HMGA1 chromatin regulators induce transcriptional networks involved in GATA2 and proliferation during MPN progression.

Myeloproliferative neoplasms (MPNs) transform to myelofibrosis (MF) and highly lethal acute myeloid leukemia (AML), although the actionable mechanisms driving progression remain elusive. Here, we elucidate the role of the high mobility group A1 (HMGA1) chromatin regulator as a novel driver of MPN progression. HMGA1 is upregulated in MPN, with highest levels after transformation to MF or AML. To define HMGA1 function, we disrupted gene expression via CRISPR/Cas9, short hairpin RNA, or genetic deletion in MPN models. HMGA1 depletion in JAK2V617F AML cell lines disrupts proliferation, clonogenicity, and leukemic engraftment. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2V617F mice, decreasing erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, and expansion of stem and progenitors, while preventing splenomegaly and fibrosis within the spleen and BM. RNA-sequencing and chromatin immunoprecipitation sequencing revealed HMGA1 transcriptional networks and chromatin occupancy at genes that govern proliferation (E2F, G2M, mitotic spindle) and cell fate, including the GATA2 master regulatory gene. Silencing GATA2 recapitulates most phenotypes observed with HMGA1 depletion, whereas GATA2 re-expression partially rescues leukemogenesis. HMGA1 transactivates GATA2 through sequences near the developmental enhancer (+9.5), increasing chromatin accessibility and recruiting active histone marks. Further, HMGA1 transcriptional networks, including proliferation pathways and GATA2, are activated in human MF and MPN leukemic transformation. Importantly, HMGA1 depletion enhances responses to the JAK2 inhibitor, ruxolitinib, preventing MF and prolonging survival in murine models of JAK2V617F AML. These findings illuminate HMGA1 as a key epigenetic switch involved in MPN transformation and a promising therapeutic target to treat or prevent disease progression.

Two clinical phenotypes in polycythemia vera.

BACKGROUND: Polycythemia vera is the ultimate phenotypic consequence of the V617F mutation in Janus kinase 2 (encoded by JAK2), but the extent to which this mutation influences the behavior of the involved CD34+ hematopoietic stem cells is unknown. METHODS: We analyzed gene expression in CD34+ peripheral-blood cells from 19 patients with polycythemia vera, using oligonucleotide microarray technology after correcting for potential confounding by sex, since the phenotypic features of the disease differ between men and women. RESULTS: Men with polycythemia vera had twice as many up-regulated or down-regulated genes as women with polycythemia vera, in a comparison of gene expression in the patients and in healthy persons of the same sex, but there were 102 genes with differential regulation that was concordant in men and women. When these genes were used for class discovery by means of unsupervised hierarchical clustering, the 19 patients could be divided into two groups that did not differ significantly with respect to age, neutrophil JAK2 V617F allele burden, white-cell count, platelet count, or clonal dominance. However, they did differ significantly with respect to disease duration; hemoglobin level; frequency of thromboembolic events, palpable splenomegaly, and splenectomy; chemotherapy exposure; leukemic transformation; and survival. The unsupervised clustering was confirmed by a supervised approach with the use of a top-scoring-pair classifier that segregated the 19 patients into the same two phenotypic groups with 100% accuracy. CONCLUSIONS: Removing sex as a potential confounder, we identified an accurate molecular method for classifying patients with polycythemia vera according to disease behavior, independently of their JAK2 V617F allele burden, and identified previously unrecognized molecular pathways in polycythemia vera outside the canonical JAK2 pathway that may be amenable to targeted therapy. (Funded by the Department of Defense and the National Institutes of Health.).

Disruption of the ASXL1 gene is frequent in primary, post-essential thrombocytosis and post-polycythemia vera myelofibrosis, but not essential thrombocytosis or polycythemia vera: analysis of molecular genetics and clinical phenotypes.

