Development Of Myeloproliferative Neoplasms Research Articles

Association between Inflammatory Factors and Myeloproliferative Neoplasm: A Bidirectional Mendelian Randomization Study

Introduction : Mounting epidemiological and observational evidence supports the association between chronic inflammation and the development of myeloproliferative neoplasm (MPN). Despite ongoing research, the causal or biased nature of genetic associations with specific inflammatory biomarkers remains unclear. The objective of this study is to evaluate the impact of C-reactive protein (CRP) and systemic regulators of inflammation on MPN using a bidirectional Mendelian randomization approach. Methods: Genetic associations with MPN were derived from a publicly available genome-wide association study (GWAS) comprising 1,086 cases and 407,155 controls of European ancestry. Additionally, data on inflammation were extracted from two GWASs focusing on CRP and cytokines. The primary analysis employed inverse-variance weighted Mendelian randomization (MR) as the statistical method. To validate the findings, various sensitivity analyses, such as MR-Egger, weighted median, and MR-pleiotropy residual sum and outlier (MR-PRESSO), were conducted simultaneously. Results: The findings of our study indicate that decreased levels of macrophage-migration inhibitory factor (MIF, IVW estimate odds ratio [ORIVW] per SD genetic cytokines change: 0.641; 95% CI: 0.427-0.964; P=0.032) and elevated levels of interleukin-2 receptor α (lL2Rα,1.377,1.006-1.883; P=0.046) are associated with an elevated risk of MPN. Moreover, genetically predicted MPN is correlated with heightened levels of RANTES (IVW estimate β: 0.043, 95% CI: 0.002-0.084; P=0.039) and interleukin-10 (IL-10, 0.030, 0.001-0.060; P=0.041). Conclusion: Our study provides evidence supporting the significant association between MIF and lL2Rαwith the etiology of MPN, while RANTES and interleukin-10 are more likely to be involved in MPN development. These findings offer valuable insights into the causal relationship between systemic inflammatory regulators and MPN, contributing to a better understanding of the etiology, prevention, and prognosis of MPN.

Revisiting Circulating Extracellular Matrix Fragments as Disease Markers in Myelofibrosis and Related Neoplasms.

Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) arise due to acquired somatic driver mutations in stem cells and develop over 10-30 years from the earliest cancer stages (essential thrombocythemia, polycythemia vera) towards the advanced myelofibrosis stage with bone marrow failure. The JAK2V617F mutation is the most prevalent driver mutation. Chronic inflammation is considered to be a major pathogenetic player, both as a trigger of MPN development and as a driver of disease progression. Chronic inflammation in MPNs is characterized by persistent connective tissue remodeling, which leads to organ dysfunction and ultimately, organ failure, due to excessive accumulation of extracellular matrix (ECM). Considering that MPNs are acquired clonal stem cell diseases developing in an inflammatory microenvironment in which the hematopoietic cell populations are progressively replaced by stromal proliferation-"a wound that never heals"-we herein aim to provide a comprehensive review of previous promising research in the field of circulating ECM fragments in the diagnosis, treatment and monitoring of MPNs. We address the rationales and highlight new perspectives for the use of circulating ECM protein fragments as biologically plausible, noninvasive disease markers in the management of MPNs.

Different niches for stem cells carrying the same oncogenic driver affect pathogenesis and therapy response in myeloproliferative neoplasms

Aging facilitates the expansion of hematopoietic stem cells (HSCs) carrying clonal hematopoiesis-related somatic mutations and the development of myeloid malignancies, such as myeloproliferative neoplasms (MPNs). While cooperating mutations can cause transformation, it is unclear whether distinct bone marrow (BM) HSC-niches can influence the growth and therapy response of HSCs carrying the same oncogenic driver. Here we found different BM niches for HSCs in MPN subtypes. JAK–STAT signaling differentially regulates CDC42-dependent HSC polarity, niche interaction and mutant cell expansion. Asymmetric HSC distribution causes differential BM niche remodeling: sinusoidal dilation in polycythemia vera and endosteal niche expansion in essential thrombocythemia. MPN development accelerates in a prematurely aged BM microenvironment, suggesting that the specialized niche can modulate mutant cell expansion. Finally, dissimilar HSC-niche interactions underpin variable clinical response to JAK inhibitor. Therefore, HSC-niche interactions influence the expansion rate and therapy response of cells carrying the same clonal hematopoiesis oncogenic driver.

Association of Agent Orange and myeloproliferative neoplasms, thrombosis, and bleeding among veterans.

