Novel germline JAK2R715T mutation causing PV-like erythrocytosis in 3 generations. Amelioration by Ropeg-Interferon.

Polycythemia vera (PV) is a clonal disorder arising from the acquired somatic mutations of the JAK2 gene, including JAK2V617F or several others in exon 12. A 38-year-old female had a stroke at age 32 and found to have elevated hemoglobin, normal leukocytes, normal platelets, and tested negative for JAK2V617F and exon 12 mutations. Next generation sequencing revealed a novel mutation: JAK2R715T in the pseudokinase domain (JH2) at 47.5%. Its presence in her nail DNA confirmed a germline origin. Her mother and her son similarly had erythrocytosis and a JAK2R715T mutation. Computer modeling indicated gain-of-function JAK2 activity. The propositus and her mother had polyclonal myelopoiesis, ruling out another somatic mutation-derived clonal hematopoiesis. Some erythroid progenitors of all three generations grew without erythropoietin, a hallmark of PV. The in vitro reporter assay confirmed increased activity of the JAK2R715T kinase. Similar to PV, the JAK2R715T native cells have increased STAT5 phosphorylation, augmented transcripts of prothrombotic and inflammatory genes, and decreased KLF2 transcripts. The propositus was not controlled by hydroxyurea, and JAK2 inhibitors were not tolerated; however, Ropeginterferon-alfa-2b (Ropeg-IFN-α) induced a remission. Ropeg-IFN-α treatment also reduced JAK2 activity in the propositus, her mother and JAK2V617F PV subjects. We report dominantly inherited erythrocytosis secondary to a novel germline JAK2R715T gain-of-function mutation with many but not all comparable molecular features to JAK2V617F PV. We also document a previously unreported inhibitory mechanism of JAK2 signaling by Ropeg-IFN-α.

Allogeneic stem cell transplant for multiple myeloma & myelofibrosis with split-dose busulfan, fludarabine & cyclophosphamide.

Allogeneic stem cell transplant can have high morbidity and mortality in patients with myelofibrosis (MF) and multiple myeloma (MM). This phase 2 study used a novel myeloablative regimen of split-dose busulfan, fludarabine, and then post-transplant cyclophosphamide. Four patients with MF and 2 with MM were enrolled. At 1 year, non-relapse mortality was 33.3%, and overall survival was 50%. Incidence of acute and chronic GVHD was 33.3% and 16.7%, respectively. Those surviving beyond 1 year (MF = 1, MM = 2) had durable remissions with a median follow-up of 42 months. This small study demonstrates relative safety & favorable key outcomes using this novel approach.

Increased transferrin protects from thrombosis in Chuvash erythrocytosis.

Von Hippel-Lindau protein (VHL) is essential to hypoxic regulation of cellular processes. VHL promotes proteolytic clearance of hypoxia-inducible transcription factors (HIFs) that have been modified by oxygen-dependent HIF-prolyl hydroxylases. A homozygous loss-of-function VHLR200W mutation causes Chuvash erythrocytosis, a congenital disorder caused by augmented hypoxia-sensing. Homozygous VHLR200W results in accumulation of HIFs that increase transcription of the erythropoietin gene and raise hematocrit. Phlebotomies reduce hematocrit and hyperviscosity symptoms. However, the major cause of morbidity and mortality in Chuvash erythrocytosis is thrombosis. Phlebotomies cause iron deficiency, which may further elevate HIF activity and transferrin, the HIF-regulated plasma iron transporter recently implicated in thrombogenesis. We hypothesized that transferrin is elevated in Chuvash erythrocytosis, and that iron deficiency contributes to its elevation and to thrombosis. We studied 155 patients and 154 matched controls at steady state and followed them for development of thrombosis. Baseline transferrin was elevated, and ferritin reduced in patients. VHLR200W homozygosity and lower ferritin correlated with higher erythropoietin and transferrin. During 11 years of follow-up, risk of thrombosis increased 8.9-fold in patients versus controls. Erythropoietin elevation, but not hematocrit or ferritin, correlated with thrombosis risk. Unexpectedly, transferrin elevation associated with reduced rather than increased thrombosis risk. The A allele of the promoter EPO single nucleotide polymorphisms (SNP), rs1617640, associated with elevated erythropoietin and increased thrombosis risk, whereas the A allele of the intronic TF SNP, rs3811647, associated with higher transferrin and protection from thrombosis in patients. Our findings suggest an unexpected causal relationship between increased transferrin and protection from thrombosis in Chuvash erythrocytosis.

