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author:("behmlander, S")
1.  CATS (FAM64A) abnormal expression reduces clonogenicity of hematopoietic cells 
Oncotarget  2016;7(42):68385-68396.
The CATS (FAM64A) protein interacts with CALM (PICALM) and the leukemic fusion protein CALM/AF10. CATS is highly expressed in leukemia, lymphoma and tumor cell lines and its protein levels strongly correlates with cellular proliferation in both malignant and normal cells. In order to obtain further insight into CATS function we performed an extensive analysis of CATS expression during differentiation of leukemia cell lines. While CATS expression decreased during erythroid, megakaryocytic and monocytic differentiation, a markedly increase was observed in the ATRA induced granulocytic differentiation. Lentivirus mediated silencing of CATS in U937 cell line resulted in somewhat reduced proliferation, altered cell cycle progression and lower migratory ability in vitro; however was not sufficient to inhibit tumor growth in xenotransplant model. Of note, CATS knockdown resulted in reduced clonogenicity of CATS-silenced cells and reduced expression of the self-renewal gene, GLI-1. Moreover, retroviral mediated overexpression of the murine Cats in primary bone marrow cells lead to decreased colony formation. Although our in vitro data suggests that CATS play a role in cellular processes important for tumorigenesis, such as cell cycle control and clonogenicity, these effects were not observed in vivo.
doi:10.18632/oncotarget.11724
PMCID: PMC5356563  PMID: 27588395
CATS (FAM64A); proliferation; clonogenicity; CALM/AF10; leukemogenesis
2.  An ETV6-ABL1 fusion in a patient with chronic myeloproliferative neoplasm: Initial response to Imatinib followed by rapid transformation into ALL 
We report the case of a 26 year-old patient presenting with a persistent leukocytosis and CML-like marrow but no evidence of a BCR/ABL1 fusion. Molecular cytogenetics revealed that a portion of the ETV6 locus was inserted into the ABL1 locus. An ETV6/ABL1 fusion transcript could subsequently be confirmed. The patient was started on imatinib and went into complete cytomorphological remission. QRT-PCR measurements showed a 4 log reduction of the ETV6/ABL1 fusion. 15 months later, the disease transformed into ALL and the patient expired. Thus, an ETV6/ABL1 fusion positive MPN has the potential to transform very rapidly into ALL.
doi:10.1016/j.lrr.2016.09.002
PMCID: PMC5078675  PMID: 27812500
ETV6/ABL1 fusion; CML; ALL; MRD assay; TKI treatment
3.  ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation 
Nature Communications  2016;7:11733.
The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion.
The t(8;21) translocation is often found in acute myeloid leukaemia but is not sufficient for development of the disease. In this study, the authors identify frequent mutations in the transcriptional repressor, ZBTB7A, in these patients and show that the mutations reduce DNA binding activity.
doi:10.1038/ncomms11733
PMCID: PMC4895769  PMID: 27252013
4.  miR-22 has a potent anti-tumour role with therapeutic potential in acute myeloid leukaemia 
Nature Communications  2016;7:11452.
MicroRNAs are subject to precise regulation and have key roles in tumorigenesis. In contrast to the oncogenic role of miR-22 reported in myelodysplastic syndrome (MDS) and breast cancer, here we show that miR-22 is an essential anti-tumour gatekeeper in de novo acute myeloid leukaemia (AML) where it is significantly downregulated. Forced expression of miR-22 significantly suppresses leukaemic cell viability and growth in vitro, and substantially inhibits leukaemia development and maintenance in vivo. Mechanistically, miR-22 targets multiple oncogenes, including CRTC1, FLT3 and MYCBP, and thus represses the CREB and MYC pathways. The downregulation of miR-22 in AML is caused by TET1/GFI1/EZH2/SIN3A-mediated epigenetic repression and/or DNA copy-number loss. Furthermore, nanoparticles carrying miR-22 oligos significantly inhibit leukaemia progression in vivo. Together, our study uncovers a TET1/GFI1/EZH2/SIN3A/miR-22/CREB-MYC signalling circuit and thereby provides insights into epigenetic/genetic mechanisms underlying the pathogenesis of AML, and also highlights the clinical potential of miR-22-based AML therapy.
Mir-22 has been shown to be an oncogenic microRNA in breast cancer and myelodysplastic syndrome. Here, the authors show that mir-22 functions as a tumour suppressor in de novo acute myeloid leukaemia by inhibiting the expression of several oncogenes and that restoring mir-22 expression suppresses AML progression.
doi:10.1038/ncomms11452
PMCID: PMC5477496  PMID: 27116251
5.  A 4‐gene expression score associated with high levels of Wilms Tumor‐1 (WT1) expression is an adverse prognostic factor in acute myeloid leukaemia 
British Journal of Haematology  2015;172(3):401-411.
Summary
Wilms Tumor‐1 (WT1) expression level is implicated in the prognosis of acute myeloid leukaemia (AML). We hypothesized that a gene expression profile associated with WT1 expression levels might be a good surrogate marker. We identified high WT1 gene sets by comparing the gene expression profiles in the highest and lowest quartiles of WT1 expression in two large AML studies. Two high WT1 gene sets were found to be highly correlated in terms of the altered genes and expression profiles. We identified a 17‐probe set signature of the high WT1 set as the optimal prognostic predictor in the first AML set, and showed that it was able to predict prognosis in the second AML series after adjustment for European LeukaemiaNet genetic groups. The gene signature also proved to be of prognostic value in a third AML series of 163 samples assessed by RNA sequencing, demonstrating its cross‐platform consistency. This led us to derive a 4‐gene expression score, which faithfully predicted adverse outcome. In conclusion, a short gene signature associated with high WT1 expression levels and the resultant 4‐gene expression score were found to be predictive of adverse prognosis in AML. This study provides new clues to the molecular pathways underlying high WT1 states in leukaemia.
doi:10.1111/bjh.13836
PMCID: PMC4833185  PMID: 26597595
WT1; gene signature; expression score; AML; prognosis
6.  The Establishment of an On-Campus Neurotraining Center at a Small University: Internship Experience Using Neuropsychological Techniques 
In 2015, Wilkes University’s Neuroscience Program and Psychology Department established a unique training and learning center on a small liberal arts undergraduate campus - The NeuroTraining & Research Center. This paper shares the purpose of the Center, as a learning tool for Neuroscience majors who engage in internships and research opportunities, and as a means of promoting well-being on the campus by offering training in techniques such as Neurofeedback, Biofeedback and Audio-Visual Entrainment to the college community. The role that the center plays in connecting real world applications to concepts in Neuroscience, and the approach that the authors have taken to assess student learning is presented in this article.
PMCID: PMC5105966  PMID: 27980472
neuroscience internship; neuroscience education; experiential learning; applied clinical neuroscience
7.  Mutational spectrum of adult T-ALL 
Oncotarget  2014;6(5):2754-2766.
Novel target discovery is warranted to improve treatment in adult T-cell acute lymphoblastic leukemia (T-ALL) patients. We provide a comprehensive study on mutations to enhance the understanding of therapeutic targets and studied 81 adult T-ALL patients. NOTCH1 exhibitedthe highest mutation rate (53%). Mutation frequencies of FBXW7 (10%), WT1 (10%), JAK3 (12%), PHF6 (11%), and BCL11B (10%) were in line with previous reports. We identified recurrent alterations in transcription factors DNM2, and RELN, the WNT pathway associated cadherin FAT1, and in epigenetic regulators (MLL2, EZH2). Interestingly, we discovered novel recurrent mutations in the DNA repair complex member HERC1, in NOTCH2, and in the splicing factor ZRSR2. A frequently affected pathway was the JAK/STAT pathway (18%) and a significant proportion of T-ALL patients harboured mutations in epigenetic regulators (33%), both predominantly found in the unfavourable subgroup of early T-ALL. Importantly, adult T-ALL patients not only showed a highly heterogeneous mutational spectrum, but also variable subclonal allele frequencies implicated in therapy resistance and evolution of relapse. In conclusion, we provide novel insights in genetic alterations of signalling pathways (e.g. druggable by γ-secretase inhibitors, JAK inhibitors or EZH2 inhibitors), present in over 80% of all adult T-ALL patients, that could guide novel therapeutic approaches.