7011 Background: The Myeloproliferative Neoplasms (MPNs) which include Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (MF) are acquired stem cell disorders with constitutively activated pathways from shared mutations leading to thrombotic and bleeding complications. Agent Orange (AO) is a chemical herbicide that was utilized during the Vietnam War and was associated with sarcomas and B-cell lymphomas. There is inadequate evidence of the association between AO exposure and development of MPNs, increased risk of bleeding, arterial thrombosis (AT), and venous thrombosis (VT). Methods: Utilizing the Veterans Affairs Informatics and Computing Infrastructure (VINCI) database, we performed a case control study from January 1, 2006 - January 26, 2023. We utilized ICD-9 and 10 codes to identify MPN patients, any AT, VT, and bleeding events. Age-matched controls (1:1) were selected from VINCI database. Qualitative data was compared by chi-square test. Results: We included all 95,768 MPN patients (0.7% MPN crude prevalence rate), 43,263 ET, 48,854 PV, and 3,651 MF patients. Among all MPNs, 6.4% were female, 93.6% were male, 76% were White, 13.8% Black, 0.8% American Indian, 0.9% Pacific Islander, and 8.5% unknown race. 86.8% of patients with MPN were aged 60 years or older. When comparing MPNs vs. controls, there was increased AT (29.5% vs. 24.5%, p < 0.0001), VT (6.7% vs. 3.8%, p < 0.0001), and both AT & VT (8.1% vs. 4.2%, p < 0.0001). Among female MPN patients, there were 16.2% AT, 6.1% VT, 45.5% bleeding events vs. 30.4% AT, 6.7% VT, and 39% bleeding events among male MPN patients, respectively, p < 0.0001. Among MPN patients who are greater than 60 years old, 95.4% had AT, 88% had VT, 94.4% had both AT & VT, and 89.4% had bleeding events. Bleeding events were higher in ET (44%), PMF (41.5%), PV (35%) vs. the control group (24.6%), p < 0.0001, and gastrointestinal bleeding was the most common site for both controls and MPNs followed by genitourinary or pulmonary. Among the different races, there is higher AT (range 22.6-31.5%) compared to VT (range 5.3-7.8%). There is a significant association between AO exposure and development of MPNs (odds ratio 1.61, 95% CI 1.57 to 1.65, p < 0.0001). There is a higher AT (36%) among MPNs with AO exposure compared to 28.2% in MPNs without AO exposure, p < 0.0001. There is a higher AT (28.7%) among controls with AO exposure compared to 24% in controls without AO exposure, p < 0.0001. There was 41.2% bleeding event with MPN with AO exposure compared to 39% in MPN without AO exposure, p < 0.0001. Conclusions: This is the largest database evaluating MPNs, thrombosis, and bleeding in veterans exposed to AO. There is an association of increased risk of development of MPNs, increased AT, and increased bleeding with AO exposure. Further studies including JAK2 mutation, CHIPs, and cardiovascular risk factors will be needed to evaluate contribution to thrombosis and bleeding risk.

Putative Role of Neutrophil Extracellular Trap Formation in Chronic Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs) are hematologic malignancies characterized by gene mutations that promote myeloproliferation and resistance to apoptosis via constitutively active signaling pathways, with Janus kinase 2-signal transducers and the activators of transcription (JAK-STAT) axis as a core part. Chronic inflammation has been described as a pivot for the development and advancement of MPNs from early stage cancer to pronounced bone marrow fibrosis, but there are still unresolved questions regarding this issue. The MPN neutrophils are characterized by upregulation of JAK target genes, they are in a state of activation and with deregulated apoptotic machinery. Deregulated neutrophil apoptotic cell death supports inflammation and steers them towards secondary necrosis or neutrophil extracellular trap (NET) formation, a trigger of inflammation both ways. NETs in proinflammatory bone marrow microenvironment induce hematopoietic precursor proliferation, which has an impact on hematopoietic disorders. In MPNs, neutrophils are primed for NET formation, and even though it seems obvious for NETs to intervene in the disease progression by supporting inflammation, no reliable data are available. We discuss in this review the potential pathophysiological relevance of NET formation in MPNs, with the intention of contributing to a better understanding of how neutrophils and neutrophil clonality can orchestrate the evolution of a pathological microenvironment in MPNs.

Deepening Our Understanding of the Factors Affecting Landscape of Myeloproliferative Neoplasms: What Do We Know about Them?