Downregulated KLF2 in polycythemia vera and essential thrombocythemia induces prothrombotic gene expression.

Thromboses are major causes of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET) diseases associated with JAK2V617F mutation. However, the molecular mechanism(s) of increased thrombosis in PV and ET remain unknown. Kruppel-like factor 2 (KLF2) is a transcription factor that regulates expression of genes associated with inflammation and thrombosis; the absence of KLF2 in neutrophils causes thrombosis by inducing tissue factor. We studied the role of KLF2 in regulating prothrombotic gene expression in PV and ET. Neutrophils and platelets KLF2 expression in PV and ET was lower than the controls. Furthermore, in patients with thromboses, KLF2 transcripts were lower in platelets than those without thromboses. JAK2V617F allelic burden was inversely correlated with KLF2 transcript levels, suggesting JAK-STAT pathway may downregulate KLF2 expression. Whole transcriptome analyses of neutrophils and platelets showed that a lower KLF2 expression was associated with an upregulation of KLF2-regulated thrombotic genes. In addition, low KLF2 expression in platelets positively correlated with thrombotic events. In patients with PV and ET, KLF2 expression was induced by pegylated interferon alfa (PegINF-α) but not by hydroxyurea treatments. These data suggest that KLF2 may be a regulator of PV and ET thrombosis and a novel therapeutic target to prevent thrombosis.

Developmental changes in iron metabolism and erythropoiesis in mice with human gain-of-function erythropoietin receptor.

Iron availability for erythropoiesis is controlled by the iron-regulatory hormone hepcidin. Increased erythropoiesis negatively regulates hepcidin synthesis by erythroferrone (ERFE), a hormone produced by erythroid precursors in response to erythropoietin (EPO). The mechanisms coordinating erythropoietic activity with iron homeostasis in erythrocytosis with low EPO are not well defined as exemplified by dominantly inherited (heterozygous) gain-of-function mutation of human EPO receptor (mtHEPOR) with low EPO characterized by postnatal erythrocytosis. We previously created a mouse model of this mtHEPOR that develops fetal erythrocytosis with a transient perinatal amelioration of erythrocytosis and its reappearance at 3-6 weeks of age. Prenatally and perinatally, mtHEPOR heterozygous and homozygous mice (differing in erythrocytosis severity) had increased Erfe transcripts, reduced hepcidin, and iron deficiency. Epo was transiently normal in the prenatal life; then decreased at postnatal day 7, and remained reduced in adulthood. Postnatally, hepcidin increased in mtHEPOR heterozygotes and homozygotes, accompanied by low Erfe induction and iron accumulation. With aging, the old, especially mtHEPOR homozygotes had a decline of erythropoiesis, myeloid expansion, and local bone marrow inflammatory stress. In addition, mtHEPOR erythrocytes had a reduced lifespan. This, together with reduced iron demand for erythropoiesis, due to its age-related attenuation, likely contributes to increased iron deposition in the aged mtHEPOR mice. In conclusion, the erythroid drive-mediated inhibition of hepcidin production in mtHEPOR mice in the prenatal/perinatal period is postnatally abrogated by increasing iron stores promoting hepcidin synthesis. The differences observed in studied characteristics between mtHEPOR heterozygotes and homozygotes suggest dose-dependent alterations of downstream EPOR stimulation.

A randomized phase 3 trial of interferon-α vs hydroxyurea in polycythemia vera and essential thrombocythemia.

