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Philadelphia chromosome-positive (Ph+) lymphoid blast crisis (BC), emanating from chronic myeloid leukemia (CML), is a fatal disease with limited treatment options. Asciminib (ABL001) is a novel selective allosteric inhibitor of the ABL kinase with high efficacy against TKI-resistant BCR::ABL1. In this study, we demonstrate significant suppression of an aggressive B-lymphoblastic disease and restoration of normal hematopoiesis in an inducible transgenic mouse model of p210-BCR::ABL1-positive CML-BC. Molecularly, asciminib treatment significantly reduced BCR::ABL1transcripts to background levels, demonstrating its ability to suppress BCR::ABL1-induced disease. Furthermore, asciminib treatment normalized the long-term repopulating hematopoietic stem cell (LT-HSC) population in the BM, suggesting the selective targeting of malignant LT-HSCs. This was supported by secondary transplantation experiments, resulting in absence of BC in a proportion of mice. Importantly, none of the secondary transplanted mice that received further asciminib treatment developed leukemia. Sanger sequencing of the BCR::ABL1myristoyl pocket region of both treatment-naïve and treated mice demonstrated a high mutational load. However, there was no indication of asciminib-specific mutations. These promising findings highlight the potential of asciminib as a drug that targets BC stem cells and as an alternative stand-alone or combinatorial therapy for first-line treatment of CML BC or Ph+ acute lymphoblastic leukemia.

New options for medical therapy and risk scoring systems containing molecular data are leading to increased complexity in the management of patients with myelofibrosis. To inform patients’ optimal care, we updated the 2015 guidelines on indications for and management of allogeneic haematopoietic stem-cell transplantation (HSCT) with the support of the European Society for Blood and Marrow Transplantation (EBMT) and European LeukemiaNet (ELN). New recommendations were produced using a consensus-building methodology after a comprehensive review of articles released from January, 2015 to December, 2022. Seven domains and 18 key questions were selected through a series of questionnaires using a Delphi process. Key recommendations in this update include: patients with primary myelofibrosis and an intermediate-2 or high-risk Dynamic International Prognostic Scoring System score, or a high-risk Mutation-Enhanced International Prognostic Score Systems (MIPSS70 or MIPSS70-plus) score, or a low-risk or intermediate-risk Myelofibrosis Transplant Scoring System score should be considered candidates for allogeneic HSCT. All patients who are candidates for allogeneic HSCT with splenomegaly greater than 5 cm below the left costal margin or splenomegaly-related symptoms should receive a spleen-directed treatment, ideally with a JAK-inhibitor; HLA-matched sibling donors remain the preferred donor source to date. Reduced intensity conditioning and myeloablative conditioning are both valid options for patients with myelofibrosis. Regular post-transplantation driver mutation monitoring is recommended to detect and treat early relapse with donor lymphocyte infusion. In a disease where evidence-based guidance is scarce, these recommendations might help clinicians and patients in shared decision making.

The persistence of leukemic stem cells (LSCs) represents a problem in the therapy of chronic myeloid leukemia (CML). Hence, it is of utmost importance to explore the underlying mechanisms to develop new therapeutic approaches to cure CML. Using the genetically engineered ScltTA/TRE-BCR::ABL1mouse model for chronic phase CML, we previously demonstrated that the loss of the docking protein GAB2 counteracts the infiltration of mast cells (MCs) in the bone marrow (BM) of BCR::ABL1 positive mice. Here, we show for the first time that BCR::ABL1 drives the cytokine independent expansion of BM derived MCs and sensitizes them for FcεRI triggered degranulation. Importantly, we demonstrate that genetic mast cell deficiency conferred by the Cpa3Creallele prevents BCR::ABL1 induced splenomegaly and impairs the production of pro-inflammatory cytokines. Furthermore, we show in CML patients that splenomegaly is associated with high BM MC counts and that upregulation of pro-inflammatory cytokines in patient serum samples correlates with tryptase levels. Finally, MC-associated transcripts were elevated in human CML BM samples. Thus, our study identifies MCs as essential contributors to disease progression and suggests considering them as an additional target in CML therapy.