Myeloproliferative neoplasms (MPNs) arise from the uncontrolled proliferation of hematopoietic stem and progenitor cells in bone marrow. As with all tumors, the development of MPNs is a consequence of alterations in malignant cells and their interaction with other extrinsic factors that support and promote tumor progression. Since the discovery of driver mutations, much work has focused on studying and reviewing the genomic features of the disease but has neglected to delve into the important role that many other mechanisms may play. This review discusses the genetic component of MPNs but focuses mainly on some of the most relevant work investigating other non-genetic factors that may be crucial for the disease. The studies summarized here address MPN cell-intrinsic or -extrinsic factors and the interaction between them through transcriptomic, proteomic and microbiota studies, among others.

Antibodies to calnexin and mutated calreticulin are common in human sera

Purpose of the studyCalreticulin is an endoplasmic reticulum chaperone protein, which is involved in protein folding and in peptide loading of major histocompatibility complex class I molecules together with its homolog calnexin. Mutated calreticulin is associated with a group of hemopoietic disorders, especially myeloproliferative neoplasms. Currently only the cellular immune response to mutated calreticulin has been described, although preliminary findings have indicated that antibodies to mutated calreticulin are not specific for myeloproliferative disorders. These findings have prompted us to characterize the humoral immune response to mutated calreticulin and its chaperone homologue calnexin. Patients and methodsWe analyzed sera from myeloproliferative neoplasm patients, healthy donors and relapsing-remitting multiple sclerosis patients for the occurrence of autoantibodies to wild type and mutated calreticulin forms and to calnexin by enzyme-linked immunosorbent assay. ResultsAntibodies to mutated calreticulin and calnexin were present at similar levels in serum samples of myeloproliferative neoplasm and multiple sclerosis patients as well as healthy donors. Moreover, a high correlation between antibodies to mutated calreticulin and calnexin was seen for all patient and control groups. Epitope binding studies indicated that cross-reactive antibodies bound to a three-dimensional epitope encompassing a short linear sequence in the C-terminal of mutated calreticulin and calnexin. ConclusionCollectively, these findings indicate that calreticulin mutations may be common and not necessarily lead to onset of myeloproliferative neoplasm, possibly due to elimination of cells with mutations. This, in turn, may suggest that additional molecular changes may be required for development of myeloproliferative neoplasm.

Recurrent germline variant in ATM associated with familial myeloproliferative neoplasms.

Genetic predisposition (familial risk) in the myeloproliferative neoplasms (MPNs) is more common than the risk observed in most other cancers, including breast, prostate, and colon. Up to 10% of MPNs are considered to be familial. Recent genome-wide association studies have identified genomic loci associated with an MPN diagnosis. However, the identification of variants with functional contributions to the development of MPN remains limited. In this study, we have included 630 MPN patients and whole genome sequencing was performed in 64 individuals with familial MPN to uncover recurrent germline predisposition variants. Both targeted and unbiased filtering of single nucleotide variants (SNVs) was performed, with a comparison to 218 individuals with MPN unselected for familial status. This approach identified an ATM L2307F SNV occurring in nearly 8% of individuals with familial MPN. Structural protein modeling of this variant suggested stabilization of inactive ATM dimer, and alteration of the endogenous ATM locus in a human myeloid cell line resulted in decreased phosphorylation of the downstream tumor suppressor CHEK2. These results implicate ATM, and the DNA-damage response pathway, in predisposition to MPN.

Haploinsufficiency of Calreticulin Causes Dysregulation of Hematopoiesis and Results in a Myeloproliferative Phenotype in an in-Vivo Mouse Model