The goal of therapy for patients with essential thrombocythemia (ET) and polycythemia vera (PV) is to reduce thrombotic events by normalizing blood counts. Hydroxyurea (HU) and interferon-α (IFN-α) are the most frequently used cytoreductive options for patients with ET and PV at high risk for vascular complications. Myeloproliferative Disorders Research Consortium 112 was an investigator-initiated, phase 3 trial comparing HU to pegylated IFN-α (PEG) in treatment-naïve, high-risk patients with ET/PV. The primary endpoint was complete response (CR) rate at 12 months. A total of 168 patients were treated for a median of 81.0 weeks. CR for HU was 37% and 35% for PEG (P = .80) at 12 months. At 24 to 36 months, CR was 20% to 17% for HU and 29% to 33% for PEG. PEG led to a greater reduction in JAK2V617F at 24 months, but histopathologic responses were more frequent with HU. Thrombotic events and disease progression were infrequent in both arms, whereas grade 3/4 adverse events were more frequent with PEG (46% vs 28%). At 12 months of treatment, there was no significant difference in CR rates between HU and PEG. This study indicates that PEG and HU are both effective treatments for PV and ET. With longer treatment, PEG was more effective in normalizing blood counts and reducing driver mutation burden, whereas HU produced more histopathologic responses. Despite these differences, both agents did not differ in limiting thrombotic events and disease progression in high-risk patients with ET/PV. This trial was registered at www.clinicaltrials.gov as #NCT01259856.

α-Ketoglutarate Inhibits Thrombosis and Inflammation by Prolyl Hydroxylase-2 Mediated Inactivation of Phospho-Akt.

BACKGROUND: Phospho-Akt1 (pAkt1) undergoes prolyl hydroxylation at Pro125 and Pro313 by the prolyl hydroxylase-2 (PHD2) in a reaction decarboxylating α-ketoglutarate (αKG). We investigated whether the αKG supplementation could inhibit Akt-mediated activation of platelets and monocytes, in vitro as well as in vivo, by augmenting PHD2 activity. METHODS: We treated platelets or monocytes isolated from healthy individuals with αKG in presence of agonists in vitro and assessed the signalling molecules including pAkt1. We supplemented mice with dietary αKG and estimated the functional responses of platelets and monocytes ex vivo. Further, we investigated the impact of dietary αKG on inflammation and thrombosis in lungs of mice either treated with thrombosis-inducing agent carrageenan or infected with SARS-CoV-2. FINDINGS: Octyl αKG supplementation to platelets promoted PHD2 activity through elevated intracellular αKG to succinate ratio, and reduced aggregation in vitro by suppressing pAkt1(Thr308). Augmented PHD2 activity was confirmed by increased hydroxylated-proline and enhanced binding of PHD2 to pAkt in αKG-treated platelets. Contrastingly, inhibitors of PHD2 significantly increased pAkt1 in platelets. Octyl-αKG followed similar mechanism in monocytes to inhibit cytokine secretion in vitro. Our data also describe a suppressed pAkt1 and reduced activation of platelets and leukocytes ex vivo from mice supplemented with dietary αKG, unaccompanied by alteration in their number. Dietary αKG significantly reduced clot formation and leukocyte accumulation in various organs including lungs of mice treated with thrombosis-inducing agent carrageenan. Importantly, in SARS-CoV-2 infected hamsters, we observed a significant rescue effect of dietary αKG on inflamed lungs with significantly reduced leukocyte accumulation, clot formation and viral load alongside down-modulation of pAkt in the lung of the infected animals. INTERPRETATION: Our study suggests that dietary αKG supplementation prevents Akt-driven maladies such as thrombosis and inflammation and rescues pathology of COVID19-infected lungs. FUNDING: Study was funded by the Department of Biotechnology (DBT), Govt. of India (grants: BT/PR22881 and BT/PR22985); and the Science and Engineering Research Board, Govt. of India (CRG/000092).

Anticoagulation management post-transjugular intrahepatic portosystemic shunt in portal hypertension associated with myeloproliferative neoplasms.

Portal hypertension (pHTN) complicates myeloproliferative neoplasms (MPNs), and usually occurs due to Budd-Chiari syndrome or splanchnic vein thrombosis. Current management modalities for MPN-associated pHTN include anticoagulation, transjugular intrahepatic portosystemic shunt (TIPS), and orthotopic liver transplant. Data on the thrombotic and bleeding outcomes of this practice is of poor quality, and whether direct oral anticoagulants (DOACs) are effective in this setting is unknown. We describe failure of DOACs to prevent post-TIPS complications in two case reports of patients with MPN-associated pHTN and review the associated literature. We conducted a comprehensive search in Embase (embase.com), Scopus (scopus.org), and PubMed for existing data on MPN-associated pHTN post-TIPS procedure. Four studies (n = 251) of patients with pHTN post-TIPS were eligible (MPN, n = 143). A review of the literature suggests that patients with MPN-associated pHTN may be at higher risk for post-TIPS complications including stent thrombosis and stenosis, compared with other causes of thrombotic pHTN. DOAC use has not been studied in this setting. While further studies to guide optimal management of MPN-associated pHTN post-TIPS are needed, available evidence suggests that life-long anticoagulation is warranted. DOACs should not be considered standard of care because of lack of evidence of efficacy.