BACKGROUND: The myeloproliferative neoplasms, essential thrombocytosis, polycythemia vera and primary myelofibrosis, share the same acquired genetic lesion, but the concept of JAK2 V617F serving as the sole lesion responsible for these neoplasms is under question, and there has been interest in identifying additional mutations that may contribute to disease pathogenesis. Because ASXL1 lesions have been increasingly identified in myeloid neoplasms, we examined the relationships of ASXL1 mutation or deletion to both clinical phenotype and associated molecular features in 166 patients with myeloproliferative neoplasms. DESIGN AND METHODS: Exon 12 of ASXL1 was amplified from neutrophil genomic DNA and bidirectionally sequenced in 77 patients with myelofibrosis (including patients with primary and post-essential thrombocytosis or post-polycythemia myelofibrosis), 42 patients with polycythemia vera, 41 with essential thrombocytosis and 6 with post-myelofibrosis acute myeloid leukemia. Pyrosequencing assays were designed to determine the allele percentages of JAK2 V617F (G5073770T), ASXL1 2475dupA, and ASXL1 2846_2847del in neutrophil genomic DNA samples. Clinical and laboratory characteristics of patients with wild-type and ASXL1 mutations were then compared. RESULTS: We identified nonsense mutations or hemizygous deletion of ASXL1 in 36% of the patients with myelofibrosis, but very rarely among those with polycythemia vera or essential thrombocytosis. Among the patients with myelofibrosis, those with ASXL1 lesions were not distinguished from their wild-type counterparts with regard to JAK2 V617F status, exposure to chemotherapy or evolution to leukemia. Myelofibrosis patients with ASXL1 lesions were more likely to have received anemia-directed therapy compared to those without lesions [15/26 (58%) versus 11/39 (23%); P=0.02]. Using serial banked samples and quantitative ASXL1 mutant allele burden assays, we observed the acquisition and accumulation of ASXL1 mutations over time in two patients with post-essential thrombocytosis myelofibrosis. CONCLUSIONS: ASXL1 haploinsufficiency is associated with a myelofibrosis phenotype in the context of other known and unknown lesions, and disruption of ASXL1 function may contribute to the disease pathogenesis of myelofibrosis.

Sex differences in the JAK2 V617F allele burden in chronic myeloproliferative disorders.

BACKGROUND: The JAK2(V617F) allele burden is a variable measure, determined by the frequency of mitotic recombination events and the expansion of JAK2(V617F) clones. Since variability in the JAK2(V617F) allele burden is partly responsible for the distinct phenotypes seen in the myeloproliferative disorders, the objective of this study was to identify modifiers of the allele burden. DESIGN AND METHODS: Blood samples were obtained between May 2005 and January 2009 from 272 patients with essential thrombocytosis, polycythemia vera, and myelofibrosis. The JAK2(V617F) allele burden was measured by an allele-specific quantitative polymerase chain reaction using DNA from purified neutrophils. Repeated measures, on average 2 years apart, were available for 104 patients. RESULTS: Sex, age at diagnosis, and disease duration all independently influenced the JAK2(V617F) allele burden. When considering all patients with myeloproliferative disorders, women had significantly lower allele burdens than men (P=0.04). In those patients with repeated measures, the increase in allele burden per year between the first and second evaluations was significantly less in females than in males. Among those who experienced disease evolution, females were 4.5 times more likely to have evolution from essential thrombocytosis to polycythemia vera, but 0.23 times as likely to have evolution from essential thrombocytosis to myelofibrosis. CONCLUSIONS: Sex is an independent factor accounting for variability in the JAK2(V617F) allele burden. We speculate that lower allele burdens in females reflect a lower frequency of mitotic recombination events in females than in males, and should be considered when evaluating the relationship of allele burden to disease phenotype and also in evaluating responses to JAK2(V617F)-inhibitors. Because sex may influence genotype and/or clonal expansion, underpinning the variability in JAK2(V617F) allele burden, it will be important to explore factors that determine susceptibility to mitotic recombination events.

Thrombopoietin is required for full phenotype expression in a JAK2V617F transgenic mouse model of polycythemia vera.