Background.JAK2V617F, MPLW515K/L and CALR exon9 mutations drive the development of Philadelphia negative Myeloproliferative Neoplasms (MPN). While JAK2 and MPL knock out (KO) leads to severe defects of hematopoiesis, the role of Calreticulin (CALR) deregulation is not fully understood, however recent reports demonstrated that CALR deletion leads to neoplastic transformation (Shide K et al Blood, 2020; Balliu M et al Blood Adv., 2021). Aim. To investigate the role of CALR in hematopoiesis and MPN development, we generated and characterized a Calr KO mouse model. Methods. We developed a Calr+/- mouse model by crossing mice carrying a floxed allele targeting Calr intron 3 and 7 (Calr+/flx) with Vav-Cre mice. The phenotypic characterization was performed by blood counts analysis, bone marrow (BM) and spleen histology, flow-cytometry and clonogenic assay by plating 2x104 cells for CFU-GEMM, CFU-GM and BFU-E and 2.5x105 for CFU-Mk. Proteome profiler membrane-based array and RNA-sequencing were employed to profiling BM protein and gene expression. Results. Sanger sequencing of BM cells of Calr+/- mice confirmed the deletion of Calr exons 4-7, resulting in the downregulation of Calr mRNA (-0.56-fold ±0.1) and decreased protein content (-0.47-fold ±0.21) compared to Calr+/flx mice (hence, controls). The productivity, body weight and lifespan were comparable between Calr+/- and controls. Calr+/- mice showed increased platelet count (median: 1360, range: 1156-1986 x109/L), 1.2 to 1.5-fold compared to controls, starting from month 4 (p=0.007) up to the latest time point available at 12 months (p=0.03). Erythrocytes count of Calr+/- increased after month 6 up to month 12 (median:10.3, range:9.9-10.6 x1012/L)(P=0.004) paralleled by an upward trend of hematocrit (median:47%, range:42-51%) and hemoglobin (median:18.3, range:14.2-20.1 g/L) levels. The spleen index (spleen:body weight ratio x100) resulted increased in Calr+/- (mean: 0.38±0.09) compared to controls (mean: 0.3±0.08) (P=0.039). Histology revealed an augmented number of poly-lobulated megakaryocytes, most of which found in clusters, in BM of Calr+/- mice. Most Calr+/- mice showed a progressive loss of spleen architecture with resulting loss of clear demarcation between red and white pulp combined with focal fibrosis, especially in aged mice. Calr haploinsufficiency conferred an augmented clonogenic capacity to BM and, to a lesser extent, splenic progenitors, in semisolid culture assay. BM Calr+/- CFU-GEMM, CFU-GM and CFU-Mk were in median 8.9 (5-15), 102.7 (49-157) and 16 (9.7-13.6) and 5.7 (3 -10) (P=0.007), 80.2 (33-159) (P=0.0031) and 11.9 (12.7-22) (P=0.048) in Calr+/flx, respectively. Of note, BM BFU-E count was 1.3-fold higher in Calr +/- than controls (P=0.009). Calr+/- splenic progenitors showed an enhanced clonogenic potential, strikingly for CFU-GEMM (P=0.0064). We observed that Calr+/- myeloid progenitors were significantly expanded when compared to controls: BM Mk precursors (FcγRlow/cKIT+/CD41+) and both BM and spleen LSK (Lin-/Sca1+/cKIT+) were, respectively, +1.6-fold (P=0.048), +1.4-fold (P=0.042) and +2-fold (P=0.008) than controls. Proteomic array performed on BM cells identified a set of deregulated proteins, mainly represented by cytokines (IL1α, IL4, IL6), chemokines (CXCL5, CXCL12, C1qR, VEGF, Endoglin) and cell adhesion molecules (VCAM, ICAM). These observations were strengthened by the result of transcriptomics analysis on bulk RNA-sequencing from Calr+/- and Calr+/flx mice BM cells highlighting a number of differentially expressed RNAs (increased n=385, decreased n=350) that resulted enriched in signaling pathways regulating chemokines and cell adhesion molecules, including "cell chemotaxis” (GO:0060326), "leukocyte migration” (GO:0050900), "regulation of cell-cell adhesion” (GO:0022407); cytokines, as determined by "Cytokine-cytokine receptor interaction” (KEGG:mmu04060); and Ca2+ homeostasis, including "cellular calcium ion homeostasis” (GO:0006874), "regulation of cytosolic calcium ion concentration” (GO:0051480 ). Conclusion. Finding from this in-vivo model suggest that haploinsufficiency of Calr induces dysregulation of hematopoiesis, ultimately resulting in a mild-ET phenotype. These findings will be validated in larger series and integrated with ongoing experiments to outline the molecular mechanisms underlying the CALR-driven MPN phenotype.