Large-Scale Identification of Clonal Hematopoiesis and Mutations Recurrent in Blood Cancers.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by detectable hematopoietic-associated gene mutations in a person without evidence of hematologic malignancy. We sought to identify additional cancer-presenting mutations useable for CHIP detection by performing a data mining analysis of 48 somatic mutation studies reporting mutations at diagnoses of 7,430 adult and pediatric patients with hematologic malignancies. Following extraction of 20,141 protein-altering mutations, we identified 434 significantly recurrent mutation hotspots, 364 of which occurred at loci confidently assessable for CHIP. We then performed an additional large-scale analysis of whole exome sequencing data from 4,538 persons belonging to three non-cancer cohorts for clonal mutations. We found the combined cohort prevalence of CHIP with mutations identical to those reported at blood cancer mutation hotspots to be 1.8%, and that some of these CHIP mutations occurred in children. Our findings may help to improve CHIP detection and pre-cancer surveillance for both children and adults.

Thrombotic, inflammatory, and HIF-regulated genes and thrombosis risk in polycythemia vera and essential thrombocythemia.

Thrombosis is a major cause of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET). The pathophysiology of thrombosis in these disorders remains unclear, and we hypothesized that upregulation of thrombotic, inflammatory, and hypoxia-inducible factor (HIF)-regulated genes may play a role in it. We performed unbiased RNA sequencing in granulocytes and platelets of PV patients and found differential expression of several thrombotic, inflammatory, and HIF-regulated genes. The expression of many of these genes positively correlated with JAK2 expression and JAK2V617F allelic burden. We then validated these findings by quantitative polymerase chain reaction analyses of selected gene transcripts in a larger number of PV and ET granulocytes and platelets (58 patients) and in 28 controls, and we compared these findings in patients with and without thrombosis. The study included 29 females and 29 males; of these, 28 had a history of thrombosis. We found that transcripts of several selected genes were upregulated in patients with PV or ET compared with controls. In granulocytes, the expression levels of F3, SELP, VEGFA, and SLC2A1 were significantly higher in patients with a history of thrombosis compared with those who did not have thrombosis. Patients with a history of thrombosis have significantly higher expression of IL1RAP (P < .05) in platelets compared with those without thrombosis. Our study confirms the presence of a thrombo-inflammatory state and augmented HIF activity in PV and ET and its role in thrombosis. These data may provide the background for targeted therapies in PV and ET.

Clonal hematopoiesis in hematological disorders: Three different scenarios.

Clonality studies can establish the single-cell origin of tumors and thus differentiate clonal malignant and premalignant processes from reactive polyclonal processes. Detection of clonal cells may be based on direct tracking of cell lineage-specific sequences or disease-specific somatic mutations identifying the clonal population. Historically, clonal hematopoiesis was defined using the principle of X-chromosome inactivation based on observation that in circulating clonal cells, only one of the active chromosomes was expressed. In myeloproliferative neoplasms (MPNs) virtually all circulating erythrocytes, platelets, and granulocytes are products of single mutated stem cells that preferentially differentiate into the myeloid rather than lymphoid lineage. Thus, clonal differentiated myeloid cells co-exist in circulation with polyclonal long-lived T lymphocytes that originated before the MPN-initiating somatic clonal event. Chronic lymphocytic leukemia (CLL) starts in a differentiating B cell, but other lymphoid lineages and myeloid cells remain polyclonal. Normal T and B cells co-exist with the CLL clone, but are diluted by the massively expanded CLL population, which outnumbers the residual normal cells. Clonal hematopoiesis of undetermined potential (CHIP) has been identified by whole-genome sequencing of healthy individuals. These clones contain a specific somatic mutation previously considered to be disease defining but are detected in only a small proportion of circulating leukocytes, and there is no obvious suppression of normal hematopoietic stem cells. However, more studies are needed to properly define these clones, their persistence or disappearance, and their relative propensity for transforming into leukemias, myeloproliferative neoplasms, or other clonal hematological malignancies.

