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Background: Differential leukocyte counts are usually measured based on cellular morphology or surface marker expression. It has recently been shown that leukocyte counts can also be determined by cell-type-specific DNA methylation (DNAm). Such epigenetic leukocyte counting is applicable to small blood volumes and even frozen material, but for clinical translation, the method needs to be further refined and validated., Methods: We further optimized and validated targeted DNAm assays for leukocyte deconvolution using 332 venous and 122 capillary blood samples from healthy donors. In addition, we tested 36 samples from ring trials and venous blood from 266 patients diagnosed with different hematological diseases. Deconvolution of cell types was determined with various models using DNAm values obtained by pyrosequencing or digital droplet PCR (ddPCR)., Results: Relative leukocyte quantification correlated with conventional blood counts for granulocytes, lymphocytes, B cells, T cells (CD4 or CD8), natural killer cells, and monocytes with pyrosequencing (r = 0.84; r = 0.82; r = 0.58; r = 0.50; r = 0.70; r = 0.61; and r = 0.59, respectively) and ddPCR measurements (r = 0.65; r = 0.79; r = 0.56; r = 0.57; r = 0.75; r = 0.49; and r = 0.46, respectively). In some patients, particularly with hematopoietic malignancies, we observed outliers in epigenetic leukocyte counts, which could be discerned if relative proportions of leukocyte subsets did not sum up to 100%. Furthermore, absolute quantification was obtained by spiking blood samples with a reference plasmid of known copy number., Conclusions: Targeted DNAm analysis by pyrosequencing or ddPCR is a valid alternative to quantify leukocyte subsets, but some assays require further optimization., (© American Association for Clinical Chemistry 2022.)

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 × 10 9 white blood cells per L), progressive leukocytosis (at least 100% increase if baseline count is <10 × 10 9 cells per L or at least 50% increase if baseline count is >10 × 10 9 cells per L), extreme thrombocytosis (>1500 × 10 9 platelets 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., Competing Interests: Declaration of interests MM has received consulting fees from Gilead Sciences, speaker fees from Amgen, support for attending meetings from Takeda, and is a member of the guideline committee of the Italian Society of Hematology. AMV has received speaker fees from Novartis, AOP Health, Incyte, AbbVie, GlaxoSmithKline (GSK), and Bristol Myers Squibb (BMS); and has participated on the advisory boards of Novartis, Incyte, AOP Orphan Pharmaceuticals, AbbVie, GSK, BMS, and Roche. MG has received consulting and speaker fees, support for attending meetings, and participated on advisory boards for AOP Health, Novartis, Celgene, Amgen, AstraZeneca, CTI BioPharma, Shire, Pfizer, Roche, Janssen Pharmaceuticals, and Gilead Sciences. CH has received grants or contracts with payment to her institution from Novartis, Celgene, and Constellation Pharmaceuticals; consulting fees from Keros Therapeutics, Galecto Biotech, and Roche; speaker fees from Novartis, Celgene, CTI BioPharma, AbbVie, Gilead Sciences, Janssen Pharmaceuticals, Promedior, and Geron Corporation; support for attending meetings from Novartis and Celgene, and has participated on advisory boards for Roche, CTI Biopharma, Geron Corporation, Promedior, AbbVie, AOP Orphan Pharmaceuticals, and Galecto. SK has received research grants from AOP Health, Novartis, Janssen Pharmaceuticals, and Imago BioScience; consulting fees from Pfizer, CTI BioPharma, Sanofi, Novartis, BMS/Celgene, Incyte/ARIAD, Roche, AOP Orphan Pharmaceuticals, and Janssen Pharmaceuticals; speaker fees from Pfizer, CTI BioPharma, Sanofi, Novartis, BMS/Celgene, Incyte/ARIAD, Roche, AOP Orphan Pharmaceuticals, Janssen Pharmaceuticals, Kartos Therapeutics, and Imago BioScience; support for attending meetings from Alexion Pharmaceuticals, Novartis, BMS/Celgene, Incyte/ARIAD, AOP Orphan Pharmaceuticals, CTI BioPharma, Pfizer, Sanofi, Janssen Pharmaceuticals, Geron Corporation, Kartos Therapeutics, Sierra Oncology, Imago BioScience; has participated on advisory boards for Pfizer, CTI BioPharma, Sanofi, Novartis, BMS/Celgene, Incyte/ARIAD, Roche, AOP Orphan Pharmaceuticals, Kartos Therapeutics, and Imago BioScience; has patents planned, issued, or pending from Rheinisch-Westfalische Technische Hochschule Aachen; and has a leadership role in Deutsche Gesellschaft fur Hamatologie und Medizinische Onkologie. HG has received research grants and support for attending meetings from AOP Orphan Pharmaceuticals and Novartis; consulting fees from AOP Orphan Pharmaceuticals, Novartis, and BMS/Celgene; and speaker fees from AOP Orphan Pharmaceuticals, Novartis, BMS/Celgene, and Janssen. AÁ-L has participated on advisory boards and has received speaker fees from Novartis, AOP Orphan Pharmaceuticals, and Celgene; and has received support for attending meetings from Novartis and Pfizer. VDS has received speaker fees from AbbVie and Novartis, and participated on advisory boards of AOP Health and Novartis. PG has received speaker fees from AbbVie and Novartis, and support for attending meetings from Sanofi. FPal has received speaker fees from Novartis and Incyte; consulting fees from AOP Health, CTI BioPharma, Novartis, and Celgene/BMS; and support for attending meetings from Celgene/BMS. FPas has received consulting fees from AbbVie, Novartis, BMS, and Amomed Pharma; and speaker fees from AbbVie, Janssen Pharmaceuticals, Novartis, and BMS. RTS has received research grants from the Cancer Research & Treatment Fund and Johns Family Fund, received speaker fees and payment for expert testimony from PharmaEssentia, participated on an advisory board for PharmaEssentia, and has a leadership role (Vice-President and Medical Director) at the Cancer Research & Treatment Fund. J-JK received consulting fees from AbbVie and Novartis; speaker fees from AOP Orphan Pharmaceuticals, Novartis, and BMS; and has participated on advisory boards for Incyte. TB has received research grants and speaker fees from AOP Orphan Pharmaceuticals and Novartis, and consultancy fees from AOP Orphan Pharmaceuticals. GB and RH declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The receptor tyrosine kinase c-KIT (CD117) has a key role in hematopoiesis and is a marker for endothelial and cardiac progenitor cells. In vivo, deficiency of c-KIT is lethal and therefore using CRISPR/Cas9 editing we generated heterozygous and homozygous c-KIT knockout human embryonic stem cell (ES cell) lines. The c-KIT knockout left ES cell pluripotency unaffected as shown by immunofluorescence and trilineage differentiation potential. Heterozygous and homozygous c-KIT knockouts showed complete loss of exon 17, resulting in ablation of c-KIT protein from the cell surface. c-KIT knockout ES cells provide a valuable tool for further investigating c-KIT biology., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hereditary pyrimidine 5-nucleotidase (P5'N-1) deficiency is a very rare disorder. Here, we describe a new mutation in a Turkish family. Although functional tests have not been performed, our findings confirm that the homozygous mutational state leads to clinical manifest P5'N-1 deficiency, while heterozygosity does not lead to hemolysis or anemia., Competing Interests: None., (© 2022 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd.)

Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematologic malignancies, including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), as well as post-PV-MF and post-ET-MF. Progression to more symptomatic disease, such as overt MF or acute leukemia, represents one of the major causes of morbidity and mortality. There are clinically evident but also subclinical types of MPN progression. Clinically evident progression includes evolution from ET to PV, ET to post-ET-MF, PV to post-PV-MF, or pre-PMF to overt PMF, and transformation of any of these subtypes to myelodysplastic neoplasms or acute leukemia. Thrombosis, major hemorrhage, severe infections, or increasing symptom burden (e.g., pruritus, night sweats) may herald progression. Subclinical types of progression may include increases in the extent of bone marrow fibrosis, increases of driver gene mutational allele burden, and clonal evolution. The underlying causes of MPN progression are diverse and can be attributed to genetic alterations and chronic inflammation. Particularly, bystander mutations in genes encoding epigenetic regulators or splicing factors were associated with progression. Finally, comorbidities such as systemic inflammation, cardiovascular diseases, and organ fibrosis may augment the risk of progression. The aim of this review was to discuss types and mechanisms of MPN progression and how their knowledge might improve risk stratification and therapeutic intervention. In view of these aspects, we discuss the potential benefits of early diagnosis using molecular and functional imaging and exploitable therapeutic strategies that may prevent progression, but also highlight current challenges and methodological pitfalls.

Myeloproliferative neoplasms (MPN), comprising essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), are hematological disorders of the myeloid lineage characterized by hyperproliferation of mature blood cells. The prediction of the clinical course and progression remains difficult and new therapeutic modalities are required. We conducted a CD34 + gene expression study to identify signatures and potential biomarkers in the different MPN subtypes with the aim to improve treatment and prevent the transformation from the rather benign chronic state to a more malignant aggressive state. We report here on a systematic gene expression analysis (GEA) of CD34 + peripheral blood or bone marrow cells derived from 30 patients with MPN including all subtypes (ET (n = 6), PV (n = 11), PMF (n = 9), secondary MF (SMF; post-ET-/post-PV-MF; n = 4)) and six healthy donors. GEA revealed a variety of differentially regulated genes in the different MPN subtypes vs. controls, with a higher number in PMF/SMF (200/272 genes) than in ET/PV (132/121). PROGENγ analysis revealed significant induction of TNFα/NF-κB signaling (particularly in SMF) and reduction of estrogen signaling (PMF and SMF). Consistently, inflammatory GO terms were enriched in PMF/SMF, whereas RNA splicing-associated biological processes were downregulated in PMF. Differentially regulated genes that might be utilized as diagnostic/prognostic markers were identified, such as AREG, CYBB, DNTT, TIMD4, VCAM1, and S100 family members (S100A4/8/9/10/12). Additionally, 98 genes (including CLEC1B, CMTM5, CXCL8, DACH1, and RADX) were deregulated solely in SMF and may be used to predict progression from early to late stage MPN., (© 2021. The Author(s).)

Calreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem cells (iPSCs) to establish an MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in granulocytic cells derived from homozygous CALR mutant iPSCs, rescued by repairing the mutation using CRISPR/Cas9. iPSC-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelet protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated versus unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis dependent on its zygosity, and found novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2021 by The American Society of Hematology.)

Lipocalin 2 (LCN2), a proinflammatory mediator, is involved in the pathogenesis of myeloproliferative neoplasms (MPN). Here, we investigated the molecular mechanisms of LCN2 overexpression in MPN. LCN2 mRNA expression was 20-fold upregulated in peripheral blood (PB) mononuclear cells of chronic myeloid leukemia (CML) and myelofibrosis (MF) patients vs. healthy controls. In addition, LCN2 serum levels were significantly increased in polycythemia vera (PV) and MF and positively correlated with JAK2V617F and mutated CALR allele burden and neutrophil counts. Mechanistically, we identified endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as a main driver of LCN2 expression in BCR-ABL- and JAK2V617F-positive 32D cells. The UPR inducer thapsigargin increased LCN2 expression >100-fold, and this was not affected by kinase inhibition of BCR-ABL or JAK2V617F. Interestingly, inhibition of the UPR regulators inositol-requiring enzyme 1 (IRE1) and c-Jun N -terminal kinase (JNK) significantly reduced thapsigargin-induced LCN2 RNA and protein expression, and luciferase promoter assays identified nuclear factor kappa B (NF-κB) and CCAAT binding protein (C/EBP) as critical regulators of mLCN2 transcription. In conclusion, the IRE1-JNK-NF-κB-C/EBP axis is a major driver of LCN2 expression in MPN, and targeting UPR and LCN2 may represent a promising novel therapeutic approach in MPN.

Inflammation-induced thrombosis represents a severe complication in patients with myeloproliferative neoplasms (MPN) and in those with kidney dysfunction. Overlapping disease-specific attributes suggest common mechanisms involved in MPN pathogenesis, kidney dysfunction, and thrombosis. Data from 1420 patients with essential thrombocythemia (ET, 33.7%), polycythemia vera (PV, 38.5%), and myelofibrosis (MF, 27.9%) were extracted from the bioregistry of the German Study Group for MPN. The total cohort was subdivided according to the calculated estimated glomerular filtration rate (eGFR, (mL/min/1.73 m 2 )) into eGFR1 (≥90, 21%), eGFR2 (60-89, 56%), and eGFR3 (<60, 22%). A total of 29% of the patients had a history of thrombosis. A higher rate of thrombosis and longer MPN duration was observed in eGFR3 than in eGFR2 and eGFR1. Kidney dysfunction occurred earlier in ET than in PV or MF. Multiple logistic regression analysis identified arterial hypertension, MPN treatment, increased uric acid, and lactate dehydrogenase levels as risk factors for kidney dysfunction in MPN patients. Risk factors for thrombosis included arterial hypertension, non-excessive platelet counts, and antithrombotic therapy. The risk factors for kidney dysfunction and thrombosis varied between MPN subtypes. Physicians should be aware of the increased risk for kidney disease in MPN patients, which warrants closer monitoring and, possibly, early thromboprophylaxis.

The chemokine CXCL4/platelet factor 4 (PF4) gene, a key player in myelofibrosis, was knocked out by CRISPR/Cas9 in induced pluripotent stem cells (iPS cells) of a polycythemia vera (PV) patient with JAK2 V617F mutation. Two CXCL4 KO iPS cell lines with and without JAK2 V617F mutation (UKAi002-B-1 and UKAi002-A-1, respectively) were generated. CXCL4 KO iPS cells showed deletion of exon 1 and complete loss of CXCL4 protein. Pluripotency of iPS cells was confirmed by expression of pluripotency markers and trilineage differentiation. CXCL4 KO iPS cells are expected to provide a valuable tool for investigating the role of CXCL4 in human diseases., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Dendritic cells (DC) are professional antigen-presenting cells that develop from hematopoietic stem cells. Different DC subsets exist based on ontogeny, location and function, including the recently identified proinflammatory DC3 subset. DC3 have the prominent activity to polarize CD8 + T cells into CD8 + CD103 + tissue resident T cells. Here we describe human DC3 differentiated from induced pluripotent stem cells (iPS cells). iPS cell-derived DC3 have the gene expression and surface marker make-up of blood DC3 and polarize CD8 + T cells into CD8 + CD103 + tissue-resident memory T cells in vitro . To test the impact of malignant JAK2 V617F mutation on DC3, we differentiated patient-specific iPS cells with JAK2 V617F het and JAK2 V617F hom mutations into JAK2 V617F het and JAK2 V617F hom DC3. The JAK2 V617F mutation enhanced DC3 production and caused a bias toward erythrocytes and megakaryocytes. The patient-specific iPS cell-derived DC3 are expected to allow studying DC3 in human diseases and developing novel therapeutics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Satoh, Toledo, Boehnke, Olschok, Flosdorf, Götz, Küstermann, Sontag, Seré, Koschmieder, Brümmendorf, Chatain, Tagawa and Zenke.)