Hintergrund: Die essenzielle Thrombozythämie (ET) und die Polycythaemia vera (PV) gehören zu den myeloproliferativen Neoplasien. Durch eine verbesserte molekulare und pathologische Diagnostik werden heutzutage beide Erkrankungen zunehmend auch bei jüngeren Patienten diagnostiziert. Die Prognose wird durch arterielle und venöse thromboembolische Komplikationen und im Spätstadium durch das Auftreten einer sekundären Myelofibrose oder einer akuten myeloischen Leukämie bestimmt. Ziel: Neben Hintergründen zur Ätiologie und Pathogenese beider Erkrankungen wird auf die aktuelle Diagnostik nach Weltgesundheitsorganisation (WHO), das klinische Bild, die Risikostratifikation und die Therapie eingegangen. Material und Methoden: Diese Arbeit basiert auf einer selektiven Literaturrecherche in der Datenbank PubMed zum Thema MPN, ET und PV. Ergebnisse und Schlussfolgerung: Im klinischen Alltag ist die differenzialdiagnostische Abklärung einer Thrombozytose oder einer Polyglobulie häufig und relevant. Anhand der WHO-Kriterien kann heute eine ET oder eine PV von einer sekundären Thrombozytose oder Erythrozytose abgegrenzt werden. Hierbei kommt sowohl der Klinik als auch der Molekulardiagnostik und der Knochenmarkhistologie eine besondere Rolle zu. Liegt dann die seltenere Diagnose einer ET oder einer PV vor, ist im nächsten Schritt eine Risikostratifikation notwendig, um Patienten zu identifizieren, die zusätzlich zu den Basismaßnahmen (Acetylsalicylsäure, Aderlasstherapie) eine zytoreduktive Therapie benötigen. Neben den bisher bewährten Medikamenten Hydroxyurea und Anagrelid haben heute moderne Therapieansätze mit Interferon-alpha und dem Januskinase(JAK)-Hemmer Ruxolitinib eine besondere Bedeutung.

Janus kinase (JAK) inhibitors approved for myelofibrosis provide spleen and symptom improvements but do not meaningfully improve anaemia. Momelotinib, a first-in-class inhibitor of activin A receptor type 1 as well as JAK1 and JAK2, has shown symptom, spleen, and anaemia benefits in myelofibrosis. We aimed to confirm the differentiated clinical benefits of momelotinib versus the active comparator danazol in JAK-inhibitor-exposed, symptomatic patients with anaemia and intermediate-risk or high-risk myelofibrosis.

Polycythaemia vera is associated with a reduced quality of life, a high rate of vascular events, and an intrinsic risk of disease evolution. The results of several randomised trials for the treatment of this disorder are now available, and both a new ropegylated formulation of interferon alfa-2b (ropeginterferon alfa-2b; 2018) and ruxolitinib (2015) have been approved in Europe. European LeukemiaNet (ELN) investigators have therefore deemed it appropriate to provide recommendations for the use of these drugs in clinical practice. An expert panel of 14 senior haematologists from ELN centres that had actively participated in previous ELN projects or relevant randomised trials, chaired by a member of the ELN Steering Committee, developed a list of clinical questions, and a methodologist established three patient, intervention, comparator, outcome (PICO) questions and systematically reviewed the evidence. Recommendations were approved by six Delphi consensus rounds and two virtual meetings (on Jan 26, 2021, and June 24, 2021). The expert panel recommended that patients with polycythaemia vera who are younger than 60 years and have not had previous thrombotic events should start cytoreductive drug therapy if at least one of the following criteria are fulfilled: strictly defined intolerance to phlebotomy, symptomatic progressive splenomegaly, persistent leukocytosis (>15 × 109white blood cells per L), progressive leukocytosis (at least 100% increase if baseline count is <10 × 109cells per L or at least 50% increase if baseline count is >10 × 109cells per L), extreme thrombocytosis (>1500 × 109platelets per L), inadequate haematocrit control requiring phlebotomies, persistently high cardiovascular risk, and persistently high symptom burden. Recombinant interferon alfa, either in the form of ropeginterferon alfa-2b or pegylated interferon alfa-2a, is the recommended cytoreductive treatment for these patients. The expert panel suggested that either interferon alfa or ruxolitinib should be considered for patients who are being treated with hydroxyurea but require a therapy change.