PMCID: PMC4413615  PMID: 25595890
Acute lymphoblastic leukemia; targeted therapy; T-ALL next generation sequencing; pathways; gene panel
8.  Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia 
Oncotarget  2014;5(23):12371-12382.
Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients.
PMCID: PMC4323011  PMID: 25365263
MELK; AML; OTS167
9.  Identification and characterization of OSTL (RNF217) encoding a RING-IBR-RING protein adjacent to a translocation breakpoint involving ETV6 in childhood ALL 
Scientific Reports  2014;4:6565.
Genomic aberrations involving ETV6 on band 12p13 are amongst the most common chromosomal abnormalities in human leukemia. The translocation t(6;12)(q23;13) in a childhood B-cell acute lymphoblastic leukemia (ALL) cell line fuses ETV6 with the putative long non-coding RNA gene STL. Linking STL properties to leukemia has so far been difficult. Here, we describe a novel gene, OSTL (annotated as RNF217 in Genbank), which shares the first exon and a CpG island with STL but is transcribed in the opposite direction. Human RNF217 codes for a highly conserved RING finger protein and is mainly expressed in testis and skeletal muscle with different splice variants. RNF217 shows regulated splicing in B cell development, and is expressed in a number of human B cell leukemia cell lines, primary human chronic myeloid leukemia, acute myeloid leukemia with normal karyotype and acute T-ALL samples. Using a yeast two-hybrid screen, we identified the anti-apoptotic protein HAX1 to interact with RNF217. This interaction could be mapped to the C-terminal RING finger motif of RNF217. We propose that some of the recurring aberrations involving 6q might deregulate the expression of RNF217 and result in imbalanced apoptosis signalling via HAX1, promoting leukemia development.
doi:10.1038/srep06565
PMCID: PMC4190505  PMID: 25298122
10.  Combined Molecular and Clinical Prognostic Index for Relapse and Survival in Cytogenetically Normal Acute Myeloid Leukemia 
Journal of Clinical Oncology  2014;32(15):1586-1594.
Purpose
Cytogenetically normal (CN) acute myeloid leukemia (AML) is the largest and most heterogeneous cytogenetic AML subgroup. For the practicing clinician, it is difficult to summarize the prognostic information of the growing number of clinical and molecular markers. Our purpose was to develop a widely applicable prognostic model by combining well-established pretreatment patient and disease characteristics.
Patients and Methods
Two prognostic indices for CN-AML (PINA), one regarding overall survival (OS; PINAOS) and the other regarding relapse-free survival (RFS; PINARFS), were derived from data of 572 patients with CN-AML treated within the AML Cooperative Group 99 study (www.aml-score.org).
Results
On the basis of age (median, 60 years; range, 17 to 85 years), performance status, WBC count, and mutation status of NPM1, CEBPA, and FLT3-internal tandem duplication, patients were classified into the following three risk groups according to PINAOS and PINARFS: 29% of all patients and 32% of 381 responding patients had low-risk disease (5-year OS, 74%; 5-year RFS, 55%); 56% of all patients and 39% of responding patients had intermediate-risk disease (5-year OS, 28%; 5-year RFS, 27%), and 15% of all patients and 29% of responding patients had high-risk disease (5-year OS, 3%; 5-year RFS, 5%), respectively. PINAOS and PINARFS stratified outcome within European LeukemiaNet genetic groups. Both indices were confirmed on independent data from Cancer and Leukemia Group B/Alliance trials.
Conclusion
We have developed and validated, to our knowledge, the first prognostic indices specifically designed for adult patients of all ages with CN-AML that combine well-established molecular and clinical variables and that are easily applicable in routine clinical care. The integration of both clinical and molecular markers could provide a basis for individualized patient care through risk-adapted therapy of CN-AML.
doi:10.1200/JCO.2013.52.3480
PMCID: PMC4876345  PMID: 24711548
11.  Identification of a 24-Gene Prognostic Signature That Improves the European LeukemiaNet Risk Classification of Acute Myeloid Leukemia: An International Collaborative Study 
Journal of Clinical Oncology  2013;31(9):1172-1181.
