New insights in AML biology from genomic analysis.

New Insights in AML Biology From Genomic Analysis Ashley M. Perry and Eyal C. Attar Advancements in sequencing techniques have led to the discovery of numerous genes not previously implicated in acute myeloid leukemia (AML) biology. Further in vivo studies are necessary to discern the biological impact of these mutations. Murine models, the most commonly used in vivo system, provide a physiologic context for the study of specific genes. These systems have provided deep insights into the role of genetic translocations, mutations, and dysregulated gene expression on leukemia pathogenesis. This review focuses on the phenotype of newly identified genes, including NPM1, IDH1/2, TET2, MLL, DNMT3A, EZH2, EED, and ASXL1, in mouse models and the implications on AML biology. Semin Hematol 51:282–297. C 2014 Elsevier Inc. All rights reserved.

C

ancer biology research has entered an exciting era, as next-generation sequencing (NGS) has uncovered a large number of genetic lesions with potential roles in cancer pathogenesis. However, identifying the functional role of these genes, and the impact of the mutations, presents a significant scientific challenge. In vitro and in vivo techniques provide powerful tools to examine the functional effects of oncogenes, tumor suppressor genes, and other genes relevant to the pathogenesis of acute myeloid leukemia (AML). In vivo strategies allow for the study of gene function in complex cellular and environmental contexts. Among the in vivo techniques, mouse systems are the most extensively used because of similarities between the mouse and human genome, hematopoietic systems, and bone marrow microenvironment. In addition, mice have favorable housing and breeding characteristics that provide practical advantages over other species. The 3 types of murine systems primarily used to study gene function in hematopoietic cells are transgenesis, retroviral transduction/transplantation, and xenotransplantation (reviewed by Fortier and Graubert1). Each of these strategies offers advantages and disadvantages. Transgenic models, which involve introduction of a gene of interest in a fertilized oocyte, are resource intensive and susceptible to integration site effects (ie, mutation or dysregulated expression of genes at the site of transgene integration). In the technique of retroviral transduction and transplantation, murine hematopoietic cells are infected with a retrovirus (or lentivirus) Massachusetts General Hospital Cancer Center, Boston, MA. Conflicts of interest: none. Address correspondence to: Eyal C. Attar, MD, Massachusetts General Hospital, 100 Blossom Street, Boston, MA 02114. E-mail: eattar@ partners.org 0037-1963/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1053/j.seminhematol.2014.08.005

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encoding a gene of interest and then transplanted into recipient mice; the effect of expression of the gene on hematopoiesis can then be studied in vivo. This technique permits medium-throughput study of candidate genes, but because the retroviral promoter drives gene expression and the transgene is inserted randomly into the mouse genome (where it may be “captured” by local regulatory elements), gene expression may not be physiologic. Furthermore, recipient cells are susceptible to insertional mutagenesis, as with any randomly integrated transgene. Lastly, xenotransplantation of human leukemic cells into immunocompromised mice allows for the study of primary human cells, but this system incompletely recapitulates the phenotype of human AML. For example, mice frequently develop extramedullary deposition of AML cells in the lungs, liver, and spleen. In addition, there are significant technical challenges with this system, as human AML cells from the same patient can have variable engraftment in recipient mice, and there is significant variability in engraftment among samples from different patients. The transgenic and transduction/transplantation murine model systems have been used to assess whether mutation or aberrant expression of a few (most often just one) genes recapitulates AML phenotypes. However, our understanding that AML results from multiple genetic lesions challenges these single gene model systems. Furthermore, studies of human AML cells in xenotransplant systems indicate that AML is more complex on a cellular level than previously appreciated, as leukemias have significant intratumoral heterogeneity. It has long been known that only a minority of human AML cells demonstrate heightened engraftment and self-renewal capabilities characteristic of leukemia stem cells.2 A recent study further highlights the functional heterogeneity of various genetic subclones in this respect, demonstrating that although most AML subclones have equal propensity to circulate from the bone marrow to the peripheral blood

Seminars in Hematology, Vol 51, No 4, October 2014, pp 282–297

AML biology and genomic analysis

in patients, they can have distinct cellular phenotypes, unique functional properties in vitro, and disparate subclone engraftment capabilities in immunodeficient mice.3 The first genetic studies of human AML in mice involved genes whose function was perturbed as a consequence of chromosomal translocation, often involving transcription factors. Recently, advanced DNA sequencing techniques have revealed mutations in genes involved in pathways not previously implicated in leukemia or other cancers (as discussed by Mazzarella et al in this issue of the Journal). These studies highlight the potential importance of genes implicated in DNA methylation, RNA splicing, and glucose metabolism. These results have significantly increased the number of genes implicated in AML and revealed new concepts in AML pathogenesis. These findings challenge the 2-hit model system proposed by Gilliland and Griffin4 that leukemias arise from mutations in genes belonging to 1 of only 2 general categories: class I mutations that activate signal transduction pathways and confer a proliferation advantage, and class II mutations that affect transcription factors and block differentiation. Although our existing models continue to provide useful functional information in the postgenomic era, our increased understanding of the molecular and cellular complexity of AML and the identification of novel gene families involved in pathogenesis require new tools and techniques above and beyond our existing model systems to decipher this increasingly complex field.

FIRST-GENERATION MOUSE MODELS AML With Recurrent Cytogenetic Translocations Initial mouse models examined the effects of expressing gene fusions, which resulted from cytogenetic translocations detected in human AML samples, in murine hematopoietic cells and assessed for development of disease. For example, in vivo models have been developed to assess the biologic effects of inv(16),5 t(8;21),6 t(15;17),7 and t(6;9),8 among others. Mouse embryos that express CBFB/MYH11, the product of inv(16), have impaired hematopoiesis9 but rarely develop leukemia without additional cooperating mutations.10–12 Similarly, several transgenic studies have demonstrated that RUNX1/ RUNX1T1, resulting from the t(8;21) translocation, is not sufficient to initiate leukemia but may confer an increased tendency to develop leukemia when mice are exposed to genotoxic insult, reinforcing the observation that AML develops as a consequence of multiple mutations.12 The DEK/CAN fusion protein, generated by t(6;9), induces leukemia in the retroviral transduction transplantation model when specifically expressed in long-term (but not in short-term) hematopoietic stem cells (HSCs), suggesting that genetic lesions require a specific cellular context to cause a disease phenotype.8 Finally, expression of the t (15;17) gene product PML/RARA in promyelocytes results

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in altered myeloid development and an accumulation of myeloid precursors, ultimately resulting in AML in 30% of animals in an early model7 and with higher penetrance in later models.13,14

Cytogenetically Normal AML The observation that 40% to 50% of patients with AML have a normal karyotype led to the hypothesis that additional genetic factors could contribute to the development of leukemia apart from chromosomal translocations. This finding led to additional research examining mutations in individual genes and analysis of gene expression levels to determine their contribution to AML pathogenesis. Indeed, targeted sequencing approaches identified recurrent mutations in FLT3, NPM1, KIT, CEBPA, TET2, and other genes, whereas gene expression profiling has revealed that overexpression of BAALC, ERG, EVI1, and MN1 are each associated with an adverse prognosis. Animal model systems have been used to study the functions of these various genetic lesions. For example, an internal tandem duplication (ITD) of the FLT3 gene is capable of inducing a myeloproliferative disorder (MPD) in mice but not AML unless combined with other genetic mutations,15 underscoring its role in myeloid malignancy and suggesting that molecular mutations, such as recurrent chromosomal translocations, require cooperating events to cause AML. The development of an MPD rather than AML in this model system also points to the limited ability of existing murine models to accurately model human AML, as FLT3-ITD mutations are rarely observed in human MPDs. In the case of the Wilms’ tumor gene (WT1), which is highly expressed in most AML cases, overexpression impairs myeloid differentiation but does not induce leukemia in transgenic mice.16 However, transgenic mice that overexpress both WT1 and RUNX1/RUNX1T1 develop a highly penetrant leukemia, demonstrating how enforced expression of 2 genes who are each incapable of causing leukemia alone can cooperate to cause a rapidly developing leukemia.17 Similarly, BAALC (brain and acute leukemia, cytoplasmic) overexpression does not enhance self-renewal or proliferation of murine bone marrow cells but cooperates with other oncogenes such as MN1 (meningioma 1) to induce leukemia.18 MN1 is a transcription coregulator whose expression in transgenic mice potently induces leukemia by enhancing self-renewal and blocking proliferation.19 Similarly, transgenic mice expressing the ETS transcription factor ERG develop leukemia by 5 months of age.20 Lastly, overexpression of the ecotropic virus integration site 1 protein homolog (EVI1) leads to disease in mice that recapitulates some features of the myelodysplastic syndromes (MDS).21 These examples highlight how aberrant expression of some genes, even absent mutations, can result in leukemia, underscoring the need to account for gene dosage levels in murine models of human AML. These studies reveal several characteristic features important to the study of leukemia biology. First, as in

284 the case of the DEK/CAN translocation, cell context is important, indicating that the cell of origin is highly relevant to leukemia potential. Second, leukemiaassociated chromosomal translocations such as inv(16) may have broader effects apart from driving leukemia, such as inducing a developmental block at the onset of definitive hematopoiesis. Third, both genetic (mutations) and epigenetic (aberrant expression) alterations are relevant for AML pathogenesis. Finally, these studies illustrate that, when studied in mice, many genes that are recurrently translocated, mutated, or abnormally expressed in human leukemia require additional cooperating mutations to induce the leukemia phenotype.15

FUNCTIONAL ANALYSIS OF NOVEL MUTATIONS As more advanced, high-throughput DNA sequencing techniques have become available, the landscape of AML mutations has become increasingly complex. NGS has revealed mutations in novel genes belonging to pathways known to be involved in AML. Importantly, NGS has also revealed mutations in genes belonging to pathways previously unknown in AML, such as metabolism and RNA splicing. A comprehensive NGS analysis of 200 AML genomes reported by The Cancer Genome Atlas Network revealed mutations in such diverse gene classes as tumor suppressors, DNA methylation, signaling pathway activators, myeloid transcriptions factors, chromatin modifiers, and cohesin and spliceosome complex genes22 (see also the chapter by Mazzarella et al in this issue of the Journal). This review focuses on biologic insights from recently identified molecules (Table 1).

Nucleophosmin Nucleophosmin (NPM1), also known as nucleolar phosphoprotein B23 or numatrin, is a protein associated with ribonucleoprotein structures that normally shuttles between the nucleus and cytoplasm. NPM1 has reported roles in ribosome biogenesis, in genomic stability and DNA repair, and as a histone chaperone. It directly binds the TP53 and ARF tumor suppressor proteins. Mutations in NPM1 are among the most common in AML, occurring in 35% of patients. The canonical NPM1 mutation (NPM1c) results in preferential localization of NPM1 to the cytoplasm, perturbing its known function of preventing BRD4-dependent transcription.23 The contribution of NPM1 mutations to leukemogenesis is still unclear. Mice lacking NPM1 die (during embryogenesis) of developmental abnormalities and anemia, whereas heterozygous Npm1þ/– mice demonstrate increased erythroid cellularity and dyserythropoiesis reminiscent of MDS.24 Mutations in NPM1 are believed to occur early in the development of AML based on studies demonstrating the presence of NPM1c in immunophenotypically primitive AML cells capable of initiating leukemia in xenotransplant models.25–27

A.M. Perry and E.C. Attar

Knock-in Npm1c mice were generated by inserting TCTG after nucleotide c.857 (c.854_857dupTCTG) to mimic the human mutation without any "humanized" sequence. Data from mice ages 2 to 24 months showed that 11 (36%) of 36 mice developed MPD (“MPDNpm1c”).28 In cobblestone assays (a technique used to assess HSC/progenitor function in vitro), bone marrow cells from MPD-NPM1c mice had significantly lower activity compared with wild-type or NPM1c mice (without MPD). Gene expression analysis showed downregulation of the bone marrow homing and retention genes CXCL12 and CXCR4 in NPM1c mice, indicating that mutations in NPM1 may result in mobilization of HSCs from the bone marrow and subsequent HSC depletion. A conditional, targeted knock-in NPM1c model designed to minimize interference with the native Npm1 locus was generated by flanking exon 11 with loxP sites, followed by a humanized mutant exon 11.29 These mice were crossed to Mx1-Cre transgenics and treated with pI: pC to induce recombination. At 8 weeks after Cre induction, the mutant mice had higher mean red blood cell and platelet volumes compared with control mice, with no significant differences in white blood cell count, hemoglobin, or platelet counts. In addition, although the mutant mice did not exhibit differences in marrow stem and progenitor cell compartment sizes, they did demonstrate expansion of mature myeloid (Gr1þMac1þ) and reduction in late (B220þCD19þ) B cells. Survival of conditional NPM1c mice was reduced compared with wild-type littermates (617 vs 769 days; P ¼ .018) as a result of excess deaths due to AML (13 vs 0 deaths; P ¼ .0001). The Sleeping Beauty transposon was then used to induce cooperating mutations in an attempt to reduce the latency of AML in this model. Sleeping Beauty induces insertional mutagenesis by introducing a transposon sequence into TA sites throughout the genome and thus interrupts gene function. Indeed, the latency of leukemia development was shortened by using this system. When the transposon integration sites were mapped in hematopoietic tumors to determine the genetic mutations collaborating with NPM1c to cause AML, it was determined that each tumor had a unique integration site, suggesting that interruption of multiple different genes could cooperate with NPM1c to accelerate AML. Interestingly, insertions at the Csf2, Flt3, Rasgrp1, and Kras loci were found to be mutually exclusive, suggesting that mutation of a single component of this signaling pathway is sufficient to cooperate with NPM1c to induce AML. Mice with a conditional NPM1c allele generated by using a similar strategy developed thrombocytopenia and larger platelet size compared with control mice.30 Although white blood cell count and hemoglobin levels were preserved, NPM1c-expressing mice had decreased numbers of granulocyte-monocyte progenitors but similar levels of other hematopoietic progenitors compared with control mice. They also exhibited an increase in microRNA (miRNA) 10, associated with megakaryocyte


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Table 1. Mouse Models and Phenotype of Selected Genes Implicated in AML

Gene NPM1

Mouse Model Homozygous knock-out (NPM1

Phenotype –/– 24

)

Heterozygous knock-out (NPM1þ/–)24 Knock-in TCTG into mouse NPM1 to mimic human NPM1c28 Knock-in of human NPM1c into mouse NPM1 locus, conditional expression at 8 weeks29 Knock-in of human NPM1c into mouse NPM1 locus, conditional expression30

IDH1

Conditional knock-in of R132H-mutant IDH146 Retroviral transduction/transplantation of human R132C mutant IDH1 in to C57BL/6J mice36

TET2

Knock-out of TET264

MLL

Knock-in of exons 5 through 11 of the murine MLL gene targeted to intron 4 of the endogenous MLL locus (MLL-PTD)71 MLL-PTD knock in crossed with MLL-null (MLLPTD/–)80 Double knock-in (MllPTD/WT:Flt3ITD/WT)84

DNMT3A

Conditional DNMT3A knock-out94

Embryonic lethal (E11.5–E16.5) due to anemia from defective primary hematopoiesis Aberrant organogenesis MDS-like disease with increased erythroid cellularity and dyserythropoiesis 36% develop MPD Downregulation of bone marrow– homing genes CXCL12 and CXCR4 Increased platelet and red blood cell volumes Diminished survival due to increased risk of developing AML NPM1c/WT heterozygous mice develop thrombocytopenia, increase in the megakaryocyte compartment but no AML Anemia, splenomegaly, and extramedullary hematopoiesis but no AML Peripheral blood counts and spleen weights were similar to controls; no AML. However, when expressed in HOXA9 immortalized cells, mice developed severe leukocytosis, anemia, and thrombocytopenia after week 4 of transplantation and early death compared with control mice Tet2þ/ mice were similar to weight with respect to survival, hematopoietic cellularity, and lineage composition at 4 months Tet2–/– had monocytosis, neutrophilia, anemia, splenomegaly, and hepatomegaly reminiscent of CMML Skeletal defects, increased number and self-renewal of hematopoietic progenitors, but no AML Death at birth, fetal liver cells show increased HOXA9 expression 100% developed AML, mice with homozygous FLT3-ITD with a much shorter latency than heterozygous FLT3-ITD No difference in blood counts in primary recipients after induced deletion at 4 weeks Secondary transplants performed to reset DNA methylation pattern showed HSC but not progenitor expansion, suggesting differentiation block. No AML


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Table 1 (continued ) Gene

Mouse Model

Phenotype

EZH2

EZH2 knock-out

Essential for fetal hematopoiesis. Embryos die due to anemia from defective erythropoiesis in fetal liver.95 Deletion in adult cells perturbs lymphopoiesis but does not affect other hematopoiesis96 In MLL-AF9–transformed cells, deletion of EZH2 decreased proliferation, retarded cell-cycle entry, and resulted in increased apoptotic cell death. Deletion of EZH2 decreased progression of leukemia and significantly prolonged survival of recipient mice (60 vs 76 days; P o.0001). However, all of the mice eventually died of leukemia In MLL-AF9–transformed cells, deletion of EED impaired leukemia development and improved survival (ie, PRC is required for MLL-AF9 leukemia) No defects in HSCs or PB. No development of MDS nor AML Reduced survival of embryos, developmental abnormalities, leukopenia, thrombocytopenia, multilineage dysplasia consistent with MDS Germline ASXL1Δ/Δ embryonic lethal hematopoietic-specific deletion of Asxl1 (Vav-cre ASXL1Δ/Δ) developed progressive BM and splenic hypocellularity beginning at 6 wk and progressive leukopenia and anemia that was most apparent at 6–12 mo of age, consistent with MDS. Vav-creASXL1Δ/Δ have impaired HSC self-renewal that is rescued with deletion of Tet2. Mx1-cre ASXL1Δ/Δ Tet2Δ/Δ double knock-outs have increased frequency of MDS 80% embryonic lethality. Survivors have impaired HSC pool and self-renewal, dysplastic neutrophils and multilineage cytopenias, and death by 42 days

EZH2 knock-out in an MLL-AF9 model100

EED

EED knock-out in an MLL-AF9 model101

ASXL1

Transgenic targeted disruption (ASXL1tm1Bc/tm1Bc)116 Homozygous knock-out (ASXL1–/–)

Transgenic targeted disruption of ASXL165

ASXL1 knock-out118

Abbreviations: AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; CMML, chronic myelomonocytic leukemia; DNMT, DNA methyltransferase; HSC, hematopoietic stem cells; ITD, internal tandem duplication; MDS, myelodysplastic syndromes; MLL, mixed lineage leukemia; MPD, myeloproliferative disorder; NPM1, nucleophosmin; PRC, polycomb repressor complex; PTD, partial tandem duplication; WT, wild type.

differentiation, and an increase in the flanking HoxB4 and HoxB5 genes. Some of the mice eventually developed AML after a long latency period. On a molecular level, the mechanism by which NPM1c contributes to the MPD and AML phenotypes seems to involve the bromodomain-containing protein 4 (BRD4), a

bromodomain and extra-terminal (BET) family member that binds acetylated histones. BRD4 is involved in G1 gene transcription and progression to the S-phase by binding to G1 genes31 and by binding to positivetranscription elongation factor,32 which activates RNA pol II. In addition, by recruiting the condensin II


chromatin remodeling complex to acetylated histones, BRD4 inhibits DNA damage response signaling.33 Nuclear NPM1 inhibits BRD4-dependent transcription of core genes such as BCL2 and cMYC.23 This function is perturbed in NPM1c AML, in which NPM1 is confined to the cytoplasm. In fact, blocking nuclear export of NPM1 impairs the growth of leukemia, as does treatment of cells with I-BET, an inhibitor of BRD4.23 Murine NPM1c cells treated with I-BET showed increased apoptosis and decreased cell cycling, as well as reversal of a BET-driven core transcriptional program. Murine models of NPM1c AML treated with I-BET showed decreased colony formation, lower spleen weight and white blood cell counts, and increased survival compared with vehicle-treated controls. Similar results were observed in xenotransplants using human AML samples harboring the NPM1c mutation. Indeed, mice treated with I-BET had prolonged survival, compared with mock-treated animals.34 BRD4 can be inhibited via small molecules and thus represents a potential therapeutic target in AML.29

Isocitrate Dehydrogenase Isocitrate dehydrogenase (IDH) proteins catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG [also known as 2-oxoglutarate]), releasing CO2 and nicotinamide adenine dinucleotide phosphate (reduced). In humans, there are 3 IDH isozymes, each encoded by separate genes. IDH1 is located in the cytoplasm and peroxisomes, and IDH2 is found in the mitochondrial matrix. IDH1 and 2 are structurally, functionally, and evolutionarily distinct from the NAD-dependent IDH3, which functions in the tricarboxylic acid cycle to produce nicotinamide adenine dinucleotide required for oxidative phosphorylation.35 Mutations in IDH1 and IDH2 are found in a variety of cancers, including gliomas, chondrosarcoma, lymphoma, and AML. In cytogenetically normal AML (CN-AML), 11% to 14% of patients have an IDH1 mutation and 12% to 19% have an IDH2 mutation.36,37 As with NPM1c, IDH mutations are believed to occur early in the development of AML.38 Mutant IDH differs from the wild-type enzyme in that it produces a novel metabolite, 2-hydroxyglutarate (2-HG) from α-KG,39 resulting in increased levels of 2-HG and, importantly, decreased levels of reduced nicotinamide adenine dinucleotide.40 Thus, in addition to producing α-KG, wild-type IDH is critical in maintaining the cell’s ability to participate in redox reactions and respond to oxidative stress. Interestingly, 2-HG has emerged as a biochemical marker of IDH-mutant leukemia.40 In a prospective study of patients with AML, 2-HG levels in serum, urine, marrow aspirate, and myeloblasts were significantly higher in IDH-mutant patients, with a correlation between baseline serum and urine 2-HG levels. Serum and urine 2-HG, along with IDH1/2-mutant allele burden in

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marrow, decreased in responders and occurred more rapidly with induction chemotherapy, compared with treatment with DNA methyltransferase (DNMT) inhibitors. Elevated levels of 2-HG are associated with an adverse prognosis in patients with AML,41 whereas low levels of 2-HG after induction are correlated with improved survival.42 The cellular consequences of elevated levels of 2-HG and the role of this metabolite in oncogenesis are still under investigation. 2-HG is not known to participate in metabolic processes, but it competitively inhibits a broad range of enzymes dependent on α-KG. There are 480 α-KG–dependent enzymes, all of which rely on oxygen, ascorbate, and iron as cofactors and convert α-KG to succinate and CO2. These enzymes are involved in such diverse processes as collagen biosynthesis, fatty acid metabolism, DNA repair, RNA and chromatin modifications, and hypoxic sensing.43 The enzymes under the greatest study as potential targets of 2-HG inhibition include the ten-eleven-twelve (TET) proteins involved in DNA methylation, the Jumonji C domain–containing histone demethylases, the prolyl hydroxylases and lysyl hydroxylases required for collagen folding and maturation, and the prolyl hydroxylase proteins that regulate hypoxiainducible factor (HIF) signaling.44 In vitro, cells transfected with mutant IDH or cultured in the presence of 2-HG exhibit impaired differentiation and decreased levels of differentiation factors, such as lysine-specific demethylase 4C.45 Elevated levels of 2-HG are correlated with increased histone methylation. Even in the absence of an IDH mutation, elevated levels of 2-HG can impair adipocyte differentiation in vitro by impairing function of the α-KG–dependent demethylase KDM4C. In a conditional knock-in murine model of mutant IDH1 involving the most frequently detected mutation (R132H), mutant mice had increased numbers of early hematopoietic progenitors and developed anemia, splenomegaly, and extramedullary hematopoiesis.46 Analysis of IDH1 (R132H) mutant cells revealed increased levels of 2-HG relative to wild-type cells, but similar levels of ROS, nicotinamide adenine dinucleotide phosphate (reduced), and HIF1α target genes, suggesting these mechanisms could not account for the observed phenotypes. However, IDH1 (R132H) mutant cells demonstrated increased DNA methylation, especially at CpG sites, and increased methylation at histone H3 lysine residues, similar observations in human IDH1- or IDH2-mutant AML. These findings suggest that the mechanism by which IDH mutations contribute to the leukemia phenotype may involve altered gene expression as a consequence of DNA and histone methylation changes, but that IDH mutations are not sufficient to induce leukemia. In a separate study, the leukemogenic activity of mutant IDH1 (R132C) was assessed in a retroviral transduction/ transplantation assay.36 Competitive repopulation assays demonstrated similar engraftment, peripheral blood counts, and spleen weights compared with control subjects. Again,

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mutant IDH1 was insufficient to induce leukemia, suggesting that additional cooperating mutations are required or that critical pathways are not conserved between mice and humans. Because human Ieukemia samples harboring IDH1/2 mutations frequently overexpress HOX genes (HOXA9, in particular), HOXA9-transduced mouse bone marrow cells were engineered to express R132C mutant IDH1. Indeed, compared with HOXA9-transduced cells alone, the cells co-expressing HOXA9 and mutant (R132C) IDH1 demonstrated increased engraftment and stem cell self-renewal, leukocytosis, anemia, thrombocytopenia, and MPD with diminished survival compared with control subjects. Although immortalized mutant IDH1/ HOXA9 cells demonstrated upregulation and activation of mitogen-activated protein kinase pathway genes, they were resistant to a variety of MEK1/2 inhibitors. They were, however, sensitive to a novel inhibitor of mutant IDH1, 2-[2-[3-(4-fluorophenyl)pyrrolidin-1-yl]ethyl]-1,4-dimethylpiperazine (HMS-101), a novel small molecular inhibitor of mutant IDH1. Treatment with HMS-101 resulted in decreased levels of intracellular 2-HG and increased cellular apoptosis. In addition, gene expression analysis of transduced mouse bone marrow 4 weeks after transplantation demonstrated downregulation of several CDK inhibitors in IDH1 (R132C) mice compared with wild-type mice, providing an explanation for the increased cell cycling seen in mutant cells. Downregulation was due to transcriptional repression and not promoter methylation, supporting the hypothesis that mutant IDH results in epigenetic modulation and altered gene transcription via histone modification.36 In another model system, an inhibitor of mutant IDH2 (ie, AG-6780) induced differentiation of AML cells in vitro.47 A variety of additional IDH inhibitors are in clinical development, such as AG120 for mutant IDH1 (NCT02074839) and AG-221 for mutant IDH2 (NCT01915498). In summary, mutant IDH produces a novel metabolite, 2-HG, that serves as a biochemical marker and interferes with the function of the α-KG–dependent enzymes involved in cellular function. The specific contributors to oncogenesis likely include changes in epigenetic and histone methylation, collagen synthesis, HIF1a signaling, and metabolism (as reviewed by Cairns and Mak48). IDH1/IDH2 inhibitors have entered clinical investigation.

TET Methylcytosine Dioxygenase 2 The TET family of genes encodes proteins that, as with the Jumonji C domain–containing histone demethylases, provide a link between intracellular metabolism and epigenetic gene regulation. TET enzymes are α-KG– dependent dioxygenase enzymes involved in DNA demethylation.49,50 The TET1 gene on chromosome 10q22 was initially identified as a fusion partner with the MLL (mixed lineage leukemia) gene on chromosome 11q23 in AML with a t(10;11)(q22;q23) translocation.51,52 The


different TET enzymes exhibit distinct expression patterns in vivo. TET1 is expressed in embryonic stem cells, whereas TET2 and TET3 are expressed more ubiquitously; TET2 expression is observed in a variety of differentiated tissues, especially in hematopoietic and neuronal lineages.53 Mutations in TET2 occur in 7% to 23% of AML cases54 and may be associated with negative prognosis in CN-AML.55 DNA methylation represents an important epigenetic mechanism that controls gene expression in normal cells and, when perturbed, may contribute to neoplasia. DNA methylation status represents a balance between methylation and demethylation enzymes. DNMTs methylate DNA, whereas TET enzymes participate in DNA demethylation. During DNA methylation, a methyl group is added to carbon 5 of cytosine residues by DNMTs, generating 5-methyl-cytosine (5-mC). DNMT1 preferably methylates hemimethylated DNA and is referred to as the “maintenance” methyltransferase because it is believed to be responsible for copying methylation patterns after DNA replication. In contrast, DNMT3 family members have equal preference for unmethylated and hemimethylated DNA and are considered the “de novo” methyltransferases. Despite these distinctions, overlap in DNMT functions exists. TET enzymes, conversely, catalyze the oxidation of the 5-methyl group to 5-hydroxy-methylcytosine (5-hmC). Subsequent demethylation may occur passively during DNA replication56 or via further TETmediated oxidation of 5-hmC to 5-formyl-cytosine and 5-carboxyl-cytosine; the latter may be recognized and excised by thymine DNA glycosylase to form cytosine. In wild-type cells, TET2 is primarily localized to the nucleus, and 5-hmC is detectable. In cells harboring TET2 mutations typically seen in AML, nuclear localization of TET2 is maintained; however, levels of 5-hmC are markedly reduced, indicating that these mutations are likely loss-of-function.49 In fact, bone marrow DNA from patients with myeloid malignancies and TET2 mutations demonstrate decreased levels of 5-hmC compared with normal control subjects.57 Furthermore, the identification of 5-hmC as a sixth base within the genome raises the possibility that it may play additional roles as a stable epigenetic mark beyond an intermediary in DNA demethylation. Specifically, 5-hmC recruits DNA-binding proteins such as DNA-repair proteins and transcription factors that can influence gene expression. In mouse embryonic stem cells, 5-hmC localizes to gene-rich areas of the genome and is found within gene bodies and promoter regions.58 However, quantitative mass spectrometry–based proteomic studies have found that protein readers for distinct cytosine modifications exhibit limited overlap, suggesting that, at least from a biochemical perspective, 5-mC, 5-hmC, 5-formyl-cytosine, and 5-carboxyl-cytosine behave differently.59–61 For example, whereas 5-hmC appears resistant to DNMT1,62,63 the de novo DNMT3 demethylases appear capable of dehydroxymethylating 5-hmC.

AML biology and genomic analysis The impact of Tet2 deletion on hematopoiesis in mice has been reported.64 Tet2/ mice had lower genomic 5-hmC and higher 5-mC levels in bone marrow (BM) cells compared with wild-type and Tet2þ/ BM cells. In addition, by 2 to 4 months, the majority of Tet2/ mice exhibited elevated white blood cell counts consisting of monocytes and neutrophils in addition to anemia compared with wild-type and Tet2þ/ mice. The Tet2/ mice also had increased BM cellularity and splenomegaly, and moderately enlarged livers. Approximately one third (21 of 62) of the Tet2/ mice and 8% (5 of 66) of the Tet2þ/ mice died or became moribund within 1 year, and necropsy revealed hepatomegaly, splenomegaly, and anemia. The development of this chronic myelomonocytic leukemia–like disease is accelerated when Tet2 mutations were combined with other known leukemia genetic lesions, including Asxl165 and Ezh266 (both discussed later in this article). TET2 mutations are mutually exclusive with IDH1/2 mutations in patients with myeloid malignancies, providing an important clue to their convergent mechanism of action. In a cohort of AML patients, 28 (7.3%) of 385 had Z1 TET2 mutation, whereas IDH1 R132 mutations were observed in 6.2% of patients and IDH2 mutations in 8.6% of patients (6.3% R140Q and 2.3% R172K).50 Mutations in IDH1 and IDH2 were mutually exclusive, and no cases with TET2 mutations had mutations in either IDH1 or IDH2. The dependence of TET2 on α-KG provides a mechanistic link to the IDH enzymes. As such, mutations in IDH generate 2-HG, which acts as a competitive inhibitor of α-KG and thus impairs TET2 activity,67,68 resulting in hematopoietic transformation via impaired hydroxymethylation and increased DNA hypermethylation.

Mixed Lineage Leukemia The MLL gene is located on chromosome 11q and is frequently translocated in patients with therapy-related leukemia, particularly those who previously received treatment with a topoisomerase inhibitor. MLL is an H3K4 methyltransferase,69 and translocations that replace the methyltransferase domain are seen in therapy-related, de novo, and infant leukemias.70,71 The mechanism by which MLL-translocated proteins participate in oncogenesis is under investigation, but several possibilities include stimulating transcriptional elongation via recruitment of complexes that lead to deregulated transcription72 and enhanced H3K79 methylation.73,74 The partial tandem duplication (PTD) mutation of MLL (MLL-PTD), found in 5% to 7% of cytogenetically normal AML, was first identified in CNAML and AML with trisomy 11.75 PTD results from an inframe duplication of N-terminal exons of MLL in the 5'-3' orientation, resulting in a translatable sequence.75,76 MLLPTD retains the C-terminus of the protein, which contains the activation and SET domains (responsible for histone methyltransferase activity), and partially duplicates an

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N-terminal region containing the AT-hook DNA-binding and repression domains.77 In a mouse knock-in model of the MLL-PTD, the mutant mice had axial skeletal defects and increased numbers of hematopoietic progenitor cells.71 The frequency of hematopoietic stem/progenitor cells (HSPCs) and LSK cells were increased, HSCs had a competitive advantage in repopulating assays, and progenitors had increased self-renewal, but the mice did not develop AML.78 Hematopoietic cells from these mice also had aberrant HoxA gene promoter methylation and increased expression of HoxA genes. In patients with MLL-PTD, the remaining wild-type allele is frequently silenced.79 For this reason, mice with an MLL-PTD allele were crossed to MLL null mice.80 MLLPTD/- mice died at birth, although analyses of fetal liver cells demonstrated similarly increased HoxA expression in MLLPTD/WT mice, suggesting the PTD exerts a dominant gain of function role. Interestingly, 25% of patients with an MLL mutation have a concurrent FLT3 mutation.81 To examine the combined effects of these mutations in murine hematopoiesis, MLL-PTD knock-in mice71 were crossed with FLT3-ITD mice, generated by knocking in a human ITD (W51, which encodes a duplication of 7 amino acid REYEYDL82) into exon 14 of murine Flt3 by homologous recombination in embryonic stem cells,83 resulting in MLLPTD/WT:Flt3ITD/WT double knock-in (DKI) mice. Although neither MllPTD/WT nor Flt3ITD/WT mice developed AML, the DKI mice developed leukemia with 100% penetrance.84 There was evidence of cross-talk between the mutant alleles, with MLL-PTD driving FLT3-ITD expression and FLT3-ITD decreasing MLL expression. The mean latency of development of acute leukemia was reduced with increased dosage of the FLT3ITD allele. MLL-PTD, Flt3-ITD DKI mice with a heterozygous Flt3-ITD mutation developed acute leukemia after an average of 49 weeks, whereas mice with a homozygous Flt3-ITD mutation developed acute leukemia after just 19 weeks. The miRNA 29b regulates expression of DNMT3A and DNMT3B in lung cancers.85 In AML cell lines, enforced expression of miRNA-29b resulted in global hypomethylation via direct effects on DNMT3A and DNMT3B and by indirect effects on DNMT1.86 The MLL-PTD, Flt3-ITD DKI mice have decreased levels of miRNA-29b that were not observed in mice harboring either mutation alone.87 The proteasome inhibitor, bortezomib, induces DNA hypomethylation via direct effects on DNMT and by increasing levels of miRNA-29b.88 Mice treated with bortezomib had reduced spleen weight and increased overall survival in DKI mice, suggesting that bortezomib may be a promising therapeutic agent for patients with these mutations.87 In summary, Mll-PTD mutations induce histone acetylation and methylation but alone are not sufficient to induce leukemia in mice. However, combining MLL-PTD with Flt3-ITD effectively induces leukemia. Preclinical

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evidence provides a rationale to investigate use of bortezomib in this subtype of AML.

DNA Methyltransferase 3A Mutations in DNMT3A are found in 12% to 22% of AML cases89 and are associated with poor prognosis and decreased overall survival.90 Several lines of evidence suggest that DNMT3A mutations occur early in leukemogenesis. First, DNMT3A mutations were detected in flowsorted preleukemic HSCs from patients with AML.91 Second, DNTM3A mutations have been detected in in vitro–expanded peripheral blood T cells from patients with AML.92 DNMT3A mutations are stable throughout disease evolution; that is, after acquisition, they persist from diagnosis to relapse. In a study of 98 patients with secondary and therapy-related AML, 23 patients had DNMT3A mutations that were detectable in the antecedent disorders in all cases.93 DNMT3A is believed to play a role in the epigenetic silencing of genes regulating HSCs. The important role of DNMT3A in hematopoietic cell differentiation has been demonstrated in a conditional knockout model.94 Here, mice carrying a loxP-flanked (floxed) copy of DNMT3A were crossed with Mx1-Cre mice. Purified HSCs were transplanted into wild-type recipients before the induction of DNMT3A deletion by using pI:pC at 4 weeks. Monthly analysis of peripheral blood revealed no differences in the contribution of hematopoietic cells from DNMT3A-null mice compared with control mice. Reasoning that the lack of observable difference may be due to the presence of an already established DNA-methylation pattern, HSC turnover was forced by performing a secondary transplant. After transplantation, recipient mice demonstrated a significant expansion of HSCs compared with control mice that could not be accounted for by enhanced proliferation or resistance to apoptosis. Furthermore, despite the increase in HSCs, the progenitor pool was not similarly increased, suggesting a differentiation defect. These mice also exhibited differential methylation at various loci. Hypomethylation occurred across genes responsible for HSC self-renewal, whereas hypermethylation occurred across genes responsible for regulating differentiation factors. However, none of the mutant mice in the initial report developed overt leukemia, suggesting that other cooperating mutations are necessary for the development of leukemia. These data suggest a crucial role for DNMT3A in the choice between differentiation and self-renewal.

Enhancer of Zeste Homolog 2 and the Polycomb Repressor Complex 2 Polycomb repressor complex 2 (PRC2) is responsible for maintaining a transcriptional repressive state, or gene silencing, through histone methylation. This multiprotein complex is made of several components, including the


histone-lysine N-methyltransferase, zeste homolog 2 (EZH2), the catalytic component, and EED. EZH2 accomplishes this role by adding 3 methyl groups to lysine 27 of histone 3, which results in chromatin condensation. EED, in contrast, mediates repression of gene activity through histone deacetylation. EZH2 is essential for fetal, but not adult, HSCs. EZH2-deficient embryos die of anemia because of insufficient expansion of HSPCs and defective erythropoiesis in the fetal liver,95 whereas deletion of EZH2 in adult BM perturbs lymphopoiesis but does not otherwise affect hematopoiesis.96 Both overexpression97 and loss of function98,99 mutations in EZH2 have been detected in MDS and AML, suggesting that EZH2 can function as both a tumor suppressor and an oncogene.100 In an MLL-AF9 murine model of AML, either EZH2 or EED was inactivated by using an inducible Cre system to determine whether either of these components of PRC2 participate in leukemogenesis.100 When EZH2 was inactivated, PRC2 function (as assessed by the methylation status of histone H3K27), was decreased but not absent, suggesting that EZH1 has the potential to compensate for the loss of function of EZH2. When EED was inactivated, PRC2 function was completely suppressed. With inactivation of either component of the PRC2 complex, spleen and BM cells of MLL-AF9 mice cycled less frequently and demonstrated upregulation of PRC2-target genes. Mice with MLL-AF9 leukemias lacking EED lived longer and had decreased engraftment of leukemia cells; similar results were found in MLL-AF9 mice lacking EZH2 but only in secondary recipients.101 It is proposed that EZH2 augments leukemogenic activity by reinforcing differentiation blockage in AML, maintaining cells in a primitive state with enhanced selfrenewal capacity.100 Ectopic expression of EZH2 in MllAF9 cells resulted in extensive serial replating capacity of progenitors, compared with controls. Conversely, deletion of EZH2 in MLL-AF9 cells resulted in less proliferation, fewer colonies, decreased cell cycling, and more maturation. Interestingly, EZH2 null leukemias more closely resemble chronic myelomonocytic leukemia than AML. The early growth response protein 1 is a transcription factor and positive regulator of myeloid differentiation that suppresses leukemia by abrogating the E2F-1–mediated block in terminal myeloid differentiation.100a-c The early growth response protein 1 is among many genes that are de-repressed when EZH2 function is lost. As such, MllAF9 leukemia cells from which EZH2 was genetically deleted had higher expression of EGR1. Consistent with this function, EGR1 overexpression in EZH-2–containing MLL-AF9 cells results in more differentiated colonies and significantly decreased colony replication efficiency, suggesting a reduction in the transformed phenotype as measured by using this assay. Thus, it seems that EZH2 is necessary for the formation of MLL-AF9 leukemia and, when absent, leukemogenesis is impaired, possibly due to the upregulation of EGR1.

AML biology and genomic analysis These findings illustrate the important role of PRC2 in the development of leukemia in the MLL-AF9 mouse model and the role of epigenetic modification on expression levels of genes involved in myeloid differentiation.

Additional Sex Combs–Like 1 Additional sex combs–like 1 (ASXL1) is a member of the polycomb group of proteins and, in normal cells, ASXL1 functions as a crucial mediator of PRC2.102,103 Germline mutations of ASXL1104 are associated with the developmental disorder Bohring-Opitz syndrome,105 which is characterized by facial anomalies, including prominent frontal suture, exophthalmos, hypertelorism, cleft lip and palate, flexion deformities of the upper limbs, and other anomalies. Somatic mutations in ASXL1 are associated with myeloid malignancies. Interestingly, ASXL1 maps to chromosome 20q, a region commonly deleted in human cancers.102 Mutations in ASXL1 have been noted in a variety of myeloid malignancies, including MDS, myeloproliferative neoplasms,106 chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia,107 and AML.108–110 In addition, alterations in ASXL1, which are typically frameshift, and nonsense mutations that result in C-terminal truncation, are associated with adverse prognosis across myeloid diseases.54,111–114 ASXL1 mutations result in reduced or absent protein expression in human AML cell lines and primary cells.115 Knockdown of ASXL1 in primary human AML cells is associated with increased HOXA gene expression. Furthermore, ASXL1 loss is associated with decreased H3K27me3, which is attributable to a direct effect of ASXL1 on PRC2. Depletion of ASXL1 using shorthairpin RNA in combination with expression of mutant NRAS (G12D) results in accelerated myeloproliferation and impaired survival compared with NRAS (G12D) alone. A murine knockout model was created by inserting a PGK promoter–driven neomycin-resistance (neo) gene into exon 5 in reverse orientation to the ASXL1 direction of transcription.116 In this model, mice had axial skeleton abnormalities,117 but no effect was observed on HSCs, and the animals did not develop MDS or leukemia. In a conditional knockout model system generated by inserting LoxP sites flanking exons 5 to 10 of ASXL1, hematopoietic-specific deletion of ASXL1 resulted in progressive, multilineage cytopenias and dysplasia consistent with MDS.65 Serial transplantation of whole BM cells from these mice resulted in shorter disease latency in recipients. In competitive transplantation assays, ASXL1deleted marrow demonstrated a significant disadvantage relative to control subjects that was further accentuated on serial transplantation, consistent with a defect in selfrenewal. This defect in self-renewal was restored, however, in cells with concomitant deletion of Tet2, although compound-deficient mice also exhibited reduced survival due to accelerated MDS.

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In an independently generated loss-of-function model, ASXL1–/– embryos had reduced survival, with viable mice demonstrating developmental abnormalities, including dwarfism and anophthalmia.118 Surviving ASXL1/ mice lived for up to 42 days and had features of MDS that included leukopenia, thrombocytopenia, small megakaryocytes with hypolobation, dysplastic neutrophils, and multilineage dysplasia. Haploinsufficiency of ASXL1 was sufficient to cause disease, as ASXL1þ/– mice had cytopenias and multilineage dysplasia that worsened with age. BM cells from ASXL1/ mice exhibited increased proliferation and apoptosis, a hallmark of human MDS. ASXL1/ mice had a reduced HSC pool, and ASXL1/ HSCs were at a disadvantage in competitive repopulation experiments. Lastly, BM cells from ASXL1/ mice had increased histone 3 K27 and K4 trimethylation and increased expression of HoxA genes. Together, these data point to the role of ASXL1 as a tumor suppressor in myeloid malignancies, whose loss results in increase hematopoietic apoptosis, mitosis, and histone trimethylation in addition to features of MDS such as cytopenias and dysplasia.

Plant Homeodomain Finger Containing Protein–6 Plant homeodomain finger containing protein–6 (PHF6) is an X-linked gene encoding a protein with 2 zinc finger domains, suggesting a role in transcriptional regulation. PHF6 mutations are observed in 3% (10 of 353) of AMLs119 and 6% of pediatric and 38% of adult primary T-cell acute lymphoblastic leukemias (ALLs).120–123 This protein seems to function as a tumor suppressor, and knockdown of PHF6 by using a miRNA (128-3p) accelerated T-cell ALL in a mouse model of NOTCH1induced ALL.124 In a T-cell ALL study, PHF6 mutations were present in 32% (29 of 92) of male patients and in only 2.5% (1 of 39) of female patients (P o0.001). Of the 10 patients with AML and PHF6 mutations in this study, 9 were male, supporting the theory that PHF6 is an X-linked tumor suppressor gene.119,120 The role of this mutation in leukemia pathogenesis is not well understood and, to date, there are no published animal models exploring the effect of mutant PHF6 in leukemia.

Spliceosome Genes The spliceosome is a complex molecular machine located within the nucleus and comprises small nuclear RNAs and proteins whose central function is to remove introns from transcribed pre-messenger RNAs. Research into mutations involving RNA-splicing genes such as SF3B1, SRSF2, ZRSR2, and U2AF1 has primarily focused on their presence in MDS. These mutations occur most commonly in intermediate-1, RAEB-1, and RAEB-2 MDS. In a study of 221 patients with MDS assessed for mutations in 4 splicing genes (SF3B1, SRSF2, ZRSR2, and U2AF1),


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mutations were found in 95 patients (43%).125 Mutations were mutually exclusive and less likely to occur in patients with complex karyotype and TP53 mutations. In a study of patients with MDS, U2AF1 mutations were identified in 8.7% of patients with de novo disease.126 However, in patients who progressed to AML, U2AF1 mutations were seen in 15.2% of patients compared with only 5.8% of patients who did not progress, suggesting this mutation increases the risk of progression to overt leukemia. The mechanism by which spliceosome mutations contribute to neoplasia is unclear. However, the phenomenon of aberrantly spliced genes in AML seems to be highly prevalent. One study showed that 29% of expressed genes were differentially and recurrently spliced in patients with AML compared with normal donor marrow CD34þ cells.127 NOTCH2 and FLT3, which both encode myeloid surface proteins, were among the most commonly misspliced genes, seen in 470% of samples.128 Additional research and animal models are required to dissect the role of RNA-splicing gene mutations in AML and malignancies in general.

mutations act through alternative mechanisms. One such possibility is via transcriptional control, as cohesin genes have been found to bind to CCCTC-binding factor,130 a sequence-specific transcription factor known to interact with NPM1 and tumor suppressor loci,131 although cohesins can affect transcription independent of the CCCTC-binding factor.132

Cohesin Complex

REFERENCES

The cohesin complex forms a protein ring that keeps sister chromatids connected with each other during metaphase, facilitates spindle attachment to chromosomes, and is involved in multiple other cellular functions. The multimeric cohesin protein complex is made up of 4 subunits: SCC1, SCC3, SMC1, and SMC3. SMC1 and SMC3 are members of the structural maintenance of chromosomes (SMC) family, whereas SCC1 and SCC3 bind the ATPase domains of SMC1 and SMC3, thus stabilizing the ring structure. In a study of 389 uniformly treated patients with AML analyzed by using NGS, cohesin complex mutations were found in 23 (5.9%) patients. Mutations in STAG1, a member of the SCC3 family, occurred most commonly, followed by SMC3 and STAG2. Mutations in SMC1A and RAD21 (SCC1) were less common, and cohesin family mutations were mutually exclusive. Of the 23 patients with cohesin complex mutations, 17 (74%) had intermediate-risk karyotype and 15 had a normal karyotype, suggesting that cohesin mutations do not contribute to leukemogenesis via inducing aneuploidy. Cohesin complex mutations were strongly associated with mutations in NPM1, as 57% of patients with cohesin mutations had an NPM1 mutation compared with 34% of patients without cohesin mutations (P ¼ .029). However, even when occurring in combination with NPM1, mutations in the cohesin complex were not prognostically significant.129 Consequently, although mutated in a portion of patients with AML, the mechanism by which cohesin complex mutations contribute to leukemogenesis is not well understood. The association between cohesin mutations and normal karyotype suggests that chromosomal destabilization is not the primary mechanism of action, raising the possibility that cohesin

CONCLUSIONS NGS has significantly enhanced our knowledge of AML pathogenesis. These techniques have expanded our knowledge of pathways in AML, enhanced our understanding of the origin and evolution of mutations, provided knowledge regarding the temporal sequence and co-occurrence of mutations, and, perhaps most dramatically, enforced the genetic and functional heterogeneity of AML. These advances call for the development of more sophisticated animal systems for the purpose of more accurately modeling AML and generating effective therapies.

1. Fortier JM, Graubert TA. Murine models of human acute myeloid leukemia. Cancer Treat Res. 2010;145:183-96. 2. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367:645-8. 3. Klco JM, Spencer DH, Miller CA, Griffith M, Lamprecht TL, O'Laughlin M, et al. Functional heterogeneity of genetically defined subclones in acute myeloid leukemia. Cancer Cell. 2014;25:379-92. 4. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood. 2002;100:1532-42. 5. Castilla LH, Wijmenga C, Wang Q, Stacy T, Speck NA, Eckhaus M, et al. Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knocked-in leukemia gene CBFB-MYH11. Cell. 1996; 87:687-96. 6. Fenske TS, Pengue G, Mathews V, Hanson PT, Hamm SE, Riaz N, et al. Stem cell expression of the AML1/ETO fusion protein induces a myeloproliferative disorder in mice. Proc Natl Acad Sci U S A. 2004;101:15184-9. 7. Grisolano JL, Wesselschmidt RL, Pelicci PG, Ley TJ. Altered myeloid development and acute leukemia in transgenic mice expressing PML-RAR alpha under control of cathepsin G regulatory sequences. Blood. 1997;89:376-87. 8. Oancea C, Ruster B, Henschler R, Puccetti E, Ruthardt M. The t(6;9) associated DEK/CAN fusion protein targets a population of long-term repopulating hematopoietic stem cells for leukemogenic transformation. Leukemia. 2010;24:1910-9. 9. Kundu M, Chen A, Anderson S, Kirby M, Xu L, Castilla LH, et al. Role of Cbfb in hematopoiesis and perturbations resulting from expression of the leukemogenic fusion gene Cbfb-MYH11. Blood. 2002;100:2449-56. 10. Moreno-Miralles I, Pan L, Keates-Baleeiro J, DurstGoodwin K, Yang C, Kim HG, et al. The inv(16)


11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

cooperates with ARF haploinsufficiency to induce acute myeloid leukemia. J Biol Chem. 2005;280:40097-103. Castilla LH, Perrat P, Martinez NJ, Landrette SF, Keys R, Oikemus S, et al. Identification of genes that synergize with Cbfb-MYH11 in the pathogenesis of acute myeloid leukemia. Proc Natl Acad Sci U S A. 2004;101:4924-9. Yang Y, Wang W, Cleaves R, Zahurak M, Cheng L, Civin CI, et al. Acceleration of G(1) cooperates with core binding factor beta-smooth muscle myosin heavy chain to induce acute leukemia in mice. Cancer Res. 2002;62:2232-5. Westervelt P, Lane AA, Pollock JL, Oldfather K, Holt MS, Zimonjic DB, et al. High-penetrance mouse model of acute promyelocytic leukemia with very low levels of PMLRARalpha expression. Blood. 2003;102:1857-65. Welch JS, Klco JM, Varghese N, Nagarajan R, Ley TJ. Rara haploinsufficiency modestly influences the phenotype of acute promyelocytic leukemia in mice. Blood. 2011;117: 2460-8. Kelly LM, Kutok JL, Williams IR, Boulton CL, Amaral SM, Curley DP, et al. PML/RARalpha and FLT3-ITD induce an APL-like disease in a mouse model. Proc Natl Acad Sci U S A. 2002;99:8283-8. Tsuboi A, Oka Y, Ogawa H, Elisseeva OA, Tamaki H, Oji Y, et al. Constitutive expression of the Wilms' tumor gene WT1 inhibits the differentiation of myeloid progenitor cells but promotes their proliferation in response to granulocyte-colony stimulating factor (G-CSF). Leuk Res. 1999;23:499-505. Nishida S, Hosen N, Shirakata T, Kanato K, Yanagihara M, Nakatsuka S, et al. AML1-ETO rapidly induces acute myeloblastic leukemia in cooperation with the Wilms tumor gene, WT1. Blood. 2006;107:3303-12. Heuser M, Berg T, Kuchenbauer F, Lai CK, Park G, Fung S, et al. Functional role of BAALC in leukemogenesis. Leukemia. 2012;26:532-6. Heuser M, Argiropoulos B, Kuchenbauer F, Yung E, Piper J, Fung S, et al. MN1 overexpression induces acute myeloid leukemia in mice and predicts ATRA resistance in patients with AML. Blood. 2007;110:1639-47. Goldberg L, Tijssen MR, Birger Y, Hannah RL, Kinston SJ, Schutte J, et al. Genome-scale expression and transcription factor binding profiles reveal therapeutic targets in transgenic ERG myeloid leukemia. Blood. 2013;122:2694-703. Buonamici S, Li D, Chi Y, Zhao R, Wang X, Brace L, et al. EVI1 induces myelodysplastic syndrome in mice. J Clin Invest. 2004;114:713-9. Cancer Genome Atlas Research Network. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013;368:2059-74. Dawson MA, Gudgin EJ, Horton SJ, Giotopoulos G, Meduri E, Robson S, et al. Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia. Leukemia. 2013;28: 311-20. Grisendi S, Bernardi R, Rossi M, Cheng K, Khandker L, Manova K, et al. Role of nucleophosmin in embryonic development and tumorigenesis. Nature. 2005;437: 147-53. Taussig DC, Vargaftig J, Miraki-Moud F, Griessinger E, Sharrock K, Luke T, et al. Leukemia-initiating cells from some acute myeloid leukemia patients with mutated

293

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

nucleophosmin reside in the CD34(-) fraction. Blood. 2010;115:1976-84. Martelli MP, Pettirossi V, Thiede C, Bonifacio E, Mezzasoma F, Cecchini D, et al. CD34þ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice. Blood. 2010;116:3907-22. Jan M, Snyder TM, Corces-Zimmerman MR, Vyas P, Weissman IL, Quake SR, et al. Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia. Sci Transl Med. 2012;4149ra118. 2012 Chou SH, Ko BS, Chiou JS, Hsu YC, Tsai MH, Chiu YC, et al. A knock-in Npm1 mutation in mice results in myeloproliferation and implies a perturbation in hematopoietic microenvironment. PLoS One. 2012;7: e49769. Vassiliou GS, Cooper JL, Rad R, Li J, Rice S, Uren A, et al. Mutant nucleophosmin and cooperating pathways drive leukemia initiation and progression in mice. Nat Genet. 2011;43:470-5. Sportoletti P, Varasano E, Rossi R, Bereshchenko O, Cecchini D, Gionfriddo I, et al. The human NPM1 mutation A perturbs megakaryopoiesis in a conditional mouse model. Blood. 2013;121:3447-58. Mochizuki K, Nishiyama A, Jang MK, Dey A, Ghosh A, Tamura T, et al. The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase. J Biol Chem. 2008;283:9040-8. Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K, et al. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol Cell. 2005;19:535-45. Floyd SR, Pacold ME, Huang Q, Clarke SM, Lam FC, Cannell IG, et al. The bromodomain protein Brd4 insulates chromatin from DNA damage signalling. Nature. 2013;498:246-50. Dawson MA, Gudgin EJ, Horton SJ, Giotopoulos G, Meduri E, Robson S, et al. Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia. Leukemia. 2014;28: 311-20. Corpas FJ, Barroso JB, Sandalio LM, Palma JM, Lupianez JA, del Rio LA. Peroxisomal NADP-dependent isocitrate dehydrogenase. characterization and activity regulation during natural senescence. Plant Physiol. 1999;121:921-8. Chaturvedi A, Araujo Cruz MM, Jyotsana N, Sharma A, Yun H, Gorlich K, et al. Mutant IDH1 promotes leukemogenesis in vivo and can be specifically targeted in human AML. Blood. 2013;122:2877-87. Marcucci G, Maharry K, Wu YZ, Radmacher MD, Mrozek K, Margeson D, et al. IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B study. J Clin Oncol. 2010;28: 2348-55. Kosmider O, Gelsi-Boyer V, Slama L, Dreyfus F, BeyneRauzy O, Quesnel B, et al. Mutations of IDH1 and IDH2 genes in early and accelerated phases of myelodysplastic syndromes and MDS/myeloproliferative neoplasms. Leukemia. 2010;24:1094-6.

294

39. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature. 2009;462:739-44. 40. Fathi AT, Sadrzadeh H, Borger DR, Ballen KK, Amrein PC, Attar EC, et al. Prospective serial evaluation of 2-hydroxyglutarate, during treatment of newly diagnosed acute myeloid leukemia, to assess disease activity and therapeutic response. Blood. 2012;120:4649-52. 41. Wang JH, Chen WL, Li JM, Wu SF, Chen TL, Zhu YM, et al. Prognostic significance of 2-hydroxyglutarate levels in acute myeloid leukemia in China. Proc Natl Acad Sci U S A. 2014;110:17017-22. 42. Janin M, Mylonas E, Saada V, Micol JB, Renneville A, Quivoron C, et al. Serum 2-hydroxyglutarate production in IDH1- and IDH2-mutated de novo acute myeloid leukemia: a study by the Acute Leukemia French Association group. J Clin Oncol. 2014;32:297-305. 43. Rose NR, McDonough MA, King ON, Kawamura A, Schofield CJ. Inhibition of 2-oxoglutarate dependent oxygenases. Chem Soc Rev. 2011;40:4364-97. 44. Chowdhury R, Yeoh KK, Tian YM, Hillringhaus L, Bagg EA, Rose NR, et al. The oncometabolite 2hydroxyglutarate inhibits histone lysine demethylases. EMBO Rep. 2011;12:463-9. 45. Lu C, Ward PS, Kapoor GS, Rohle D, Turcan S, AbdelWahab O, et al. IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature. 2012;483:474-8. 46. Sasaki M, Knobbe CB, Munger JC, Lind EF, Brenner D, Brustle A, et al. IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics. Nature. 2012;488:656-9. 47. Wang F, Travins J, DeLaBarre B, Penard-Lacronique V, Schalm S, Hansen E, et al. Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation. Science. 2013;340:622-6. 48. Cairns RA, Mak TW. Oncogenic isocitrate dehydrogenase mutations: mechanisms, models, and clinical opportunities. Cancer Discov. 2013;3:730-41. 49. Chang YI, Damnernsawad A, Allen LK, Yang D, Ranheim EA, Young KH, et al. Evaluation of allelic strength of human TET2 mutations and cooperation between Tet2 knockdown and oncogenic Nras mutation. Br J Haematol. 2014;166:461-5. 50. Figueroa ME, Abdel-Wahab O, Lu C, Ward PS, Patel J, Shih A, et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell. 2010;18:553-67. 51. Ono R, Taki T, Taketani T, Taniwaki M, Kobayashi H, Hayashi Y. LCX, leukemia-associated protein with a CXXC domain, is fused to MLL in acute myeloid leukemia with trilineage dysplasia having t(10;11)(q22;q23). Cancer Res. 2002;62:4075-80. 52. Lorsbach RB, Moore J, Mathew S, Raimondi SC, Mukatira ST, Downing JR. TET1, a member of a novel protein family, is fused to MLL in acute myeloid leukemia containing the t(10;11)(q22;q23). Leukemia. 2003;17:637-41. 53. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, et al. Conversion of 5-methylcytosine to 5hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009;324:930-5.


54. Shih AH, Abdel-Wahab O, Patel JP, Levine RL. The role of mutations in epigenetic regulators in myeloid malignancies. Nat Rev Cancer. 2012;12:599-612. 55. Chou WC, Chou SC, Liu CY, Chen CY, Hou HA, Kuo YY, et al. TET2 mutation is an unfavorable prognostic factor in acute myeloid leukemia patients with intermediate-risk cytogenetics. Blood. 2011;118:3803-10. 56. Bhutani N, Burns DM, Blau HM. DNA demethylation dynamics. Cell. 2011;146:866-72. 57. Ko M, Huang Y, Jankowska AM, Pape UJ, Tahiliani M, Bandukwala HS, et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature. 2010;468:839-43. 58. Wu H, D'Alessio AC, Ito S, Wang Z, Cui K, Zhao K, et al. Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev. 2011;25: 679-84. 59. Frauer C, Hoffmann T, Bultmann S, Casa V, Cardoso MC, Antes I, et al. Recognition of 5-hydroxymethylcytosine by the Uhrf1 SRA domain. PLoS One. 2011;6:e21306. 60. Yildirim O, Li R, Hung JH, Chen PB, Dong X, Ee LS, et al. Mbd3/NURD complex regulates expression of 5hydroxymethylcytosine marked genes in embryonic stem cells. Cell. 2011;147:1498-510. 61. Spruijt CG, Gnerlich F, Smits AH, Pfaffeneder T, Jansen PW, Bauer C, et al. Dynamic readers for 5-(hydroxy) methylcytosine and its oxidized derivatives. Cell. 2013; 152:1146-59. 62. Valinluck V, Sowers LC. Endogenous cytosine damage products alter the site selectivity of human DNA maintenance methyltransferase DNMT1. Cancer Res. 2007;67: 946-50. 63. Hashimoto H, Liu Y, Upadhyay AK, Chang Y, Howerton SB, Vertino PM, et al. Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation. Nucleic Acids Res. 2012;40:4841-9. 64. Li Z, Cai X, Cai CL, Wang J, Zhang W, Petersen BE, et al. Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies. Blood. 2011;118:4509-18. 65. Abdel-Wahab O, Gao J, Adli M, Dey A, Trimarchi T, Chung YR, et al. Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo. J Exp Med. 2013;210:2641-59. 66. Muto T, Sashida G, Oshima M, Wendt GR, MochizukiKashio M, Nagata Y, et al. Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders. J Exp Med. 2013;210:2627-39. 67. Koivunen P, Lee S, Duncan CG, Lopez G, Lu G, Ramkissoon S, et al. Transformation by the (R)-enantiomer of 2-hydroxyglutarate linked to EGLN activation. Nature. 2012;483:484-8. 68. Xu W, Yang H, Liu Y, Yang Y, Wang P, Kim SH, et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases. Cancer Cell. 2011;19:17-30. 69. Whitman SP, Hackanson B, Liyanarachchi S, Liu S, Rush LJ, Maharry K, et al. DNA hypermethylation and epigenetic silencing of the tumor suppressor gene, SLC5A8, in acute myeloid leukemia with the MLL partial tandem duplication. Blood. 2008;112:2013-6.


70. Meyer C, Kowarz E, Hofmann J, Renneville A, Zuna J, Trka J, et al. New insights to the MLL recombinome of acute leukemias. Leukemia. 2009;23:1490-9. 71. Dorrance AM, Liu S, Yuan W, Becknell B, Arnoczky KJ, Guimond M, et al. Mll partial tandem duplication induces aberrant Hox expression in vivo via specific epigenetic alterations. J Clin Invest. 2006;116:2707-16. 72. Mohan M, Lin C, Guest E, Shilatifard A. Licensed to elongate: a molecular mechanism for MLL-based leukaemogenesis. Nat Rev Cancer. 2010;10:721-8. 73. Bernt KM, Zhu N, Sinha AU, Vempati S, Faber J, Krivtsov AV, et al. MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell. 2011;20:66-78. 74. Deshpande AJ, Chen L, Fazio M, Sinha AU, Bernt KM, Banka D, et al. Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l. Blood. 2013;121:2533-41. 75. Caligiuri MA, Schichman SA, Strout MP, Mrozek K, Baer MR, Frankel SR, et al. Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations. Cancer Res. 1994;54:370-3. 76. Caligiuri MA, Strout MP, Lawrence D, Arthur DC, Baer MR, Yu F, et al. Rearrangement of ALL1 (MLL) in acute myeloid leukemia with normal cytogenetics. Cancer Res. 1998;58:55-9. 77. Zeleznik-Le NJ, Harden AM, Rowley JD. 11q23 translocations split the “AT-hook” cruciform DNA-binding region and the transcriptional repression domain from the activation domain of the mixed-lineage leukemia (MLL) gene. Proc Natl Acad Sci U S A. 1994;91:10610-4. 78. Zhang Y, Yan X, Sashida G, Zhao X, Rao Y, Goyama S, et al. Stress hematopoiesis reveals abnormal control of selfrenewal, lineage bias, and myeloid differentiation in Mll partial tandem duplication (Mll-PTD) hematopoietic stem/ progenitor cells. Blood. 2012;120:1118-29. 79. Whitman SP, Liu S, Vukosavljevic T, Rush LJ, Yu L, Liu C, et al. The MLL partial tandem duplication: evidence for recessive gain-of-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy. Blood. 2005;106:345-52. 80. Dorrance AM, Liu S, Chong A, Pulley B, Nemer D, Guimond M, et al. The Mll partial tandem duplication: differential, tissue-specific activity in the presence or absence of the wild-type allele. Blood. 2008;112:2508-11. 81. Whitman SP, Ruppert AS, Marcucci G, Mrozek K, Paschka P, Langer C, et al. Long-term disease-free survivors with cytogenetically normal acute myeloid leukemia and MLL partial tandem duplication: a Cancer and Leukemia Group B study. Blood. 2007;109:5164-7. 82. Kelly LM, Liu Q, Kutok JL, Williams IR, Boulton CL, Gilliland DG. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model. Blood. 2002;99:310-8. 83. Lee BH, Tothova Z, Levine RL, Anderson K, Buza-Vidas N, Cullen DE, et al. FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia. Cancer Cell. 2007;12:367-80.

295

84. Zorko NA, Bernot KM, Whitman SP, Siebenaler RF, Ahmed EH, Marcucci GG, et al. Mll partial tandem duplication and Flt3 internal tandem duplication in a double knock-in mouse recapitulates features of counterpart human acute myeloid leukemias. Blood. 2012;120: 1130-6. 85. Fabbri M, Garzon R, Cimmino A, Liu Z, Zanesi N, Callegari E, et al. MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B. Proc Natl Acad Sci U S A. 2007;104:15805-10. 86. Garzon R, Liu S, Fabbri M, Liu Z, Heaphy CE, Callegari E, et al. MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1. Blood. 2009;113:6411-8. 87. Bernot KM, Nemer JS, Santhanam R, Liu S, Zorko NA, Whitman SP, et al. Eradicating acute myeloid leukemia in a Mll(PTD/wt):Flt3(ITD/wt) murine model: a path to novel therapeutic approaches for human disease. Blood. 2013;122:3778-83. 88. Liu S, Wu LC, Pang J, Santhanam R, Schwind S, Wu YZ, et al. Sp1/NFkappaB/HDAC/miR-29b regulatory network in KIT-driven myeloid leukemia. Cancer Cell. 2010;17: 333-47. 89. Ley TJ, Ding L, Walter MJ, McLellan MD, Lamprecht T, Larson DE, et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med. 2010;363:2424-33. 90. Thol F, Damm F, Ludeking A, Winschel C, Wagner K, Morgan M, et al. Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia. J Clin Oncol. 2011;29:2889-96. 91. Corces-Zimmerman MR, Hong WJ, Weissman IL, Medeiros BC, Majeti R. Preleukemic mutations in human acute myeloid leukemia affect epigenetic regulators and persist in remission. Proc Natl Acad Sci U S A. 2014;111: 2548-53. 92. Shlush LI, Zandi S, Mitchell A, Chen WC, Brandwein JM, Gupta V, et al. Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature. 2014;506: 328-33. 93. Fried I, Bodner C, Pichler MM, Lind K, Beham-Schmid C, Quehenberger F, et al. Frequency, onset and clinical impact of somatic DNMT3A mutations in therapy-related and secondary acute myeloid leukemia. Haematologica. 2012;97:246-50. 94. Challen GA, Sun D, Jeong M, Luo M, Jelinek J, Berg JS, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet. 2012;44:23-31. 95. Mochizuki-Kashio M, Mishima Y, Miyagi S, Negishi M, Saraya A, Konuma T, et al. Dependency on the polycomb gene Ezh2 distinguishes fetal from adult hematopoietic stem cells. Blood. 2011;118:6553-61. 96. Su IH, Basavaraj A, Krutchinsky AN, Hobert O, Ullrich A, Chait BT, et al. Ezh2 controls B cell development through histone H3 methylation and Igh rearrangement. Nat Immunol. 2003;4:124-31. 97. Xu F, Li X, Wu L, Zhang Q, Yang R, Yang Y, et al. Overexpression of the EZH2, RING1 and BMI1 genes is common in myelodysplastic syndromes: relation to adverse epigenetic alteration and poor prognostic scoring. Ann Hematol. 2011;90:643-53.

296

98. Ernst T, Chase AJ, Score J, Hidalgo-Curtis CE, Bryant C, Jones AV, et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet. 2010;42:722-6. 99. Nikoloski G, Langemeijer SM, Kuiper RP, Knops R, Massop M, Tonnissen ER, et al. Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet. 2010;42:665-7. 100. Tanaka S, Miyagi S, Sashida G, Chiba T, Yuan J, Mochizuki-Kashio M, et al. Ezh2 augments leukemogenicity by reinforcing differentiation blockage in acute myeloid leukemia. Blood. 2012;120:1107-17. 100a. Nguyen HQ, Hoffman-Liebermann B, Liebermann DA. The zinc finger transcription factor Egr-1 is essential for and restricts differentiation along the macrophage lineage. Cell. 1993;72:197-209. 100b. Krishnaraju K, Hoffman B, Libermann DA. Early growth response gene 1 stimulates development of hematopoietic progenitor cells along the macrophage lineage at the expense of the granulocyte and erythroid lineages. Blood. 2001;97:1298-305. 100c. Gibbs JD, Libermann DA, Hoffman B. Egr-1 abrogates the E2F-1 block in terminal myeloid differentiation and suppresses leukemia. Oncogene. 2008;27:98-106. 101. Neff T, Sinha AU, Kluk MJ, Zhu N, Khattab MH, Stein L, et al. Polycomb repressive complex 2 is required for MLL-AF9 leukemia. Proc Natl Acad Sci U S A. 2012;109: 5028-33. 102. Fisher CL, Berger J, Randazzo F, Brock HW. A human homolog of additional sex combs, ADDITIONAL SEX COMBS-LIKE 1, maps to chromosome 20q11. Gene. 2003;306:115-26. 103. Fisher CL, Randazzo F, Humphries RK, Brock HW. Characterization of Asxl1, a murine homolog of additional sex combs, and analysis of the Asx-like gene family. Gene. 2006;369:109-18. 104. Hoischen A, van Bon BW, Rodriguez-Santiago B, Gilissen C, Vissers LE, de Vries P, et al. De novo nonsense mutations in ASXL1 cause Bohring-Opitz syndrome. Nat Genet. 2011;43:729-31. 105. Bohring A, Silengo M, Lerone M, Superneau DW, Spaich C, Braddock SR, et al. Severe end of Opitz trigonocephaly (C) syndrome or new syndrome? Am J Med Genet. 1999;85:438-46. 106. Makishima H, Jankowska AM, McDevitt MA, O'Keefe C, Dujardin S, Cazzolli H, et al. CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations and additional chromosomal aberrations constitute molecular events in chronic myelogenous leukemia. Blood. 2011;117:e198-206. 107. Sugimoto Y, Muramatsu H, Makishima H, Prince C, Jankowska AM, Yoshida N, et al. Spectrum of molecular defects in juvenile myelomonocytic leukaemia includes ASXL1 mutations. Br J Haematol. 2010;150:83-7. 108. Carbuccia N, Trouplin V, Gelsi-Boyer V, Murati A, Rocquain J, Adelaide J, et al. Mutual exclusion of ASXL1 and NPM1 mutations in a series of acute myeloid leukemias. Leukemia. 2010;24:469-73. 109. Gelsi-Boyer V, Trouplin V, Adelaide J, Bonansea J, Cervera N, Carbuccia N, et al. Mutations of polycombassociated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. Br J Haematol. 2009; 145:788-800.


110. Boultwood J, Perry J, Pellagatti A, Fernandez-Mercado M, Fernandez-Santamaria C, Calasanz MJ, et al. Frequent mutation of the polycomb-associated gene ASXL1 in the myelodysplastic syndromes and in acute myeloid leukemia. Leukemia. 2010;24:1062-5. 111. Gelsi-Boyer V, Trouplin V, Roquain J, Adelaide J, Carbuccia N, Esterni B, et al. ASXL1 mutation is associated with poor prognosis and acute transformation in chronic myelomonocytic leukaemia. Br J Haematol. 2010;151:365-75. 112. Bejar R, Stevenson K, Abdel-Wahab O, Galili N, Nilsson B, Garcia-Manero G, et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med. 2011;364:2496-506. 113. Stein BL, Williams DM, O'Keefe C, Rogers O, Ingersoll RG, Spivak JL, et al. Disruption of the ASXL1 gene is frequent in primary, post-essential thrombocytosis and post-polycythemia vera myelofibrosis, but not essential thrombocytosis or polycythemia vera: analysis of molecular genetics and clinical phenotypes. Haematologica. 2011;96: 1462-9. 114. Gelsi-Boyer V, Brecqueville M, Devillier R, Murati A, Mozziconacci MJ, Birnbaum D. Mutations in ASXL1 are associated with poor prognosis across the spectrum of malignant myeloid diseases. J. Hematol Oncol. 2012; 5:12. 115. Abdel-Wahab O, Adli M, LaFave LM, Gao J, Hricik T, Shih AH, et al. ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression. Cancer Cell. 2012;22:180-93. 116. Fisher CL, Pineault N, Brookes C, Helgason CD, Ohta H, Bodner C, et al. Loss-of-function additional sex combs like 1 mutations disrupt hematopoiesis but do not cause severe myelodysplasia or leukemia. Blood. 2010;115:38-46. 117. Fisher CL, Lee I, Bloyer S, Bozza S, Chevalier J, Dahl A, et al. Additional sex combs-like 1 belongs to the enhancer of trithorax and polycomb group and genetically interacts with Cbx2 in mice. Dev Biol. 2010;337:9-15. 118. Wang J, Li Z, He Y, Pan F, Chen S, Rhodes S, et al. Loss of Asxl1 leads to myelodysplastic syndrome-like disease in mice. Blood. 2014;123:541-53. 119. Van Vlierberghe P, Patel J, Abdel-Wahab O, Lobry C, Hedvat CV, Balbin M, et al. PHF6 mutations in adult acute myeloid leukemia. Leukemia. 2011;25:130-4. 120. Van Vlierberghe P, Palomero T, Khiabanian H, Van der Meulen J, Castillo M, Van Roy N, et al. PHF6 mutations in T-cell acute lymphoblastic leukemia. Nat Genet. 2010; 42:338-42. 121. Grossmann V, Haferlach C, Weissmann S, Roller A, Schindela S, Poetzinger F, et al. The molecular profile of adult T-cell acute lymphoblastic leukemia: mutations in RUNX1 and DNMT3A are associated with poor prognosis in T-ALL. Genes Chromosomes Cancer. 2013; 52:410-22. 122. Huh HJ, Lee SH, Yoo KH, Sung KW, Koo HH, Jang JH, et al. Gene mutation profiles and prognostic implications in Korean patients with T-lymphoblastic leukemia. Ann Hematol. 2013;92:635-44. 123. Wang Q, Qiu H, Jiang H, Wu L, Dong S, Pan J, et al. Mutations of PHF6 are associated with mutations of NOTCH1, JAK1 and rearrangement of SET-NUP214 in


124.

125.

126.

127.

T-cell acute lymphoblastic leukemia. Haematologica. 2011;96:1808-14. Mets E, Van Peer G, Van der Meulen J, Boice M, Taghon T, Goossens S, et al. MicroRNA-128-3p is a novel oncomiR targeting PHF6 in T-cell acute lymphoblastic leukemia. Haematologica. 2014;99:1326-33. Damm F, Kosmider O, Gelsi-Boyer V, Renneville A, Carbuccia N, Hidalgo-Curtis C, et al. Mutations affecting mRNA splicing define distinct clinical phenotypes and correlate with patient outcome in myelodysplastic syndromes. Blood. 2012;119:3211-8. Graubert TA, Shen D, Ding L, Okeyo-Owuor T, Lunn CL, Shao J, et al. Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes. Nat Genet. 2012;44:53-7. Adamia S, Haibe-Kains B, Pilarski PM, Bar-Natan M, Pevzner S, Avet-Loiseau H, et al. A genome-wide aberrant RNA splicing in patients with acute myeloid leukemia identifies novel potential disease markers and therapeutic targets. Clin Cancer Res. 2014;20:1135-45.

297

128. Adamia S, Bar-Natan M, Haibe-Kains B, Pilarski PM, Bach C, Pevzner S, et al. NOTCH2 and FLT3 gene missplicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML. Blood. 2014;123:2816-25. 129. Thol F, Bollin R, Gehlhaar M, Walter C, Dugas M, Suchanek KJ, et al. Mutations in the cohesin complex in acute myeloid leukemia: clinical and prognostic implications. Blood. 2014;123:914-20. 130. Lee BK, Iyer VR. Genome-wide studies of CCCTCbinding factor (CTCF) and cohesin provide insight into chromatin structure and regulation. J Biol Chem. 2012;287:30906-13. 131. Zlatanova J, Caiafa P. CTCF and its protein partners: divide and rule? J Cell Sci. 2009;122:1275-84. 132. Schmidt D, Schwalie PC, Ross-Innes CS, Hurtado A, Brown GD, Carroll JS, et al. A CTCF-independent role for cohesin in tissue-specific transcription. Genome Res. 2010;20:578-88.

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