Patients with cancer, both hematologic and solid malignancies, are at increased risk for thrombosis and thromboembolism. In addition to general risk factors such as immobility and major surgery, shared by non-cancer patients, cancer patients are exposed to specific thrombotic risk factors. These include, among other factors, cancer-induced hypercoagulation, and chemotherapy-mediated endothelial dysfunction as well as tumor-cell-derived microparticles. After an episode of thrombosis in a cancer patient, secondary thromboprophylaxis to prevent recurrent thromboembolism has long been established and is typically continued as long as the cancer is active or actively treated. On the other hand, primary prophylaxis, even though firmly established in hospitalized cancer patients, has only recently been studied in ambulatory patients. This recent change is mostly due to the emergence of direct oral anticoagulants (DOACs). DOACs have a shorter half-life than vitamin K antagonists (VKA), and they overcome the need for parenteral application, the latter of which is associated with low-molecular-weight heparins (LMWH) and can be difficult for the patient to endure in the long term. Here, first, we discuss the clinical trials of primary thromboprophylaxis in the population of cancer patients in general, including the use of VKA, LMWH, and DOACs, and the potential drug interactions with pre-existing medications that need to be taken into account. Second, we focus on special situations in cancer patients where primary prophylactic anticoagulation should be considered, including myeloma, major surgery, indwelling catheters, or immobilization, concomitant diseases such as renal insufficiency, liver disease, or thrombophilia, as well as situations with a high bleeding risk, particularly thrombocytopenia, and specific drugs that may require primary thromboprophylaxis. We provide a novel algorithm intended to aid specialists but also family practitioners and nurses who care for cancer patients in the decision process of primary thromboprophylaxis in the individual patient.

Twenty-five years after the approval of the first anticancer nanodrug, we have to start re(de)fining tumor-targeted drug delivery alongside advances in immuno-oncology. Given that cancer is characterized by an immunological imbalance that goes beyond the primary tumor, we should focus on targeting, engaging, and modulating cancer-associated immune cells in the tumor microenvironment (TME), circulation, and immune cell-enriched tissues. When designed and applied rationally, nanomedicines will assist in restoring the immunological equilibrium at the whole-body level, which holds potential not only for cancer therapy, but also for the treatment of a range of other disorders., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The central nervous system (CNS) responds to diverse neurologic injuries with a vigorous activation of astrocytes. In addition to their role in the maintenance of CNS homeostasis and neuronal function, astrocytes are thought to participate in the regulation of innate and adaptive immune responses in the CNS. Following antigen recognition, reactive astrocytes may participate in the initiation of innate immune responses, and modulate adaptive immune response leading to the recruitment of peripheral immune cells. Among activation, astrocytes undergo morphological changes and express several molecules, e.g., chemokines. Lipocalin 2 (LCN2) is involved in the control of innate immune responses, regulation of excess iron, and reactive oxygen production. Here, we investigated the influence of LCN2 on basic astrocytic functions linked to inflammatory responses. In vitro studies revealed a similar chemokine expression pattern in wild-type and Lcn2-deficient astrocyte cultures after treatment with lipopolysaccharides (LPS). Increased wound closure and morphological changes upon LPS treatment are independent of Lcn2 expression. We conclude that LCN2 is not necessary for basic astrocytic functions in the context of inflammation. However, CNS-derived LCN2 might have a regulatory effect on other cells, e.g., endothelial cells of the blood-brain barrier.

Dyskeratosis Congenita (DKC) is a systemic disorder caused by mutations resulting in impaired telomere maintenance. Clinical features include bone marrow failure and an increased risk of developing hematological malignancies. There are conflicting data whether androgen derivatives (AD) can elongate telomeres in vivo and whether AD treatment enhances the risk of gaining myelodysplastic syndrome-related mutations. Seven TERC or TERT-mutated DKC patients underwent AD treatment. All patients revealed hematological response. Telomere length of lymphocytes and granulocytes increased significantly and no MDS-related mutations were detected. Pending longer follow-up, treatment with AD seems to represent an efficient and safe therapy for DKC patients., (© 2020 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

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., (© 2021 by The American Society of Hematology.)

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., (© 2021 by The American Society of Hematology.)

Functional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The calreticulin ( CALR ) exon 9 mutations that are identified in 20% of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN) generate immunogenic antigens. Thus, therapeutic cancer vaccination against mutant CALR could be a new treatment modality in CALR -mutant MPN., Methods: The safety and efficacy of vaccination with the peptide CALRLong36 derived from the CALR exon 9 mutations was tested in a phase I clinical vaccination trial with montanide as adjuvant. Ten patients with CALR mut MPN were included in the trial and received 15 vaccines over the course of one year. The primary end point was evaluation of safety and toxicity of the vaccine. Secondary endpoint was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT03566446)., Results: Patients had a median age of 59.5 years and a median disease duration of 6.5 years. All patients received the intended 15 vaccines, and the vaccines were deemed safe and tolerable as only two grade three AE were detected, and none of these were considered to be related to the vaccine. A decline in platelet counts relative to the platelets counts at baseline was detected during the first 100 days, however this did not translate into neither a clinical nor a molecular response in any of the patients. Immunomonitoring revealed that four of 10 patients had an in vitro interferon (IFN)-γ ELISPOT response to the CALRLong36 peptide at baseline, and four additional patients displayed a response in ELISPOT upon receiving three or more vaccines. The amplitude of the immune response increased during the entire vaccination schedule for patients with essential thrombocythemia. In contrast, the immune response in patients with primary myelofibrosis did not increase after three vaccines., Conclusion: Therapeutic cancer vaccination with peptide vaccines derived from mutant CALR with montanide as an adjuvant, is safe and tolerable. The vaccines did not induce any clinical responses. However, the majority of patients displayed a marked T-cell response to the vaccine upon completion of the trial. This suggests that vaccines directed against mutant CALR may be used with other cancer therapeutic modalities to enhance the anti-tumor immune response., Competing Interests: MOH, HH, and MA have filed a patent regarding the CALR exon 9 mutations as a target for cancer immune therapy. The patent has been transferred to University Hospital Zealand, Zealand Region and Copenhagen University Hospital at Herlev, Capital Region according to Danish Law concerning inventions made at public research institutions. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Handlos Grauslund, Holmström, Jørgensen, Klausen, Weis-Banke, El Fassi, Schöllkopf, Clausen, Gjerdrum, Breinholt, Kjeldsen, Hansen, Koschmieder, Chatain, Novotny, Petersen, Kjær, Skov, Met, Svane, Hasselbalch and Andersen.)

