/granulocytic progenitors [GMP]: LKCD34+CD16/32+) were evaluated in BM by flow cytometry. BM stroma was evaluated according to frequencies of macrophages (F4/80+CD11b+), inflammatory macrophages (M1) (F4/80+CD11b+CD11c+), arterioles (Cd45/Ter119-CD31+Sca-1+), osteoblasts (Cd45/Ter119-CD51+Sca-1-) and mesenchymal stem cell (Cd45/Ter119-CD51+Sca-1+). BM c-Kit cells were enriched using AutoMacs separator with anti-CD117 magnetics beads (Miltenyi) and used for RNA extraction and PCR array for cell cycle and apoptosis (PAMM-020Z and PAMM-012Z, respectively-Qiagen). Genes with fold-change ≥2 in both directions were selected. All experiments were approved by the Animal Ethics Committee. For comparisons, unpaired T-test or Mann-Whitney test were used as appropriated. All P-values were two-sided with a significance level of 0.05. Results: At randomization, mice of both groups were similar according PB chimerism, body weight and blood counts (all P>0.05). After 6 weeks of metformin treatment mice had no evidence of hematological toxicity or weight loss. Metformin treated mice presented lower platelets counts in PB (mean for vehicle vs. metformin: 4370×103/uL vs. 3451×103/uL; P=0.04) and reduction of splenomegaly (P=0.02). Metformin reduced CD45.2 chimerism (92.7% vs. 88.9%; P=0.03), and showed a trend in reducing pro-erythroblasts (0.91% vs. 0.53%; P=0.06) and early erythroid progenitors (37.4% vs. 32.4%; P=0.11), while increased late erythroid progenitors (16.2% vs. 27.2%; P=0.008) in spleen. Metformin significantly increased ST-HSC frequency (0.06% vs. 0.29%; P=0.001) compared to vehicle. Conversely, bone marrow mononuclear cells from metformin treated mice showed decreased clonogenic capacity (2.5 colonies/1000 BMMC vs. 8.6 colonies/1000 BMMC; P=0.001). Competitive secondary transplant into lethally irradiated mice was performed and results will be show at meeting. Metformin treatment did not modulate the frequency of erythroid progenitors, LT-HSC, MPP, MEP, GMP and CMP in BM nor of the stromal cells evaluated. In c-Kit enriched cells, PCR-array identified 31 downregulated and 64 upregulated genes by metformin treatment. Metformin reduced expression of anti-apoptotic genes (Bcl2 [Fold-change: 0.33] and Xiap [0.48]), cell cycle regulators (Cdkn2a [0.21], Ccnd1 [0.27], Ccn1a [0.42]), while increased expression of TP53 family members (Trp63 [4.1] and Trp53 [2.1]), pro-apoptotic (Bid [2.5] and Bcl10 [1.9]) and cellular-stress response genes (Atm [66.9] and Cdkn2a [3.1]). Conclusions: In a conditional Jak2V617F-induced MPN murine model, daily treatment with metformin reduced tumor burden, extramedullary hematopoiesis, and clonogenic capacity of HSPC by acting on molecular processes related to maintenance and proliferation of MPN stem cell. Disclosures Figueiredo-Pontes: Novartis: Honoraria.

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

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