Purpose
To identify a robust prognostic gene expression signature as an independent predictor of survival of patients with acute myeloid leukemia (AML) and use it to improve established risk classification.
Patients and Methods
Four independent sets totaling 499 patients with AML carrying various cytogenetic and molecular abnormalities were used as training sets. Two independent patient sets composed of 825 patients were used as validation sets. Notably, patients from different sets were treated with different protocols, and their gene expression profiles were derived using different microarray platforms. Cox regression and Kaplan-Meier methods were used for survival analyses.
Results
A prognostic signature composed of 24 genes was derived from a meta-analysis of Cox regression values of each gene across the four training sets. In multivariable models, a higher sum value of the 24-gene signature was an independent predictor of shorter overall (OS) and event-free survival (EFS) in both training and validation sets (P < .01). Moreover, this signature could substantially improve the European LeukemiaNet (ELN) risk classification of AML, and patients in three new risk groups classified by the integrated risk classification showed significantly (P < .001) distinct OS and EFS.
Conclusion
Despite different treatment protocols applied to patients and use of different microarray platforms for expression profiling, a common prognostic gene signature was identified as an independent predictor of survival of patients with AML. The integrated risk classification incorporating this gene signature provides a better framework for risk stratification and outcome prediction than the ELN classification.
doi:10.1200/JCO.2012.44.3184
PMCID: PMC3595425  PMID: 23382473
12.  Activating FLT3 Mutants Show Distinct Gain-of-Function Phenotypes In Vitro and a Characteristic Signaling Pathway Profile Associated with Prognosis in Acute Myeloid Leukemia 
PLoS ONE  2014;9(3):e89560.
About 30% of patients with acute myeloid leukemia (AML) harbour mutations of the receptor tyrosine kinase FLT3, mostly internal tandem duplications (ITD) and point mutations of the second tyrosine kinase domain (TKD). It was the aim of this study to comprehensively analyze clinical and functional properties of various FLT3 mutants.
In 672 normal karyotype AML patients FLT3-ITD, but not FLT3-TKD mutations were associated with a worse relapse free and overall survival in multivariate analysis. In paired diagnosis-relapse samples FLT3-ITD showed higher stability (70%) compared to FLT3-TKD (30%). In vitro, FLT3-ITD induced a strong activating phenotype in Ba/F3 cells. In contrast, FLT3-TKD mutations and other point mutations – including two novel mutations – showed a weaker but clear gain-of-function phenotype with gradual increase in proliferation and protection from apoptosis. The pro-proliferative capacity of the investigated FLT3 mutants was associated with cell surface expression and tyrosine 591 phosphorylation of the FLT3 receptor. Western blot experiments revealed STAT5 activation only in FLT3-ITD positive cell lines, in contrast to FLT3-non-ITD mutants, which displayed an enhanced signal of AKT and MAPK activation. Gene expression analysis revealed distinct difference between FLT3-ITD and FLT3-TKD for STAT5 target gene expression as well as deregulation of SOCS2, ENPP2, PRUNE2 and ART3.
FLT3-ITD and FLT3 point mutations show a gain-of-function phenotype with distinct signalling properties in vitro. Although poor prognosis in AML is only associated with FLT3-ITD, all activating FLT3 mutations can contribute to leukemogenesis and are thus potential targets for therapeutic interventions.
doi:10.1371/journal.pone.0089560
PMCID: PMC3946485  PMID: 24608088
13.  FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia 
Blood Cancer Journal  2011;1(11):e42-.
The t(10;11)(p13;q14) translocation results in the fusion of the CALM (clathrin assembly lymphoid myeloid leukemia protein) and AF10 genes. This translocation is observed in acute myeloblastic leukemia (AML M6), acute lymphoblastic leukemia (ALL) and malignant lymphoma. Using a yeast two-hybrid screen, the four and a half LIM domain protein 2 (FHL2) was identified as a CALM interacting protein. Recently, high expression of FHL2 in breast, gastric, colon, lung as well as in prostate cancer was shown to be associated with an adverse prognosis. The interaction between CALM and FHL2 was confirmed by glutathione S-transferase-pulldown assay and co-immunoprecipitation experiments. The FHL2 interaction domain of CALM was mapped to amino acids 294–335 of CALM. The transcriptional activation capacity of FHL2 was reduced by CALM, but not by CALM/AF10, which suggests that regulation of FHL2 by CALM might be disturbed in CALM/AF10-positive leukemia. Extremely high expression of FHL2 was seen in acute erythroid leukemia (AML M6). FHL2 was also highly expressed in chronic myeloid leukemia and in AML with complex aberrant karyotype. These results suggest that FHL2 may play an important role in leukemogenesis, especially in the case of AML M6.