The myeloproliferative neoplasms, polycythemia vera, essential thrombocytosis and primary myelofibrosis are hematopoietic stem cell disorders and share driver mutations that either directly activate the thrombopoietin receptor, MPL, or activate it indirectly through gain-of-function mutations in the gene for JAK2, its cognate tyrosine kinase. Paradoxically, MPL surface expression in hematopoietic stem cells is also reduced in the myeloproliferative neoplasms due to abnormal post-translational glycosylation and premature destruction of JAK2, suggesting that the myeloproliferative neoplasms are disorders of MPL processing since MPL is the only hematopoietic growth factor receptor in hematopoietic stem cells. To examine this possibility, we genetically manipulated MPL expression and maturation in a JAK2V617F transgenic mouse model of polycythemia vera. Elimination of MPL expression completely abrogated the polycythemia vera phenotype in this JAK2V617F transgenic mouse model, which could only be partially restored by expression of one MPL allele. Most importantly, elimination of thrombopoietin gene expression abrogated the polycythemia vera phenotype in this JAK2V617F transgenic mouse model, which could be completely restored by expression of a single thrombopoietin allele. These data indicate that polycythemia vera is in part a thrombopoietin-dependent disorder and that targeting the MPL-thrombopoietin axis could be an effective, nonmyelotoxic therapeutic strategy in this disorder.

Sex determines the presentation and outcomes in MPN and is related to sex-specific differences in the mutational burden.

The factors underlying the variable presentation and clinical course of myeloproliferative neoplasms (MPNs) remain unclear. The aim of this study was to evaluate the independent effect of sex on MPN presentation and outcomes. A total of 815 patients with essential thrombocytosis, polycythemia vera, or primary myelofibrosis were evaluated between 2005 and 2019, and the association of sex with presenting phenotype, JAK2 V617F burden, progression, and survival was examined. Men presented more often with primary myelofibrosis vs essential thrombocytosis (relative risk, 3.2; P < .001) and polycythemia vera (relative risk, 2.1; P < .001), had higher rates of transformation to secondary myelofibrosis (hazard ratio [HR], 1.55; P = .013) and acute myeloid leukemia (HR, 3.67; P < .001), and worse survival (HR, 1.63; P = .001) independent of age, phenotype at diagnosis, and MPN-specific mutation. Men had higher JAK2 V617F allele burdens in their CD34+ cells (P = .001), acquired more somatic mutations (P = .012) apart from the MPN-specific mutations, and had an increased frequency of 1 (odds ratio, 2.35; P = .017) and 2 (odds ratio, 20.20; P = .011) high-risk mutations independent of age, phenotype, and driver mutation. Male sex is an independent predictor of poor outcomes in MPNs. This seems to be due to an increased risk of non-MPN-specific somatic mutations, particularly high-risk mutations, rather than MPN-specific mutation allele frequency. Conversely, disease progression in female subjects is more dependent on JAK2 mutation allele burden than on acquisition of other somatic mutations. Sex should be considered in prognostic models and when evaluating therapeutic strategies in MPNs.

Ascites specific inhibition of CD1d-mediated activation of natural killer T cells.

PURPOSE: Natural killer T (NKT) cells recognize lipid antigen presented by CD1 molecules. NKT cells can both directly, through cytotoxicity, and indirectly, through activation of other effector cells, mediate antitumor immunity. It has been shown, however, that tumor-associated lipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. Given that ovarian cancer-associated ascites has been reported to have increased levels of gangliosides, we examined the effect of tumor-associated and other ascites on CD1d-mediated antigen presentation to NKT cells. EXPERIMENTAL DESIGN: To investigate the effects of ascites on NKT cell activation, we pretreated CD1d-expressing cells with the ascites and measured their ability to stimulate cytokine production in NKT cells. To determine whether antigen processing or editing was necessary, CD1d-immunoglobulin-based artificial antigen presenting cells (aAPC) were also incubated with ascites. In addition, to examine specificity, we analyzed whether ascites fluid could influence the activation of classic CD8+ T cells. RESULTS: Pretreatment of CD1d-expressing cells with ascites from the majority of patients inhibited the ability of the cells to stimulate/activate NKT cells in a dose-dependent manner. Ascites treatment also partially blocked the ability of alpha-galactosylceramide-loaded CD1d-immunoglobulin-based aAPC to activate NKT cells. In addition, our data show that treatment with ascites does not inhibit HLA-A2-mediated activation of classic CD8+ T cells. CONCLUSIONS: Together, these data suggest that ovarian and other cancers may have developed immune evasion mechanisms specifically targeting the CD1/NKT cell system.