The molecular causes of myeloproliferative neoplasms (MPNs) have not yet been fully elucidated. Approximately 7% to 8% of the patients carry predisposing genetic germline variants that lead to driver mutations, which enhance JAK-STAT signaling. To identify additional predisposing genetic germline variants, we performed whole-exome sequencing in 5 families, each with parent-child or sibling pairs affected by MPNs and carrying the somatic JAK2 V617F mutation. In 4 families, we detected rare germline variants in known tumor predisposition genes of the DNA repair pathway, including the highly penetrant BRCA1 and BRCA2 genes. The identification of an underlying hereditary tumor predisposition is of major relevance for the individual patients as well as for their families in the context of therapeutic options and preventive care. Two patients with essential thrombocythemia or polycythemia vera experienced progression to acute myeloid leukemia, which may suggest a high risk of leukemic transformation in these familial MPNs. Our study demonstrates the relevance of genetic germline diagnostics in elucidating the causes of MPNs and suggests novel therapeutic options (eg, PARP inhibitors) in MPNs. Furthermore, we uncover a broader tumor spectrum upon the detection of a germline mutation in genes of the DNA repair pathway.

The molecular causes of myeloproliferative neoplasms (MPNs) have not yet been fully elucidated. Approximately 7% to 8% of the patients carry predisposing genetic germline variants that lead to driver mutations, which enhance JAK-STAT signaling. To identify additional predisposing genetic germline variants, we performed whole-exome sequencing in 5 families, each with parent-child or sibling pairs affected by MPNs and carrying the somatic JAK2V617F mutation. In 4 families, we detected rare germline variants in known tumor predisposition genes of the DNA repair pathway, including the highly penetrant BRCA1and BRCA2genes. The identification of an underlying hereditary tumor predisposition is of major relevance for the individual patients as well as for their families in the context of therapeutic options and preventive care. Two patients with essential thrombocythemia or polycythemia vera experienced progression to acute myeloid leukemia, which may suggest a high risk of leukemic transformation in these familial MPNs. Our study demonstrates the relevance of genetic germline diagnostics in elucidating the causes of MPNs and suggests novel therapeutic options (eg, PARP inhibitors) in MPNs. Furthermore, we uncover a broader tumor spectrum upon the detection of a germline mutation in genes of the DNA repair pathway.

Recommendations regarding management of essential thrombocythaemia rely on studies done before the discovery of the CALRmutation. On May 20, 2020, the European LeukemiaNet annual meeting was held with the goal to identify unmet clinical needs in myeloproliferative neoplasms. Because patients with a CALRmutation have specific clinical characteristics, treatment of CALR-mutated essential thrombocythaemia was considered an unmet clinical need by the European LeukemiaNet. The elaboration of a consensus document with recommendations according to current evidence was proposed as a solution for resolving uncertainties in the treatment of CALR-mutated essential thrombocythaemia. A steering committee comprising four European LeukemiaNet members was then formed and a panel of ten experts in the field was recruited. The experts proposed 51 potential unmet clinical needs in the management of CALR-mutated essential thrombocythaemia and were asked to score the relevance of each topic. Those topics that obtained the highest scores as relevant unmet clinical needs were identified, including antiplatelet therapy in patients at low risk, definition of extreme thrombocytosis and its management in patients at low risk, indications of cytoreduction and targets of therapy, first-line treatment of choice in young patients (<60 years), and management of pregnancy. After the steering committee revised the available evidence for each topic, a consensus on management and proposal for improving knowledge was achieved by use of an email-based, two round, Delphi approach. Consensus was achieved when 90% of the panellists agreed with a statement and included 14 recommendations and six solution proposals. Key recommendations included careful observation for asymptomatic patients with classical, low-risk, CALR-mutated essential thrombocythaemia without cardiovascular risk factors; caution in the use of antiplatelet therapy for symptomatic patients at low risk with platelet counts of 1000–1500 × 109platelets per L, in such cases cytoreduction is an adequate option, especially if adquired Von Willebrand disease is present; cytoreduction is recommended for extreme thrombocytosis (platelet count >1500 × 109platelets per L) with pegylated interferon alfa being the preferred option for younger patients; both hydroxycarbamide and anagrelide might be given to patients ineligible for pegylated interferon alfa; and treatment algorithms for patients with high-risk pregnancies should not be changed according to genotype. The European LeukemiaNet proposes to use these recommendations in the routine management of patients with CALR-mutated essential thrombocythaemia, and designing new clinical studies in this field might be useful.