Mutational Profile of Children with Myeloproliferative Neoplasms

Introduction Myeloproliferative neoplasms (MPNs) are clonal bone marrow disorders of over proliferation driven largely by mutations in the JAK2, CALR, and MPL genes. Mutational profiles including driver and secondary mutations in adult subjects with MPNs have been well studied and recent work has determined that driver mutations occur as early as in utero. There is little described about mutational profiles in pediatric patients with MPNs. Given the importance of clonal evolution and the relevance of MPN-associated mutations for risk assessment, clinical features, and outcomes, it is important to develop an understanding of the mutational spectrum in young subjects. Therefore, we sought to evaluate a pediatric MPN cohort for potential MPN-associated mutations. Methods We isolated mononuclear cells from the blood and/or bone marrow of 29 pediatric patients (median age 10 years, range 2-21) with MPNs. DNA was extracted and sequenced on the HEMEPACT platform, a gene panel including almost 600 genes focused on myeloid malignancies. Variants were called using a combination of variant callers. For single-nucleotide variants (SNV), we used CaVEMan, Mutect, and Strelka, and for small insertions and deletions also used Pindel. VAF was uniformly reported across all called variants using a realignment procedure. Likely artifact variants were filtered out based on: (i) The number of callers calling a given variant and the combination of filters from the triple callers. (ii) Variants with VAF <2% in all samples or less than 3 mutant supporting reads were excluded. Results In this cohort, 18 subjects had essential thrombocytosis (ET), 8 subjects had polycythemia vera (PV), 2 had pre-fibrotic myelofibrosis, and 1 had myelofibrosis. Clinical sequencing identified 9 subjects with ET with a JAK2V617F mutation, 3 ET subjects with CALR mutations, and 6 ET subjects who were "triple negative" (TN) for driver mutations. Six subjects with PV were found to have a JAK2V617F mutation, while 2 had JAK2 exon 12 mutations. Both subjects with pre-fibrotic myelofibrosis had CALR mutations, and the subject with myelofibrosis had a MPL mutation. HEMEPACT identified mutations in 10 additional cancer-associated genes (Figure 1A). After JAK2 and CALR, the most frequently mutated genes were TET2 and SETBP1. Two subjects had TET2 mutations that co-occurred with JAK2V617F (Figure 1B), and both had very low TET2 variant allele frequencies (VAFs). Two subjects had SETBP1 mutations, one co-occurring with JAK2V617F, and 1 with CALR mutation (Figure 1B), both of whom had high SETBP1 VAFs, possibly implying a germline mutation. No mutations in ASXL1, EZH2, or DNMT3A were identified. Of the 6 TN subjects, 3 had mutations detected on HEMEPACT (in CHEK2, CUX1, and RB1). Eight of the 29 subjects (28%) had more than 1 mutation detected, and the average number of mutations was 1.2 per subject. In contrast, in a small cohort of adult (median age 41 years, range 21-82) subjects with ET (n=2) and PV (n=7), 6 of 9 subjects (67%) had more than 1 mutation identified, and the average number of mutations per subject was 2. JAK2V617F variant allele frequency of the adult cohort was 4-times higher than in the pediatric cohort (median=0.16 vs. median=0.04). Conclusion This is one of the largest analyses to date of the mutational spectrum of pediatric MPNs. TET2 and SETBP1 mutations co-occurred with driver mutations, and no DNMT3A or ASXL1 mutations were seen in these young patients. While fewer pediatric than adult subjects had multiple mutations, there were some young patients who had already developed multiple mutations in childhood and it is important to better understand the role clonal hematopoiesis may play in pediatric MPNs. Buccal samples in our pediatric patients showed evidence of contamination and identifying the best way to sample the germline from children with these disorders is crucial for the study of MPNs in young patients. There is much to learn about the mutational spectrum of pediatric MPNs, including the role of germline mutations in disease pathogenesis, drivers of MPN development in triple-negative MPNs, mutation acquisition over time, and role of various mutations in MPN progression and outcomes. With this initial evaluation we have begun the process of answering some of these questions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms

AbstractMutant calreticulin (CALR) proteins resulting from a −1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

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.

Inferring the initiation and development of myeloproliferative neoplasms

The developmental history of blood cancer begins with mutation acquisition and the resulting malignant clone expansion. The two most prevalent driver mutations found in myeloproliferative neoplasms-JAK2V617F and CALRm-occur in hematopoietic stem cells, which are highly complex to observe invivo. To circumvent this difficulty, we propose a method relying on mathematical modeling and statistical inference to determine disease initiation and dynamics. Our findings suggest that CALRm mutations tend to occur later in life than JAK2V617F. Our results confirm the higher proliferative advantage of the CALRm malignant clone compared to JAK2V617F. Furthermore, we illustrate how mathematical modeling and Bayesian inference can be used for setting up early screening strategies.

JAK2-V617F is a negative regulation factor of SHIP1 protein and thus influences the AKT signaling pathway in patients with Myeloproliferative neoplasm (MPN).

