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With emerging new drugs in myelofibrosis (MF), a robust and harmonized framework for defining the severity of anemia and response to treatment will enhance clinical investigation and facilitate inter-study comparisons. Accordingly, the lead authors on the 2013 edition of the International Working Group-European LeukemiaNet (IWG-ELN) response criteria in MF were summoned to revise their document with the intent to i) account for gender-specific differences in determining hemoglobin levels for eligibility criteria, ii) revise definition of transfusion-dependent anemia (TDA) based on current restrictive transfusion practices, and iii) provide a structurally simple and easy to apply response criteria that are sensitive enough to detect efficacy signals (minor response) and also account for major responses. The initial draft of the 2024 IWG-ELN proposed criteria was subsequently circulated around a wider group of international experts and their feedback incorporated. The proposed articles include new definitions for TDA (≥3 units in the 12 weeks prior to study enrollment) and hemoglobin thresholds for eligibility criteria (<10 g/dL for women and <11 g/dL for men). The revised document also provides separate (TDA vs. non-TDA) and graded (major vs. minor response) response criteria while preserving the requirement for a 12-week period of screening and observation on treatment., (Copyright © 2024 American Society of Hematology.)

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.)

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.)

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.)

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.)

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.)

Mutations in FMS-like tyrosine kinase 3 (FLT3), such as internal tandem duplications (ITDs), can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors (FLT3i's), which are rarely curative. PARP1 inhibitors (PARP1i's) have been successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying homologous recombination (HR) deficiency. We show here that inhibition of FLT3(ITD) activity by the FLT3i AC220 caused downregulation of DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in inhibition of 2 major DNA double-strand break (DSB) repair pathways, HR, and nonhomologous end-joining. PARP1i, olaparib, and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells, thus mimicking synthetic lethality. Moreover, the combination of FLT3i and PARP1i eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells, as well as leukemic progenitors, from human and mouse leukemia samples. Notably, the combination of AC220 and BMN673 significantly delayed disease onset and effectively reduced leukemia-initiating cells in an FLT3(ITD)-positive primary AML xenograft mouse model. In conclusion, we postulate that FLT3i-induced deficiencies in DSB repair pathways sensitize FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP1i's. Therefore, FLT3(ITD) could be used as a precision medicine marker for identifying AML patients that may benefit from a therapeutic regimen combining FLT3 and PARP1i's., (© 2018 by The American Society of Hematology.)

Hematopoietic stem and progenitor cell (HSPC) functions are regulated by a specialized microenvironment in the bone marrow - the hematopoietic stem cell niche - of which the extracellular matrix (ECM) is an integral component. We describe here the localization of ECM molecules, in particular the laminin α4, α3 and α5 containing isoforms in the bone marrow. Laminin 421 (composed of laminin α4, β2, γ1 chains) is identified as a major component of the bone marrow ECM, occurring abundantly surrounding venous sinuses and in a specialized reticular fiber network of the intersinusoidal spaces of murine bone marrow (BM) in close association with HSPC. Bone marrow from Lama4 -/- mice is significantly less efficient in reconstituting the hematopoietic system of irradiated wildtype (WT) recipients in competitive bone marrow transplantation assays and shows reduced colony formation in vitro. This is partially due to retention of Lin - c-kit + Sca-1 + CD48 - long-term and short-term hematopoietic stem cells (LT-HSC/ST-HSC) in the G0 phase of the cell cycle in Lama4 -/- bone marrow and hence a more quiescent phenotype. In addition, the extravasation of WT BM cells into Lama4 -/- bone marrow is impaired, influencing the recirculation of HSPC. Our data suggest that these effects are mediated by a compensatory expression of laminin α5 containing isoforms (laminin 521/522) in Lama4 -/- bone marrow. Collectively, these intrinsic and extrinsic effects lead to reduced HSPC numbers in Lama4 -/- bone marrow and reduced hematopoietic potential., (Copyright © 2018 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F) + murine MPN-like disease and also against JAK2(V617F) + , CALR(del52) + , and MPL(W515L) + primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors., (© 2017 by The American Society of Hematology.)

Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.

With the use of ChIP on microarray assays in primary leukemia samples, we report that acute myeloid leukemia (AML) blasts exhibit significant alterations in histone H3 acetylation (H3Ac) levels at > 1000 genomic loci compared with CD34(+) progenitor cells. Importantly, core promoter regions tended to have lower H3Ac levels in AML compared with progenitor cells, which suggested that a large number of genes are epigenetically silenced in AML. Intriguingly, we identified peroxiredoxin 2 (PRDX2) as a novel potential tumor suppressor gene in AML. H3Ac was decreased at the PRDX2 gene promoter in AML, which correlated with low mRNA and protein expression. We also observed DNA hypermethylation at the PRDX2 promoter in AML. Low protein expression of the antioxidant PRDX2 gene was clinically associated with poor prognosis in patients with AML. Functionally, PRDX2 acted as inhibitor of myeloid cell growth by reducing levels of reactive oxygen species (ROS) generated in response to cytokines. Forced PRDX2 expression inhibited c-Myc-induced leukemogenesis in vivo on BM transplantation in mice. Taken together, epigenome-wide analyses of H3Ac in AML led to the identification of PRDX2 as an epigenetically silenced growth suppressor, suggesting a possible role of ROS in the malignant phenotype in AML.

Recent evidence suggests chronic myeloid leukemia (CML) stem cells are insensitive to kinase inhibitors and responsible for minimal residual disease in treated patients. We investigated whether CML stem cells, in a transgenic mouse model of CML-like disease or derived from patients, are dependent on Bcr-Abl. In the transgenic model, after retransplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off were able to persist in vivo and reinitiate leukemia in secondary recipients on Bcr-Abl reexpression. Bcr-Abl knockdown in human CD34(+) CML cells cultured for 12 days in physiologic growth factors achieved partial inhibition of Bcr-Abl and downstream targets p-CrkL and p-STAT5, inhibition of proliferation and colony forming cells, but no reduction of input cells. The addition of dasatinib further inhibited p-CrkL and p-STAT5, yet only reduced input cells by 50%. Complete growth factor withdrawal plus dasatinib further reduced input cells to 10%; however, the surviving fraction was enriched for primitive leukemic cells capable of growth in a long-term culture-initiating cell assay and expansion on removal of dasatinib and addition of growth factors. Together, these data suggest that CML stem cell survival is Bcr-Abl kinase independent and suggest curative approaches in CML must focus on kinase-independent mechanisms of resistance.

Introduction: The functions of large noncoding RNAs (ncRNAs) have remained elusive in many cases. Metastasis-Associated-in-Lung-Adenocarcinoma-Transcript-1 (MALAT-1) is an ncRNA that is highly expressed in several tumor types., Methods: Overexpression and RNA interference (RNAi) approaches were used for the analysis of the biological functions of MALAT-1 RNA. Tumor growth was studied in nude mice. For prognostic analysis, MALAT-1 RNA was detected on paraffin-embedded non-small cell lung cancer (NSCLC) tissue probes (n = 352) using in situ hybridization., Results: MALAT-1 was highly expressed in several human NSCLC cell lines. MALAT-1 expression was regulated by an endogenous negative feedback loop. In A549 NSCLCs, RNAi-mediated suppression of MALAT-1 RNA suppressed migration and clonogenic growth. Forced expression of MALAT-1 in NIH 3T3 cells significantly increased migration. Upon injection into nude mice, NSCLC xenografts with decreased MALAT-1 expression were impaired in tumor formation and growth. In situ hybridization on paraffin-embedded lung cancer tissue probes revealed that high MALAT-1 RNA expression in squamous cell carcinoma of the lung was associated with a poor prognosis. On genetic level, MALAT-1 displays the strongest association with genes involved in cancer like cellular growth, movement, proliferation, signaling, and immune regulation., Conclusions: These data indicate that MALAT-1 expression levels are associated with patient survival and identify tumor-promoting functions of MALAT-1.

Background: XIAP (X-linked inhibitor of apoptosis protein) is an inhibitor of caspases 3 and 9 that is overexpressed in acute myeloid leukemia (AML) and may contribute to chemoresistance. We report an open-label randomized phase II trial of reinduction chemotherapy with and without the XIAP antisense oligonucleotide AEG35156 in patients with AML who did not achieve remission with initial induction chemotherapy., Methods: Twenty-seven patients with AML who were refractory to initial induction chemotherapy were randomized and treated with AEG35156 (650 mg) in combination with high-dose cytarabine and idarubicin. Thirteen patients were randomized and treated with high-dose cytarabine and idarubicin alone. The rates of response and toxicity were determined., Results: Of the 27 patients assigned to AEG35156 in combination with high-dose cytarabine and idarubicin, 3 died during reinduction chemotherapy, 5 achieved complete remission (CR), and 6 achieved CR with incomplete platelet count recovery (CRp), for an overall response rate of 41%. Of the 13 patients assigned to the control arm of the study, none died during reinduction, 6 achieved CR, and 3 achieved CRp, for an overall response rate of 69%. The differences in response rates between patients in the AEG35156 and control arms were not statistically different (P = 0.18 by the χ(2) test)., Conclusions: The addition of AEG35156 to reinduction chemotherapy was well tolerated but did not improve rates of remission. Therefore alternative therapeutic strategies should be explored in patients with AML refractory to induction chemotherapy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein, which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis, we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells, as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients, even in the absence of t(8;21). On a functional level, knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly, self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies, serial replating capacity of primary cells, and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML.

The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However, because most HES patients lack FIP1L1-PDGFRA, we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA-negative HES patients revealed several novel PDGFRA point mutations (R481G, L507P, I562M, H570R, H650Q, N659S, L705P, R748G, and Y849S). When cloned into 32D cells, N659S and Y849S and-on selection for high expressors-also H650Q and R748G mutants induced growth factor-independent proliferation, clonogenic growth, and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity, we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion, our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA-negative HES patients.

Background: About 50% of patients (age ≥60 years) who have acute myeloid leukaemia and are otherwise medically healthy (ie, able to undergo intensive chemotherapy) achieve a complete remission (CR) after intensive chemotherapy, but with a substantially increased risk of early death (ED) compared with younger patients. We verified the association of standard clinical and laboratory variables with CR and ED and developed a web-based application for risk assessment of intensive chemotherapy in these patients., Methods: Multivariate regression analysis was used to develop risk scores with or without knowledge of the cytogenetic and molecular risk profiles for a cohort of 1406 patients (aged ≥60 years) with acute myeloid leukaemia, but otherwise medically healthy, who were treated with two courses of intensive induction chemotherapy (tioguanine, standard-dose cytarabine, and daunorubicin followed by high-dose cytarabine and mitoxantrone; or with high-dose cytarabine and mitoxantrone in the first and second induction courses) in the German Acute Myeloid Leukaemia Cooperative Group 1999 study. Risk prediction was validated in an independent cohort of 801 patients (aged >60 years) with acute myeloid leukaemia who were given two courses of cytarabine and daunorubicin in the Acute Myeloid Leukaemia 1996 study., Findings: Body temperature, age, de-novo leukaemia versus leukaemia secondary to cytotoxic treatment or an antecedent haematological disease, haemoglobin, platelet count, fibrinogen, and serum concentration of lactate dehydrogenase were significantly associated with CR or ED. The probability of CR with knowledge of cytogenetic and molecular risk (score 1) was from 12% to 91%, and without knowledge (score 2) from 21% to 80%. The predicted risk of ED was from 6% to 69% for score 1 and from 7% to 63% for score 2. The predictive power of the risk scores was confirmed in the independent patient cohort (CR score 1, from 10% to 91%; CR score 2, from 16% to 80%; ED score 1, from 6% to 69%; and ED score 2, from 7% to 61%)., Interpretation: The scores for acute myeloid leukaemia can be used to predict the probability of CR and the risk of ED in older patients with acute myeloid leukaemia, but otherwise medically healthy, for whom intensive induction chemotherapy is planned. This information can help physicians with difficult decisions for treatment of these patients., Funding: Deutsche Krebshilfe and Deutsche Forschungsgemeinschaft., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34(+) cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

Although the potential role of Pim2 as a cooperative oncogene has been well described in lymphoma, its role in leukemia has remained largely unexplored. Here we show that high expression of Pim2 is observed in patients with acute promyelocytic leukemia (APL). To further characterize the cooperative role of Pim2 with promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha), we used a well-established PML-RARalpha (PRalpha) mouse model. Pim2 coexpression in PRalpha-positive hematopoietic progenitor cells (HPCs) induces leukemia in recipient mice after a short latency. Pim2-PRalpha cells were able to repopulate mice in serial transplantations and to induce disease in all recipients. Neither Pim2 nor PRalpha alone was sufficient to induce leukemia upon transplantation in this model. The disease induced by Pim2 overexpression in PRalpha cells contained a slightly higher fraction of immature myeloid cells, compared with the previously described APL disease induced by PRalpha. However, it also clearly resembled an APL-like phenotype and showed signs of differentiation upon all-trans retinoic acid (ATRA) treatment in vitro. These results support the hypothesis that Pim2, which is also a known target of Flt3-ITD (another gene that cooperates with PML-RARalpha), cooperates with PRalpha to induce APL-like disease.

In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL(+) Lin(-)Sca-1(+)c-kit(+) (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin(-)Sca-1(-)c-kit(+)), nor mature granulocytes (CD11b(+)Gr-1(+)), nor potential stem cell niche cells (CD45(-)Ter119(-)) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL(+) LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity.

2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of tumor cells in vitro and in vivo. Here we assessed the effects of CDDO on CCAAT enhancer-binding protein alpha (CEBPA), a transcription factor critical for granulocytic differentiation. In HL60 acute myeloid leukemia (AML) cells, CDDO (0.01 to 2 muM) induces apoptosis in a dose-dependent manner. Conversely, subapoptotic doses of CDDO promote phagocytic activity and granulocytic-monocytic differentiation of HL60 cells through increased de novo synthesis of p42 CEBPA protein. CEBPA translational up-regulation is required for CDDO-induced granulocytic differentiation and depends on the integrity of the CEBPA upstream open reading frame (uORF). Moreover, CDDO increases the ratio of transcriptionally active p42 and the inactive p30 CEBPA isoform, which, in turn, leads to transcriptional activation of CEBPA-regulated genes (eg, GSCFR) and is associated with dephosphorylation of eIF2alpha and phosphorylation of eIF4E. In concordance with these results, CDDO induces a CEBPA ratio change and differentiation of primary blasts from patients with acute myeloid leukemia (AML). Because AML is characterized by arrested differentiation, our data suggest the inclusion of CDDO in the therapy of AML characterized by dysfunctional CEBPA expression.

In acute myeloid leukemia (AML), mutational activation of the receptor tyrosine kinase (RTK) Flt3 is frequently involved in leukemic transformation. However, little is known about a possible role of highly expressed wild-type Flt3 in AML. The proto-oncogene c-Cbl is an important regulator of RTK signaling, acting through its ubiquitin ligase activity and as a platform for several signaling adaptor molecules. Here, we analyzed the role of c-Cbl in Flt3 signal transduction and myeloid transformation. C-Cbl physically interacted with Flt3 and was tyrosine phosphorylated in the presence of Flt3-ligand (FL). Overexpression of a dominant-negative form of c-Cbl (Cbl-70Z) inhibited FL-induced Flt3 ubiquitylation and internalization, indicating involvement of c-Cbl in Flt3 signaling. DNA sequencing of AML bone marrow revealed a case with a c-Cbl point mutation (Cbl-R420Q). Cbl-R420Q inhibited Flt3 internalization and ubiquitylation. Coexpression of Cbl-R420Q or Cbl-70Z with Flt3 induced cytokine-independent growth and survival of 32Dcl3 cells in the absence of FL. Also, the mutant Cbl proteins altered the amplitude and duration of Flt3-dependent signaling events. Our results indicate an important role of Cbl proteins in Flt3 signal modulation. Also, the data suggest a novel mechanism of leukemic transformation in AML by mutational inactivation of negative RTK regulators.

Aberrant DNA methylation is the most frequent molecular alteration in acute myeloid leukemia (AML). To identify methylation-silenced genes in AML, we performed microarray analyses in U937 cells exposed to the demethylating agent 5-aza-deoxy-cytidine. Overall, 274 transcripts were significantly induced. Interestingly, C/EBPdelta expression was significantly induced (more than 10-fold) by demethylation whereas expression of all other C/EBP family members remained unchanged. The C/EBPdelta promoter was strongly methylated in different leukemic cell lines and showed signs of a repressed chromatin state. Analyses of the promoter regions of the entire C/EBP family (alpha, beta, gamma, delta, epsilon, zeta) in bone marrow samples from AML patients (n = 80) and controls (n = 15) by mass spectrometry revealed that C/EBPdelta is the most commonly hypermethylated C/EBP gene in AML. Hypermethylation occurred in more than 35% of AML patients at primary diagnosis. A significant correlation (P = .016) was observed between hypermethylation of the C/EBPdelta promoter and low expression of C/EBPdelta in AML patients. C/EBPdelta promoter activity was strongly repressed by methylation in vitro, and transcriptional repression partially depended on MeCP2 activity. C/EBPdelta exhibited growth-inhibitory properties in primary progenitor cells as well as in Flt3-ITD-transformed cells. Taken together, C/EBPdelta is a novel tumor suppressor gene in AML that is silenced by promoter methylation.

Increasing evidence suggests that leukemias are sustained by leukemic stem cells. However, the molecular pathways underlying the transformation of normal cells into leukemic stem cells are still poorly understood. The involvement of a small group of key transcription factors into this process was suggested by their frequent mutation or down-regulation in patients with acute myeloid leukemia (AML). Recent findings in mice with hypomorphic transcription-factor genes demonstrated that leukemic stem-cell formation in AML could directly be caused by reduced transcription-factor activity beyond a critical threshold. Most interestingly, those experimental models and the paucity of biallelic null mutations or deletions in transcription-factor genes in patients suggest that AML is generally associated with graded down-regulation rather than complete disruption of transcription factors. Here, we discuss the effects of transcription-factor concentrations on hematopoiesis and leukemia, with a focus on the regulation of transcription-factor gene expression as a major mechanism that alters critical threshold levels during blood development and cancer.

To develop murine models of leukemogenesis, a series of transgenic mice expressing BCR-ABL in different hematopoietic cell subsets was generated. Here we describe targeted expression of P210 BCR-ABL in stem and progenitor cells of murine bone marrow using the tet-off system. The transactivator protein tTA was placed under the control of the murine stem cell leukemia (SCL) gene 3' enhancer. Induction of BCR-ABL resulted in neutrophilia and leukocytosis, and the mice became moribund within 29 to 122 days. Autopsy of sick mice demonstrated splenomegaly, myeloid bone marrow hyperplasia, and extramedullary myeloid cell infiltration of multiple organs. BCR-ABL mRNA and protein were detectable in the affected organs. Fluorescence-activated cell sorter (FACS) analysis demonstrated a significant increase in mature and immature myeloid cells in bone marrow and spleen, together with increased bilineal B220+/Mac-1+ cells in the bone marrow. tTA mRNA was expressed in FACS-sorted hematopoietic stem cells expanded 26-fold after BCR-ABL induction. Thirty-one percent of the animals demonstrated a biphasic phenotype, consisting of neutrophilia and subsequent B-cell lymphoblastic disease, reminiscent of blast crisis. In summary, this mouse model recapitulates many characteristics of human chronic myeloid leukemia (CML) and may help elucidate basic leukemogenic mechanisms in CML stem cells during disease initiation and progression.

The BCR/ABL fusion protein is found in more than 90% of patients with chronic myeloid leukemia (CML) as well as in a subset of patients with acute B-cell leukemia. We have previously described a transgenic model for an inducible and reversible acute B-cell leukemia caused by p210 BCR/ABL. Here, we describe a new model of an inducible BCR/ABL disease by directing the expression of the oncogene to megakaryocytic progenitor cells within the murine bone marrow using the tetracycline-responsive expression system under the control of human CD34 regulatory elements. The predominant feature was the development of a chronic thrombocytosis. The condition progressed with the development of splenomegaly accompanied by lymphadenopathy in some mice. Affected animals demonstrated a dramatic increase in the number of megakaryocytes in the bone marrow and the spleen. Immunohistochemistry demonstrated that the reporter gene was expressed in hematopoietic stem cells (HSCs), common myeloid progenitor (CMP) cells, as well as in megakaryocytic/erythroid progenitor cells (MEPs). Although these mice did not display the increase in granulopoiesis commonly found in chronic myeloid leukemia (CML), the phenotype closely resembles a myeloproliferative disorder affecting the megakaryocytic lineage observed in some patients with the BCR/ABL P210 translocation.

Histone deacetyrase (HDAC) inhibitors induce growth arrest and differentiation of leukemia cell lines and tumor cells derived from a large variety of human tissues. Here we showed that HDAC inhibitors sodium butyrate, TSA, and valproate regulated the expression of Interleukin-18 (IL-18), a cytokine with antitumor and proinflammatory properties, in human acute myeloid leukemia cell lines U937 and HEL. Sodium butyrate increased expression of IL-18 protein and mRNA and activated 1357bp IL-18 gene promoter construct. IL-18 mRNA level was up-regulated by TSA or valproate, which also activated IL-18 full-length promoter. While sodium butyrate or TSA stimulated the 108-bp IL-18 minimal promoter, valproate failed to activate it, indicating that valproate may use a distinct mechanism from sodium butyrate and TSA to activate IL-18 gene expression., ((c)2002 Elsevier Science (USA).)