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Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA sequencing revealed that CALRdel52 led to the expansion of TGFβ1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGFβ antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, whereas T-cell depletion abrogated the protective effects conferred by neutralizing TGFβ. TGFβ1 reduced perforin and TNFα production by T cells in vitro. TGFβ1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGFβ1 expression. In four independent patient cohorts, TGFβ1 expression was increased in the BM of patients with MPN compared with healthy individuals, and the BM of patients with MPN contained a higher frequency of Treg compared with healthy individuals. Together, this study identified an ERK/Sp1/TGFβ1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity. Significance: Targeting the mutant calreticulin/TGFβ1 axis increases T-cell activity and glycolytic capacity, providing the rationale for conducting clinical trials on TGFβ antagonists as an immunotherapeutic strategy in patients with myeloproliferative neoplasms., (©2024 American Association for Cancer Research.)

Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting T cell (Tc) immunoglobulin and mucin-containing molecule 3 (TIM-3) for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti-TIM-3 Ab treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti-TIM-3 treatment resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ Tc enhanced Tc activation, proliferation, and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion, and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and activation in vitro, suggesting that anti-TIM-3 treatment-mediated GVL effects are Tc induced. In contrast to anti-programmed cell death protein 1 (anti-PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) treatment, anti-TIM-3-treatment did not enhance acute graft-versus-host disease (aGVHD). TIM-3 and its ligands were frequently expressed in acute myeloid leukemia (AML) cells of patients with post-allo-HCT relapse. We decipher the connections between oncogenic mutations found in AML and TIM-3 ligand expression and identify anti-TIM-3 treatment as a strategy for enhancing GVL effects via metabolic and transcriptional Tc reprogramming without exacerbation of aGVHD. Our findings support clinical testing of anti-TIM-3 Ab in patients with AML relapse after allo-HCT.

Leukemia stem cells (LSCs) share numerous features with healthy hematopoietic stem cells (HSCs). G-protein coupled receptor family C group 5 member C (GPRC5C) is a regulator of HSC dormancy. However, GPRC5C functionality in acute myeloid leukemia (AML) is yet to be determined. Within patient AML cohorts, high GPRC5C levels correlated with poorer survival. Ectopic Gprc5c expression increased AML aggression through the activation of NF-κB, which resulted in an altered metabolic state with increased levels of intracellular branched-chain amino acids (BCAAs). This onco-metabolic profile was reversed upon loss of Gprc5c, which also abrogated the leukemia-initiating potential. Targeting the BCAA transporter SLC7A5 with JPH203 inhibited oxidative phosphorylation and elicited strong antileukemia effects, specifically in mouse and patient AML samples while sparing healthy bone marrow cells. This antileukemia effect was strengthened in the presence of venetoclax and azacitidine. Our results indicate that the GPRC5C-NF-κB-SLC7A5-BCAAs axis is a therapeutic target that can compromise leukemia stem cell function in AML., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Ruxolitinib (RUX) is a Janus kinase 1/2 inhibitor (JAKi) approved in the EU for treating disease‑related splenomegaly or symptoms in adults patients with myelofibrosis (MF). This is an interim analysis of JAKoMo, a prospective, non‑interventional, phase IV study in MF. Between 2012-2019 (cutoff March 2021), 928 patients (JAKi-naïve and -pretreated) enrolled from 122 German centers. This analysis focuses on JAKi-naïve patients. RUX was administered according to the Summary of Product Characteristics. Compared to the COMFORT-I, -II, and JUMP trials, patients in JAKoMo were older (median 73 years), had poorer Eastern Cooperative Oncology Group (ECOG) performance statuses (16.5% had ECOG ≥ 2), and were more transfusion dependent (48.5%). JAKoMo represents the more challenging patients with MF encountered outside of interventional studies. However, patients with low-risk International Prognostic Scoring System (IPSS) scores or without palpable splenomegaly were also included. Following RUX treatment, 82.5% of patients experienced rapid (≤ 1 month), significant decreases in palpable spleen size, which remained durable for 24 months (60% patients). Symptom assessment scores improved significantly in Month 1 (median -5.2) up to Month 12 (-6.2). Common adverse events (AEs) were anemia (31.2%) and thrombocytopenia (28.6%). At cutoff, 54.3% of patients had terminated the study due to, death, AEs, or deterioration of health. No new safety signals were observed. Interim analysis of the JAKoMo study confirms RUX safety and efficacy in a representative cohort of real-world, elderly, JAKi-naïve patients with MF. Risk scores were used in less than half of the patients to initiate RUX treatment.Trial registration: NCT05044026; September 14, 2021., (© 2023. The Author(s).)