We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.

We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hivs CD41loHSCs compared with wild-type controls. An increase in CD41hiHSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hiHSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41loHSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hiHSCs, whereas CD41loHSCs showed higher gene expression of interferon and the JAK/STATand TNFα/NFκBsignaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hiHSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hiHSCs and reduced the number of JAK2-V617F+HSCs in mice and patients with MPN. The shift toward the CD41hisubset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2mutant clone.

The KIT D816V mutation is found in >80% of patients with systemic mastocytosis (SM) and is key to neoplastic mast cell (MC) expansion and accumulation in affected organs. Therefore, KIT D816V represents a prime therapeutic target for SM. Here, we generated a panel of patient-specific KIT D816V induced pluripotent stem cells (iPSCs) from patients with aggressive SM and mast cell leukemia to develop a patient-specific SM disease model for mechanistic and drug-discovery studies. KIT D816V iPSCs differentiated into neoplastic hematopoietic progenitor cells and MCs with patient-specific phenotypic features, thereby reflecting the heterogeneity of the disease. CRISPR/Cas9n-engineered KIT D816V human embryonic stem cells (ESCs), when differentiated into hematopoietic cells, recapitulated the phenotype observed for KIT D816V iPSC hematopoiesis. KIT D816V causes constitutive activation of the KIT tyrosine kinase receptor, and we exploited our iPSCs and ESCs to investigate new tyrosine kinase inhibitors targeting KIT D816V. Our study identified nintedanib, a US Food and Drug Administration–approved angiokinase inhibitor that targets vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, as a novel KIT D816V inhibitor. Nintedanib selectively reduced the viability of iPSC-derived KIT D816V hematopoietic progenitor cells and MCs in the nanomolar range. Nintedanib was also active on primary samples of KIT D816V SM patients. Molecular docking studies show that nintedanib binds to the adenosine triphosphate binding pocket of inactive KIT D816V. Our results suggest nintedanib as a new drug candidate for KIT D816V–targeted therapy of advanced SM.

The KITD816V mutation is found in >80% of patients with systemic mastocytosis (SM) and is key to neoplastic mast cell (MC) expansion and accumulation in affected organs. Therefore, KITD816V represents a prime therapeutic target for SM. Here, we generated a panel of patient-specific KITD816V induced pluripotent stem cells (iPSCs) from patients with aggressive SM and mast cell leukemia to develop a patient-specific SM disease model for mechanistic and drug-discovery studies. KITD816V iPSCs differentiated into neoplastic hematopoietic progenitor cells and MCs with patient-specific phenotypic features, thereby reflecting the heterogeneity of the disease. CRISPR/Cas9n-engineered KITD816V human embryonic stem cells (ESCs), when differentiated into hematopoietic cells, recapitulated the phenotype observed for KITD816V iPSC hematopoiesis. KITD816V causes constitutive activation of the KIT tyrosine kinase receptor, and we exploited our iPSCs and ESCs to investigate new tyrosine kinase inhibitors targeting KIT D816V. Our study identified nintedanib, a US Food and Drug Administration–approved angiokinase inhibitor that targets vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, as a novel KIT D816V inhibitor. Nintedanib selectively reduced the viability of iPSC-derived KITD816V hematopoietic progenitor cells and MCs in the nanomolar range. Nintedanib was also active on primary samples of KIT D816V SM patients. Molecular docking studies show that nintedanib binds to the adenosine triphosphate binding pocket of inactive KIT D816V. Our results suggest nintedanib as a new drug candidate for KIT D816V–targeted therapy of advanced SM.

1.An international panel of 38 experts and clinical trialists in myelofibrosis participated in the preparation of the current document.2.New definitions of transfusion status, gender-specific hemoglobin thresholds, and criteria for major and minor responses are submitted.