In a multicenter European retrospective study including 162 patients with COVID-19 occurring in essential thrombocythemia (ET, n = 48), polycythemia vera (PV, n = 42), myelofibrosis (MF, n = 56), and prefibrotic myelofibrosis (pre-PMF, n = 16), 15 major thromboses (3 arterial and 12 venous) were registered in 14 patients, of whom all, but one, were receiving LMW-heparin prophylaxis. After adjustment for the competing risk of death, the cumulative incidence of arterial and venous thromboembolic events (VTE) reached 8.5% after 60 days follow-up. Of note, 8 of 12 VTE were seen in ET. Interestingly, at COVID-19 diagnosis, MPN patients had significantly lower platelet count (p < 0.0001) than in the pre-COVID last follow-up.This decline was remarkably higher in ET (-23.3%, p < 0.0001) than in PV (-16.4%, p = 0.1730) and was associated with higher mortality rate (p = 0.0010) for pneumonia. The effects of possible predictors of thrombosis, selected from those clinically relevant and statistically significant in univariate analysis, were examined in a multivariate model. Independent risk factors were transfer to ICU (SHR = 3.73, p = 0.029), neutrophil/lymphocyte ratio (SHR = 1.1, p = 0.001) and ET phenotype (SHR = 4.37, p = 0.006). The enhanced susceptibility to ET-associated VTE and the associated higher mortality for pneumonia may recognize a common biological plausibility and deserve to be delved to tailor new antithrombotic regimens including antiplatelet drugs.

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.

Bone marrow (BM) fibrosis in myeloproliferative neoplasms (MPNs) is associated with a poor prognosis. The development of myelofibrosis and differentiation of mesenchymal stromal cells to profibrotic myofibroblasts depends on macrophages. Here, we compared macrophage frequencies in BM biopsies of MPN patients and controls (patients with non-neoplastic processes), including primary myelofibrosis (PMF, n = 18), essential thrombocythemia (ET, n = 14), polycythemia vera (PV, n = 12), and Philadelphia chromosome-positive chronic myeloid leukemia (CML, n = 9). In PMF, CD68-positive macrophages were greatly increased compared to CML (p = 0.017) and control BM (p < 0.001). Similar findings were observed by CD163 staining (PMF vs. CML: p = 0.017; PMF vs. control: p < 0.001). Moreover, CD68-positive macrophages were increased in PV compared with ET (p = 0.009) and reactive cases (p < 0.001). PMF had higher frequencies of macrophages than PV (CD68: p < 0.001; CD163: p < 0.001) and ET (CD68: p < 0.001; CD163: p < 0.001). CD163 and CD68 were often co-expressed in macrophages with stellate morphology in Philadelphia chromosome-negative MPN, resulting in a sponge-like reticular network that may be a key regulator of unbalanced hematopoiesis in the BM space and may explain differences in cellularity and clinical course.

COVID-19 carries a high risk of severe disease course, particularly in patients with comorbidities. Therapy of severe COVID-19 infection has relied on supportive intensive care measures. More specific approaches including drugs that limit the detrimental "cytokine storm", such as Janus-activated kinase (JAK) inhibitors, are being discussed. Here, we report a compelling case of a 55-yo patient with proven COVID-19 pneumonia, who was taking the JAK1/2 inhibitor ruxolitinib in-label for co-existing primary myelofibrosis for 15 months prior to coronavirus infection. The patient had significant comorbidities, including chronic kidney disease, arterial hypertension, and obesity, and our previous cohort suggested that he was thus at high risk for acute respiratory distress syndrome (ARDS) and death from COVID-19. Since abrupt discontinuation of ruxolitinib may cause fatal cytokine storm and ARDS, ruxolitinib treatment was continued and was well tolerated, and the patient´s condition remained stable, without the need for mechanical ventilation or vasopressors. The patient became negative for SARS-CoV-2 and was discharged home after 15 days. In conclusion, our report provides clinical evidence that ruxolitinib treatment is feasible and can be beneficial in patients with COVID-19 pneumonia, preventing cytokine storm and ARDS., (© 2020 The Authors. European Journal of Haematology published by John Wiley & Sons Ltd.)

Prostate cancer (PCa) is one of the most common and deadly cancers in men worldwide. The surrounding tumor microenvironment (TME) is important in tumor progression, as cytokines and soluble mediators including tumor necrosis factor (TNF-α) or lipocalin-2 (LCN2) can influence tumor growth and formation of metastasis. The exact mechanisms on how these pleiotropic factors affect PCa are still unknown. In this study, we showed for the first time that LCN2 mRNA and protein expression are strongly inducible by TNF-α in the highly metastatic human PCa cell line PC-3. In addition, we observed higher levels of secreted LCN2 in cell culture medium of TNF-α-treated PC-3 cells. We found that different signaling pathways such as p38, NF-κB or JNK were activated shortly after TNF-α treatment. Moreover, the mRNA levels of IL-1β and IL-8 were also significantly increased after 24 h stimulation. Mechanistically, the NF-κB pathway and the JNK signaling axis are directly responsible for LCN2 upregulation. This was shown by the fact that pretreatment with the JNK inhibitors SP600125 or JNK-IN-8 strongly downregulated phosphorylation of c-Jun protein and markedly reduced TNF-α-mediated LCN2 upregulation in PC-3 cells. Likewise, the NF-κB inhibitor QNZ was able to repress TNF-α-induced LCN2 expression in PC-3 cells. Taking into consideration that LCN2 has been described as a tumor promoting factor in PCa, our results indicate that JNK regulates LCN2 expression and unmasks the JNK signaling axis as a possible therapeutic target for patients with PCa., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

Kindlin-3 (K3)-mediated integrin adhesion controls homing and bone marrow (BM) retention of normal hematopoietic cells. However, the role of K3 in leukemic stem cell (LSC) retention and growth in the remodeled tumor-promoting BM is unclear. We report that loss of K3 in a mouse model of chronic myeloid leukemia (CML) triggers the release of LSCs from the BM into the circulation and impairs their retention, proliferation, and survival in secondary organs, which curbs CML development, progression, and metastatic dissemination. We found de novo expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) on CML-LSCs but not normal hematopoietic stem cells and this enabled us to specifically deplete K3 with a CTLA-4-binding RNA aptamer linked to a K3-siRNA (small interfering RNA) in CTLA-4 + LSCs in vivo, which mobilized LSCs in the BM, induced disease remission, and prolonged survival of mice with CML. Thus, disrupting interactions of LSCs with the BM environment is a promising strategy to halt the disease-inducing and relapse potential of LSCs., Competing Interests: The authors declare no competing interest.

Hematopoiesis is a highly regulated and complex process involving hematopoietic stem cells (HSCs), cell surface adhesion molecules, and cytokines as well as cells of the hematopoietic niche in the bone marrow (BM). Myeloproliferative neoplasms (MPNs) are characterized by clonal expansion of HSCs involving one or more blood cell lineages. Philadelphia-negative MPNs (Ph-neg MPNs) comprise polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). In nearly all patients with Ph-neg MPN, mutations in the genes encoding janus kinase 2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL) can be detected and, together with additional mutations in epigenetic modifier genes, these genetic aberrations contribute to the clonal expansion of the cells. In addition to these intracellular changes in the malignant clone, inflammatory processes involving both the clonal and the non-clonal cells contribute to the signs and symptoms of the patients, as well as to progression of the disease to myelofibrosis (MF) or acute leukemia, and to thrombotic complications. This contribution has been corroborated in preclinical studies including mouse models and patient-derived iPS cells, and in clinical trials, using anti-inflammatory drugs such as JAK inhibitors and steroids, or immunomodulatory drugs such as IMiDs and interferon-alpha (IFNa), all of which change the (im)balance of circulating inflammatory factors (e.g., TNFa, IL-1b, and TGFβ) in MPN. Currently, allogeneic hematopoietic (stem) cell transplantation (allo-HCT) remains the only curative treatment for Ph-neg MPN and is the treatment of choice in intermediate-2 and high-risk MF. HCT can reverse inflammatory changes induced by MPN as well as fibrosis in a large proportion of patients, but it also induces itself profound changes in inflammatory cells and cytokines in the patient, which may help to eradicate the disease but also in part cause significant morbidity (e.g., by graft-versus-host disease). In this review, we focus on the contribution of aberrant inflammation to disease pathogenesis in Ph-neg MPN as well as the current understanding of its alterations after allogeneic HCT.

The muscle specific isoform of the supervillin protein (SV2), encoded by the SVIL gene, is a large sarcolemmal myosin II- and F-actin-binding protein. Supervillin (SV2) binds and co-localizes with costameric dystrophin and binds nebulin, potentially attaching the sarcolemma to myofibrillar Z-lines. Despite its important role in muscle cell physiology suggested by various in vitro studies, there are so far no reports of any human disease caused by SVIL mutations. We here report four patients from two unrelated, consanguineous families with a childhood/adolescence onset of a myopathy associated with homozygous loss-of-function mutations in SVIL. Wide neck, anteverted shoulders and prominent trapezius muscles together with variable contractures were characteristic features. All patients showed increased levels of serum creatine kinase but no or minor muscle weakness. Mild cardiac manifestations were observed. Muscle biopsies showed complete loss of large supervillin isoforms in muscle fibres by western blot and immunohistochemical analyses. Light and electron microscopic investigations revealed a structural myopathy with numerous lobulated muscle fibres and considerable myofibrillar alterations with a coarse and irregular intermyofibrillar network. Autophagic vacuoles, as well as frequent and extensive deposits of lipoproteins, including immature lipofuscin, were observed. Several sarcolemma-associated proteins, including dystrophin and sarcoglycans, were partially mis-localized. The results demonstrate the importance of the supervillin (SV2) protein for the structural integrity of muscle fibres in humans and show that recessive loss-of-function mutations in SVIL cause a distinctive and novel myopathy., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

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., (© 2020 by The American Society of Hematology.)

What is the role of inflammation in Myeloproliferative Neoplasms? This is currently a topic of much debate. In this review, we will discuss experimental results and basic concepts of inflammatory processes in the pathogenesis of myeloproliferative neoplasms (MPN). So, which are the players involved? First, these are the clonal hematopoietic stem cells (HSC) and their normal stem cell counterparts in the bone marrow (BM), as well as their more mature progeny in the BM and the peripheral blood (PB), including neutrophils, macrophages, erythrocytes, and platelets, but also other cell lineages. Second, these cells produce a plethora of inflammatory cytokines, such as interleukin 6 (IL6), interleukin 8 (IL8), TNF-alpha (TNFa), interferon-alpha (IFNa), and others. Third, these cells and cytokines act in concert with non-hematopoietic cells, including endothelial cells and mesenchymal stromal cells (MSCs). The latter cells, in particular GLI1 positive or leptin receptor (LepR) positive stromal cells, may become activated by the hematopoietic clone to give rise to myofibroblasts, producing excessive fibrosis in the bone marrow (myelofibrosis). Ultimately, the inflammatory and fibrotic circuit involving these three key players may lead to progression of the disease, resulting in BM failure and transformation into acute leukemia, also termed blast crisis. Here, we review the role of these three effectors in the pathogenesis of MPN., Competing Interests: Declaration of Competing Interest SK is a member of the Advisory Boards of Pfizer, Incyte/Ariad, Novartis, AOP, BMS, Roche and CTI; Honoraria: Novartis, BMS, Pfizer, Incyte/Ariad, Shire; Scientific Research Support: Novartis Foundation, BMS, Novartis; Others: Alexion. NC reports no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Structural analogues of PFI-1 varying at the sulfur core were prepared, and their activities as BET inhibitors in myeloid cell lines and primary cells from patients with acute myeloid leukemia were studied. Docking calculations followed by molecular dynamics simulations revealed the binding mode of the newly prepared inhibitors, suggesting explanations for the observed high enantiospecificity of the inhibitory activity., Competing Interests: The authors declare no competing financial interest.

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.

Patients with myeloproliferative neoplasms (MPNs), such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are at increased risk for arterial and venous thrombosis/thromboembolism. In particular, the risk of splanchnic venous thrombosis, such as portal vein thrombosis or Budd-Chiari syndrome, is significantly higher in patients with MPN than in the normal population. At the same time, MPN patients are at increased risk for severe bleeding. Therefore, the treatment of patients with MPN must be based on their suspected probability of thrombosis/thromboembolism and bleeding. For this purpose, patient and MPN-specific risk factors are used. Patients at expected high risk of thrombosis should receive adequate primary or secondary thromboprophylaxis in addition to cytoreductive therapy. This may consist of antiplatelet agents and/or anticoagulant agents and must be balanced with the individual bleeding risk. The goal is to increase the quality of life and life span of patients with MPNs by preventing (re-)thrombosis and severe bleeding., Competing Interests: S.K. reports research funding from Novartis, Janssen, AOP Orphan Pharmaceuticals AG, and Bristol-Myers Squibb, as well as consultancy honoraria from Novartis, Incyte/Ariad, Bristol-Myers Squibb, AOP Orphan Pharmaceuticals AG, Pfizer, Celgene, Bayer, Roche, CTI, and Shire., (Georg Thieme Verlag KG Stuttgart · New York.)

Treatment of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute leukemia (Ph+ ALL) has been revolutionized with the advent of tyrosine kinase inhibitors (TKIs). Most patients with CML achieve long-term survival similar to individuals without CML due to treatment with TKIs not only in frontline but also in further lines of therapy. The third-generation TKI ponatinib has demonstrated efficacy in patients with refractory CML and Ph+ ALL. Ponatinib is currently the most potent TKI in this setting demonstrating activity against T315I mutant clones. However, ponatinib's safety data revealed a dose-dependent, increased risk of serious cardiovascular (CV) events. Guidance is needed to evaluate the benefit-risk profile of TKIs, such as ponatinib, and safety measures to prevent treatment-associated CV events. An expert panel of German hematologists and cardiologists summarize current evidence regarding ponatinib's efficacy and CV safety profile. We propose CV management strategies for patients who are candidates for ponatinib., (© 2019 The Author(s) Published by S. Karger AG, Basel.)

Hypereosinophilia (HE) is caused by a variety of disorders, ranging from parasite infections to autoimmune diseases and cancer. Only a small proportion of HE cases are clonal malignancies, and one of these, the group of eosinophilia-associated tyrosine kinase fusion-driven neoplasms, is sensitive to tyrosine kinase inhibitors, while most subtypes lack specific treatment. Eosinophil functions are highly dependent on actin polymerization, promoting priming, shape change, and infiltration of inflamed tissues. Therefore, we investigated the role of the actin-binding protein lymphocyte cytosolic protein 1 (LCP1) in malignant and nonmalignant eosinophil differentiation. We use the protein kinase C-β (PKCβ) selective inhibitor enzastaurin (Enza) to dephosphorylate and inactivate LCP1 in FIP1L1-platelet-derived growth factor receptor α (PDGFRA)-positive Eol-1 cells, and this was associated with reduced proliferation, metabolic activity, and colony formation as well as enhanced apoptosis and impaired migration. While Enza did not alter FIP1L1-PDGFRA-induced signal transducer and activator of transcription 3 (STAT3), STAT5, and ERK1/2 phosphorylation, it inhibited STAT1 Tyr701 and AKT Ser473 (but not AKT Thr308 ) phosphorylation, and short hairpin RNA knockdown experiments confirmed that this process was mediated by LCP1 and associated mammalian target of rapamycin complex 2 (mTORC2) activity loss. Homeobox protein HoxB8 immortalized murine bone marrow cells showed impaired eosinophilic differentiation upon Enza treatment or LCP1 knockdown. Furthermore, Enza treatment of primary HE samples reduced eosinophil differentiation and survival. In conclusion, our data show that HE involves active LCP1, which interacts with mTOR and triggers mTORC2 activity, and that the PKCβ inhibitor Enza as well as targeting of LCP1 may provide a novel treatment approach to hypereosinophilic disorders., (© 2019 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.)

Philadelphia negative (Ph-neg) myeloproliferative neoplasms (MPN) are a heterogenous group of clonal stem cell disorders. Approved treatment options include hydroxyurea, anagrelide, and ruxolitinib, which are not curative. The concept of synthetic lethality may become an additional therapeutic strategy in these diseases. In our study, we show that DNA repair is altered in classical Ph-neg MPN, as analyzed by gene expression analysis of 11 genes involved in the homologous recombination repair pathway (HRR), the non-homologous end-joining pathway (NHEJ), and the single-strand break repair pathway (SSB). Altogether, peripheral blood-derived cells from 57 patients with classical Ph-neg MPN and 13 healthy controls were analyzed. LIG3 as an essential part of the SSB was significantly lower expressed compared to controls in all three entities (essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF)). In addition, while genes of other DNA-repair pathways showed-possibly compensatory-increased expression in ET (HRR, NHEJ) and PV (NHEJ), MF samples displayed downregulation of all genes involved in NHEJ. With regard to the JAK2 mutational status (analyzed in ET and MF only), no upregulation of the HRR was detected. Though further studies are needed, based on these findings, we conclude that synthetic lethality may become a promising strategy in treating patients with Ph-neg MPN.

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., (© 2019 by The American Society of Hematology.)

Tyrosine kinase inhibitor (TKI) therapy effectively blocks oncogenic Bcr-Abl signaling and induces molecular remission in the majority of CML patients. However, the disease-driving stem cell population is not fully targeted by TKI therapy in the majority of patients, and leukemic stem cells (LSCs) capable of re-inducing the disease can persist. In TKI-resistant CML, STAT3 inhibition was previously shown to reduce malignant cell survival. Here, we show therapy-resistant cell-extrinsic STAT3 activation in TKI-sensitive CML cells, using cell lines, HoxB8-immortalized murine BM cells, and primary human stem cells. Moreover, we identified JAK1 but not JAK2 as the STAT3-activating kinase by applying JAK1/2 selective inhibitors and genetic inactivation. Employing an IL-6-blocking peptide, we identified IL-6 as a mediator of STAT3 activation. Combined inhibition of Bcr-Abl and JAK1 further reduced CFUs from murine CML BM, human CML MNCs, as well as CD34 + CML cells, and similarly decreased LT-HSCs in a transgenic CML mouse model. In line with these observations, proliferation of human CML CD34 + cells was strongly reduced upon combined Bcr-Abl and JAK1 inhibition. Remarkably, the combinatory therapy significantly induced apoptosis even in quiescent LSCs. Our findings suggest JAK1 as a potential therapeutic target for curative CML therapies.

The 60th American Society of Hematology (ASH) held in San Diego in December 2018 was followed by the 13th Post-ASH chronic myeloproliferative neoplasms (MPNs) workshop on December 4 and 5, 2018. This closed annual workshop, first introduced in 2006 by Goldman and Mughal, was organized in collaboration with Alpine Oncology Foundation and allowed experts in preclinical and clinical research in the chronic MPNs to discuss the current scenario, including relevant presentations at ASH, and address pivotal open questions that impact translational research and clinical management. This review is based on the presentations and deliberations at this workshop, and rather than provide a resume of the proceedings, we have selected some of the important translational science and treatment issues that require clarity. We discuss the experimental and observational evidence to support the intimate interaction between aging, inflammation, and clonal evolution of MPNs, the clinical impact of the unfolding mutational landscape on the emerging targets and treatment of MPNs, new methods to detect clonal heterogeneity, the challenges in managing childhood and adolescent MPN, and reflect on the treatment of systemic mastocytosis (SM) following the licensing of midostaurin., (© 2019 John Wiley & Sons, Ltd.)