JAK2 ex13InDel drives oncogenic transformation and is associated with chronic eosinophilic leukemia and polycythemia vera.

The V617F mutation in the JH2 domain of Janus kinase 2 (JAK2) is an oncogenic driver in several myeloproliferative neoplasms (MPNs), including essential thrombocythemia, myelofibrosis, and polycythemia vera (PV). Other mutations in JAK2 have been identified in MPNs, most notably exon 12 mutations in PV. Here, we describe a novel recurrent mutation characterized by a common 4-amino-acid deletion and variable 1-amino-acid insertion (Leu583-Ala586DelInsSer/Gln/Pro) within the JH2 domain of JAK2. All 4 affected patients had eosinophilia, and both patients with Leu583-Ala586DelInsSer fulfilled diagnostic criteria of both PV and chronic eosinophilic leukemia (CEL). Computational and functional studies revealed that Leu583-Ala586DelInsSer (herein referred to as JAK2ex13InDel) deregulates JAK2 through a mechanism similar to JAK2V617F, activates signal transducer and activator of transcription 5 and extracellular signal-regulated kinase, and transforms parental Ba/F3 cells to growth factor independence. In contrast to JAK2V617F, JAK2ex13InDel does not require an exogenous homodimeric type 1 cytokine receptor to transform Ba/F3 cells and is capable of activating ß common chain family cytokine receptor (interleukin-3 receptor [IL-3R], IL-5R, and granulocyte-macrophage colony stimulating factor receptor) signaling in the absence of ligand, with the maximum effect observed for IL-5R, consistent with the clinical phenotype of eosinophilia. Recognizing this new PV/CEL-overlap MPN has significant clinical implications, as both PV and CEL patients are at high risk for thrombosis, and concomitant cytoreduction of red cells, neutrophils, and eosinophils may be required for prevention of thromboembolic events. Targeted next-generation sequencing for genes recurrently mutated in myeloid malignancies in patients with unexplained eosinophilia may reveal additional cases of Leu583-Ala586DelInsSer/Gln/Pro, allowing for complete characterization of this unique MPN.

Experimental Modeling of Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are genetically very complex and heterogeneous diseases in which the acquisition of a somatic driver mutation triggers three main myeloid cytokine receptors, and phenotypically expresses as polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). The course of the diseases may be influenced by germline predispositions, modifying mutations, their order of acquisition and environmental factors such as aging and inflammation. Deciphering these contributory elements, their mutual interrelationships, and their contribution to MPN pathogenesis brings important insights into the diseases. Animal models (mainly mouse and zebrafish) have already significantly contributed to understanding the role of several acquired and germline mutations in MPN oncogenic signaling. Novel technologies such as induced pluripotent stem cells (iPSCs) and precise genome editing (using CRISPR/Cas9) contribute to the emerging understanding of MPN pathogenesis and clonal architecture, and form a convenient platform for evaluating drug efficacy. In this overview, the genetic landscape of MPN is briefly described, with an attempt to cover the main discoveries of the last 15 years. Mouse and zebrafish models of the driver mutations are discussed and followed by a review of recent progress in modeling MPN with patient-derived iPSCs and CRISPR/Cas9 gene editing.

Iron chelation and 2-oxoglutarate-dependent dioxygenase inhibition suppress mantle cell lymphoma's cyclin D1.