We report the clinical presentation and risk factors for survival in 175 patients with myeloproliferative neoplasms (MPN) and COVID-19, diagnosed between February and June 2020. After a median follow-up of 50 days, mortality was higher than in the general population and reached 48% in myelofibrosis (MF). Univariate analysis, showed a significant relationship between death and age, male gender, decreased lymphocyte counts, need for respiratory support, comorbidities and diagnosis of MF, while no association with essential thrombocythemia (ET), polycythemia vera (PV), and prefibrotic-PMF (pre-PMF) was found. Regarding MPN-directed therapy ongoing at the time of COVID-19 diagnosis, Ruxolitinib (Ruxo) was significantly more frequent in patients who died in comparison with survivors (p= 0.006). Conversely, multivariable analysis found no effect of Ruxo alone on mortality, but highlighted an increased risk of death in the 11 out of 45 patients who discontinued treatment. These findings were also confirmed in a propensity score matching analysis. In conclusion, we found a high risk of mortality during COVID-19 infection among MPN patients, especially in MF patients and/or discontinuing Ruxo at COVID-19 diagnosis. These findings call for deeper investigation on the role of Ruxo treatment and its interruption, in affecting mortality in MPN patients with COVID-19.

Despite the successes achieved with molecular targeted inhibition of the oncogenic driver Bcr-Abl in chronic myeloid leukemia (CML), the majority of patients still require lifelong tyrosine kinase inhibitor (TKI) therapy. This is primarily caused by resisting leukemic stem cells (LSCs), which prevent achievement of treatment-free remission in all patients. Here we describe the ITIM (immunoreceptor tyrosine-based inhibition motif)-containing Fc gamma receptor IIb (FcγRIIb, CD32b) for being critical in LSC resistance and show that targeting FcγRIIb downstream signaling, by using a Food and Drug Administration-approved BTK inhibitor, provides a successful therapeutic approach. First, we identified FcγRIIb upregulation in primary CML stem cells. FcγRIIb depletion caused reduced serial re-plaiting efficiency and cell proliferation in malignant cells. FcγRIIb targeting in both a transgenic and retroviral CML mouse model provided in vivo evidence for successful LSC reduction. Subsequently, we identified BTK as a main downstream mediator and targeting the Bcr-Abl-FcγRIIb-BTK axis in primary CML CD34+cells using ibrutinib, in combination with standard TKI therapy, significantly increased apoptosis in quiescent CML stem cells thereby contributing to the eradication of LSCs.. As a potential curative therapeutic approach, we therefore suggest combining Bcr-Abl TKI therapy along with BTK inhibition.

The sonidegib and ruxolitinib combination was assessed in an open-label study in JAK inhibitor-naive patients with myelofibrosis (MF). The primary objective of phase 1b was to establish the maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) and phase 2 was to assess spleen volume reduction at weeks 24 and 48. Fifty patients were enrolled. In the dose-escalation phase (n = 23), doses for sonidegib once daily/ruxolitinib twice daily were 400/10 mg (level 1, n = 8), 400/15 mg (level 2, n = 10), and 400/20 mg (level 3, n = 5). Two patients had dose-limiting toxicity at level 2: increased blood creatine phosphokinase (grades 3 and 4, n = 1 each). MTD/RP2D was determined as sonidegib 400 mg daily + ruxolitinib 20 mg twice daily. In phase 1b expansion and phase 2 stage 1 (n = 27), by weeks 24 and 48, ≥35% reduction in spleen volume was observed in 44.4% and 29.6% patients, respectively. By weeks 24 and 48, 42.0% and 26.0% patients had ≥50% reduction in Myelofibrosis Symptom Assessment Form total symptom score, respectively. Most common treatment-related adverse events (grade 3/4) were increased blood creatine phosphokinase (18%), anemia (14%), and thrombocytopenia (12%). Four deaths were reported due to multiple organ dysfunction syndrome (on-treatment; no relationship with study treatment), acute myeloid leukemia, MF progression, and aspiration pneumonia. Although well tolerated, this combination will not be further developed in MF patients due to modest overall benefit compared with historical ruxolitinib monotherapy. This trial was registered at www.clinicaltrials.gov as #NCT01787552.