The transcription factor NFE2 is overexpressed in most patients with myeloproliferative neoplasms (MPN). Moreover, mutations in NFE2, found in a subset of MPN patients, strongly predispose for transformation to acute leukemia. Transgenic mice overexpressing NFE2 as well as mice harboring NFE2 mutations display an MPN phenotype and spontaneously develop leukemia. However, the molecular mechanisms effecting NFE2-driven leukemic transformation remain incompletely understood. Here we show that the pro-leukemic histone demethylase JMJD2C constitutes a novel NFE2 target gene. JMJD2C expression is elevated in MPN patients as well as in NFE2 transgenic mice. Moreover, we show that loss of JMJD2C selectively impairs proliferation of JAK2 V617F mutated cells. Our data suggest that JMJD2C represents a promising drug target in MPN and provide a rationale for further investigation in preclinical and clinical settings., (© 2023. The Author(s).)

Histone methylation tightly regulates chromatin accessibility, transcription, proliferation, and cell differentiation, and its perturbation contributes to oncogenic reprogramming of cells. In particular, many myeloid malignancies show evidence of epigenetic dysregulation. Jumonji C (JmjC) domain-containing proteins comprise a large and diverse group of histone demethylases (KDMs), which remove methyl groups from lysines in histone tails and other proteins. Cumulating evidence suggests an emerging role for these demethylases in myeloid malignancies, rendering them attractive targets for drug interventions. In this review, we summarize the known functions of Jumonji C (JmjC) domain-containing proteins in myeloid malignancies. We highlight challenges in understanding the context-dependent mechanisms of these proteins and explore potential future pharmacological targeting.

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.

Even after development of the JAK1/JAK2 inhibitor ruxolitinib, myeloproliferative neoplasm (MPN) patients require novel therapeutic options. While ruxolitinib can considerably improve quality of life and prolong survival, it does not modify the natural disease course in most patients. Moreover, resistance develops with prolonged use. Therefore, various combination treatments are currently being investigated. Published data provide a compelling rationale for the inhibition of insulin growth factor-1 receptor (IGF-1R) signaling in MPN. Here we report that genetic and pharmacological inhibition of IGF-1R selectively reduced Jak2 V617F -driven cytokine-independent proliferation ex vivo. Two different structurally unrelated IGF-1R inhibitors ameliorated disease phenotype in a murine MPN model and significantly prolonged survival. Moreover, in mice, low-dose ruxolitinib synergized with IGF-1R inhibition to increase survival. Our data demonstrate preclinical efficacy of IGF-1R inhibition in a murine MPN model., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Of BCR-ABL negative myeloproliferative neoplasm (MPN) patients, 3-14 % display a concomitant monoclonal gammopathy (MGUS). Nonetheless, literature on co-occurring MPN and MGUS is scarce, the molecular underpinnings are unknown and it is unclear whether patients require a specific management. Here, we compared the clinical and genetic features of MPN patients with and without concomitant MGUS. Of 114 MPN patients prospectively studied by serum immunofixation (median age, 67 years; 36.0 % essential thrombocythemia [ET], 24.6 % polycythemia vera [PV], 11.4 % secondary myelofibrosis [sMF], 28.1 % primary myelofibrois [PMF]; 73.7 % JAK2 V617F positive), 10 (9 %) harbored an M-protein. No relevant clinical differences existed between MPN patients with or without M-protein. Seven additional MPN/MGUS patients were retrospectively identified in our MPN registry, yielding a total of 17 patients (7 ET, 3 PV, 3 sMF, 4 PMF). One patient developed multiple myeloma (MM) and one smoldering MM. Seven of 12 patients analyzed carried mutations (e.g. in ASXL1 or TET2) in addition to those in JAK2 or CALR, and 4 of 10 patients showed aberrant cytogenetics. M-protein was mainly IgG (12/17), followed by IgM (4/17). In the two patients that underwent allogeneic stem cell transplantation mutant JAK2 and M-protein were no longer detectable post-transplant. In conclusion, MGUS prevalence in our cohort was in the range of previous reports and at most slightly higher than expected in the general population. MGUS presence did not correlate with a specific MPN entity, clinical features or genetic alterations. Our observations suggest that there is no strong clinical or biological relationship between the occurrence of MGUS and MPN., (Copyright © 2020. Published by Elsevier Ltd.)