Myeloproliferative neoplasms (MPN) are a group of chronic haematological disorders. At the molecular level of MPN cells, the gain-of-function mutation V617F of the Janus kinase 2 (JAK2) leads to a constitutive activation of the downstream signaling cascade and is a conventional criteria for diagnosis. Here, the functional role of the tumor suppressor SHIP1 (SH2 domain containing inositol-5 phosphatase 1) in the pathogenesis of MPNs was investigated. Primary blood samples of MPN-patients were analysed using Western Blot technique regarding the level of SHIP1 expression. Moreover, SHIP1 and SHIP1-mutations were lentivirally transduced in the JAK2-V617F-positive UKE-1 cell line and expression was monitored over time. In addition, we examined SHIP1 reconstitution by inhibition of JAK2-V617F. Furthermore, we transfected SHIP1-expressing cells with a JAK2-V617F respectively a BCR-ABL construct and investigated changes in SHIP1 expression. Four out of five MPN-patient samples showed a loss or a reduction in SHIP1 expression. We identified JAK2 as a negative regulator of SHIP1 expression in MPN cells and inhibition of JAK2-V617F implicates a reconstituted SHIP1 expression. This is significant because SHIP1 negatively regulates the AKT signaling pathway and in consequence the reconstitution of SHIP1 expression leads to a decreased cell growth. Moreover, we examined the impact of SHIP1 and patient-derived SHIP1-mutations on AKT phosphorylation and show the benefit of a combined therapy in MPN cells with inhibitors of the AKT/mTOR pathway. In summary, the data suggest that SHIP1 may play a role during the development of MPNs and could be the basis for establishing a targeted therapy.

OBESITY AND MYELOPROLIFERATIVE NEOPLASMS: A SYSTEMATIC REVIEW

Objective: Obesity is a known trigger for carcinogenesis and its involvement is proven in the development of solid and blood cancers. However, the involvement of obesity in myeloproliferative neoplasms (MPNs), namely polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), has not been assessed so far. Design and method: Systematic review in PubMed/MEDLINE from its inception to March 13th, 2022. The database was searched using specific keywords/word combinations to identify relevant papers which were further evaluated if they were original articles and investigated the role of obesity in MPNs. Reviews, case reports, meeting abstracts, letters to the editor, in vivo/in vitro studies were excluded. Results: The initial search yielded 88 results of which 62 remained after the exclusion of reviews/case reports (n = 26). After screening of titles/abstracts, 25 full-texts were assessed of which 18 were entered into the qualitative analysis. Most conclusions were drawn from singular studies. Two papers pointed out that obesity affects the evaluation of red cell mass in PV due to the hypovascular nature of the adipose tissue, arguing for new reference ranges in obese subjects. Adults with obesity during adolescence had an increased risk of MPNs development during adulthood. Obesity was particularly linked to the development of ET. Obese MPNs adults had an elevated number of bone marrow adipocytes, higher total symptom burden (U-shaped association with BMI), higher rate of cerebrovascular accidents (ET) and venous thrombosis, lower risk of post-PV myelofibrosis and stroke, higher survival rates. Obesity did not influence anxiety in MPNs or the occurrence of thrombosis (ET). However, lower survival rates and thrombosis-free survival were noted in MPNs patients with multiple cardiovascular risk factors. PMF patients treated with ruxolitinib who had higher BMI values registered better outcomes. Ruxolitinib-treated subjects exhibited an increase in BMI. Obesity did not influence the outcome of allogeneic bone marrow transplantation in PMF, nor did it influence the outcome of this procedure in elderly patients transplanted for myeloid malignancies. Conclusions: Obesity can influence the development and evolution of MPNs and should be carefully monitored. Further studies should assess the obesity paradox in MPNs.

Genetic Knock-out of TNFR1 and TNFR2 in a JAK2-V617F Polycythemia Vera Mouse Model.

Genetic Knock-out of TNFR1 and TNFR2 in a JAK2-V617F Polycythemia Vera Mouse Model.

Insights into the Potential Mechanisms of JAK2V617F Somatic Mutation Contributing Distinct Phenotypes in Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are a group of blood cancers in which the bone marrow (BM) produces an overabundance of erythrocyte, white blood cells, or platelets. Philadelphia chromosome-negative MPN has three subtypes, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The over proliferation of blood cells is often associated with somatic mutations, such as JAK2, CALR, and MPL. JAK2V617F is present in 95% of PV and 50–60% of ET and PMF. Based on current molecular dynamics simulations of full JAK2 and the crystal structure of individual domains, it suggests that JAK2 maintains basal activity through self-inhibition, whereas other domains and linkers directly/indirectly enhance this self-inhibited state. Nevertheless, the JAK2V617F mutation is not the only determinant of MPN phenotype, as many normal individuals carry the JAK2V617F mutation without a disease phenotype. Here we review the major MPN phenotypes, JAK-STAT pathways, and mechanisms of development based on structural biology, while also describing the impact of other contributing factors such as gene mutation allele burden, JAK-STAT-related signaling pathways, epigenetic modifications, immune responses, and lifestyle on different MPN phenotypes. The cross-linking of these elements constitutes a complex network of interactions and generates differences in individual and cellular contexts that determine the phenotypic development of MPN.