Phenotypic variability within the JAK2 V617F-positive MPD: roles of progenitor cell and neutrophil allele burdens.

OBJECTIVE: The myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), differ phenotypically, but share the same JAK2(V617F) mutation. We examined the relationship of the quantitative JAK2(V617F) allele burden to MPD disease phenotype among the three MPD classes and within PV. MATERIALS AND METHODS: We measured the JAK2(V617F) allele percentage in genomic DNA from neutrophils, CD34(+) cells, and cloned progenitors in 212 JAK2(V617F)-positive MPD patients and correlated the allele burdens to both disease class and disease features. RESULTS: In ET and PV, mean CD34(+) cell JAK2(V617F) allele burdens were lower than the corresponding neutrophil allele burdens, but these were equivalent in PMF. JAK2(WT) progenitors were present in ET and PV when the CD34(+) JAK2(V617F) allele burden was lower than the neutrophil allele burden, but not in PV and PMF subjects in whom the CD34(+) cell and neutrophil allele burdens were similar. CD34(+) cell JAK2(V617F) clonal dominance, defined as coherence between the CD34(+) cell and neutrophil JAK2(V617F) allele burdens, was present in 24% of ET, 56% of PV, and 93% of PMF patients, and was independent of the CD34(+) cell JAK2(V617F) genotype. Clonally dominant PV patients had significantly longer disease durations, higher white cell counts, and larger spleens than nondominant PV patients. CONCLUSIONS: We conclude that the extent of JAK2(V617F) CD34(+) cell clonal dominance is associated with disease phenotype within the MPD and, in PV, is associated with extramedullary disease, leukocytosis, and disease duration.

Phenotypic variations and new mutations in JAK2 V617F-negative polycythemia vera, erythrocytosis, and idiopathic myelofibrosis.

OBJECTIVE: The chronic myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis, and idiopathic myelofibrosis (IMF), are characterized by a spectrum of clinical features and linked by common genetic lesions in JAK2 and MPL. However, the clinical phenotypes in genetically undefined MPD patients are similar to those patients with JAK2 and MPL lesions. We, therefore, sought to determine whether there were JAK2 or MPL lesions in a well-defined, JAK2 V617F-negative MPD cohort, and to determine if clinical associations could be identified based on variations identified in these genes. METHODS: We examined the JAK2 and MPL genes in JAK2 V617F-negative PV, IMF, and idiopathic erythrocytosis patients for sequence variations. RESULTS: We identified two previously unrecognized JAK2 mutations and three previously unrecognized MPL mutations in JAK2 V617F-negative PV, erythrocytosis, and IMF patients. We identified JAK2 exon 12 lesions in 30% of JAK2 V617F-negative PV patients, and either JAK2 V617F or JAK2 exon 12 lesions in 9% of erythrocytosis patients. In IMF, in addition to the MPL gene mutation, W515K, we identified three additional mutations: 204P and two intervening sequence transitions, IVS 11/12 and 10/11. CONCLUSIONS: While the clinical phenotype of JAK2 exon 12 lesions in the MPD was predominantly erythroid, there was significant disease spectrum overlap between JAK2 V617F and JAK2 exon 12 mutations. By contrast, MPL gene mutations were not associated with erythrocytosis, but segregated primarily with the phenotypes of thrombocytosis, extramedullary disease, myelofibrosis, and osteosclerosis.

Disease burden at the progenitor level is a feature of primary myelofibrosis: a multivariable analysis of 164 JAK2 V617F-positive myeloproliferative neoplasm patients.