Activity of the transcription factor NFE2 is elevated in the majority of patients with myeloproliferative neoplasms (MPNs), either by overexpression of the wild-type alleles or by the presence of an activating mutation. In murine models, enhanced NFE2 activity causes an MPN phenotype with spontaneous transformation to acute leukemia. However, little is known about the downstream target genes activated by augmented NFE2 levels. Here, we describe that NFE2 regulates expression of the hematopoietic master regulators GATA2 and SCL/TAL1, which are in turn overexpressed in primary MPN cells, suggesting that concomitant aberrant activation of several transcription factors coordinately contributes to the cellular expansion characteristic of these disorders., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) carries a 10-100 fold lower mutational burden than other neoplastic entities. Mechanistic explanations for why a low number of mutations suffice to induce leukemogenesis are therefore required. Here we demonstrate that transgenic overexpression of the wild type sphingosine-1-phosphate receptor 3 (S1P 3 ) in murine hematopoietic stem cells is sufficient to induce a transplantable myeloid leukemia. In contrast, S1P 3 expression in more mature compartments does not cause malignant transformation. Treatment with the sphingosine phosphate receptor modulator Fingolimod, which prevents receptor signaling, normalized peripheral blood cell counts and reduced spleen sizes in S1P 3 expressing mice. Gene expression analyses in AML patients revealed elevated S1P 3 expression specifically in two molecular subclasses. Our data suggest a previously unrecognized contribution of wild type S1P 3 signaling to leukemogenesis that warrants the exploration of S1P 3 antagonists in preclinical AML models.

The histone demethylase JMJD1C is overexpressed in patients with myeloproliferative neoplasms (MPNs) and has been implicated in leukemic stem cell function of MLL-AF9 and HOXA9-driven leukemia. In the emerging field of histone demethylase inhibitors, JMJD1C therefore became a potential target. Depletion of Jmjd1c expression significantly reduced cytokine-independent growth in an MPN cell line, indicating a role for JMJD1C in MPN disease maintenance. Here, we investigated a potential role for the demethylase in MPN disease initiation. We introduced a Cre-inducible JAK2V617F mutation into Jmjd1c knockout mice. We show that Jmjd1c is dispensable, both for healthy hematopoiesis as well as for JAK2V617F-driven MPN disease initiation. Jmjd1c knockout mice did not show any significant changes in peripheral blood composition. Likewise, introduction of JAK2V617F into Jmjd1c-/- mice led to a similar MPN phenotype as JAK2V617F in a Jmjd1c wt background. This indicates that there is a difference between the role of JMJD1C in leukemic stem cells and in MPN. In the latter, JMJC domain-containing family members may serve redundant roles, compensating for the loss of individual proteins., Competing Interests: The authors have declared that no competing interests exist.

In acute myeloid leukemia (AML), acquired genetic aberrations carry prognostic implications and guide therapeutic decisions. Clinical algorithms have been improved by the incorporation of novel aberrations. Here, we report the presence and functional characterization of mutations in the transcription factor NFE2 in patients with AML and in a patient with myelosarcoma. We previously described NFE2 mutations in patients with myeloproliferative neoplasms and demonstrated that expression of mutant NFE2 in mice causes a myeloproliferative phenotype. Now, we show that, during follow-up, 34% of these mice transform to leukemia presenting with or without concomitant myelosarcomas, or develop isolated myelosarcomas. These myelosarcomas and leukemias acquired AML-specific alterations, including the murine equivalent of trisomy 8, loss of the AML commonly deleted region on chromosome 5q, and mutations in the tumor suppressor Trp53 Our data show that mutations in NFE2 predispose to the acquisition of secondary changes promoting the development of myelosarcoma and/or AML., (© 2019 by The American Society of Hematology.)

Scientifically active medical doctors are required for successful translation of novel basic findings into the clinic. However, there is an increasing tendency of young medical doctors to primarily follow a more clinically and not scientifically orientated career pathway. Therefore, the establishment of novel career education structures and career perspectives in university medicine are important to stop this development. Here, we will discuss the current situation and ongoing attempts to design novel structural programs that allow a better combination of clinical and scientific work by highlighting also current developments at the Faculty of Medicine at the University of Freiburg., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (© Georg Thieme Verlag KG Stuttgart · New York.)

Knowledge of the molecular and clonal characteristics in the myelodysplastic syndromes (MDS) and during progression to acute myeloid leukaemia (AML) is essential to understand the disease dynamics and optimize treatment. Sequencing serial bone marrow samples of eight patients, we observed that MDS featured a median of 3 mutations. Mutations in genes involved in RNA-splicing or epigenetic regulation were most frequent, and exclusively present in the major clone. Minor subclones were distinguishable in three patients. As the MDS progressed, a median of one mutation was gained, leading to clonal outgrowth. No AML developed genetically independent of a pre-existing clone. The gained mutation mostly affected genes encoding signalling proteins. Additional acquisition of genomic aberrations frequently occurred. Upon treatment, emergence of new clones could be observed. As confirmed by single-cell sequencing, multiple mutations in identical genes in different clones were present within individual patients. DNA-methylation profiling in patients without identification of novel mutations in AML revealed methylation changes in individual genes. In conclusion, our data complement previous observations on the mutational and clonal characteristics in MDS and at progression. Moreover, DNA-methylation changes may be associated with progression in single patients. Redundancy of mutated genes in different clones suggests fertile grounds promoting clonal selection or acquisition., (© 2018 British Society for Haematology and John Wiley & Sons Ltd.)