The roles of sex and genetics in the MPN.

The Philadelphia chromosome negative myeloproliferative neoplasms(MPNs), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are acquired hematopoietic stem cell disorders driven by activating mutations of intracellular signal transduction pathways that control the production of circulating blood cells. The MPN are characterized clinically by marked variation in degrees of vascular risk, familial clustering, and evolution to myelofibrosis and acute leukemia. MPN disease presentations and outcomes are highly variable, and are markedly influenced by both sex and germline genetic variation. This chapter will focus on the evidence of sex and germline genetic background as modifiers of MPN development and outcomes. Large population genome wide association studies in both clonal hematopoiesis and MPN have revealed novel mechanisms, including inflammatory pathways and genomic instability, which further our understanding of how sex and genetic background mediate MPN risk. Recent advances in our understanding of clonal hematopoiesis and MPN development in various contexts informs the mechanisms by which sex, inflammation, exposures and genetics influence MPN incidence and outcomes, and provide opportunities to develop new strategies for prognostics and therapeutics in the MPN.

Whole Genome Sequencing Uncovers a Rare Germline Variant in ATM Associated with Familial Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are sporadic diseases characterized by a somatic driver mutation in the JAK2, CALR or MPL gene. Although it is generally considered a sporadic disease, approximately 10% of MPN cases display familial clustering, and there is a 5 to 7-fold increased risk of developing an MPN among first degree relatives of MPN patients. In contrast to other myeloid malignancies, investigation of large pedigrees with familial clustering of MPN has failed to identify high-risk predisposition genes relevant to the general MPN population. Genome wide association studies (GWAS) have identified common, low penetrance risk alleles for MPN predisposition in multiple genes including JAK2, TERT, TET2, ATM and SH2B3. In order to identify novel germline predisposition variants in MPN, an unbiased whole genome sequencing (WGS) approach was utilized to examine genomic structure and germline variations in a cohort of individuals with familial MPN.Methods: The study cohort was comprised of 67 individuals with familial MPN enrolled in a prospective research registry at Johns Hopkins Hospital. Familial MPN was defined as a diagnosis of MPN in an individual with a family history of MPN or related myeloid malignancy (myelodysplastic syndrome and chronic myelomonocytic leukemia) in a first or second degree relative. Neutrophil genomic DNA was subjected to WGS using Illumina HiSeq platform and sequenced to 60x depth. We performed germline variant calling using HaplotypeCaller and following the GATK best practices. The variants detected were further enriched for germline by allele frequency 40-60% or >90% and presence in the gnomAD database. Non-synonymous coding variants that occurred in the study cohort at a statistically higher frequency that in the general population (gnomAD) were selected for further analysis. Prediction of variant deleteriousness was assessed by 4 algorithms (Provean, SIFT, Polyphen-2, CADD).Results: Filtering of 32,788 non-synonymous, likely germline variants produced 148 that occurred at a higher frequency in our cohort than in the general population (p < 0.01, Fisher's exact test). Of these, 29 were predicted to be pathogenic in 3 out of 4 algorithms. Five unrelated individuals were found to harbor a heterozygous p.Leu2307Phe variant in the ATM gene (chr11:108326169 C>T). The clinical characteristics of these individuals are presented in Table 1. The structure prediction of ATM indicates that Ser2306 is a potential phosphorylation site of protein kinase A, suggesting that the methionine-aromatic bond between M2026 and the mutated F2307 may block the phosphorylation of S2306 (Figure 1).Conclusions: We identified a rare ATM germline variant (chr11:108326169 C>T; p.Leu2307Phe) present in 5 individuals with familial MPN. ATM is involved in DNA damage repair and important in the maintenance of genomic integrity. Heterozygous germline variants in ATM are known to predispose to multiple cancer types, including breast, prostate, pancreatic and melanoma. Further, common polymorphisms in ATM have been found to be associated with the MPN phenotype via GWAS. Our data suggests that this variant may impact ATM activation and its function in DNA damage repair, and functional studies are in progress. These data implicate a rare germline ATM variant as a novel risk factor for development of MPN. [Display omitted] DisclosuresHourigan: Sellas: Research Funding.