OBJECTIVE: Suppression of normal hematopoiesis by the neoplastic clone (clonal dominance) is a feature of the myeloproliferative neoplasms, but the determinants that predict clonal dominance are unknown. The objective of this study was to identify clinical and laboratory variables that associate with the JAK2 V617F CD34(+) progenitor allele burden and clonal dominance, which was defined by congruence of the JAK2 V617F CD34(+) progenitor and neutrophil allele burdens. MATERIALS AND METHODS: A cross-sectional analysis was performed on 164 consecutive JAK2 V617F-positive patients: 30 with essential thrombocytosis (ET), 100 with polycythemia vera (PV), and 34 with myelofibrosis (MF), including 8 post-ET MF and 3 post-PV MF. The JAK2 V617F CD34(+) progenitor and neutrophil allele burdens were measured using an allele-specific, quantitative real-time polymerase chain reaction assay. RESULTS: After adjusting for genotype, sex, age at diagnosis, and disease duration, disease type was the strongest predictor of clonal dominance, with the odds ratio being nearly 61.9 times higher for MF patients when compared with ET patients (p < 0.001), and 9.7 times higher when compared with PV patients (p = 0.002). Additionally, clonal dominance was associated with a clinical phenotype of an increased spleen size (p = 0.006), increased white blood cell count (p = 0.009), and lower hemoglobin (p < 0.001), even after adjusting for disease type and duration. CONCLUSIONS: These data indicate that loss of wild-type clones at the progenitor level is a feature of MF (primary MF, post-ET MF, and post-PV MF), presumably due to expansion of the JAK2 V617F clone and that this characteristic is surprisingly independent of JAK2 V617F homozygosity, suggesting that additional genomic lesions may contribute to this unique molecular process that distinguishes MF from ET and PV.

Ovarian cancer-associated ascites demonstrates altered immune environment: implications for antitumor immunity.

BACKGROUND: To identify immunosuppressive elements present in ovarian cancer associated ascites. PATIENTS AND METHODS: Ascites and plasma were obtained from ovarian, primary peritoneal or fallopian tube cancer patients. Surface markers were identified by fluorescence-activated cell sorting (FACS). Cytokine and chemokine concentrations were measured with LINCOplex microarrays. Antigen-specific T-lymphocytes from ascites and plasma were expanded with artificial antigen-presenting cells (aAPC). Cell-mediated immune response was assessed with chromium release assays. RESULTS: Samples were collected from 37 patients with advanced ovarian cancer. FACS was performed on 27 ascites specimens. A low CD4/CD8 ratio (<1.6) was seen in 13 patient samples and associated with significantly improved overall survival (p=0.040). LINCOplex evaluation of 22 paired ascites and plasma samples demonstrated significantly elevated levels of IL-6, IL-8, IL-10, IL-15, IP-10, MCP-1, MIP-1beta and VEGF and significantly reduced levels of IL-2, IL-5, IL-7, IL-17, PDGF-BB, and RANTES in ascites compared to plasma (p<0.05). Autologous ovarian cancer cell lysis with T-lymphocytes from ascites was limited. Although aAPC stimulation resulted in effective expansion of antigen specific T-cells from peripheral lymphocytes (35-fold), only limited expansion was noted from ascites-derived lymphocytes (10-fold). CONCLUSION: Ovarian cancer-associated ascites may provide an immunosuppressive environment. A high CD4/CD8 ratio, which may indicate the presence of regulatory T-cells, is associated with poor outcome. Reduced IL-2 and elevated IL-6 and IL-10 levels favor a Th2 inhibitory immune response. This immunosuppressive climate may explain our observation of non responsiveness in ascites derived T-cells.

Molecular mimicry in the chronic myeloproliferative disorders: reciprocity between quantitative JAK2 V617F and Mpl expression.

An activating JAK2 mutation (JAK2 V617F) is present in the chronic myeloproliferative disorders (MPDs), polycythemia vera (PV), idiopathic myelofibrosis (IMF), and essential thrombocytosis (ET). JAK2 is also a chaperone for Mpl and responsible for its cell-surface expression. We observed a reciprocal relationship between neutrophil JAK2 V617F allele percentage and platelet Mpl expression in JAK2 V617F-positive PV, IMF, and ET patients. However, severely impaired platelet Mpl expression was present in JAK2 V617F-negative MPD patients. While JAK2 V617F allele status did not necessarily correlate with the clinical MPD phenotype, the degree of impaired platelet Mpl expression did. We conclude that multiple molecular abnormalities are involved in the pathogenesis of the MPDs and that aberrant Mpl expression may be a common denominator of aberrant signaling in both the JAK2 V617F-positive and JAK2 V617F-negative MPDs.