Despite recent advances, the myeloproliferative neoplasms (MPNs) are attended by considerable morbidity and mortality. Janus kinase (Jak) inhibitors such as ruxolitinib manage symptoms but do not substantially change the natural history of the disease. In this report, we show the effects of IMG-7289, an irreversible inhibitor of the epigenetically active lysine-specific demethylase 1 (LSD1) in mouse models of MPN. Once-daily treatment with IMG-7289 normalized or improved blood cell counts, reduced spleen volumes, restored normal splenic architecture, and reduced bone marrow fibrosis. Most importantly, LSD1 inhibition lowered mutant allele burden and improved survival. IMG-7289 selectively inhibited proliferation and induced apoptosis of JAK2 V617F cells by concomitantly increasing expression and methylation of p53, and, independently, the pro-apoptotic factor PUMA and by decreasing the levels of its antiapoptotic antagonist BCL XL . These data provide a molecular understanding of the disease-modifying activity of the LSD1 inhibitor IMG-7289 that is currently undergoing clinical evaluation in patients with high-risk myelofibrosis. Moreover, low doses of IMG-7289 and ruxolitinib synergize in normalizing the MPN phenotype in mice, offering a rationale for investigating combination therapy., (Copyright © 2018 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

The transcription factor "nuclear factor erythroid 2" (NFE2) is overexpressed in the majority of patients with myeloproliferative neoplasms (MPNs). In murine models, elevated NFE2 levels cause an MPN phenotype with spontaneous leukemic transformation. However, both the molecular mechanisms leading to NFE2 overexpression and its downstream targets remain incompletely understood. Here, we show that the histone demethylase JMJD1C constitutes a novel NFE2 target gene. JMJD1C levels are significantly elevated in polycythemia vera (PV) and primary myelofibrosis patients; concomitantly, global H3K9me1 and H3K9me2 levels are significantly decreased. JMJD1C binding to the NFE2 promoter is increased in PV patients, decreasing both H3K9me2 levels and binding of the repressive heterochromatin protein-1α (HP1α). Hence, JMJD1C and NFE2 participate in a novel autoregulatory loop. Depleting JMJD1C expression significantly reduced cytokine-independent growth of an MPN cell line. Independently, NFE2 is regulated through the epigenetic JAK2 pathway by phosphorylation of H3Y41. This likewise inhibits HP1α binding. Treatment with decitabine lowered H3Y41ph and augmented H3K9me2 levels at the NFE2 locus in HEL cells, thereby increasing HP1α binding, which normalized NFE2 expression selectively in JAK2 V617F -positive cell lines., (© 2018 by The American Society of Hematology.)

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2 V617F -mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2 V617F -mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2 V617F -myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2 V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient-derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2 V617F -mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1-mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2 V617F -mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Myelofibrosis is a myeloproliferative neoplasm associated with progressive cytopenias and high symptom burden. MF patients with thrombocytopenia have poor prognosis but the presence of thrombocytopenia frequently precludes the use of JAK2 inhibitors. In this study, we assessed quality of life and symptom burden in 418 MF patients with (n=89) and without (n=329) thrombocytopenia using prospective data from the MPN-QOL study group database, including the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) and Total Symptom Score (MPN10). Thrombocytopenia, defined as platelet count <100×10 9 /L (moderate 51-100×10 9 /L; severe ≤50×10 9 /L), was associated with anemia (76% vs. 45%, p<0.001), leukopenia (29% vs. 11%, p<0.001), and need for red blood cell transfusion (35% vs. 19%, p=0.002). Thrombocytopenic patients had more fatigue, early satiety, inactivity, dizziness, sad mood, cough, night sweats, itching, fever, and weight loss; total symptom scores were also higher (33 vs. 24, p<0.001). Patients with severe thrombocytopenia were more likely to have anemia (86% vs. 67%, p=0.04), leukopenia (40% vs. 20%, p=0.04), and transfusion requirements (51% vs. 20%, p=0.002) but few differences in symptoms when compared to patients with moderate thrombocytopenia. These results suggest that MF patients with thrombocytopenia experience greater symptomatic burden than MF patients without thrombocytopenia and may benefit from additional therapies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