C-MYC Augments the Proliferation and Survival of Hematopoietic Stem Cells and Multipotent Progenitors to Drive Myeloproliferative Neoplasms

Human genomic studies have identified frequent MYC amplification and copy number gains in myeloid malignancies, and previous studies have shown that MYC plays important roles in survival of Myeloproliferative Neoplasms (MPN) and Acute Myeloid Leukemia (AML) cells. Notably, our recent studies have shown that MYC impairs myeloid cell differentiation and promotes proliferation of myeloid progenitors and AML cells by controlling genomic methylation. However, it is unclear if increased levels of MYC in hematopoietic stem cells (HSCs) and myeloid progenitors is sufficient to provoke the development of MPN or AML and, if so, how this occurs.To addresses these questions we generated Mx1-Cre;Rosa26-LSL-MYC transgenic mouse model that inducibly overexpress MYC following polyinosinic:polycytidylic acid (pIpC) injection and Cre-mediated deletion of loxp-stop-loxp cassette. MYC overexpression was confirmed by qRT-PCR and immunoblot. Complete blood counts (CBC) with differential in the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice vs. -MYC +/-or -wild type (WT) littermate mice at week 23 revealed worsening anemia (Hb, 9.6 vs. 16.3 vs. 15.5g/dL, p<0.0001), lymphopenia (73.2 vs. 84.3 vs. 84.5%, p<0.0001), and monocytosis (7.4 vs. 1.8 vs. 0.9%, p=0.0097). Also, bone marrow (BM) cells from the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice showed increased monocyte- and granulomonocyte-colony forming potential (CFU-M and CFU-GM), but with limited self-renewal capacity ex vivo (i.e., no CFU after 5 serial plating). Further, inducible MYC overexpression promotes expansion of HSCs (Lin -Sca-1 +cKit + [LSK]), multipotent progenitors (MPPs; LSK CD48 +CD150 -), common myeloid progenitors (CMPs; Lin -Sca1 -cKit +), granulocyte-monocyte progenitors (GMPs; Lin -Sca-1 -cKit +CD34 +FCγR +), and Gr-1/CD11b+ mature myeloid cells, with concomitant reduction of B220+ or CD3+ cells in the BM and spleen. In addition, MYC overexpression provokes splenomegaly (565 vs. 150 vs. 100mg at week 18~22, p<0.0001), extramedullary hematopoiesis with markedly atypical megakaryopoiesis and myeloid preponderance akin to MPN that reduces overall survival (median OS, 157 days vs. not reached vs. not reached, p<0.0001). Collectively, these findings suggest MYC confers enhanced proliferation and survival properties to HSCs and MPPs leading to MPN-like disease.We have shown MYC oncogenic functions in AML cells requires its suppression of TFEB, an mTORC1 regulated bHLH-LZ transcription factor, and that TFEB functions as a tumor suppressor by inducing IDH1/2-TET2 signaling, thus promoting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) conversion in key genes that drive myeloid differentiation and cell death. Similarly, inducible overexpression of MYC in the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice significantly reduces the expression of Tfeb, Idh1 and Idh2, and 5hmC levels in both c-Kit + and Cd11b + BM cells. Further, 4-OHT-mediated silencing of Myc in ex vivo cultured BM cells from the Rosa26-CreER T2+/-;Myc fl/fl mice impairs myeloid cell proliferation and robustly induces the expression of Tfeb, Idh1, and Idh2 as well as levels of 5hmC. Finally, inducible TFEB expression in normal 32D.3 myeloid progenitor cells impairs cell proliferation and upregulates 5hmC levels, and these responses are partially reversed by treatment with 2-hydroxyglutarate, an oncometabolite that inhibits 5mC-to-5hmC conversion. Collectively, these findings suggest that the MYC-TFEB-IDH1/2 epigenetic circuit plays a pivotal role in promoting myeloid proliferation to drive the malignant transformation of HSCs to the MPN. DisclosuresKuykendall: Pharmaessentia: Honoraria; Abbvie: Honoraria; Protagonist: Consultancy, Research Funding; Incyte: Consultancy; Blueprint: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria, Speakers Bureau. Komrokji: Agios: Honoraria, Speakers Bureau; Acceleron: Honoraria; Geron: Honoraria; Novartis: Honoraria; Abbvie: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; JAZZ: Honoraria, Speakers Bureau.