The sonidegib and ruxolitinib combination was assessed in an open-label study in JAK inhibitor-naive patients with myelofibrosis (MF). The primary objective of phase 1b was to establish the maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) and phase 2 was to assess spleen volume reduction at weeks 24 and 48. Fifty patients were enrolled. In the dose-escalation phase (n = 23), doses for sonidegib once daily/ruxolitinib twice daily were 400/10 mg (level 1, n = 8), 400/15 mg (level 2, n = 10), and 400/20 mg (level 3, n = 5). Two patients had dose-limiting toxicity at level 2: increased blood creatine phosphokinase (grades 3 and 4, n = 1 each). MTD/RP2D was determined as sonidegib 400 mg daily + ruxolitinib 20 mg twice daily. In phase 1b expansion and phase 2 stage 1 (n = 27), by weeks 24 and 48, ≥35% reduction in spleen volume was observed in 44.4% and 29.6% patients, respectively. By weeks 24 and 48, 42.0% and 26.0% patients had ≥50% reduction in Myelofibrosis Symptom Assessment Form total symptom score, respectively. Most common treatment-related adverse events (grade 3/4) were increased blood creatine phosphokinase (18%), anemia (14%), and thrombocytopenia (12%). Four deaths were reported due to multiple organ dysfunction syndrome (on-treatment; no relationship with study treatment), acute myeloid leukemia, MF progression, and aspiration pneumonia. Although well tolerated, this combination will not be further developed in MF patients due to modest overall benefit compared with historical ruxolitinib monotherapy. This trial was registered at www.clinicaltrials.govas #NCT01787552.

Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50–60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.

The PROUD-PV and CONTINUATION-PV trials aimed to compare the novel monopegylated interferon ropeginterferon alfa-2b with hydroxyurea, the standard therapy for patients with polycythaemia vera, over 3 years of treatment.

Increased energy requirement and metabolic reprogramming are hallmarks of cancer cells. We show that metabolic alterations in hematopoietic cells are fundamental to the pathogenesis of mutant JAK2–driven myeloproliferative neoplasms (MPNs). We found that expression of mutant JAK2 augmented and subverted metabolic activity of MPN cells, resulting in systemic metabolic changes in vivo, including hypoglycemia, adipose tissue atrophy, and early mortality. Hypoglycemia in MPN mouse models correlated with hyperactive erythropoiesis and was due to a combination of elevated glycolysis and increased oxidative phosphorylation. Modulating nutrient supply through high-fat diet improved survival, whereas high-glucose diet augmented the MPN phenotype. Transcriptomic and metabolomic analyses identified numerous metabolic nodes in JAK2-mutant hematopoietic stem and progenitor cells that were altered in comparison with wild-type controls. We studied the consequences of elevated levels of Pfkfb3, a key regulatory enzyme of glycolysis, and found that pharmacological inhibition of Pfkfb3 with the small molecule 3PO reversed hypoglycemia and reduced hematopoietic manifestations of MPNs. These effects were additive with the JAK1/2 inhibitor ruxolitinib in vivo and in vitro. Inhibition of glycolysis by 3PO altered the redox homeostasis, leading to accumulation of reactive oxygen species and augmented apoptosis rate. Our findings reveal the contribution of metabolic alterations to the pathogenesis of MPNs and suggest that metabolic dependencies of mutant cells represent vulnerabilities that can be targeted for treating MPNs.

Increased energy requirement and metabolic reprogramming are hallmarks of cancer cells. We show that metabolic alterations in hematopoietic cells are fundamental to the pathogenesis of mutant JAK2–driven myeloproliferative neoplasms (MPNs). We found that expression of mutant JAK2 augmented and subverted metabolic activity of MPN cells, resulting in systemic metabolic changes in vivo, including hypoglycemia, adipose tissue atrophy, and early mortality. Hypoglycemia in MPN mouse models correlated with hyperactive erythropoiesis and was due to a combination of elevated glycolysis and increased oxidative phosphorylation. Modulating nutrient supply through high-fat diet improved survival, whereas high-glucose diet augmented the MPN phenotype. Transcriptomic and metabolomic analyses identified numerous metabolic nodes in JAK2-mutant hematopoietic stem and progenitor cells that were altered in comparison with wild-type controls. We studied the consequences of elevated levels of Pfkfb3, a key regulatory enzyme of glycolysis, and found that pharmacological inhibition of Pfkfb3 with the small molecule 3PO reversed hypoglycemia and reduced hematopoietic manifestations of MPNs. These effects were additive with the JAK1/2 inhibitor ruxolitinib in vivo and in vitro. Inhibition of glycolysis by 3PO altered the redox homeostasis, leading to accumulation of reactive oxygen species and augmented apoptosis rate. Our findings reveal the contribution of metabolic alterations to the pathogenesis of MPNs and suggest that metabolic dependencies of mutant cells represent vulnerabilities that can be targeted for treating MPNs.