DNA methylation differences between normal tissue and cancerous tissue resulting in differential expression of genes are a hallmark of acute myeloid leukemia (AML) and can provide malignant cells with a growth advantage via silencing of specific genes, for example, transcription factors. Oligodendrocyte lineage transcription factor 2 (OLIG2) was reported to be differentially methylated and associated with prognosis in AML and, as reported for acute lymphoblastic leukemia and malignant glioma, may play a role in malignant transformation. We report that DNA methylation of OLIG2 is associated with decreased expression of mRNA in AML cell lines and patients. Moreover, in cell lines, decreased mRNA expression also translated into decreased OLIG2 protein expression. Treatment of non-expressing cell lines PL-21 and U-937 with the demethylating agent decitabine resulted in robust re-expression of OLIG2 on mRNA and protein levels. Furthermore, stable overexpression of OLIG2 in non-expressing cell lines Kasumi-1 and U-937, using a lentiviral vector system, led to moderate growth inhibition after 4 days and resulted in signs of differentiation in U-937 cells. Interestingly, although CD34 + cells from healthy donors and 10 of 12 AML patients exhibited no protein expression, OLIG2 was expressed in two patients, both bearing the translocation t(15;17), corresponding to OLIG2 expression in NB-4 cells, also harboring t(15;17). In conclusion, we provide first evidence that OLIG2 is epigenetically regulated via DNA methylation and expressed in a subset of AML patients. OLIG2 may exert antiproliferative activity in leukemia cell lines, and its potential leukemia-suppressing role in AML warrants further investigation., (Copyright © 2017 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Tissue microarray (TMA) technique is an established high-throughput method to analyze multiple tissue specimens in parallel. However, in order to obtain reliable results from immunohistochemical analyses of TMA blocks, cell composition of TMA spots must correspond to whole tissue sections (WTS) particularly in tissues with a heterogeneous cell composition as it is the case in myeloproliferative neoplasms (MPN). The aim of this study was to validate TMA of bone marrow biopsies from MPN patients. TMAs of MPN bone marrow biopsies (ET: n = 26, PV: n = 26, and PMF: n = 29) were compiled in triplicates and MPN-specific histological parameters were assessed. Results of TMA spots were compared with WTS' results using the intra-class correlation coefficient (ICC). Immunohistochemical NFE2 and calreticulin stainings of the TMA with quantitative evaluation were performed. TMA construction was technically successful with a loss of 10 % of all spots. ICC calculation revealed high to moderate correlations of TMA with WTS, especially the parameters that are typically affected in MPN tissue, e.g. cellularity of hematopoiesis (ICC 0.62-0.89), number of megakaryocytes (ICC 0.50-0.71), micro-vessel density (ICC 0.56-0.91), or grade of myelofibrosis (ICC 0.56-0.89). Results of NFE2 and calreticulin immunohistochemistry of MPN TMAs are consistent with previously published data. Overall, our results show moderate to good correlation between histological data of WTS and TMA spots illustrating that the TMA technique is applicable to bone marrow biopsies of MPN patients. However, TMA construction in triplicates is necessary to reach sufficient correlation. MPN TMAs can be applied for serial immunohistochemical surveys of archived tissues to assess the mutation status or to further sub-classify MPN cases.

The myeloproliferative neoplasms, including polycythemia vera, essential thrombocythemia and myelofibrosis, are distinguished by their debilitating symptom profiles, life-threatening complications and profound impact on quality of life. The role gender plays in the symptomatology of myeloproliferative neoplasms remains under-investigated. In this study we evaluated how gender relates to patients' characteristics, disease complications and overall symptom expression. A total of 2,006 patients (polycythemia vera=711, essential thrombocythemia=830, myelofibrosis=460, unknown=5) were prospectively evaluated, with patients completing the Myeloproliferative Neoplasm-Symptom Assessment Form and Brief Fatigue Inventory Patient Reported Outcome tools. Information on the individual patients' characteristics, disease complications and laboratory data was collected. Consistent with known literature, most female patients were more likely to have essential thrombocythemia (48.6% versus 33.0%; P<0.001) and most male patients were more likely to have polycythemia vera (41.8% versus 30.3%; P<0.001). The rate of thrombocytopenia was higher among males than females (13.9% versus 8.2%; P<0.001) and males also had greater red-blood cell transfusion requirements (7.3% versus 4.9%; P=0.02) with shorter mean disease duration (6.4 versus 7.2 years, P=0.03). Despite there being no statistical differences in risk scores, receipt of most therapies or prior complications (hemorrhage, thrombosis), females had more severe and more frequent symptoms for most individual symptoms, along with overall total symptom score (22.8 versus 20.3; P<0.001). Females had particularly high scores for abdominal-related symptoms (abdominal pain/discomfort) and microvascular symptoms (headache, fatigue, insomnia, concentration difficulties, dizziness; all P<0.01). Despite complaining of more severe symptom burden, females had similar quality of life scores to those of males. The results of this study suggest that gender contributes to the heterogeneity of myeloproliferative neoplasms by influencing phenotypic profiles and symptom expression., (Copyright© Ferrata Storti Foundation.)

