Catherine L. Carmichael

Heritable GATA2 mutations associated with familial myelodysplastic syndrome and acute myeloid leukemia

We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies.

Integrative analysis of RUNX1 downstream pathways and target genes

BackgroundThe 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.ResultsHere 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.ConclusionThis 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.

Novel RUNX1 mutations in familial platelet disorder with enhanced risk for acute myeloid leukemia: clues for improved identification of the FPD/AML syndrome.

Novel RUNX1 mutations in familial platelet disorder with enhanced risk for acute myeloid leukemia: clues for improved identification of the FPD/AML syndrome

Trisomy of Erg is required for myeloproliferation in a mouse model of Down syndrome.

Down syndrome is characterized by multiple phenotypic manifestations associated with trisomy of chromosome 21. The transient myeloproliferative disorder and acute megakaryocytic leukemia associated with Down syndrome are uniquely associated with mutations in the transcription factor GATA1; however, the identity of trisomic genes on chromosome 21 that predispose to these hematologic disorders remains unknown. Using a loss-of-function allele, we show that specific reduction to functional disomy of the Erg gene corrects the pathologic and hematologic features of myeloproliferation in the Ts(17(16))65Dn mouse model of Down syndrome, including megakaryocytosis and progenitor cell expansion. Our data provide genetic evidence establishing the need for Erg trisomy for myeloproliferation in Ts(17(16))65Dn mice and imply that increased ERG gene dosage may be a key consequence of trisomy 21 that can predispose to malignant hematologic disorders in Down syndrome.

Hematopoietic defects in the Ts1Cje mouse model of Down syndrome.

Down syndrome (DS) persons are born with various hematopoietic abnormalities, ranging from relatively benign, such as neutrophilia and macrocytosis, to a more severe transient myeloproliferative disorder (TMD). In most cases, these abnormalities resolve in the first few months to years of life. However, sometimes the TMD represents a premalignant disease that develops into acute megakaryocytic leukemia (AMKL), usually in association with acquired GATA1 mutations. To gain insight into the mechanisms responsible for these abnormalities, we analyzed the hematopoietic development of the Ts1Cje mouse model of DS. Our analyses identified defects in mature blood cells, including macrocytosis and anemia, as well as abnormalities in fetal liver and bone marrow stem and progenitor cell function. Despite these defects, the Ts1Cje mice do not develop disease resembling either TMD or AMKL, and this was not altered by a loss of function allele of Gata1. Thus, loss of Gata1 and partial trisomy of chromosome 21 orthologs, when combined, do not appear to be sufficient to induce TMD or AMKL-like phenotypes in mice.

ZEB2 drives immature T-cell lymphoblastic leukaemia development via enhanced tumour-initiating potential and IL-7 receptor signalling

Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2-driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R. This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2-driven mouse model.

Hematopoietic overexpression of the transcription factor Erg induces lymphoid and erythro-megakaryocytic leukemia

The transcription factor encoded by the E-twenty-six (ETS)-related gene, ERG, is an essential regulator of hematopoietic stem cell function and a potent human oncoprotein. Enforced expression of ERG in murine hematopoietic cells leads to the development of a well-characterized lymphoid leukemia and a less well-defined non lymphoid disease. To clarify the latter, we generated murine bone marrow chimeras with enforced Erg expression in engrafted hematopoietic progenitor cells. As expected, these mice developed lymphoid leukemia. However, the previously reported non lymphoid disease that developed was shown to be a uniform, transplantable leukemia with both erythroid and megakaryocytic characteristics. In vivo, this disease had the overall appearance of an erythroleukemia, with an accumulation of immature erythroblasts that infiltrated the bone marrow, spleen, liver, and lung. However, when stimulated in vitro, leukemic cell clones exhibited both erythroid and megakaryocytic differentiation, suggesting that transformation occurred in a bipotential progenitor. Thus, in mice, Erg overexpression induces the development of not only lymphoid leukemia but also erythro-megakaryocytic leukemia.

Variability of Inducible Expression across the Hematopoietic System of Tetracycline Transactivator Transgenic Mice

The tetracycline (tet)-regulated expression system allows for the inducible overexpression of protein-coding genes, or inducible gene knockdown based on expression of short hairpin RNAs (shRNAs). The system is widely used in mice, however it requires robust expression of a tet transactivator protein (tTA or rtTA) in the cell type of interest. Here we used an in vivo tet-regulated fluorescent reporter approach to characterise inducible gene/shRNA expression across a range of hematopoietic cell types of several commonly used transgenic tet transactivator mouse strains. We find that even in strains where the tet transactivator is expressed from a nominally ubiquitous promoter, the efficiency of tet-regulated expression can be highly variable between hematopoietic lineages and between differentiation stages within a lineage. In some cases tet-regulated reporter expression differs markedly between cells within a discrete, immunophenotypically defined population, suggesting mosaic transactivator expression. A recently developed CAG-rtTA3 transgenic mouse displays intense and efficient reporter expression in most blood cell types, establishing this strain as a highly effective tool for probing hematopoietic development and disease. These findings have important implications for interpreting tet-regulated hematopoietic phenotypes in mice, and identify mouse strains that provide optimal tet-regulated expression in particular hematopoietic progenitor cell types and mature blood lineages.

Transposon mutagenesis reveals cooperation of ETS family transcription factors with signaling pathways in erythro-megakaryocytic leukemia

To define genetic lesions driving leukemia, we targeted cre-dependent Sleeping Beauty (SB) transposon mutagenesis to the blood-forming system using a hematopoietic-selective vav 1 oncogene (vav1) promoter. Leukemias of diverse lineages ensued, most commonly lymphoid leukemia and erythroleukemia. The inclusion of a transgenic allele of Janus kinase 2 (JAK2)V617F resulted in acceleration of transposon-driven disease and strong selection for erythroleukemic pathology with transformation of bipotential erythro-megakaryocytic cells. The genes encoding the E-twenty-six (ETS) transcription factors Ets related gene (Erg) and Ets1 were the most common sites for transposon insertion in SB-induced JAK2V617F-positive erythroleukemias, present in 87.5% and 65%, respectively, of independent leukemias examined. The role of activated Erg was validated by reproducing erythroleukemic pathology in mice transplanted with fetal liver cells expressing translocated in liposarcoma (TLS)-ERG, an activated form of ERG found in human leukemia. Via application of SB mutagenesis to TLS-ERG–induced erythroid transformation, we identified multiple loci as likely collaborators with activation of Erg. Jak2 was identified as a common transposon insertion site in TLS-ERG–induced disease, strongly validating the cooperation between JAK2V617F and transposon insertion at the Erg locus in the JAK2V617F-positive leukemias. Moreover, loci expressing other regulators of signal transduction pathways were conspicuous among the common transposon insertion sites in TLS-ERG–driven leukemia, suggesting that a key mechanism in erythroleukemia may be the collaboration of lesions disturbing erythroid maturation, most notably in genes of the ETS family, with mutations that reduce dependence on exogenous signals.

Transgenic, inducible RNAi in megakaryocytes and platelets in mice

Summary  Background: RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)‐regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. Objective: We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. Methods and results: Using a tet‐regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet‐on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV‐rtTA mice) display inducible reporter gene expression with unusual and near‐complete efficiency in megakaryocytes and platelets. To test whether the CMV‐rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet‐regulated short hairpin RNA (shRNA) targeting Bcl‐xL, a pro‐survival Bcl‐2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl‐xL in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl‐xL levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl‐xL, clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. Conclusions: We have established a novel transgenic strategy for inducible gene knockdown in megakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice.