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1.  Runx1 deficiency permits granulocyte lineage commitment but impairs subsequent maturation 
Oncogenesis  2013;2(11):e78-.
First-hits in the multi-hit process of leukemogenesis originate in germline or hematopoietic stem cells (HSCs), yet leukemia-initiating cells (LICs) usually have a lineage-committed phenotype. The molecular mechanisms underlying this compartment shift during leukemia evolution have not been a major focus of investigation and remain poorly understood. Here a mechanism underlying this shift was examined in the context of Runx1 deficiency, a frequent leukemia-initiating event. Lineage-negative cells isolated from the bone marrow of Runx1-haploinsufficient and wild-type control mice were cultured in granulocyte-colony-stimulating factor to force lineage commitment. Runx1-haploinsufficient cells demonstrated significantly greater and persistent exponential cell growth than wild-type controls. Not surprisingly, the Runx1-haploinsufficient cells were differentiation-impaired, by morphology and by flow-cytometric evaluation for granulocyte differentiation markers. Interestingly, however, this impaired differentiation was not because of decreased granulocyte lineage commitment, as RNA and protein upregulation of the master granulocyte lineage-commitment transcription factor Cebpa, and Hoxb4 repression, was similar in wild-type and Runx1-haploinsufficient cells. Instead, RNA and protein expression of Cebpe, a key driver of progressive maturation after lineage commitment, were significantly decreased in Runx1-haploinsufficient cells. Primary acute myeloid leukemia cells with normal cytogenetics and RUNX1 mutation also demonstrated this phenotype of very high CEBPA mRNA expression but paradoxically low expression of CEBPE, a CEBPA target gene. Chromatin-immunoprecipitation analyses suggested a molecular mechanism for this phenotype: in wild-type cells, Runx1 binding was substantially greater at the Cebpe than at the Cebpa enhancer. Furthermore, Runx1 deficiency substantially diminished high-level Runx1 binding at the Cebpe enhancer, but lower-level binding at the Cebpa enhancer was relatively preserved. Thus, Runx1-deficiency permits Cebpa upregulation and the exponential cell growth that accompanies lineage commitment, but by impairing activation of Cebpe, a key proliferation-terminating maturation gene, extends this exponential growth. These mechanisms facilitate germline cell or HSC of origin, yet evolution into LIC with lineage-committed phenotype.
PMCID: PMC3849692  PMID: 24189977
acute myeloid leukemia; myelodysplastic syndrome; leukemia stem cell; differentiation therapy; CEBPA; CEBPE
2.  RUNX1 Mutations Are Associated With Poor Outcome in Younger and Older Patients With Cytogenetically Normal Acute Myeloid Leukemia and With Distinct Gene and MicroRNA Expression Signatures 
Journal of Clinical Oncology  2012;30(25):3109-3118.
To determine the association of RUNX1 mutations with therapeutic outcome in younger and older patients with primary cytogenetically normal acute myeloid leukemia (CN-AML) and with gene/microRNA expression signatures.
Patients and Methods
Younger (< 60 years; n = 175) and older (≥ 60 years; n = 225) patients with CN-AML treated with intensive cytarabine/anthracycline-based first-line therapy on Cancer and Leukemia Group B protocols were centrally analyzed for RUNX1 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene/microRNA expression profiles were derived using microarrays.
RUNX1 mutations were found in 8% and 16% of younger and older patients, respectively (P = .02). They were associated with ASXL1 mutations (P < .001) and inversely associated with NPM1 (P < .001) and CEBPA (P = .06) mutations. RUNX1-mutated patients had lower complete remission rates (P = .005 in younger; P = .006 in older) and shorter disease-free survival (P = .058 in younger; P < .001 in older), overall survival (P = .003 in younger; P < .001 in older), and event-free survival (P < .001 for younger and older) than RUNX1 wild-type patients. Because RUNX1 mutations were more common in older patients and almost never coexisted with NPM1 mutations, RUNX1 mutation–associated expression signatures were derived in older, NPM1 wild-type patients and featured upregulation of genes normally expressed in primitive hematopoietic cells and B-cell progenitors, including DNTT, BAALC, BLNK, CD109, RBPMS, and FLT3, and downregulation of promoters of myelopoiesis, including CEBPA and miR-223.
RUNX1 mutations are twice as common in older than younger patients with CN-AML and negatively impact outcome in both age groups. RUNX1-mutated blasts have molecular features of primitive hematopoietic and lymphoid progenitors, potentially leading to novel therapeutic approaches.
PMCID: PMC3732007  PMID: 22753902
3.  Detection of CEBPA Double Mutants in Acute Myeloid Leukemia Using a Custom Gene Expression Array 
Double (bi-allelic) mutations in the gene encoding the CCAAT/enhancer-binding protein-alpha (CEBPA) transcription factor have a favorable prognostic impact in acute myeloid leukemia (AML). Double mutations in CEBPA can be detected using various techniques, but it is a notoriously difficult gene to sequence due to its high GC-content. Here we developed a two-step gene expression classifier for accurate and standardized detection of CEBPA double mutations. The key feature of the two-step classifier is that it explicitly removes cases with low CEBPA expression, thereby excluding CEBPA hypermethylated cases that have similar gene expression profiles as a CEBPA double mutant, which would result in false-positive predictions. In the second step, we have developed a 55 gene signature to identity the true CEBPA double-mutation cases. This two-step classifier was tested on a cohort of 505 unselected AML cases, including 26 CEBPA double mutants, 12 CEBPA single mutants, and seven CEBPA promoter hypermethylated cases, on which its performance was estimated by a double-loop cross-validation protocol. The two-step classifier achieves a sensitivity of 96.2% (95% confidence interval [CI] 81.1 to 99.3) and specificity of 100.0% (95% CI 99.2 to 100.0). There are no false-positive detections. This two-step CEBPA double-mutation classifier has been incorporated on a microarray platform that can simultaneously detect other relevant molecular biomarkers, which allows for a standardized comprehensive diagnostic assay. In conclusion, gene expression profiling provides a reliable method for CEBPA double-mutation detection in patients with AML for clinical use.
PMCID: PMC3634142  PMID: 23485358
4.  Cooperation between RUNX1-ETO9a and Novel Transcriptional Partner KLF6 in Upregulation of Alox5 in Acute Myeloid Leukemia 
PLoS Genetics  2013;9(10):e1003765.
Fusion protein RUNX1-ETO (AML1-ETO, RUNX1-RUNX1T1) is expressed as the result of the 8q22;21q22 translocation [t(8;21)], which is one of the most common chromosomal abnormalities found in acute myeloid leukemia. RUNX1-ETO is thought to promote leukemia development through the aberrant regulation of RUNX1 (AML1) target genes. Repression of these genes occurs via the recruitment of the corepressors N-COR and SMRT due to their interaction with ETO. Mechanisms of RUNX1-ETO target gene upregulation remain less well understood. Here we show that RUNX1-ETO9a, the leukemogenic alternatively spliced transcript expressed from t(8;21), upregulates target gene Alox5, which is a gene critically required for the promotion of chronic myeloid leukemia development by BCR-ABL. Loss of Alox5 expression reduces activity of RUNX1-ETO9a, MLL-AF9 and PML-RARα in vitro. However, Alox5 is not essential for the induction of leukemia by RUNX1-ETO9a in vivo. Finally, we demonstrate that the upregulation of Alox5 by RUNX1-ETO9a occurs via the C2H2 zinc finger transcription factor KLF6, a protein required for early hematopoiesis and yolk sac development. Furthermore, KLF6 is specifically upregulated by RUNX1-ETO in human leukemia cells. This identifies KLF6 as a novel mediator of t(8;21) target gene regulation, providing a new mechanism for RUNX1-ETO transcriptional control.
Author Summary
The 8;21 translocation is one of the most common genetic abnormalities present in acute myeloid leukemia (AML). This translocation causes expression of the fusion gene RUNX1-ETO and its splicing isoforms. RUNX1-ETO proteins then reprogram the transcriptional landscape of the cell and cooperate with further mutations to induce leukemia development. In this study, we examine the transcriptional control of the RUNX1-ETO target gene Alox5. Although Alox5 appears to be dispensable for AML development in a mouse model, it is required for some RUNX1-ETO functions. In studying the regulation of Alox5 expression, we have discovered a novel RUNX1-ETO partner protein, KLF6, which is both upregulated by RUNX1-ETO and participates in RUNX1-ETO gene regulation. This provides new insight into the under-studied mechanisms of RUNX1-ETO target gene upregulation and identifies KLF6 as a potentially important protein for further study in t(8;21) AML development.
PMCID: PMC3794898  PMID: 24130502
5.  Frequency and Prognostic Impact of CEBPA Proximal, Distal and Core Promoter Methylation in Normal Karyotype AML: A Study on 623 Cases 
PLoS ONE  2013;8(2):e54365.
The clinical impact of aberrant CEBPA promoter methylation (PM) in AML is controversially discussed. The aim of this study was to clarify the significance of aberrant CEBPA PM with regard to clinical features in a cohort of 623 cytogenetically normal (CN) de novo AML. 555 cases had wild-type CEBPA, 68 cases harbored CEBPA mutations. The distal promoter was methylated in 238/623 cases (38.2%), the core promoter in 8 of 326 cases (2.5%), whereas proximal PM was never detected. CEBPA PM and CEBPA mutations were mutually exclusive. CEBPA distal PM positive cases were characterized by reduced CEBPA mRNA expression levels and elevated white blood cell counts. CEBPA distal PM was less frequent in patients with mutations in FLT3, NPM1 and TET2 and more frequent in cases with RUNX1 and IDH2R140 mutations. Overall, no association of methylation to prognosis was seen. However CEBPA distal PM was associated with inferior outcome in cases with low FLT3-ITD ratio or TET2 mutations. A distinct gene expression profile of CEBPA distal PM positive cases compared to CEBPA mutated and CEBPA distal PM negative cases was observed. In conclusion, the presence of aberrant CEBPA PM is associated with distinct biological features but impact on outcome is weak.
PMCID: PMC3562230  PMID: 23383300
6.  Prognostic Significance of, and Gene and MicroRNA Expression Signatures Associated With, CEBPA Mutations in Cytogenetically Normal Acute Myeloid Leukemia With High-Risk Molecular Features: A Cancer and Leukemia Group B Study 
Journal of Clinical Oncology  2008;26(31):5078-5087.
To evaluate the prognostic significance of CEBPA mutations in the context of established molecular markers in cytogenetically normal (CN) acute myeloid leukemia (AML) and gain biologic insights into leukemogenesis of the CN-AML molecular high-risk subset (FLT3 internal tandem duplication [ITD] positive and/or NPM1 wild type) that has a significantly higher incidence of CEBPA mutations than the molecular low-risk subset (FLT3-ITD negative and NPM1 mutated).
Patients and Methods
One hundred seventy-five adults age less than 60 years with untreated primary CN-AML were screened before treatment for CEBPA, FLT3, MLL, WT1, and NPM1 mutations and BAALC and ERG expression levels. Gene and microRNA (miRNA) expression profiles were obtained for the CN-AML molecular high-risk patients.
CEBPA mutations predicted better event-free (P = .007), disease-free (P = .014), and overall survival (P < .001) independently of other molecular and clinical prognosticators. Among patients with CEBPA mutations, 91% were in the CN-AML molecular high-risk group. Within this group, CEBPA mutations predicted better event-free (P < .001), disease-free (P = .004), and overall survival (P = .009) independently of other molecular and clinical characteristics and were associated with unique gene and miRNA expression profiles. The major features of these profiles were upregulation of genes (eg, GATA1, ZFPM1, EPOR, and GFI1B) and miRNAs (ie, the miR-181 family) involved in erythroid differentiation and downregulation of homeobox genes.
Pretreatment testing for CEBPA mutations identifies CN-AML patients with different outcomes, particularly in the molecular high-risk group, thus improving molecular risk-based classification of this large cytogenetic subset of AML. The gene and miRNA expression profiling provided insights into leukemogenesis of the CN-AML molecular high-risk group, indicating that CEBPA mutations are associated with partial erythroid differentiation.
PMCID: PMC2652095  PMID: 18809607
7.  Acute myeloid leukemia and transcription factors: role of erythroid Krüppel-like factor (EKLF) 
We have investigated the role of erythroid transcription factors mRNA expression in patients with acute myeloid leukemia (AML) in the context of cytogenetic and other prognostic molecular markers, such as FMS-like Tyrosine Kinase 3 (FLT3), Nucleophosmin 1 (NPM1), and CCAAT/enhance-binding protein α (CEBPA) mutations. Further validation of Erythroid Krüppel-like Factor (EKLF) mRNA expression as a prognostic factor was assessed.
We evaluated GATA binding protein 1 (GATA1), GATA binding protein 2 (GATA2), EKLF and Myeloproliferative Leukemia virus oncogen homology (cMPL) gene mRNA expression in the bone marrow of 65 AML patients at diagnosis, and assessed any correlation with NPM1, FLT3 and CEBPA mutations. EKLF-positive AML was associated with lower WBC in peripheral blood (P = 0.049), a higher percentage of erythroblasts in bone marrow (p = 0.057), and secondary AMLs (P = 0.036). High expression levels of EKLF showed a trend to association with T-cell antigen expression, such as CD7 (P = 0.057). Patients expressing EKLF had longer Overall Survival (OS) and Event Free Survival (EFS) than those patients not expressing EKLF (median OS was 35.61 months and 19.31 months, respectively, P = 0.0241; median EFS was 19.80 months and 8.03 months, respectively, P = 0.0140). No correlation of GATA1, GATA2, EKLF and cMPL levels was observed with FLT-3 or NPM1 mutation status. Four of four CEBPA mutated AMLs were EKLF positive versus 10 of 29 CEBPA wild-type AMLs; three of the CEBPA mutated, EKLF-positive AMLs were also GATA2 positive. There were no cases of CEBPA mutations in the EKLF-negative AML group. In conclusion, we have validated EKLF mRNA expression as an independent predictor of outcome in AML, and its expression is not associated with FLT3-ITD and NPM1 mutations. EKLF mRNA expression in AML patients may correlate with dysregulated CEBPA.
PMCID: PMC3407786  PMID: 22676581
Acute myeloid leukemia; Transcription factors; EKLF; GATA1; GATA2; cMPL; FLT3; NPM1; CEBPA mutations
8.  C/EBPα Dysregulation in AML and ALL 
Critical reviews in oncogenesis  2011;16(1-2):93-102.
The transcription factor CCAAT/Enhancer Binding Protein α (C/EBPα) is a critical regulator of myeloid development, directing granulocyte and monocyte differentiation. As such, it is dysregulated in over half of patients with acute myeloid leukemia (AML). C/EBPα expression is suppressed as result of common leukemia-associated genetic and epigenetic alterations such as AML1-ETO, BCR-ABL, FLT3-ITD, or CEBPA promoter methylation. In addition, 10–15% of patients with AML with intermediate risk cytogenetics are characterized by mutations of the CEBPA gene. Two classes of mutations are described. N-terminal changes result in expression of a truncated dominant negative C/EBPαp30 isoform. C-terminal mutations are in-frame insertions or deletions resulting in alteration of the leucine zipper preventing dimerization and DNA binding. Often, patients carry both N- and C-terminal mutations each affecting a different allele, and a mouse model recapitulates the human phenotype. Patients with mutated CEBPA AML comprise a clinically distinct group with favorable outcome consistently seen in patients with biallelic mutations. In addition, C/EBP family members are aberrantly expressing from the immunoglobulin heavy chain locus in 2% of pre-B ALLs. This review summarizes the normal hematopoietic developmental pathways regulated by C/EBPα and discusses the molecular pathways involved in mutated CEBPA AML and ALL.
PMCID: PMC3243939  PMID: 22150310
leukemia; myeloid; differentiation; hematopoiesis
9.  BAALC expression: a suitable marker for prognostic risk stratification and detection of residual disease in cytogenetically normal acute myeloid leukemia 
Blood Cancer Journal  2014;4(1):e173-.
High brain and acute leukemia, cytoplasmic (BAALC) expression defines an important risk factor in cytogenetically normal acute myeloid leukemia (CN-AML). The prognostic value of BAALC expression in relation to other molecular prognosticators was analyzed in 326 CN-AML patients (<65 years). At diagnosis, high BAALC expression was associated with prognostically adverse mutations: FLT3 internal tandem duplication (FLT3-ITD) with an FLT3-ITD/FLT3 wild-type (wt) ratio of ⩾0.5 (P=0.001), partial tandem duplications within the MLL gene (MLL-PTD) (P=0.002), RUNX1 mutations (mut) (P<0.001) and WT1mut (P=0.001), while it was negatively associated with NPM1mut (P<0.001). However, high BAALC expression was also associated with prognostically favorable biallelic CEBPA (P=0.001). Survival analysis revealed an independent adverse prognostic impact of high BAALC expression on overall survival (OS) and event-free survival (EFS), and also on OS when eliminating the effect of allogeneic stem cell transplantation (SCT) (OSTXcens). Furthermore, we analyzed BAALC expression in 416 diagnostic and follow-up samples of 66 patients. During follow-up, BAALC expression correlated with mutational load or expression levels, respectively, of other minimal residual disease markers: FLT3-ITD (r=0.650, P<0.001), MLL-PTD (r=0.728, P<0.001), NPM1mut (r=0.599, P<0.001) and RUNX1mut (r=0.889, P<0.001). Moreover, a reduction in BAALC expression after the second cycle of induction chemotherapy was associated with improved EFS. Thus, our data underline the utility of BAALC expression as a marker for prognostic risk stratification and detection of residual disease in CN-AML.
PMCID: PMC3913940  PMID: 24413067
BAALC expression; CN-AML; prognosis; MRD
10.  Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells 
The Journal of Clinical Investigation  2013;123(9):3876-3888.
RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS) and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in AMLs with leukemogenic fusion proteins, such as core-binding factor (CBF) leukemia and MLL fusion leukemia, raising the possibility that RUNX1 could actually promote the growth of these leukemia cells. Using normal human cord blood cells and those expressing leukemogenic fusion proteins, we discovered a dual role of RUNX1 in myeloid leukemogenesis. RUNX1 overexpression inhibited the growth of normal cord blood cells by inducing myeloid differentiation, whereas a certain level of RUNX1 activity was required for the growth of AML1-ETO and MLL-AF9 cells. Using a mouse genetic model, we also showed that the combined loss of Runx1/Cbfb inhibited leukemia development induced by MLL-AF9. RUNX2 could compensate for the loss of RUNX1. The survival effect of RUNX1 was mediated by BCL2 in MLL fusion leukemia. Our study unveiled an unexpected prosurvival role for RUNX1 in myeloid leukemogenesis. Inhibiting RUNX1 activity rather than enhancing it could be a promising therapeutic strategy for AMLs with leukemogenic fusion proteins.
PMCID: PMC3754260  PMID: 23979164
11.  Integrative analysis of RUNX1 downstream pathways and target genes 
BMC Genomics  2008;9:363.
The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia.
Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes.
This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.
PMCID: PMC2529319  PMID: 18671852
12.  Recognizing familial myeloid leukemia in adults 
Germline testing for familial cases of myeloid leukemia in adults is becoming more common with the recognition of multiple genetic syndromes predisposing people to bone marrow disease. Currently, Clinical Laboratory Improvement Amendments approved testing exists for several myeloid leukemia predisposition syndromes: familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML), caused by mutations in RUNX1; familial AML with mutated CEBPA; familial myelodysplastic syndrome and acute leukemia with mutated GATA2; and the inherited bone marrow failure syndromes, including dyskeratosis congenita, a disease of abnormal telomere maintenance. With the recognition of additional families with a genetic component to their leukemia, new predisposition alleles will likely be identified. We highlight how to recognize and manage these cases as well as outline the characteristics of the major known syndromes. We look forward to future research increasing our understanding of the scope of inherited myeloid leukemia syndromes.
PMCID: PMC3734901  PMID: 23926458
familial leukemia predisposition; RUNX1; CEBPA; GATA2; dyskeratosis congenita; Fanconi anemia
13.  Mapping of MN1 Sequences Necessary for Myeloid Transformation 
PLoS ONE  2013;8(4):e61706.
The MN1 oncogene is deregulated in human acute myeloid leukemia and its overexpression induces proliferation and represses myeloid differentiation of primitive human and mouse hematopoietic cells, leading to myeloid leukemia in mouse models. To delineate the sequences within MN1 necessary for MN1-induced leukemia, we tested the transforming capacity of in-frame deletion mutants, using retroviral transduction of mouse bone marrow. We found that integrity of the regions between amino acids 12 to 458 and 1119 to 1273 are required for MN1’s in vivo transforming activity, generating myeloid leukemia with some mutants also producing T-cell lympho-leukemia and megakaryocytic leukemia. Although both full length MN1 and a mutant that lacks the residues between 12–228 (Δ12–228 mutant) repressed myeloid differentiation and increased myeloproliferative activity in vitro, the mutant lost its transforming activity in vivo. Both MN1 and Δ12–228 increased the frequency of common myeloid progentiors (CMP) in vitro and microarray comparisons of purified MN1-CMP and Δ12–228-CMP cells showed many differentially expressed genes including Hoxa9, Meis1, Myb, Runx2, Cebpa, Cebpb and Cebpd. This collection of immediate MN1-responsive candidate genes distinguishes the leukemic activity from the in vitro myeloproliferative capacity of this oncoprotein.
PMCID: PMC3634013  PMID: 23626719
14.  CEBPA-dependent HK3 and KLF5 expression in primary AML and during AML differentiation 
Scientific Reports  2014;4:4261.
The basic leucine zipper transcription factor CCAAT/enhancer binding protein alpha (CEBPA) codes for a critical regulator during neutrophil differentiation. Aberrant expression or function of this protein contributes to the development of acute myeloid leukemia (AML). In this study, we identified two novel unrelated CEBPA target genes, the glycolytic enzyme hexokinase 3 (HK3) and the krüppel-like factor 5 (KLF5) transcription factor, by comparing gene profiles in two cohorts of CEBPA wild-type and mutant AML patients. In addition, we found CEBPA-dependent activation of HK3 and KLF5 transcription during all-trans retinoic acid (ATRA) mediated neutrophil differentiation of acute promyelocytic leukemia (APL) cells. Moreover, we observed direct regulation of HK3 by CEBPA, whereas our data suggest an indirect regulation of KLF5 by this transcription factor. Altogether, our data provide an explanation for low HK3 and KLF5 expression in particular AML subtype and establish these genes as novel CEBPA targets during neutrophil differentiation.
PMCID: PMC3939455  PMID: 24584857
15.  FLT3-ITDs Instruct a Myeloid Differentiation and Transformation Bias in Lymphomyeloid Multipotent Progenitors 
Cell Reports  2013;3(6):1766-1776.
Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies.
Graphical Abstract
•Flt3-ITDs collaborate with Runx1 mutation to cause acute myeloid leukemia exclusively•Flt3-ITDs instruct myeloid lineage bias in lymphoid-primed multipotent precursors•Flt3-ITDs inhibit thymic seeding by bone marrow progenitors•Flt3-ITD-induced myeloid bias and progenitor phenotype involve upregulation of Pu.1
In this study, Mead, Jacobsen, and colleagues demonstrate that constitutive growth factor receptor (GFR) signaling through an Flt3-ITD mutation instructs a myeloid-lineage differentiation bias to multipotent hematopoietic progenitor cells. Runx1 mutation collaborated with Flt3-ITD to induce aggressive, universally myeloid-lineage leukemias, indicating that Flt3-ITD GFR signaling acts to dictate the phenotype of resulting malignancies. The Flt3-ITD-induced myeloid lineage bias involves upregulation of the transcription factor Pu.1, thus establishing how GFR signaling might elicit lineage-instructive signaling in vivo.
PMCID: PMC3701326  PMID: 23727242
16.  Wilms’ Tumor 1 Gene Mutations Independently Predict Poor Outcome in Adults With Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study 
Journal of Clinical Oncology  2008;26(28):4595-4602.
To analyze the prognostic impact of Wilms’ tumor 1 (WT1) gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML).
Patients and Methods
We studied 196 adults younger than 60 years with newly diagnosed primary CN-AML, who were treated similarly on Cancer and Leukemia Group B (CALGB) protocols 9621 and 19808, for WT1 mutations in exons 7 and 9. The patients also were assessed for the presence of FLT3 internal tandem duplications (FLT3-ITD), FLT3 tyrosine kinase domain mutations (FLT3-TKD), MLL partial tandem duplications (MLL-PTD), NPM1 and CEBPA mutations, and for the expression levels of ERG and BAALC.
Twenty-one patients (10.7%) harbored WT1 mutations. Complete remission rates were not significantly different between patients with WT1 mutations and those with unmutated WT1 (P = .36; 76% v 84%). Patients with WT1 mutations had worse disease-free survival (DFS; P < .001; 3-year rates, 13% v 50%) and overall survival (OS; P < .001; 3-year rates, 10% v 56%) than patients with unmutated WT1. In multivariable analyses, WT1 mutations independently predicted worse DFS (P = .009; hazard ratio [HR] = 2.7) when controlling for CEBPA mutational status, ERG expression level, and FLT3-ITD/NPM1 molecular-risk group (ie, FLT3-ITDnegative/NPM1mutated as low risk v FLT3-ITDpositive and/or NPM1wild-type as high risk). WT1 mutations also independently predicted worse OS (P < .001; HR = 3.2) when controlling for CEBPA mutational status, FLT3-ITD/NPM1 molecular-risk group, and white blood cell count.
We report the first evidence that WT1 mutations independently predict extremely poor outcome in intensively treated, younger patients with CN-AML. Future trials should include testing for WT1 mutations as part of molecularly based risk assessment and risk-adapted treatment stratification of patients with CN-AML.
PMCID: PMC2653131  PMID: 18559874
Seminars in oncology  2012;39(1):97-108.
Cytotoxic chemotherapy for acute myeloid leukemia (AML) usually produces only temporary remissions, at the cost of significant toxicity and risk for death. One fundamental reason for treatment failure is that it is designed to activate apoptosis genes (eg., TP53) that may be unavailable because of mutation or deletion. Unlike deletion of apoptosis genes, genes that mediate cell cycle exit by differentiation are present in myelodysplastic syndrome (MDS) and AML cells but are epigenetically repressed: MDS/AML cells express high levels of key lineage-specifying transcription factors (TF). Mutation in these TF (eg., CEBPA) or their cofactors (eg., RUNX1) affect transactivation function and produce epigenetic repression of late-differentiation genes that antagonize MYC. Importantly, this aberrant epigenetic repression can be redressed clinically by depleting DNA methyltransferase 1 (DNMT1, a central component of the epigenetic network that mediates transcription repression) using the deoxycytidine analogue decitabine (DAC) at non-cytotoxic concentrations. The DNMT1 depletion is sufficient to trigger upregulation of late-differentiation genes and irreversible cell cycle exit by p53-independent differentiation mechanisms. Fortuitously, the same treatment maintains or increases self-renewal of normal hematopoietic stem cells (HSC), which do not express high levels of lineage-specifying TF. The biological rationale for this approach to therapy appears to apply to cancers other than MDS/AML also. DAC or 5-azacytidine dose and schedule can be rationalized to emphasize this mechanism of action, as an alternative or complement to conventional apoptosis-based oncotherapy.
PMCID: PMC3655437  PMID: 22289496
Decitabine; differentiation; p53; p16; p27; CDKN2A; CDKN1B; therapy; chromatin modifying enzymes; cancer; leukemia
18.  The tumour-suppressive miR-29a/b1 cluster is regulated by CEBPA and blocked in human AML 
British Journal of Cancer  2010;103(2):275-284.
CCAAT/enhancer-binding protein-α (CEBPA) is crucial for normal granulopoiesis and is frequently disrupted in acute myeloid leukaemia (AML). Increasing evidence suggests that CEBPA exerts its effects, in parts, by regulating specific microRNAs (miRNAs), as previously shown for miR-223. The aim of this study was to investigate the genome-wide pattern of miRNAs regulated by CEBPA in myeloid cells.
In Kasumi-1 cells, conditionally expressing CEBPA, we assessed the expression of 470 human miRNAs by microarray analysis. We further investigated the microarray results by qRT-PCR, luciferase reporter assays, and chromatin immunoprecipitation assays.
In all, 18 miRNAs were more than two-fold suppressed or induced after CEBPA restoration. Among these 18 miRNAs, we focused on CEBPA-mediated regulation of the tumour-suppressive miR-29b. We observed that miR-29b is suppressed in AML patients with impaired CEBPA function or loss of chromosome 7q. We found that CEBPA selectively regulates miR-29b expression on its miR-29a/b1 locus on chromosome 7q32.3, whereas miR-29b2/c on chromosome 1q32.2 is not affected.
This study reports the activation of the tumour-suppressive miR-29b by the haematopoietic key transcription factor CEBPA. Our data provide a rationale for miR-29b suppression in AML patients with loss of chromosome 7q or CEBPA deficiency.
PMCID: PMC2906742  PMID: 20628397
AML; CEBPA; miR-29a/b/c family; transcriptional regulation
19.  CD7 in acute myeloid leukemia: correlation with loss of wild-type CEBPA, consequence of epigenetic regulation 
CD7 is a negative prognostic marker in myeloid malignancies. In acute myeloid leukemia (AML), an inverse correlation exists between expression of wild-type CEBPA and CD7. Aim of this study was to find out whether C/EBPα is a negative regulator of CD7 and which other regulatory mechanisms might be involved.
As already described for primary AML cells, the majority of AML cell lines tested were either C/EBPα+/CD7- or C/EBPα-/CD7+. However, the existence of isolated CD7+ cell lines expressing wild-type C/EBPα challenges the notion that C/EBPα acts as a unique repressor of CD7. Furthermore, ectopic expression of CEBPA did not reduce CD7 in CD7+ cells and knock-down of C/EBPα failed to induce CD7 in CD7- cells. In contrast, the DNA demethylating agent Aza-2'deoxycytidine triggered CD7 expression in CD7- AML and in T-cell lines suggesting epigenetic regulation of CD7. Bisulfite sequencing data confirmed that CpGs in the CD7 exon1 region are methylated in CD7- cell lines, and unmethylated in CD7+ cell lines.
We confirmed an inverse correlation between the expression of wild-type CEBPA and of CD7 in AML cells. Our results contradict the hypothesis that C/EBPα acts as repressor for CD7, and instead show that epigenetic mechanisms are responsible for CD7 regulation, in AML cells as well as in T-cells, the typical CD7 expressing cell type.
PMCID: PMC2873354  PMID: 20398252
20.  Myeloid malignancies: mutations, models and management 
BMC Cancer  2012;12:304.
Myeloid malignant diseases comprise chronic (including myelodysplastic syndromes, myeloproliferative neoplasms and chronic myelomonocytic leukemia) and acute (acute myeloid leukemia) stages. They are clonal diseases arising in hematopoietic stem or progenitor cells. Mutations responsible for these diseases occur in several genes whose encoded proteins belong principally to five classes: signaling pathways proteins (e.g. CBL, FLT3, JAK2, RAS), transcription factors (e.g. CEBPA, ETV6, RUNX1), epigenetic regulators (e.g. ASXL1, DNMT3A, EZH2, IDH1, IDH2, SUZ12, TET2, UTX), tumor suppressors (e.g. TP53), and components of the spliceosome (e.g. SF3B1, SRSF2). Large-scale sequencing efforts will soon lead to the establishment of a comprehensive repertoire of these mutations, allowing for a better definition and classification of myeloid malignancies, the identification of new prognostic markers and therapeutic targets, and the development of novel therapies. Given the importance of epigenetic deregulation in myeloid diseases, the use of drugs targeting epigenetic regulators appears as a most promising therapeutic approach.
PMCID: PMC3418560  PMID: 22823977
21.  Granulopoiesis Requires Increased C/EBPα Compared to Monopoiesis, Correlated with Elevated Cebpa in Immature G-CSF Receptor versus M-CSF Receptor Expressing Cells 
PLoS ONE  2014;9(4):e95784.
C/EBPα is required for the formation of granulocyte-monocyte progenitors; however, its role in subsequent myeloid lineage specification remains uncertain. Transduction of murine marrow with either of two Cebpa shRNAs markedly increases monocyte and reduces granulocyte colonies in methylcellulose or the monocyte to neutrophil ratio in liquid culture. Similar findings were found after marrow shRNA transduction and transplantation and with CEBPA knockdown in human marrow CD34+ cells. These results apparently reflect altered myeloid lineage specification, as similar knockdown allowed nearly complete 32Dcl3 granulocytic maturation. Cebpa knockdown also generated lineage-negative blasts with increased colony replating capacity but unchanged cell cycle parameters, likely reflecting complete differentiation block. The shRNA having the greatest effect on lineage skewing reduced Cebpa 3-fold in differentiating cells but 6-fold in accumulating blasts. Indicating that Cebpa is the relevant shRNA target, shRNA-resistant C/EBPα-ER rescued marrow myelopoiesis. Cebpa knockdown in murine marrow cells also increased in vitro erythropoiesis, perhaps reflecting 1.6-fold reduction in PU.1 leading to GATA-1 derepression. Global gene expression analysis of lineage-negative blasts that accumulate after Cebpa knockdown demonstrated reduction in Cebpe and Gfi1, known transcriptional regulators of granulopoiesis, and also reduced Ets1 and Klf5. Populations enriched for immature granulocyte or monocyte progenitor/precursors were isolated by sorting Lin−Sca-1−c-Kit+ cells into GCSFR+MCSFR− or GCSFR−MCSFR+ subsets. Cebpa, Cebpe, Gfi1, Ets1, and Klf5 RNAs were increased in the c-Kit+GCSFR+ and Klf4 and Irf8 in the c-Kit+MCSFR+ populations, with PU.1 levels similar in both. In summary, higher levels of C/EBPα are required for granulocyte and lower levels for monocyte lineage specification, and this myeloid bifurcation may be facilitated by increased Cebpa gene expression in granulocyte compared with monocyte progenitors.
PMCID: PMC3994156  PMID: 24752325
22.  Cell-Autonomous Function of Runx1 Transcriptionally Regulates Mouse Megakaryocytic Maturation 
PLoS ONE  2013;8(5):e64248.
RUNX1 transcription factor (TF) is a key regulator of megakaryocytic development and when mutated is associated with familial platelet disorder and predisposition to acute myeloid leukemia (FPD-AML). We used mice lacking Runx1 specifically in megakaryocytes (MK) to characterized Runx1-mediated transcriptional program during advanced stages of MK differentiation. Gene expression and chromatin-immunoprecipitation-sequencing (ChIP-seq) of Runx1 and p300 identified functional Runx1 bound MK enhancers. Runx1/p300 co-bound regions showed significant enrichment in genes important for MK and platelet homeostasis. Runx1 occupied genomic regions were highly enriched in RUNX and ETS motifs and to a lesser extent in GATA motif. Megakaryocytic specificity of Runx1/P300 bound enhancers was validated by transfection mutagenesis and Runx1/P300 co-bound regions of two key megakaryocytic genes Nfe2 and Selp were tested by in vivo transgenesis. The data provides the first example of genome wide Runx1/p300 occupancy in maturating primary FL-MK, unravel the Runx1-regulated program controlling MK maturation in vivo and identify a subset of its bona fide regulated genes. It advances our understanding of the molecular events that upon RUNX1mutations in human lead to the predisposition to familial platelet disorders and FPD-AML.
PMCID: PMC3662678  PMID: 23717578
23.  TET2 Mutations Improve the New European LeukemiaNet Risk Classification of Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study 
Journal of Clinical Oncology  2011;29(10):1373-1381.
To determine the frequency of TET2 mutations, their associations with clinical and molecular characteristics and outcome, and the associated gene- and microRNA-expression signatures in patients with primary cytogenetically normal acute myeloid leukemia (CN-AML).
Patients and Methods
Four-hundred twenty-seven patients with CN-AML were analyzed for TET2 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene- and microRNA-expression profiles were derived using microarrays.
TET2 mutations, found in 23% of patients, were associated with older age (P < .001) and higher pretreatment WBC (P = .04) compared with wild-type TET2 (TET2-wt). In the European LeukemiaNet (ELN) favorable-risk group (patients with CN-AML who have mutated CEBPA and/or mutated NPM1 without FLT3 internal tandem duplication [FLT3-ITD]), TET2-mutated patients had shorter event-free survival (EFS; P < .001) because of a lower complete remission (CR) rate (P = .007), and shorter disease-free survival (DFS; P = .003), and also had shorter overall survival (P = .001) compared with TET2-wt patients. TET2 mutations were not associated with outcomes in the ELN intermediate-I–risk group (CN-AML with wild-type CEBPA and wild-type NPM1 and/or FLT3-ITD). In multivariable models, TET2 mutations were associated with shorter EFS (P = .004), lower CR rate (P = .03), and shorter DFS (P = .05) only among favorable-risk CN-AML patients. We identified a TET2 mutation-associated gene-expression signature in favorable-risk but not in intermediate-I–risk patients and found distinct mutation-associated microRNA signatures in both ELN groups.
TET2 mutations improve the ELN molecular-risk classification in primary CN-AML because of their adverse prognostic impact in an otherwise favorable-risk patient subset. Our data suggest that these patients may be candidates for alternative therapies.
PMCID: PMC3084003  PMID: 21343549
24.  Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia 
The New England journal of medicine  2012;366(12):1079-1089.
Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. The prognostic value of recently identified somatic mutations has not been systematically evaluated in a phase 3 trial of treatment for AML.
We performed a mutational analysis of 18 genes in 398 patients younger than 60 years of age who had AML and who were randomly assigned to receive induction therapy with high-dose or standard-dose daunorubicin. We validated our prognostic findings in an independent set of 104 patients.
We identified at least one somatic alteration in 97.3% of the patients. We found that internal tandem duplication in FLT3 (FLT3-ITD), partial tandem duplication in MLL (MLL-PTD), and mutations in ASXL1 and PHF6 were associated with reduced overall survival (P = 0.001 for FLT3-ITD, P = 0.009 for MLL-PTD, P = 0.05 for ASXL1, and P = 0.006 for PHF6); CEBPA and IDH2 mutations were associated with improved overall survival (P = 0.05 for CEBPA and P = 0.01 for IDH2). The favorable effect of NPM1 mutations was restricted to patients with co-occurring NPM1 and IDH1 or IDH2 mutations. We identified genetic predictors of outcome that improved risk stratification among patients with AML, independently of age, white-cell count, induction dose, and post-remission therapy, and validated the significance of these predictors in an independent cohort. High-dose daunorubicin, as compared with standard-dose daunorubicin, improved the rate of survival among patients with DNMT3A or NPM1 mutations or MLL translocations (P = 0.001) but not among patients with wild-type DNMT3A, NPM1, and MLL (P = 0.67).
We found that DNMT3A and NPM1 mutations and MLL translocations predicted an improved outcome with high-dose induction chemotherapy in patients with AML. These findings suggest that mutational profiling could potentially be used for risk stratification and to inform prognostic and therapeutic decisions regarding patients with AML. (Funded by the National Cancer Institute and others.)
PMCID: PMC3545649  PMID: 22417203
25.  Clinical impact of gene mutations and lesions detected by SNP-array karyotyping in acute myeloid leukemia patients in the context of gemtuzumab ozogamicin treatment: Results of the ALFA-0701 trial 
Oncotarget  2014;5(4):916-932.
We recently showed that the addition of fractionated doses of gemtuzumab ozogamicin (GO) to standard chemotherapy improves clinical outcome of acute myeloid leukemia (AML) patients. In the present study, we performed mutational analysis of 11 genes (FLT3, NPM1, CEBPA, MLL, WT1, IDH1/2, RUNX1, ASXL1, TET2, DNMT3A), EVI1 overexpression screening, and 6.0 single-nucleotide polymorphism array (SNP-A) analysis in diagnostic samples of the 278 AML patients enrolled in the ALFA-0701 trial. In cytogenetically normal (CN) AML (n = 146), 38% of the patients had at least 1 SNP-A lesion and 89% of the patients had at least 1 molecular alteration. In multivariate analysis, the independent predictors of higher cumulative incidence of relapse were unfavorable karyotype (P = 0.013) and randomization in the control arm (P = 0.007) in the whole cohort, and MLL partial tandem duplications (P = 0.014) and DNMT3A mutations (P = 0.010) in CN-AML. The independent predictors of shorter overall survival (OS) were unfavorable karyotype (P < 0.001) and SNP-A lesion(s) (P = 0.001) in the whole cohort, and SNP-A lesion(s) (P = 0.006), DNMT3A mutations (P = 0.042) and randomization in the control arm (P = 0.043) in CN-AML. Interestingly, CN-AML patients benefited preferentially more from GO treatment as compared to AML patients with abnormal cytogenetics (hazard ratio for death, 0.52 versus 1.14; test for interaction, P = 0.04). Although the interaction test was not statistically significant, the OS benefit associated with GO treatment appeared also more pronounced in FLT3 internal tandem duplication positive than in negative patients.
PMCID: PMC4011594  PMID: 24659740
acute myeloid leukemia; SNP array lesions; gene mutations; gemtuzumab ozogamicin; prognosis

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