The patients with mantle cell lymphoma (MCL) have translocation t(11;14) associated with cyclin D1 overexpression. We observed that iron (an essential cofactor of dioxygenases including prolyl hydroxylases [PHDs]) depletion by deferoxamine blocked MCL cells' proliferation, increased expression of DNA damage marker γH2AX, induced cell cycle arrest and decreased cyclin D1 level. Treatment of MCL cell lines with dimethyloxalylglycine, which blocks dioxygenases involving PHDs by competing with their substrate 2-oxoglutarate, leads to their decreased proliferation and the decrease of cyclin D1 level. We then postulated that loss of EGLN2/PHD1 in MCL cells may lead to down-regulation of cyclin D1 by blocking the degradation of FOXO3A, a cyclin D1 suppressor. However, the CRISPR/Cas9-based loss-of-function of EGLN2/PHD1 did not affect cyclin D1 expression and the loss of FOXO3A did not restore cyclin D1 levels after iron chelation. These data suggest that expression of cyclin D1 in MCL is not controlled by ENGL2/PHD1-FOXO3A pathway and that chelation- and 2-oxoglutarate competition-mediated down-regulation of cyclin D1 in MCL cells is driven by yet unknown mechanism involving iron- and 2-oxoglutarate-dependent dioxygenases other than PHD1. These data support further exploration of the use of iron chelation and 2-oxoglutarate-dependent dioxygenase inhibitors as a novel therapy of MCL.

Pegylated interferon alfa-2a for polycythemia vera or essential thrombocythemia resistant or intolerant to hydroxyurea.

Prior studies have reported high response rates with recombinant interferon-α (rIFN-α) therapy in patients with essential thrombocythemia (ET) and polycythemia vera (PV). To further define the role of rIFN-α, we investigated the outcomes of pegylated-rIFN-α2a (PEG) therapy in ET and PV patients previously treated with hydroxyurea (HU). The Myeloproliferative Disorders Research Consortium (MPD-RC)-111 study was an investigator-initiated, international, multicenter, phase 2 trial evaluating the ability of PEG therapy to induce complete (CR) and partial (PR) hematologic responses in patients with high-risk ET or PV who were either refractory or intolerant to HU. The study included 65 patients with ET and 50 patients with PV. The overall response rates (ORRs; CR/PR) at 12 months were 69.2% (43.1% and 26.2%) in ET patients and 60% (22% and 38%) in PV patients. CR rates were higher in CALR-mutated ET patients (56.5% vs 28.0%; P = .01), compared with those in subjects lacking a CALR mutation. The median absolute reduction in JAK2V617F variant allele fraction was -6% (range, -84% to 47%) in patients achieving a CR vs +4% (range, -18% to 56%) in patients with PR or nonresponse (NR). Therapy was associated with a significant rate of adverse events (AEs); most were manageable, and PEG discontinuation related to AEs occurred in only 13.9% of subjects. We conclude that PEG is an effective therapy for patients with ET or PV who were previously refractory and/or intolerant of HU. This trial was registered at www.clinicaltrials.gov as #NCT01259856.

Development of a symptom assessment in patients with myelofibrosis: qualitative study findings.

BACKGROUND: The goal of the research reported here was to understand the patient experience of living with myelofibrosis (MF) and establish content validity of the Modified Myeloproliferative Neoplasm Symptom Assessment Diary (MPN-SD). METHODS: Qualitative interviews were performed in patients with MF, including both concept elicitation and cognitive debriefing. Patients with MF were asked to spontaneously report on their signs, symptoms, and impacts of MF, as well as their understanding of the MPN-SD content, and use of the tool on an electronic platform. A supplementary literature review and meetings with MF experts were also performed. RESULTS: Twenty-three patients with MF participated in qualitative interviews. Signs and symptoms most commonly reported by ruxolitinib-experienced patients (n = 16) were: fatigue and/or tiredness (n = 16, 100%), shortness of breath (n = 11, 69%), pain below the ribs on the left side and/or stomach pain and/or abdominal pain (n = 9, 56%), and enlarged spleen (n = 9, 56%) and for ruxolitinib-naïve patients (n = 7) were: fatigue and/or tiredness (n = 6, 86%), pain below the ribs on the left side (n = 6, 86%), enlarged spleen (n = 4, 57%), full quickly/filling up quickly (n = 4, 57%), night sweats and/or general sweats (n = 4, 57%), and itching (n = 4, 57%). Patients demonstrated that they were able to read, understand, and provide meaningful responses to the MPN-SD. The final version of the MPN-SD includes the 10 most commonly reported concepts from the MF patient interviews. CONCLUSIONS: The findings demonstrate the comprehensiveness of the MPN-SD in assessing MF symptoms in both ruxolitinib-experienced and ruxolitinib-naïve patients, while remaining easy for patients to understand and complete.