Background: Myelofibrosis (MF) is a BCR-ABL1-negative myeloproliferative neoplasm (MPN) characterized by progressive bone marrow fibrosis, splenomegaly, and cytopenias due to impaired hematopoiesis (Passamonti and Mora. Blood 2022). Despite the availability of comprehensive national and international guidelines for diagnosing and managing MF, gaps remain in translating these guidelines into clinical practice, particularly for patients with cytopenias, non-response or intolerance to Janus kinase (JAK) inhibitor treatment, and those ineligible for clinical trial enrollment. Consequently, an international expert consensus group was established with the objective of augmenting the existing guidance.

Introduction:Polycythemia vera (PV) is characterized by trilineage blood cell expansion, PV-associated symptoms, and the risk of thromboembolic complications, progression to post-PV myelofibrosis, and acute leukemia. Cytoreductive treatment with hydroxyurea (HU) or ropeginterferon-alpha is approved in EU for the treatment of high-risk patients (pts) with PV. In addition, ruxolitinib (RUX) is approved for HU-intolerant/-resistant PV. However, RUX vs. best available therapy (BAT) has not been investigated as first-line treatment of pts with untreated PV. We hypothesized that RUX may have higher efficacy in such early-line PV pts.

Objectives: This study aimed at investigating the therapeutic efficacy of ABL001 in the ScltTA-p210-BCR::ABL1transgenic mouse model. ABL001 (asciminib) is a novel allosteric inhibitor of ABL kinase demonstrating its particular efficacy against BCR::ABL1mutations that emerge during treatment with traditional TKIs. However, the activity and tolerability of ABL001 in a primary mouse model of BCR::ABL1disease remain unknown. In addition, resistance mutations against ABL001 were observed in the myristoyl pocket of BCR::ABL1, but their emergence has not been analyzed in BCR::ABL1+ murine hematopoietic cells. Importantly, leukemic stem cells (LSCs) expressing BCR::ABL1have developed mechanisms to evade eradication by tyrosine kinase inhibitors, underscoring the necessity for alternative therapeutic approaches.

Overt Myelofibrosis (MF) is an end-stage subtype of BCR-ABL1 negative myeloproliferative neoplasms (MPN), which is characterized by progressive bone marrow (BM) fibrosis resulting in loss of normal hematopoietic support as well as inflammatory and vascular complications. While aberrant neoangiogenesis is known to participate in the pathogenesis of MF, Erba et al. (Am J Pathol, 2017) suggested the occurrence of endothelial-to-mesenchymal transition (EndMT) in BM and spleen of MF patients (pts). Given the relevance of endothelial cells (EC) for MF development and the occurrence of JAK2V617F-mutated EC in MPN pts, targeting of the vascular niche has emerged as a promising therapeutic strategy. To untangle the impact of the JAK2V617F mutation on human vasculogenesis, we leveraged high-purity and clonal patient-specific induced pluripotent stem cell (iPSC)-derived EC (iEC) harboring either the JAK2V617F mutation in hetero- (JAK2V617F HET) or homozygosis (JAK2V617F HOM). JAK2 WT and CRISPR-repaired isogenic JAK2 WT (both referred to as JAK2 WT) were used as controls.

Background:Janus kinase inhibitors (JAKis) provide symptom improvement and spleen volume reduction in patients with primary, post-polycythemia vera, and post-essential thrombocythemia myelofibrosis (MF). There remains a substantial unmet need for therapies that alter disease trajectory, improve outcomes, and enhance survival. The COMFORT-1 and -2 studies established JAKi monotherapy as standard-of-care with spleen volume reduction of ≥35% at Week 24 (SVR 35W24) of 42% and SVR 35W48of 29%, respectively. In combination with the JAKi ruxolitinib, navitoclax, an orally available inhibitor of antiapoptotic B-cell lymphoma 2 proteins (BCL-X L, BCL-2, BCL-W), was shown to have pronounced antitumor activity in patients with MF in the phase 2 REFINE trial (NCT03222609). TRANSFORM-1 is an ongoing, phase 3, double-blind, placebo-controlled, multicenter, international study evaluating the safety and efficacy of navitoclax plus ruxolitinib (NAV + RUX) compared with placebo plus ruxolitinib (PBO + RUX) in JAK2i-naïve adults with MF.

Introduction: