Myeloproliferative Neoplasms Disease Progression Research Articles

Research status and challenges of advanced myeloproliferative neoplasms

Classical myeloproliferative neoplasms (MPN), also known as Ph-MPN, includes polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Secondary myelofibrosis (sMF) and secondary acute myeloid leukemia (sAML) are important disease progressions of MPN. After MPN disease progression, hematopoietic stem cells undergo new clonal evolution, leading to drug resistance, poor treatment effect and poor survival of patients. In recent years, the exploration of the mechanism of disease progression and the precise diagnosis and treatment of MPN have attracted much attention. This article summarizes the research status of MPN disease progression, including the pathogenesis, risk stratification, and precision treatment, in order to provide reference for exploring new diagnosis and treatment methods of MPN disease progression.

Loss of Complement Factor I (Cfi) a Negative Regulator of Complement Cascade Activity Exacerbates JAK2V617F-Dependent Phenotype

Background: The current evidence suggests that inflammatory phenotype contributes to the pathogenesis of myeloproliferative neoplasms (MPNs). While our understanding of complement cascade contributions to inflammation-mediated pathologies has expanded, role this cascade plays in etiology of myeloid malignancies has not been detailed. Previously, we uncovered an inactivating RNSV in complement factor I (CFI G119R), an inhibitor of the complement cascade, in 20% of PMF patients (n=10) using WGS, and in a separate patient cohort (n=10) we observed an increase in CH50 levels (n=3) and decrease in C3 levels (n=3) (Oakes et al., 2021, Blood, 138:1472).These findings prompted us to generate murine models allowing assessment of the Cfi loss-driven complement overactivity effects on hematopoiesis and development of MPNs. Methods: A conventional Cfi knockout (KO) mouse model was generated by targeting exon 2 of the Cfi gene using CRISPR/Cas9. Inactivation of one or two copies of Cfi was confirmed by genotyping and total loss (Cfi KO) or Cfi deficiency (Cfi HET) were confirmed in peripheral blood (PB) plasma. Complete blood counts (CBC) and the frequencies of granulocytes, B cells, plasmocytes, T cells, macrophages/monocytes, platelets, erythroid cells in the bone marrow were longitudinally assessed by flow cytometry for 40 weeks in Cfi WT, HET and KO mice. Cfi HET animals were crossed with JAK2V617F(fl/+);Mx1-cre(+) mice (obtained from crossing B6.Cg-Tg( Mx1- cre+ 1)Cgn/J; # 003556, JAX) and JAK2V617F expressing (B6N.129S6(SJL)- Jak2 tm1.2Ble/AmlyJ #031658, JAX) to generate Cfi(+/-)/JAK2V617F(fl/+);Tg(Mx1-cre(+/-) (CFI/JAK2) strain. Complete blood counts and survival were monitored for Cfi(+/+)/JAK2V617F(fl/+);Tg(Mx1-cre)(+/-) (CFI WT/JAK2 HET) Cfi(+/-)/JAK2V617F(fl/+);Tg(Mx1-cre)(+/-) (CFI HET/JAK2 HET) or Cfi(-/-)/JAK2V617F(fl/+); Tg(Mx1-cre)(+/-) (CFI KO/JAK2 HET) mice. Results:Cfi HET and KO mice were fertile and viable. Cohort of Cfi WT n=13, Het n=12, KO n=13 mice was subjected to analyses. Genotypes were confirmed via PCR and western blot. Flow cytometry demonstrated a copy loss dependent decrease in total T-cells (CD3+) and increase in mature resting B-cells (CD19+/B220+). CBC demonstrated a copy loss dependent decrease in both monocyte (p adj. KO vs. WT 0.002) and granulocyte percentage (p adj. KO vs. WT 0.003), and increase in lymphocyte percentage (p adj. KO vs. WT 0.001). In PB plasma we detected increased C3 activation in KO mice compared to WT and Het, demonstrated as decreased C3 alpha and presence of inactivated C3 due to increased proteolytical processing of C3 complex. Proteolytic cleavage of CFB compared to Cfi WT and Het was also noted in Cfi KO animals. A significant decrease in survival probability of CFI HET/JAK2 HET vs. CFI WT/JAK2 HET (p=0.03), CFI KO/JAK2 HET vs. CFI WT/JAK2 HET (p=0.003), and CFI HET/JAK2 HET vs. CFI KO/JAK2 HET (p=0.05) averaging 92 days for Cfi HET/JAK2 HET, 67 days for Cfi KO/JAK2 HET vs. 98 days for Cfi WT/JAK2 HET was noted (Fig. A). A granulocyte and monocyte percentage of CFI HET/JAK2 HET was increased as compared to CFI WT/JAK2 HET mice (Fig. B). Conclusions:An overactivation of complement cascade at the C3 level is noted in Cfi deficient animals, that is phenotypically linked to an increase in frequency of B cells. These findings may suggest involvement of Cfi directly, and/or in conjunction with generally overactive complement, in B-cell maturation and function. The absence of one copy of Cfi in JAK2V617F mice shows more severe MPN disease progression resulting in markedly decreased survival, linked to increased frequencies of monocytes and granulocytes and markedly decreased lymphocytes. In sum, these data indicate that increased complement activity may increase JAK2V617F-dependent phenotype severity. In depth mechanistic studies detailing observed phenotype are warranted.

Whole blood transcriptional profiling reveals highly deregulated atherosclerosis genes in Philadelphia-chromosome negative myeloproliferative neoplasms.

The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are associated with a huge comorbidity burden, including an increased risk of cardiovascular diseases. Recently, chronic inflammation has been suggested to be the driving force for clonal evolution and disease progression in MPN but also potentially having an impact upon the development of accelerated (premature) atherosclerosis. Since chronic inflammation, atherosclerosis, and atherothrombosis are prevalent in MPNs and we have previously shown oxidative stress genes to be markedly upregulated in MPNs, we hypothesized that genes linked to development of atherosclerosis might be highly deregulated as well. Using whole blood gene expression profiling in patients with essential thrombocythemia (ET; n = 19), polycythemia vera (PV; n = 41), or primary myelofibrosis (PMF; n = 9), we herein for the first time report aberrant expression of several atherosclerosis genes. Of 84 atherosclerosis genes, 45, 56, and 46 genes were deregulated in patients with ET, PV, or PMF, respectively. Furthermore, BCL2L1, MMP1, PDGFA, PTGS1, and THBS4 were progressively significantly upregulated and BCL2 progressively significantly downregulated from ET over PV to PMF (all FDR <0.05). We have for the first time shown massive deregulation of atherosclerosis genes in MPNs, likely reflecting the inflammatory state in MPNs in association with in vivo activation of leukocytes, platelets, and endothelial cells being deeply involved in the atherosclerotic process.

Successful Engraftment of Murine JAK2V617F MPN-like Disease into Unconditioned Mice

Background: JAK2V617F (JAK2VF) is the leading driver mutation present in hematopoietic cells of classical myeloproliferative neoplasms (MPN). The contribution of the non-hematopoietic compartment to MPN pathology remains obscure, although alterations and mutations in non-hematopoietic bone marrow (BM) cells (nonHC) can induce MPN-like diseases (reviewed in PMID32780848). Strikingly, the MPN-inducing JAK2VF mutation may be detected decades before MPN diagnosis, suggesting that the nonHC may act to support the expansion of the MPN clone. To examine this role of the nonHC within the BM during MPN disease, a model relying on low-level engraftment into an unaltered BM microenvironment is needed. Current transplantation models require myeloablative conditioning (irradiation) to ensure cell engraftment, which permanently alters the BM microenvironment (PMID24607941). We have developed a novel murine system that mimics JAK2VF+ MPN without the use of myeloablative conditioning, allowing the role of the BM microenvironment in the natural progression of MPN pathology to be studied. Methods: Floxed-JAK2VF (JAX#031658) were crossed with Mx1-Cre (JAX#003556) mice to produce a poly I:C-inducible MPN-like disease model (CD45.2). Five-week-old donor animals, designated JAK2MUT (Mx1-Cre(+);JAK2VF(+/-)) or JAK2CTRL (Mx1-Cre(-);JAK2VF(+/-)), were injected with poly I:C to activate the JAK2VF mutation. Five weeks later, BM was isolated and transplanted via single tail vein injection into age- and sex-matched C57BL/6 Ptprca (JAX#002014, CD45.1) recipient animals, at a concentration of 1.5x107 per animal. 10 mice (5 female, 5 male) were used as BM transplant (BMT) recipients of either JAK2MUT or JAK2CTRL BM. In addition, 10 total non-injected sex- and age-matched hosts were kept as a naïve (non-injected) group. Body weight (BW) of all 20 BMT recipients was monitored as an indicator of overall health. Blood from the submandibular vein was collected from all recipients every 4-6 weeks for complete blood cell counts (CBC) and flow cytometry analysis to determine alterations within the hematopoietic system and percentage of donor (CD45.2) chimerism within CD45.1 hosts. Results: At transplantation, JAK2MUT donors displayed advanced MPN-like disease (hematocrit (HCT) >70%, red blood cells (RBC) >1.9x107/µl, platelets (PLT) >1.1x106/µl). Over the course of the study, only male recipients receiving JAK2MUT donor cells showed a significantly different trend in weight change at 6 weeks post-transplantation with an average of 0.18% less BW gained. Hosts receiving JAK2CTRL cells displayed low engraftment efficiency <1% after 6 weeks post BMT, whereas hosts receiving JAK2MUT cells displayed ~4% observable engraftment within peripheral blood (Fig. A). Additionally, hosts receiving JAKMUT cells displayed reddening of extremities indictive of erythromelalgia, characteristic of JAK2VF+ MPN (Fig. A). When compared to donors, JAKMUT transplanted hosts displayed CBC abnormalities, characteristic of polycythemia (HCT >49%, RBC >25% naïve, unaltered levels of PLT) (PMID27956542). 2 of 10 recipients of JAK2MUT BMT died as the result of highly penetrant MPN 4-6 weeks after the transplant. Conclusion: While many ways to transplant hematopoietic cells within unconditioned animals have been described, we have successfully transplanted JAK2VF BM into the non-conditioned recipients and showed presence of diagnosable disease in animals displaying ~5% donor chimerism. Our model will offer advantages over existing methods for studying early MPN disease progression within an unconditioned murine BM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Interleukin-1 contributes to clonal expansion and progression of bone marrow fibrosis in JAK2V617F-induced myeloproliferative neoplasm

Chronic inflammation is frequently associated with myeloproliferative neoplasms (MPN), but the role of inflammation in the pathogenesis of MPN remains unclear. Expression of the proinflammatory cytokine interleukin-1 (IL-1) is elevated in patients with MPN as well as in Jak2V617F knock-in mice. Here, we show that genetic deletion of IL-1 receptor 1 (IL-1R1) normalizes peripheral blood counts, reduces splenomegaly and ameliorates bone marrow fibrosis in homozygous Jak2V617F mouse model of myelofibrosis. Deletion of IL-1R1 also significantly reduces Jak2V617F mutant hematopoietic stem/progenitor cells. Exogenous administration of IL-1β enhances myeloid cell expansion and accelerates the development of bone marrow fibrosis in heterozygous Jak2V617F mice. Furthermore, treatment with anti-IL-1R1 antibodies significantly reduces leukocytosis and splenomegaly, and ameliorates bone marrow fibrosis in homozygous Jak2V617F mice. Collectively, these results suggest that IL-1 signaling plays a pathogenic role in MPN disease progression, and targeting of IL-1R1 could be a useful strategy for the treatment of myelofibrosis.

The different variant allele frequencies of type I/type II mutations and the distinct molecular landscapes in CALR-mutant essential thrombocythaemia and primary myelofibrosis

ABSTRACT Objective Calreticulin (CALR) mutations have been identified as driver mutations in a quarter of patients with essential thrombocythaemia (ET) and primary myelofibrosis (PMF), which are subgroups of myeloproliferative neoplasms (MPNs). A 52-bp deletion (type I mutation) and a 5-bp insertion (type II mutation) are the most frequent variants. To better understand the impact of different CALR mutant variants, with or without nondriver mutations, on the clinical subtypes of MPN needs further investigation. Methods The clinical characteristics, laboratory parameters and genetic mutation statuses were analysed in a cohort of 77 MPN patients with CALR mutations (ET = 24, prePMF = 33, and overt PMF = 20). Targeted NGS using a 38-gene panel was performed to evaluate the variant allele frequency (VAF) of CALR type I/type II mutations and assess the molecular landscape of nondriver gene mutations. Results A lower VAF of type I vs. type II was observed in CALR-mutant ET, prePMF and overt PMF, and a higher frequency of type I vs. type II was found in CALR-mutant overt PMF. Additional somatic mutations were indicated to be useful in understanding the pathogenesis of MPN. In this study, the mutation landscape was more complex in overt PMF than in ET or in prePMF. Mutations in epigenetic regulators (ASXL1, EZH2 and TET2) were more common in overt PMF. Conclusions The two different subtypes of CALR mutations may have different impacts on MPN. A lower VAF of CALR type I indicates a greater contribution to disease progression in MPN, and increased nondriver mutations may be important in myelofibrosis progression.

Patients With Myeloproliferative Neoplasms Harbor High Frequencies of CD8 T Cell-Platelet Aggregates Associated With T Cell Suppression.

Myeloproliferative neoplasms (MPN) are chronic cancers of the hematopoietic stem cells in the bone marrow, and patients often harbor elevated numbers of circulating platelets (PLT). We investigated the frequencies of circulating PLT-lymphocyte aggregates in MPN patients and the effect of PLT-binding on CD8 T cell function. The phenotype of these aggregates was evaluated in 50 MPN patients and 24 controls, using flow cytometry. In vitro studies compared the proliferation, cytokine release, and cytoxicity of PLT-bound and PLT-free CD8 T cells. Frequencies of PLT-CD8 T cell aggregates, were significantly elevated in MPN patients. Advanced disease stage and CALR mutation associated with the highest aggregate frequencies with a predominance of PLT-binding to antigen-experienced CD8 T cells. PLT-bound CD8 T cells showed reduction in proliferation and cytotoxic capacity. Our data suggest that CD8 T cell responses are jeopardized in MPN patients. JAK2 and CALR exon 9 mutations – the two predominant driver mutations in MPN – are targets for natural T cell responses in MPN patients. Moreover, MPN patients have more infections compared to background. Thus, PLT binding to antigen experienced CD8 T cells could play a role in the inadequacy of the immune system to control MPN disease progression and prevent recurrent infections.

Type I but Not Type II Calreticulin Mutations Activate the IRE1α/XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms.

Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type-specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265.

A Murine Model with JAK2V617F Expression in Both Hematopoietic Cells and Vascular Endothelial Cells Recapitulates the Key Features of Human Myeloproliferative Neoplasm

IntroductionAlthough murine models have provided unequivocal evidence that the JAK2V617F mutation is able to cause myeloproliferative neoplasms (MPNs), there is significant heterogeneity in disease phenotypes between different murine models and none has been able to recapitulate both the myeloproliferative phenotype and cardiovascular pathology seen in patients with MPNs. In addition, these murine models were mostly followed for less than 3-9 months and how aging affects MPN disease progression has not been studied. Endothelial cells (ECs) are an essential component of the hematopoietic niche, and they have been shown to express the JAK2V617F mutation in patients with MPNs. In this study, we investigated how MPN progresses in the JAK2V617F-bearing vascular niche during aging.MethodsJAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter) were crossed with Tie2-cre mice to express JAK2V617F specifically in all hematopoietic cells (including HSPCs) and vascular ECs (Tie2FF1).ResultsThe Tie2FF1 mice developed essential thrombocythemia to primary myelofibrosisdisease transformation with extramedullary splenic hematopoiesis during 18-month follow up. No evidence of leukemia transformation was observed in the Tie2FF1 mice. (Figure 1)Hematopoietic colony formation assays, flow cytometry analysis, and in vitro culture experiments revealed that there was a loss of both HSPC number and HSPC function in the marrow of old Tie2FF1 mice during aging, mimicking the advanced phases of myelofibrosis. In contrast, the spleen of old Tie2FF1 mice was able to maintain the expansion of JAK2V617F mutant hematopoiesis during aging and MPN disease progression. (Figure 2)These differences between marrow and spleen hematopoiesis in the old Tie2FF1 mice prompted us to investigate how aging affects the JAK2V617F mutant hematopoiesis differently in the marrow and spleen. We found that, although the JAK2V617F mutant HSCs (Lin -cKit +Sca1 +CD150 +CD48 -) from old Tie2FF1 mice were more proliferative than wild-type HSCs in both the marrow and spleen, mutant marrow HSCs were more apoptotic and senescent than wild-type HSCs in the marrow while mutant spleen HSCs were relatively protected in the spleen. Examination of the hematopoietic vascular niche revealed that marrow ECs (CD45 -CD31 +) were significantly decreased in old Tie2FF1 mice compared to age-matched control mice; in contrast, spleen ECs were significantly expanded and less senescent in old Tie2FF1 mice compared to control mice. Therefore, the different vascular niche function of the marrow and spleen could contribute to the decreased marrow hematopoiesis and expanded splenic hematopoiesis we have observed in the Tie2FF1 mice during aging. (Figure 3)Previously, we reported that the Tie2FF1 mice developed spontaneous heart failure with thrombosis, vasculopathy, and cardiomyopathy at 20wk of age. Here, we followed the cardiovascular function of Tie2FF1 mice during aging. At 18mo of age, the Tie2FF1 mice continued to demonstrate a phenotype of dilated cardiomyopathy with a moderate but significant decrease in left ventricular ejection fraction and an increase in left ventricular volume and mass compared to age-matched control mice. Histology examination revealed spontaneous thrombosis in the right ventricle, pulmonary arteries, both main (epicardial) coronary arteries and scattered coronary arterioles (microvessels) in the old Tie2FF1 mice, while age-matched Tie2-cre control mice had no evidence of spontaneous thrombosis in their heart or lungs. Despite these cardiovascular dysfunctions, there was no difference in body weight nor was there any increased incidence of sudden death between the old Tie2FF1 mice and control mice. These findings suggested that there was a persistent but compensated cardiomyopathy and heart failure in the Tie2FF1 mice during aging. (Figure 4)ConclusionCompared to other MPN murine models reported so far, the Tie2FF1 mice is the first MPN murine model that faithfully recapitulated almost all the key features of the human MPN diseases. Considering the presence of the JAK2V617F mutation in microvascular ECs isolated from patients with MPNs and the recapitulation of all the key features of human MPN diseasesby the Tie2FF1 mice, the roles of endothelial dysfunction in the hematologic and cardiovascular pathogenesis of MPN shall be further investigated. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DUSP6 Mediates Resistance to JAK2 Inhibition and Drives Myeloproliferative Neoplasm Disease Progression

Myeloproliferative neoplasms (MPNs) are clonally derived from hematopoietic stem/progenitor cells (HSPCs) and typically harbor somatic mutations in one of three genes (JAK2, CALR, MPL) leading to aberrant activation of JAK-STAT signaling. While small molecule inhibitors of JAK2 provide symptomatic benefit for MPN patients, they do not eradicate the underlying malignant clone, nor do they prevent disease progression. Chronic 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.To understand alterations to the transcriptional landscape underlying MPN disease progression, we performed bulk transcriptome profiling on myelofibrosis (MF) and sAML patient CD34+ HSPCs. Differential gene expression analysis revealed upregulation of dual-specificity phosphatase 6 (DUSP6), which encodes a MAPK phosphatase that regulates ERK signaling, in sAML CD34+ cells. Elevated DUSP6 protein expression accompanying disease progression was confirmed via MPN patient bone marrow immunofluorescence and imaging mass cytometry analysis. We performed further single cell RNA sequencing (scRNA-seq) in conjunction with TotalSeq surface protein marker detection on more than 50,000 sorted CD34+ cells of serial samples from three patients at chronic MPN and sAML stages, and two healthy controls, which revealed DUSP6 among the top 21 genes elevated in all three paired samples across disease progression. Subsequent differentiation trajectory pseudotime analysis demonstrated concomitant elevation of DUSP6 across state trajectories and disease progression.Genetic and pharmacologic targeting of DUSP6 followed by biochemical and mass cytometry analysis identified signaling inhibition through S6 and JAK/STAT, establishing them as novel, non-canonical effectors of DUSP6. DUSP6 inhibition also led to potent suppression of cell proliferation, induction of apoptosis and cell cycle arrest, and reduction of inflammatory cytokine production in primary MPN samples. Furthermore, ectopic DUSP6 expression augmented proliferation and mediated JAK2 inhibitor resistance, while DUSP6 inhibition reduced colony-forming potential of JAK2 inhibitor-persistent patient cells.Mechanistically, DUSP6 suppression dampened S6 signaling via inhibition of RSK1 (RPS6KA1), which we identified as a second indispensable candidate associated with poor clinical outcome via Kaplan-Meier overall survival (Log-rank p = 0.0005) and multivariate (RPS6KA1 expression hazard ratio = 1.60, 95% confidence interval: 1.10, 2.34) analyses of the TCGA LAML cohort. Strong correlation was observed (r = 0.68; p = 0.0009) between RPS6KA1 and DUSP6 expression in CD34+ HSPCs, and pharmacologic inhibition of RSK1 with BI-D1870 suppressed proliferation and colony formation across AML cell lines and primary samples.DUSP6 inhibition in vivo via small molecule inhibitor BCI resolved pathologically elevated hematocrit and white blood cell counts and reduced splenomegaly in Jak2 V617F knock-in mice. In the MPL W515L retroviral transplant model, BCI suppressed leukocytosis while reducing reticulin fibrosis and prolonging survival. In patient-derived xenograft (PDX) model of NSGS mice engrafted with sAML patient CD34+ cells, BCI treatment or DUSP6 knockdown reduced peripheral blood hCD45+ engraftment. Importantly, BCI treatment did not pathologically cause cytopenias or decrease spleen weights in wild-type mice, nor did it reduce hCD45+ engraftment in NSGS PDX mice engrafted with healthy donor CD34+ cells. Lastly, NSGS PDX mice engrafted with MF patient CD34+ cells ectopically expressing DUSP6 demonstrated marked leukocytosis, splenomegaly, and early lethality.These findings underscore DUSP6 in driving MPN disease progression and therapeutic resistance, and highlight the DUSP6-RSK1 axis as a novel, druggable pathway in myeloid malignancies. [Display omitted] DisclosuresOh: Abbvie: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Celgene Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Constellation: Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Kartos Therapeutics: Membership on an entity's Board of Directors or advisory committees; PharamaEssentia: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Membership on an entity's Board of Directors or advisory committees.

A Murine Model With JAK2V617F Expression in Both Hematopoietic Cells and Vascular Endothelial Cells Recapitulates the Key Features of Human Myeloproliferative Neoplasm.

The myeloproliferative neoplasms (MPNs) are characterized by an expansion of the neoplastic hematopoietic stem/progenitor cells (HSPC) and an increased risk of cardiovascular complications. The acquired kinase mutation JAK2V617F is present in hematopoietic cells in a majority of patients with MPNs. Vascular endothelial cells (ECs) carrying the JAK2V617F mutation can also be detected in patients with MPNs. In this study, we show that a murine model with both JAK2V617F-bearing hematopoietic cells and JAK2V617F-bearing vascular ECs recapitulated all the key features of the human MPN disease, which include disease transformation from essential thrombocythemia to myelofibrosis, extramedullary splenic hematopoiesis, and spontaneous cardiovascular complications. We also found that, during aging and MPN disease progression, there was a loss of both HSPC number and HSPC function in the marrow while the neoplastic hematopoiesis was relatively maintained in the spleen, mimicking the advanced phases of human MPN disease. Different vascular niche of the marrow and spleen could contribute to the different JAK2V617F mutant stem cell functions we have observed in this JAK2V617F-positive murine model. These results indicate that the spleen is functionally important for the JAK2V617F mutant neoplastic hematopoiesis during aging and MPN disease progression. Compared to other MPN murine models reported so far, our studies demonstrate that JAK2V617F-bearing vascular ECs play an important role in both the hematologic and cardiovascular abnormalities of MPN.

High Mobility Group A1 Chromatin Remodeling Proteins: Potential Therapeutic Targets Involved in Polycythemia Vera Transformation to Acute Leukemia in Humans and JAK2 V617F Transgenic Mouse Models

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), although the mechanisms driving disease progression remain elusive. Polycythemia vera (PV) is the most common clinical subtype caused by acquired mutations of JAK2. While PV generally presents as an indolent process, ~25% of patients will progress to MF or AML and JAK2 mutations alone do not account for MPN transformation.HMGA1 / 2 genes encode oncogenic chromatin binding proteins that are overexpressed in acute leukemia and drive clonal expansion in murine leukemia models. In preliminary studies, we found up-regulation in gene expression of both HMGA1/2 in HSC with disease progression in MPN. We therefore sought to test the hypothesis that HMGA proteins are critical drivers of transformation and rational therapeutic targets to treat or prevent PV progression.Methods: We examined clonal evolution in HSC from 49 JAK2V617F -positive PV patients using standard and SNP-array karyotyping and a targeted resequencing panel of 163 genes linked to myeloid cancers. HSC clonal burden was assessed via JAK2V617F genotype analysis on single cells. We also measured HMGA1 / 2 in JAK2V617F -positive human AML cell lines from MPN patients (PV-AML cells), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F transgenic murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F / MPLSV ; Blood 2015;126:484) using real-time quantitative RT-PCR. To elucidate the role of HMGA1/2, we silenced HMGA1 or HMGA2 via short hairpin RNA and assessed proliferation in human PV-AML cells. Finally, to investigate Hmga1 function in vivo, we crossed mice deficient in Hmga1 onto murine models of PV and PV-AML. We also compared leukemic engraftment in JAK2V617F -positive PV-AML cells with or without HMGA1/2 silencing.Results: Both HMGA1/2 mRNA were up-regulated in all JAK2V617F -positive contexts. In primary human PV CD34+ cells, HMGA1 and HMGA2 were increased by 7- and 100-fold, respectively, compared to controls. There was a dramatic up-regulation in both HMGA1/2 in HSC of patients who transformed from PV to MF or AML compared to chronic phase PV when analyzed in cross-sectional samples from different patients or prospectively in selected patients. Overexpression of HMGA1 /2 also correlated with clonal dominance of JAK2V617F -homozygous stem cells, and additional mutations of epigenetic regulators, including EZH2 and SETBP1 . Similarly, when assessed in unfractionated bone marrow or tumor samples in transgenic mouse models for PV and PV-AML, Hmga1 / 2 were overexpressed compared to control littermates, with highest levels in the PV-AML mouse model. Silencing HMGA1 or HMGA2 in human PV-AML cells (DAMI, SET-2) dramatically halted proliferation. Moreover, leukemic engraftment was prevented in PV-AML cells after silencing HMGA1 or HMGA2. In preliminary studies with the transgenic PV-AML murine model, Hmga1 deficiency ameliorates the fulminant phenotype of aggressive leukemia and early mortality. PV-AML mice survived a median of 5 weeks (range 3-10; n=40) whereas PV-AML in Hmga1 deficient backgrounds survived a median of 8 weeks (range 7-16; n=7). The leukemic burden was decreased in mice with Hmga1 deficiency.Conclusions:HMGA1 and HMGA2 are overexpressed in MPN, and higher levels associate with disease progression to MF and AML, in both human PV and in transgenic murine models of PV. Strikingly, silencing HMGA1 or HMGA2 halted proliferation and prevented leukemic engraftment in vivo . Preliminary data in Hmga1 deficient models suggest that Hmga1 is required for PV progression to AML. These data highlight a key role for HMGA proteins as critical drivers of PV transformation and suggest that HMGA1 /2 overexpression is a consequence of aberrant JAK/STAT signaling and epigenetic dysregulation. Our findings indicate that HMGA1 /2 overexpression may function as a necessary molecular switch for PV leukemic transformation. Therefore, HMGA proteins and their transcriptional pathways offer novel therapeutic targets aimed at the prevention of PV progression to MF and AML. DisclosuresNo relevant conflicts of interest to declare.

Advanced Forms of the Myeloproliferative Neoplasms Are Associated with Chromosomal Abnormalities Involving 1q and 12q: Implicating MDM2 and MDM4 in Disease Progression

Inevitably patients (pts) with myeloproliferative neoplasms (MPNs) including polycythemia vera (PV) and essential thrombocythemia (ET) progress to more advanced forms of myelofibrosis (MF) and acute myeloid leukemia (AML). Loss of TP53 function plays a critical role in cancer biology yet, the MPNs are characterized predominately by wild type (WT) TP53 (Rampal et al, PNAS, 2014;111: 5401-10). The major protein regulators of TP53 are MDM2 and MDM 4. TP53 activity has been reported to be down-regulated in MPNs due to overexpression of MDM2 (Lu et al, Blood, 2012;120:3098-3105, Nakatake et al, Oncogene, 2012: 311: 323-33). Since the gene for MDM4 is located on chromosome 1q and the gene for MDM2 is located on chromosome 12q, we hypothesized that cytogenetic abnormalities involving these locations might lead to disease progression This hypothesis is supported by the recent observation that mice engineered to possess JAK2 V617F and lack TP53 are prone to develop AML as compared to JAK2 V617F+ mice with WT TP53 (Rampal et al, PNAS, 2014:111:5401-10). Gain of 1q (+1q) in the form of jumping translocations in pts with MPN was previously reported to be associated with transformation to AML (Najfeld et al, Br.J. Haematol., 2010; 151: 285-291).These studies were extended to 362 MPN pts enrolled in 5 different MPN-RC clinical trials involving pts with ET or PV (222), MF and MPN -Blast Phase (MPN-BP)(140) and 989 pts with MPNs, ET(177), PV (400), MF (385) and MPN-BP(27) who were studied at the Icahn School of Medicine at Mount Sinai (ISMMS) between 1986 and June 2017. 92 pts in the various MPN-RC trials (25.4%) and 344 (34.7%) of the pts from ISMMS were chromosomally abnormal. Among the abnormalities, 23 pts in the MPN-RC trials had either +1q/dup(1q)(n=16) or rearrangements of 12q (n=7) (6.3% of the total pts and 25.0% of the chromosomally abnormal pts). Similarly, the most frequently observed chromosomal abnormality at ISMMS associated with disease progression, either as a sole abnormality or as a subclone with other chromosomal abnormalities, was gain of 1q, identified in 59 pts (6.0% of total, 17.4% of the abnormal pts). Gain of 1q was observed in three forms: 1) as an unbalanced translocation with the breakpoint at 1q21. 2) a duplication of 1q region and/or 3) as a jumping +1q translocation. The most frequent unbalanced translocation identified was +der(9)t(1;9)(q21;q12) (24%) resulting in three copies of 1q and 3 copies of 9p. Rearrangement of 12q most frequently involved balanced translocations and/or inv(12q) with 50% of these pts having a breakpoint at 12q15 where the gene for MDM2 is located. Gain of 1q/dup(1q) and rearrangements of 12q were mutually exclusive with the exception of one patient with a translocation between chromosomes 1 and 12 [der(12)t(1;12)(q24;q15)]. Among PV/ET pts enrolled in MPN-RC trials, 1.3% (3/222) had +1q and 0.0% (0/222) had 12q abnormalities, while 11% (15/140) of pts enrolled in MF and MPN-BP treatment protocols had +1q and 5% (7/140) had 12q abnormalities. These findings indicate that +1q and structural abnormalities of 12q are associated with more advanced forms of MF and MPN-BP (p<0.001). In both, the MPN-RC and the ISMMS data bases, 42 cases with +1q and 13 cases with 12q abnormalities were identified where the driver mutational status was known. The JAK2 V617F mutation was observed in 83.3% (35/42)of pts with +1q while 23.1% (3/13) pts with 12q abnormalities were JAK2 V617F+.These data indicate that gain of 1q but not 12q abnormalities are associated with the JAK2 V617F mutation (p<0.001). In a limited number of pts (n=10) we were able to monitor the chronological acquisition of 1q and 12q abnormalities. The 1q abnormalities occurred 5-7 years following the initial cytogenetic evaluation of PV pts and the proportion of cells with gain of 1q increased over time being present in 100% of cells over the next 4-5 years of observation. By contrast, 12q abnormalities occurred exclusively in MF pts as the sole abnormality and appeared in 90-100% of the examined cells at presentation. In 5 MF pts with +1q, qPCR demonstrated upregulation of MDM 4 mRNA transcripts (86%) when compared to 5 pts without +1q. These data suggest that MPN disease progression is associated with specific chromosomal abnormalities that result in up regulation of MDM2 or MDM4 which further down-regulate p53 .These data provide the rationale for the use of MDM2/MDM4 antagonists as a means of halting MPN disease progression. DisclosuresMascarenhas:CTI Biopharma: Research Funding; Novartis: Other: DSMB member , Research Funding; Merck: Research Funding; Incyte: Other: Clinical Trial Steering Committee , Research Funding; Promedior: Research Funding; Janssen: Research Funding.

Cell competition between wild-type and JAK2V617F mutant cells in a murine model of a myeloproliferative neoplasm

The myeloproliferative neoplasms (MPNs) are characterized by overproduction of mature blood cells and increased risk of transformation to frank leukemia. The acquired kinase mutation JAK2V617F plays a central role in a majority of patients with these diseases. As MPNs are clonal stem cell disorders (i.e. arise from a single stem cell which eventually expands), the hematopoietic stem/progenitor cell (HSPC) compartment in MPNs is heterogeneous with the presence of both JAK2 wild-type and JAK2V617F mutant cells. Mechanisms responsible for the mutant stem cell expansion in MPNs are not fully understood. Utilizing in vitro co-culture assays and in vivo competitive transplantation assays, we show that the presence of wild-type cells alters both the gene expression profile and cellular function of JAK2V617F mutant HSPCs and inhibits the expansion of co-existing JAK2V617F mutant cells in a normal microenvironment. In contrast, we found that a microenvironment bearing the mutant kinase promotes JAK2V617F mutant HSPC expansion over wild-type cells due in part to altered CXCL12/CXCR4 signaling. Further understanding of the molecular mechanisms controlling the competitive interactions between normal and JAK2V617F mutant cells, and how these mechanisms break down during MPN disease progression hold great potential for advances in treating patients with these diseases.

Selective Targeting of Histone Deacetylase 11 Disables Metabolism of Myeloproliferative Neoplasms

Introduction: Acetylated histone and non-histone proteins are pharmacologic targets for both solid and hematological cancers including myeloproliferative neoplasms (MPNs), a group of clonal hematological malignancies driven by aberrant JAK2/STAT signaling. MPNs are characterized by epigenetic alterations, including aberrant acetylation, which makes this disease particularly interesting for targeting with HDAC inhibitors. Four classes of histone deacetylases (Class I-IV HDACs) regulate gene transcription and modulate cellular processes that drive the initiation and progression of cancer. Pan-HDAC and class I-selective HDAC inhibitors have gained traction in clinical settings, yet we reasoned that specific targeting of the 18 distinct HDAC proteins may establish roles for select HDACs as therapeutic vulnerabilities in MPNs. Methods: To explore the roles of individual HDACs in MPN, we first conducted an inhibitor screen of compounds having distinct HDAC selectivity based on electrophoretic mobility shift assays with full-length human HDAC proteins expressed in baculovirus and unique peptide substrates. Ultra-specific HDAC6 compounds were initially targeted for analysis based on its previously defined role in HSP90-mediated JAK2 stabilization and translation. Survival of MPN cell line models, MPN patient samples, leukemia cell lines, and MPN disease progression in mice transplanted with Hdac6-/-, and Hdac11-/- hematopoietic stem cells (HSCs) transduced with the MPLW515L oncogene, as well as Tg-Hdac11-eGfp mice were used to show the role of HDAC6 and HDAC11 in oncogene-driven and homeostatic hematopoiesis. As further proof of specificity, HDAC6 and HDAC11 were genetically ablated in MPN model cell lines using either RNA interference or inducible shRNA. For HDAC11 substrate identification, a combination of RNA-seq, acetylated proteome (SILAC), global metabolomics (LC-MS), Seahorse metabolic assays (Agilent Technologies), enzymatic assays, and acetylation-specific immunoblotting and mutation profiling were performed (Fig. 1). Results: Despite the established interplay between HDAC6, HSP90 and JAK2, neither a highly selective HDAC6 inhibitor, HDAC6 silencing, nor the Hdac6 deficiency suppressed MPN pathogenesis, although there were clear effects on the acetylation of α-tubulin, a well characterized HDAC6-selective substrate. Intriguingly, both inhibition of HDAC11 activity with highly-specific HDAC11 inhibitors and silencing HDAC11 using an inducible validated shRNA, identified HDAC11 as a therapeutic vulnerability for multiple human MPN cell lines. The Tg-Hdac11-eGFP reporter mice showed that HDAC11 is expressed in several hematopoietic cell types, including myeloid cells, erythroblasts, and megakaryocytes. Thus, Hdac11-/- and Hdac11+/+MPLWT bone marrow were examined for steady-state hematopoiesis and transplantation chimerism. These studies demonstrated that HDAC11 does not contribute to homeostatic or transplantated bone marrow reconstitution. However, in the oncogenic MPL model, recipient mice transplanted withoncogenic MPLW515L-expressing Hdac11-deficient HSCs displayed markedly impaired cytokine-independent colony-formation, had less fibrosis, and displayed improved survival in primary and secondary MPN hematopoietic stem cell transplantation; thus HDAC11 contributes to MPN pathogenesis (Fig. 1). Studies in additional leukemia cell lines, including THP-1, HL-60, and mantle lymphoma cell lines, but not in Ramos or K562 cells, established that HDAC11 contributes to oncogene-driven events in other cell types. Mechanistically, RNA-seq, SILAC proteomics, and metabolic profiling revealed that HDAC11 controls aerobic glycolysis by deacetylating Lys343 of the glycolytic enzyme enolase-1 (ENO1), functionally inactivating ENO1. Finally, the effects of targeting HDAC11 on metabolism were augmented by blocking compensatory pathways of oxidative phosphorylation that are induced via JAK2V617Fand MPLW515L oncogenic signaling. Conclusions: Our comprehensive screens of HDAC inhibitors, coupled with our biological, in vivo and molecular studies, indicate that HDAC11 is an attractive and potent target for disabling MPN metabolism and pathogenesis. These finding support the rationale for further development of clinical HDAC11 inhibitors for the treatment of metabolically-active cancers such as MPNs. Disclosures Pinilla Ibarz: Teva: Consultancy; TG Therapeutics: Consultancy; Sanofi: Speakers Bureau; Bayer: Speakers Bureau; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Abbvie: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Reuther:Incyte Corporation: Research Funding. Levine:Loxo: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; Lilly: Honoraria; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Gilead: Consultancy; Celgene: Consultancy, Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria. Verma:BMS: Research Funding; Janssen: Research Funding; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria. Epling-Burnette:Incyte Corporation: Research Funding; Celgene Corporation: Patents &amp; Royalties, Research Funding; Forma Therapeutics: Research Funding.

Altered Dynamics of Monocyte Subpopulations and Pro-Inflammatory Signaling Pathways in Polycythemia Vera Revealed By Mass Cytometry

Introduction: Polycythemia vera (PV) is a clonal hematologic malignancy characterized by overproduction of mature erythrocytes that exhibits a propensity for transformation to myelofibrosis (MF) and/or secondary acute myeloid leukemia (sAML). Like the majority of myeloproliferative neoplasms (MPN), the defining genetic feature of PV is the JAK2 V617F mutation, occurring in &gt;95% of patients, resulting in overactive JAK-STAT signaling which drives myeloproliferation and can contribute to inflammation. We have demonstrated that aberrant NFkB pathway signaling plays an integral role in maintaining monocyte-derived inflammatory cytokine production in MF (Fisher et al, Leukemia 2017 &amp; 2019). We have recently shown that inflammation is a hallmark of PV as well, but the signaling pathways and cell populations responsible have not been elucidated (Fowles et al, Leukemia 2019). Mass cytometry (CyTOF) enables the simultaneous measurement of 40+ markers with single cell resolution, thereby allowing comprehensive analysis of intracellular signaling networks in defined hematopoietic cell populations. We utilized mass cytometry to investigate cell populations and inflammatory signaling pathways in a cohort of PV patients including those who transformed to MF or secondary acute myeloid leukemia (sAML). Methods: Plasma cytokine levels were measured with the U-PLEX Human Cytokine 10plex assay (MSD) from 20 normal donors, 22 PV, 92 MF and 6 sAML patients, as well as serial samples from PV patients transformed to MF/sAML. Cryopreserved cells bone marrow (BM) and/or peripheral blood (PB) from 15 PV patients and 12 normal donors from were incubated with or without TPO or TNF and then stained with surface and intracellular antibodies prior to being subjected to mass cytometric analysis. Cell population frequency and intracellular signaling were identified and calculated through viSNE analysis and manual gating in Cytobank. Results: Plasma levels of inflammatory cytokines IL-6, IL-8, MIP-1a, and TNF increased with disease severity from PV to MF and sAML. For serial samples, an increased number of elevated cytokines was observed in post-transformation samples, suggesting a role for inflammation in MPN disease progression. Mass cytometry analysis revealed expansion of CD14+CD16+ intermediate monocytes and CD16+CD14-CD33+ non-classical monocytes in PV PB compared to both normal BM and PB. A reduction in CD14+CD16- classical monocytes was observed in PV compared to normal in both BM and PB, suggesting a shift in pro-inflammatory subsets in PV. Heightened NFKB activation in response to TNF stimulation was observed in lineage negative CD34+ cells in the majority of PV PB and BM samples tested compared to normal. Similarly, abnormally elevated pSTAT3/5 levels were frequently observed in response to TPO stimulation. In monocyte populations, basal elevations in several signaling markers including pNFkB, pSTAT1, and pMAPKAP2 were identified. Notably, in serial samples from patients transformed to MF or sAML, evidence of progressive NFkB hyperactivation associated with disease evolution was observed. Conclusions: Inflammatory cytokines are known to be elevated in MPN in part through constitutive JAK-STAT signaling, and recently we identified NFkB signaling as an important contributor in MF. To our knowledge NFkB has not previously been investigated in PV. This study suggests that NFkB signaling is similarly important to PV and might play a role in disease transformation. Our plasma cytokine data identified a correlation with higher cytokine levels associated with disease severity from PV to MF and sAML. This is consistent with prior studies associating IL-6 and IL-8 with leukemic transformation of MF. The observed expansion of CD14+CD16+ monocytes in PV is notable because studies have associated this population with increased endothelial cell adhesion and atherosclerosis. These associations have also been reported in JAK2 V617F-mutant red blood cells and neutrophils, raising the question of how the JAK2 mutation affects the function of pro-inflammatory monocyte subsets. Integration of this study with molecular and gene expression data to further investigate the role of inflammation in MPN progression are ongoing. Disclosures Oh: Novartis: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees.

Significance of Abnormalities of PPM1D in Myeloproliferative Neoplasms

The TP53 pathway has been shown to be dysregulated in myeloproliferative neoplasms (MPN) through several different mechanisms, including TP53 mutations, TP53 deletions and Murine Double Minute 2 (MDM2) overexpression. Downregulation of the TP53 pathway likely plays a role in MPN progression as evidenced by the association of TP53 loss of heterozygosity with transformation to acute leukemia and the presence of inactivating mutations of TP53 found in a proportion of MPN-blast phase (MPN-BP) cases (Kubesova et al, Leukemia. 2018;32(2):450-61). Furthermore, MDM2 inhibition induces TP53 pathway upregulation and consequent targeting of hematopoietic stem and progenitor cells in polycythemia vera (PV) and myelofibrosis (MF). The MDM2 inhibitor, idasanutlin, has shown activity in a Phase I trial of high-risk PV patients (Mascarenhas et al. Blood. 2019 Jun 5). An additional regulator of TP53 is protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D) which by dephosphorylating TP53 and stabilizing MDM2 regulates the DNA damage response pathway and apoptosis. Somatic activating mutations of PPM1D are present in both solid and hematologic malignancies and are specifically associated with therapy-related myeloid disorders (Hsu et al. Cell Stem Cell. 2018 Nov 1;23(5):700-13). Grinfeld et al. (N Engl J Med. 2018 Oct 11;379(15):1416-30) recently reported that in their genomic analysis of 2035 MPN patients, PPM1D was the eighth most common mutated gene in MPNs at 1.9% frequency. To determine whether there was an association of PPM1D mutations with MPNs we analyzed the genomic data of patients who participated in several clinical trials executed by the Myeloproliferative Neoplasm Research Consortium (MPN-RC). Of 89 MPN-BP patients, 5 patients harbored a PPM1D mutation with a median variant allele frequency (VAF) of 17%. In comparison, 4 out of 135 high risk PV and ET patients harbored a PPM1D mutation with a median VAF of 24%. All mutations were truncating which is consistent with previous reports of PPMID mutations in malignancies. The PPM1D gene is located on the long arm of chromosome 17 (17q23.2) and we hypothesized that cytogenetic aberrations involving this gene locus might also contribute to abnormalities of PPM1D function. Through analysis of our cytogenetic database, 16/1,124 (1.4%) MPN patients were found to have cytogenomic abnormalities involving the region containing the PPM1D gene. Eight of these patients had karyotypic abnormalities, including 3 pts with isochromosome 17q resulting in a gain of 17q and a loss of 17p, including the TP53 gene. The other 4 patients had structural variations of 17q which might lead to aberrant expression of PPM1D. One patient by FISH analysis had gain of 17q. Ten patients had cytogenomic aberrations of 17q detected through analysis of array comparative genomic hybridization and single nucleotide polymorphism (aCGH+SNP), 2 of which had concurrent karyotypic abnormalities of 17q. All patients had a gain or copy neutral loss of heterozygosity (cnLOH) of the 17q22-24.2 region. CnLOH of this region could lead to aberrant overexpression of the PPM1D gene. One of the 8 patients with cnLOH of 17q harbored a PPM1D mutation. Of the 16 patients with 17q cytogenomic abnormalities, 7 (44%) had MPN-BP and 7 (44%) patients had ET or ET that progressed to myelofibrosis (MF) or MPN-BP indicating an association of these abnormalities with advanced disease. By quantitative real time-PCR we determined the PPM1D transcript levels in mononuclear cells from 31 MPN patients without known PPM1D mutations (7 polycythemia vera (PV), 6 ET, 14 MF, 4 MPN-BP) and compared the transcript levels to mononuclear cells from healthy control patients. Forty-two percent (13/31) patients had overexpression of PPM1D (&gt;1.5 fold increase range). Fold increase ranged from 1.5-8 and overexpression was present in the following diagnoses: 4/7 PV, 1/6 ET, 8/14 MF and 0/4 MPN-BP. We have provided evidence that a number of abnormalities of PPM1D including activating mutations, cytogenetic aberrations and transcript overexpression are present in a significant proportion of MPN patients. These abnormalities in PPM1D can be additional events that lead to the downregulation of TP53 and contribute to MPN disease progression. We propose that inhibitors of PPM1D may be used in combination with other drugs that upregulate TP53 to treat MPN patients. Disclosures Rampal: Constellation, Incyte, and Stemline Therapeutics: Research Funding; Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy. Mascarenhas:CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding. Hoffman:Merus: Research Funding.

The Molecular Genetics of Myeloproliferative Neoplasms.

Activated JAK-STAT signaling is central to the pathogenesis of BCR-ABL-negative myeloproliferative neoplasms (MPNs) and occurs as a result of MPN phenotypic driver mutations in JAK2, CALR, or MPL The spectrum of concomitant somatic mutations in other genes has now largely been defined in MPNs. With the integration of targeted next-generation sequencing (NGS) panels into clinical practice, the clinical significance of concomitant mutations in MPNs has become clearer. In this review, we describe the consequences of concomitant mutations in the most frequently mutated classes of genes in MPNs: (1) DNA methylation pathways, (2) chromatin modification, (3) RNA splicing, (4) signaling pathways, (5) transcription factors, and (6) DNA damage response/stress signaling. The increased use of molecular genetics for early risk stratification of patients brings the possibility of earlier intervention to prevent disease progression in MPNs. However, additional studies are required to decipher underlying molecular mechanisms and effectively target them.

PF666 LOW JAK2 V617F ALLELE BURDEN PHI‐NEGATIVE CHRONIC MYELOPROLIFERATIVE NEOPLASMS MAY ASSOCIATE WITH ADDITIONAL CALR OR MPL MUTATIONS

Background:Jak2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9 and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Phi‐negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of prolipherative signal transduction and therefore after the emergence of one type of mutation another types basically should not have any selective advantages for clonal expansion. However simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases.Aims:To analyze cases with combined mutations in Russian cohort of MPN patientsMethods:Study includes 3267 samples, directed for genetic analysis to the National Research Center for Hematology between 2014 and 2019 from the patients with clinical evidence of MPN. DNAs and RNAs were extracted from blood using reagent kit from Interlabservice (Russia). Jak2 V617F mutation was quantitated by real‐time PCR kit from Syntol (Russia) according to manufacturers instructions. CALR exon 9 deletions/insertions were analyzed by fragment analysis (sensitivity &gt; = 3%). MPL W515L/K mutations were assessed by in‐house allele specific PCR. All cases were tested for phi‐negativity using BCR‐ABl p210 PCR kit from Interlabservice (Russia).Results:In 406 of 3267 cases (12.5%) CALR mutations were detected. In 24 of these 406 cases (6%) Jak2 V617F mutation was found in addition to CALR mutation. Matched measures were obtained at least twice at different time points during the course of disease for these cases. In 19 from 24 (80%) cases with combined CALR and Jak2 mutations V617F allele burden was lower than 3%. Also combination of Jak2 V617F with MPL W515L/K was observed in 3 out of 3267 cases only. Two of them had V617F allele burden lower than 3% (Tabl.1).Summary/Conclusion:Additional mutations may coexist over low background of JAK2 V617F allele. Therefore cases of MPNs with low initial JAK2 V617F allele should be further tested for CALR (and possibly MPL) mutations to ensure correct disease and treatment monitoring. Further dynamic observation studies are required to assess the value of additional mutations for MPN disease progression and treatment outcome.image

Advanced forms of MPNs are accompanied by chromosomal abnormalities that lead to dysregulation of TP53

AbstractThe Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and the prefibrotic form of primary myelofibrosis (PMF), frequently progress to more overt forms of MF and a type of acute leukemia termed MPN-accelerated phase/blast phase (MPN-AP/BP). Recent evidence indicates that dysregulation of the tumor suppressor tumor protein p53 (TP53) commonly occurs in the MPNs. The proteins MDM2 and MDM4 alter the cellular levels of TP53. We investigated in 1,294 patients whether abnormalities involving chromosomes 1 and 12, which harbor the genes for MDM4 and MDM2, respectively, and chromosome 17, where the gene for TP53 is located, are associated with MPN disease progression. Gain of 1q occurred not only in individuals with MPN-BP but also in patients with PV and ET, who, with further follow-up, eventually evolve to either MF and/or MPN-BP. These gains of 1q were most prevalent in patients with a history of PV and those who possessed the JAK2V617F driver mutation. The gains of 1q were accompanied by increased transcript levels of MDM4. In contrast, 12q chromosomal abnormalities were exclusively detected in patients who presented with MF or MPN-BP, but were not accompanied by further increases in MDM2/MDM4 transcript levels. Furthermore, all patients with a loss of 17p13, which leads to a deletion of TP53, had either MF or MPN-AP/BP. These findings suggest that gain of 1q, as well as deletions of 17p, are associated with perturbations of the TP53 pathway, which contribute to MPN disease progression.