We have recently demonstrated that the transcription factor nuclear factor-erythroid 2, which is critical for erythroid maturation and globin gene expression, plays an important role in the pathophysiology of myeloproliferative neoplasms. Myeloproliferative neoplasm patients display elevated levels of nuclear factor-erythroid 2 and transgenic mice overexpressing the transcription factor develop myeloproliferative neoplasm, albeit, surprisingly without erythrocytosis. Nuclear factor-erythroid 2 transgenic mice show both a reticulocytosis and a concomitant increase in iron deposits in the spleen, suggesting both enhanced erythrocyte production and increased red blood cell destruction. We therefore hypothesized that elevated nuclear factor-erythroid 2 levels may lead to increased erythrocyte destruction by interfering with organelle clearance during erythroid maturation. We have previously shown that nuclear factor-erythroid 2 overexpression delays erythroid maturation of human hematopoietic stem cells. Here we report that increased nuclear factor-erythroid 2 levels also impede murine maturation by retarding mitochondrial depolarization and delaying mitochondrial elimination. In addition, ribosome autophagy is delayed in transgenics. We demonstrate that the autophagy genes NIX and ULK1 are direct novel nuclear factor-erythroid 2 target genes, as these loci are bound by nuclear factor-erythroid 2 in chromatin immunoprecipitation assays. Moreover, Nix and Ulk1 expression is increased in transgenic mice and in granulocytes from polycythemia vera patients. This is the first report implying a role for nuclear factor-erythroid 2 in erythroid maturation by affecting autophagy., (Copyright© Ferrata Storti Foundation.)

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal disorders involving hematopoietic stem and progenitor cells and are associated with myeloproliferation, splenomegaly and constitutional symptoms. Similar signs and symptoms can also be found in patients with chronic inflammatory diseases, and inflammatory processes have been found to play an important role in the pathogenesis and progression of MPNs. Signal transduction pathways involving JAK1, JAK2, STAT3 and STAT5 are causally involved in driving both the malignant cells and the inflammatory process. Moreover, anti-inflammatory and immune-modulating drugs have been used successfully in the treatment of MPNs. However, to date, many unresoved issues remain. These include the role of somatic mutations that are present in addition to JAK2V617F, CALR and MPL W515 mutations, the interdependency of malignant and nonmalignant cells and the means to eradicate MPN-initiating and -maintaining cells. It is imperative for successful therapeutic approaches to define whether the malignant clone or the inflammatory cells or both should be targeted. The present review will cover three aspects of the role of inflammation in MPNs: inflammatory states as important differential diagnoses in cases of suspected MPN (that is, in the absence of a clonal marker), the role of inflammation in MPN pathogenesis and progression and the use of anti-inflammatory drugs for MPNs. The findings emphasize the need to separate the inflammatory processes from the malignancy in order to improve our understanding of the pathogenesis, diagnosis and treatment of patients with Philadelphia-negative MPNs.

Purpose: Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) associated with disabling symptoms and a heightened risk of life-threatening complications. Recent studies have demonstrated the effectiveness of JAK inhibitor therapy in patients with PV patients who have a history of prior hydroxyurea (HU) use (including resistance or intolerance), phlebotomy requirements, and palpable splenomegaly. We aimed to determine how these features contribute alone and in aggregate to the PV symptom burden., Patients and Methods: Through prospective evaluation of 1,334 patients with PV who had characterized symptom burden, we assessed patient demographics, laboratory data, and the presence of splenomegaly by disease feature (ie, known HU use, known phlebotomy requirements, splenomegaly)., Results: The presence of each feature in itself is associated with a moderately high symptom burden (MPN symptom assessment form [SAF] total symptom score [TSS] range, 27.7 to 29.2) that persists independent of PV risk category. In addition, symptoms incrementally increase in severity with the addition of other features. Patients with PV who had all three features (PV-HUPS) faced the highest total score (MPN-SAF TSS, 32.5) but had similar individual symptom scores to patients with known HU use (PV-HU), known phlebotomy (PV-P), and splenomegaly (PV-S)., Conclusion: The results of this study suggest that patients with PV who have any one of the features in question (known HU use, known phlebotomy, or splenomegaly) have significant PV-associated symptoms. Furthermore, it demonstrates that many PV symptoms remain severe independent of the number of features present., (© 2015 by American Society of Clinical Oncology.)

We have previously described novel histone acetyltransferase (HAT) inhibitors that block neuroblastoma cell growth in vitro. Here we show that two selected pyridoisothiazolone HAT inhibitors, PU139 and PU141, induce cellular histone hypoacetylation and inhibit growth of several neoplastic cell lines originating from different tissues. Broader in vitro selectivity profiling shows that PU139 blocks the HATs Gcn5, p300/CBP-associated factor (PCAF), CREB (cAMP response element-binding) protein (CBP) and p300, whereas PU141 is selective toward CBP and p300. The pan-inhibitor PU139 triggers caspase-independent cell death in cell culture. Both inhibitors block growth of SK-N-SH neuroblastoma xenografts in mice and the PU139 was shown to synergize with doxorubicin in vivo. The latter also reduces histone lysine acetylation in vivo at concentrations that block neoplastic xenograft growth. This is one of the very few reports on hypoacetylating agents with in vivo anticancer activity.

It has been known for some time that solid tumors, especially gastrointestinal tumors, can arise on the basis of chronic inflammation. However, the role of inflammation in the genesis of hematological malignancies has not been extensively studied. Recent evidence clearly shows that changes in the bone marrow niche can suffice to induce myeloid diseases. Nonetheless, while it has been demonstrated that myeloproliferative neoplasms (MPN) are associated with a proinflammatory state, it is not clear whether inflammatory processes contribute to the induction or maintenance of MPN. More provocatively stated: which comes first, the hen or the egg, inflammation or MPN? In other words, can chronic inflammation itself trigger an MPN? In this review, we will describe the evidence supporting a role for inflammation in initiating and promoting MPN development. Furthermore, we will compare and contrast the data obtained in gastrointestinal tumors with observations in MPN patients and models, pointing out the opportunities provided by novel murine MPN models to address fundamental questions regarding the role of inflammatory stimuli in the molecular pathogenesis of MPN.

We performed a multicenter, investigator initiated, phase I dose escalation study of the oral multi-kinase inhibitor lestaurtinib in patients with JAK2V617F positive myelofibrosis, irrespective of baseline platelet count. A total of 34 patients were enrolled. Dose-limiting toxicities were observed in three patients overall, at the 100 mg (n = 1) and 160 mg (n = 2) twice-daily dose levels. The maximum tolerated dose was 140 mg twice daily. Gastrointestinal toxicity was the most common adverse event. Sixteen patients were evaluable for response at 12 weeks. Seven patients had clinical improvement by International Working Group - Myeloproliferative Neoplasms Research and Treatment criteria. Meaningful reductions in JAK2V617F allele burden were not observed. To measure JAK2 inhibition in vivo, plasma from treated patients was assayed for its ability to inhibit phosphorylation of signal transducer and activator of transcription 5 (STAT5): doses lower than 140 mg had variable and incomplete inhibition. In this phase I study, although gastrointestinal adverse events were common, significant clinical activity with lestaurtinib was observed (ClinicalTrials.gov identifier: NCT00668421).

The antiapoptotic BCL-2 protein MCL-1, which opposes mitochondrial outer membrane permeabilization, was shown to have a crucial role in the survival of hematopoietic cells. We have previously shown that, upon loss of phosphatidylinositol 3-kinase signaling, S159 of MCL-1 is phosphorylated by glycogen synthase kinase-3 (GSK-3), earmarking MCL-1 for enhanced ubiquitylation and degradation. In this study, we introduced MCL-1(wt) or the phosphorylation-deficient mutant MCL-1(S159A) in mouse BM cells, followed by adoptive transfer to recipient mice. Mice expressing MCL-1(S159A) exhibited significantly elevated white blood cell and lymphocyte counts, whereas no effect was observed on the distribution of T and B lymphocyte subsets or the numbers of monocytes, red blood cells or platelets. Expression of MCL-1(S159A) in Eμ-Myc transgenic bone marrow significantly accelerated the onset of disease, and these mice displayed increased spleen weights compared with Eμ-Myc/MCL-1(wt) mice. Our data demonstrate that the absence of MCL-1 S159 phosphorylation provides a survival advantage for hematopoietic cells in vivo and facilitates oncogenesis.

In this issue of Blood, Lundberg et al correlate the presence of known mutations in patients with myeloproliferative neoplasms (MPNs) with clinical outcome, thereby proposing a molecular risk stratification.

The adaptor protein linker activator of T-cells 2 (LAT2) is a known AML1/ETO target gene whose function during normal hematopoiesis is unknown. We addressed the role of LAT2 during erythroid and myeloid differentiation of normal human CD34+ hematopoietic cells. LAT2 is expressed at low levels in CD34+ cells and upregulated during cytokine-induced myeloid and erythroid differentiation. Forced LAT2 expression leads to a delay of erythroid and myeloid differentiation keeping CD34+ cells in a more immature state, whereas LAT2 knockdown accelerates differentiation. It is tempting to speculate that by affecting the differentiation capacity of normal hematopoietic progenitors, LAT2 may contribute to the pathogenesis of AML., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The establishment of imatinib as the standard therapy for CML marked the beginning of a new era of treatment. Due to occurring intolerance and resistance against the drug, developing newer inhibitors was promoted. This led to the second-generation inhibitors dasatinib, nilotinib and bosutinib. Despite all achieved improvement, all first- and second-generation inhibitors are ineffective against the BCR-ABL T315I "gatekeeper" mutation. In order to overcome this issue and to further improve the inhibitory effect, the third-generation inhibitor ponatinib was developed. Various clinical trials have been launched to study the effect of ponatinib in the clinical setting. Based on positive phase 1 and phase 2 trials, ponatinib was approved for the second-line treatment of CML and Ph+ ALL in December 2012 in the United States and in July 2013 in the European Union. Further trials investigate the potential effect of ponatinib in kinase-dependent subgroups of other malignancies. In conclusion, ponatinib has proved to be a powerful BCR-ABL inhibitor, which exhibits clinical activity both in BCR-ABL wild-type and mutant CML, including activity against the T315I mutation. Despite previous TKI failure, chronic-phase CML patients can achieve sustained remissions using the novel drug, offering a new therapeutic option in the treatment for CML.

Background: Besides essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) the myeloproliferative neoplasms (MPN) defined by the World Health Organization (WHO) comprise the entity of unclassifiable MPNs (MPN, U). The exact differential diagnosis of the specific MPN entities can be challenging particularly at early stages of the diseases. So far, pathologists have had to rely only on histomorphological evaluation of bone marrow biopsies in combination with laboratory data because helpful ancillary tests are not yet available. Even molecular tests, such as JAK2 mutation analysis are not helpful particularly in the differential diagnosis of ET and PMF because both entities are associated with the V617F mutation in 50 % of the cases. Recently overexpression of the transcription factor NF-E2 in MPN was described., Materials and Methods: A collective of samples consisting of 163 bone marrow biopsies including 139 MPN cases was stained immunohistochemically for NF-E2 and analyzed regarding the subcellular localization of NF-E2 in erythroid progenitor cells. The results were compared between the MPN entities as well as the controls and statistical analyses were conducted., Results and Discussion: This study showed that NF-E2 immunohistochemistry and analysis of the proportion of nuclear positive erythroblasts of all erythroid precursor cells can help to distinguish between ET and PMF even in early stages of the diseases. An MPN, U case showing a proportion of more than 20 % nuclear positive erythroblasts can be classified as a PMF with 92 % accuracy.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

The World Health Organization (WHO) classification of myeloproliferative neoplasms (MPNs) comprises several entities including essential thrombocythemia (ET); primary myelofibrosis (PMF); and MPN, unclassifiable (MPN,U). Differential diagnosis between ET and early, prefibrotic PMF can be challenging but is critical because clinical course and outcome vary considerably between these entities. We have previously shown that the transcription factor nuclear factor erythroid 2 (NF-E2) is aberrantly expressed in MPN patients. Here we demonstrate that NF-E2 is mislocalized in PMF cells and that aberrant NF-E2 localization discriminates statistically highly significantly between ET and PMF. A threshold of 20% nuclear NF-E2 staining was cross-validated by ".682+ bootstrapping." Moreover, this cutoff correctly classifies diagnostic bone marrow biopsies of MPN,U patients specified upon follow-up as ET or PMF with 92% accuracy. Because interobserver concordance between independent pathologists was high (Spearman's rank correlation coefficient, 0.727), we propose that quantitative NF-E2 immunohistochemistry represents a diagnostic tool that can reliably support a differential diagnosis between ET and PMF.

The transcription factor nuclear factor erythroid-2 is over-expressed in patients with myeloproliferative neoplasms irrespective of the presence of the JAK2(V617F) mutation. Our transgenic mouse model over-expressing nuclear factor erythroid-2, which recapitulates many features of myeloproliferative neoplasms including transformation to acute myeloid leukemia, clearly implicates this transcription factor in the pathophysiology of myeloproliferative neoplasms. Because the targets mediating nuclear factor erythroid-2 effects are not well characterized, we conducted microarray analysis of CD34(+) cells lentivirally transduced to over-express nuclear factor erythroid-2 or to silence this transcription factor via shRNA, in order to identify novel target genes. Here, we report that the cytokine interleukin 8 is a novel target gene. Nuclear factor erythroid-2 directly binds the interleukin 8 promoter in vivo, and these binding sites are required for promoter activity. Serum levels of interleukin 8 are known to be elevated in both polycythemia vera and primary myelofibrosis patients. Recently, increased interleukin 8 levels have been shown to be predictive of inferior survival in primary myelofibrosis patients in multivariate analysis. Therefore, one of the mechanisms by which nuclear factor erythroid-2 contributes to myeloproliferative neoplasm pathology may be increased interleukin 8 expression.

The molecular etiology of myeloproliferative neoplasms (MPNs) remains incompletely understood, despite recent advances incurred through the discovery of several different mutations in MPN patients. We have recently described overexpression of the transcription factor NF-E2 in MPN patients and shown that elevated NF-E2 levels in vivo cause an MPN phenotype and predispose to leukemic transformation in transgenic mice. We report the presence of acquired insertion and deletion mutations in the NF-E2 gene in MPN patients. These result in truncated NF-E2 proteins that enhance wild-type (WT) NF-E2 function and cause erythrocytosis and thrombocytosis in a murine model. NF-E2 mutant cells acquire a proliferative advantage, witnessed by clonal dominance over WT NF-E2 cells in MPN patients. Our data underscore the role of increased NF-E2 activity in the pathophysiology of MPNs.