doi:10.1038/bcj.2011.40
PMCID: PMC3256755  PMID: 22829078
CALM; AF10; FHL2
14.  Up a lymphoid blind alley: Does CALM/AF10 disturb Ikaros during leukemogenesis? 
The Ikaros gene is required for normal development of lymphocytes and frequent intragenic deletions of Ikaros have been identified in acute lymphoblastic leukemia. However, little is known about the role of Ikaros in myeloid malignancies. Here we discuss the role of Ikaros as a lineage master regulator during the onset and progression of myeloid leukemias, namely CALM-AF10 positive acute myeloid leukemia and chronic myeloid leukemia. Alterations of Ikaros at the gene or protein level may act as a bi-directional lineage switch subverting developmental plasticity for malignant transformation. Finally, we propose that promiscuous signaling involving Ikaros and FOXO transcription factors might be a critical link between early lineage fate and uncontrolled proliferation.
doi:10.4331/wjbc.v2.i6.115
PMCID: PMC3159521  PMID: 21765976
Acute lymphoblastic leukemia; Acute myeloid leukemia; CALM/AF10; Chronic myeloid leukemia; Ikaros
15.  Elevated PIN1 expression by C/EBPα-p30 blocks C/EBPα induced granulocytic differentiation via c-Jun in AML 
The transcription factor C/EBPα plays an important role in granulopoiesis. The tumor suppressor function of C/EBPα is shown by the findings that loss of expression or function of C/EBPα in leukemic blasts contributes to a block in myeloid cell differentiation and to leukemia. C/EBPα mutations are found in around 9% of acute myeloid leukemia (AML) patients. The mechanism by which the mutant form of C/EBPα (C/EBPα-p30) exerts a differentiation block is not well understood. By using a proteomic screen, we have recently reported PIN1 as a target of C/EBPα-p30 in AML. In the present study, we show that C/EBPα-p30 induces PIN1 expression. We observed elevated PIN1 expression in leukemic patient samples. Induction of C/EBPα-p30 results in recruitment of E2F1 in the PIN1 promoter. We demonstrate that the inhibition of PIN1 leads to myeloid differentiation in primary AML blasts with C/EBPα mutations. Overexpression of PIN1 in myeloid cells leads to block of granulocyte differentiation. We also demonstrate that PIN1 increases the stability of the c-Jun protein by inhibiting c-Jun ubiquitination and c-Jun blocks granulocyte differentiation mediated by C/EBPα. Our data suggest the inhibition of PIN1 could be a potential strategy of treating AML patients with C/EBPα mutation.
doi:10.1038/leu.2010.37
PMCID: PMC2923485  PMID: 20376080
C/EBPα; PIN1; AML
16.  The CALM and CALM/AF10 interactor CATS is a marker for proliferation 
Molecular Oncology  2008;2(4):356-367.
The CATS protein was recently identified as a novel CALM interacting protein. CATS increases the nuclear and specifically the nucleolar localization of the leukemogenic CALM/AF10 fusion protein. We cloned and characterized the murine Cats gene. Detailed analysis of murine Cats expression during mouse embryogenesis showed an association with rapidly proliferating tissues. Interestingly, the Cats transcript is highly expressed in murine hematopoietic cells transformed by CALM/AF10. The CATS protein is highly expressed in leukemia, lymphoma and tumor cell lines but not in non‐proliferating T‐cells or human peripheral blood lymphocytes. CATS protein levels are cell cycle dependent and it is induced by mitogens, suggesting a role of CATS in the control of cell proliferation and possibly CALM/AF10‐mediated leukemogenesis.
doi:10.1016/j.molonc.2008.08.001
PMCID: PMC5527775  PMID: 19383357
CALM/AF10; Leukemia; Nucleolus; Proliferation; CATS
17.  Association Between a Prognostic Gene Signature and Functional Gene Sets 
Background
The development of expression-based gene signatures for predicting prognosis or class membership is a popular and challenging task. Besides their stringent validation, signatures need a functional interpretation and must be placed in a biological context. Popular tools such as Gene Set Enrichment have drawbacks because they are restricted to annotated genes and are unable to capture the information hidden in the signature’s non-annotated genes.
Methodology
We propose concepts to relate a signature with functional gene sets like pathways or Gene Ontology categories. The connection between single signature genes and a specific pathway is explored by hierarchical variable selection and gene association networks. The risk score derived from an individual patient’s signature is related to expression patterns of pathways and Gene Ontology categories. Global tests are useful for these tasks, and they adjust for other factors. GlobalAncova is used to explore the effect on gene expression in specific functional groups from the interaction of the score and selected mutations in the patient’s genome.
Results
We apply the proposed methods to an expression data set and a corresponding gene signature for predicting survival in Acute Myeloid Leukemia (AML). The example demonstrates strong relations between the signature and cancer-related pathways. The signature-based risk score was found to be associated with development-related biological processes.
Conclusions
Many authors interpret the functional aspects of a gene signature by linking signature genes to pathways or relevant functional gene groups. The method of gene set enrichment is preferred to annotating signature genes to specific Gene Ontology categories. The strategies proposed in this paper go beyond the restriction of annotation and deepen the insights into the biological mechanisms reflected in the information given by a signature.
PMCID: PMC2735947  PMID: 19812786
18.  A novel role for Lef-1, a central transcription mediator of Wnt signaling, in leukemogenesis 
Canonical Wnt signaling is critically involved in normal hematopoietic development and the self-renewal process of hematopoietic stem cells (HSCs). Deregulation of this pathway has been linked to a large variety of cancers, including different subtypes of leukemia. Lef-1 is a major transcription factor of this pathway and plays a pivotal role in lymphoid differentiation as well as in granulopoiesis. Here, we demonstrate Lef-1 expression in murine HSCs as well as its expression in human leukemia. Mice transplanted with bone marrow retrovirally transduced to express Lef-1 or a constitutive active Lef-1 mutant showed a severe disturbance of normal hematopoietic differentiation and finally developed B lymphoblastic and acute myeloid leukemia (AML). Lef-1–induced AMLs were characterized by immunoglobulin (Ig) DH-JH rearrangements and a promiscuous expression of lymphoid and myeloid regulatory factors. Furthermore, single cell experiments and limiting dilution transplantation assays demonstrated that Lef-1–induced AML was propagated by a leukemic stem cell with lymphoid characteristics displaying Ig DH-JH rearrangements and a B220+ myeloid marker− immunophenotype. These data indicate a thus far unknown role of Lef-1 in the biology of acute leukemia, pointing to the necessity of balanced Lef-1 expression for an ordered hematopoietic development.
doi:10.1084/jem.20071875
PMCID: PMC2275375  PMID: 18316418
19.  Block of C/EBPα function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations 
Mutations constitutively activating FLT3 kinase are detected in ∼30% of acute myelogenous leukemia (AML) patients and affect downstream pathways such as extracellular signal–regulated kinase (ERK)1/2. We found that activation of FLT3 in human AML inhibits CCAAT/enhancer binding protein α (C/EBPα) function by ERK1/2-mediated phosphorylation, which may explain the differentiation block of leukemic blasts. In MV4;11 cells, pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation. Differentiation of MV4;11 cells was also observed when C/EBPα mutated at serine 21 to alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was mutated to aspartate (S21D), which mimics phosphorylation of C/EBPα. Thus, our results suggest that therapies targeting the MEK/ERK cascade or development of protein therapies based on transduction of constitutively active C/EBPα may prove effective in treatment of FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies.
doi:10.1084/jem.20052242
PMCID: PMC2118199  PMID: 16446383
20.  The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice 
Journal of Clinical Investigation  2005;115(8):2159-2168.
The molecular characterization of leukemia has demonstrated that genetic alterations in the leukemic clone frequently fall into 2 classes, those affecting transcription factors (e.g., AML1-ETO) and mutations affecting genes involved in signal transduction (e.g., activating mutations of FLT3 and KIT). This finding has favored a model of leukemogenesis in which the collaboration of these 2 classes of genetic alterations is necessary for the malignant transformation of hematopoietic progenitor cells. The model is supported by experimental data indicating that AML1-ETO and FLT3 length mutation (FLT3-LM), 2 of the most frequent genetic alterations in AML, are both insufficient on their own to cause leukemia in animal models. Here we report that AML1-ETO collaborates with FLT3-LM in inducing acute leukemia in a murine BM transplantation model. Moreover, in a series of 135 patients with AML1-ETO–positive AML, the most frequently identified class of additional mutations affected genes involved in signal transduction pathways including FLT3-LM or mutations of KIT and NRAS. These data support the concept of oncogenic cooperation between AML1-ETO and a class of activating mutations, recurrently found in patients with t(8;21), and provide a rationale for therapies targeting signal transduction pathways in AML1-ETO–positive leukemias.
doi:10.1172/JCI24225
PMCID: PMC1174917  PMID: 16025155
21.  Oligomerization of the ABL tyrosine kinase by the Ets protein TEL in human leukemia. 
Molecular and Cellular Biology  1996;16(8):4107-4116.
TEL is a member of the Ets family of transcription factors which are frequently rearranged in human leukemia. The mechanism of TEL-mediated transformation, however, is unknown. We report the cloning and characterization of a chromosomal translocation associated with acute myeloid leukemia which fuses TEL to the ABL tyrosine kinase. The TEL-ABL fusion confers growth factor-independent growth to the marine hematopoietic cell line Ba/F3 and transforms Rat-1 fibroblasts and primary murine bone marrow cells. TEL-ABL is constitutively tyrosine phosphorylated and localizes to the cytoskeleton. A TEL-ABL mutant containing an ABL kinase-inactivating mutation is not constitutively phosphorylated and is nontransforming but retains cytoskeletal localization. However, constitutive phosphorylation, cytoskeletal localization, and transformation are all dependent upon a highly conserved region of TEL termed the helix-loop-helix (HLH) domain. TEL-ABL formed HLH-dependent homo-oligomers in vitro, a process critical for tyrosine kinase activation. These experiments suggest that oligomerization of TEL-ABL mediated by the TEL HLH domain is required for tyrosine kinase activation, cytoskeletal localization, and transformation. These data also suggest that oligomerization of Ets proteins through the highly conserved HLH domain may represent a previously unrecognized phenomenon.
PMCID: PMC231407  PMID: 8754809
23.  Breakpoint junctions of chromosome 9p deletions in two human glioma cell lines. 
Molecular and Cellular Biology  1994;14(11):7604-7610.
Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p21. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5' of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One- and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangement is discussed.
Images
PMCID: PMC359296  PMID: 7523863
24.  Elevated levels of glyoxylate shunt enzymes in Escherichia coli strains constitutive for fatty acid degradation. 
Journal of Bacteriology  1980;143(2):720-725.
Mutants of Escherichia coli K-12 constitutive for the synthesis of the enzymes of fatty acid degradation (fadR) have elevated levels of the glyoxylate shunt enzymes, isocitrate lyase and malate synthase. A temperature-sensitive fadR strain has high levels of glyoxylate shunt enzymes when grown at elevated temperatures but has low, inducible levels of glyoxylate shunt enzymes when grown at low temperatures. The increased activity of glyoxylate shunt enzymes did not appear to be due to the degradation of intracellular fatty acids in fadR strains or differences in allosteric effectors in fadR versus fadR+ strains. These studies suggest that the fadR gene product may be involved in the regulation of the glyoxylate operon.
PMCID: PMC294349  PMID: 7009561
25.  Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic 
Molecular Biology of the Cell  1999;10(8):2687-2702.
The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure–function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP–CALM was targeted to the plasma membrane–coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP–CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP–CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin–CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.
PMCID: PMC25500  PMID: 10436022

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