Chung H. Kok

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.

Ecotopic viral integration site 1 (EVI1) regulates multiple cellular processes important for cancer and is a synergistic partner for FOS protein in invasive tumors

Ecotropic viral integration site 1 (EVI1) is an oncogenic dual domain zinc finger transcription factor that plays an essential role in the regulation of hematopoietic stem cell renewal, and its overexpression in myeloid leukemia and epithelial cancers is associated with poor patient survival. Despite the discovery of EVI1 in 1988 and its emerging role as a dominant oncogene in various types of cancer, few EVI1 target genes are known. This lack of knowledge has precluded a clear understanding of exactly how EVI1 contributes to cancer. Using a combination of ChIP-Seq and microarray studies in human ovarian carcinoma cells, we show that the two zinc finger domains of EVI1 bind to DNA independently and regulate different sets of target genes. Strikingly, an enriched fraction of EVI1 target genes are cancer genes or genes associated with cancer. We also show that more than 25% of EVI1-occupied genes contain linked EVI1 and activator protein (AP)1 DNA binding sites, and this finding provides evidence for a synergistic cooperative interaction between EVI1 and the AP1 family member FOS in the regulation of cell adhesion, proliferation, and colony formation. An increased number of dual EVI1/AP1 target genes are also differentially regulated in late-stage ovarian carcinomas, further confirming the importance of the functional cooperation between EVI1 and FOS. Collectively, our data indicate that EVI1 is a multipurpose transcription factor that synergizes with FOS in invasive tumors.

Genetic regulators of myelopoiesis and leukemic signaling identified by gene profiling and linear modeling

Mechanisms controlling the balance between proliferation and self‐renewal versus growth suppression and differentiation during normal and leukemic myelopoiesis are not understood. We have used the bi‐potent FDB1 myeloid cell line model, which is responsive to myelopoietic cytokines and activated mutants of the granulocyte macrophage‐colony stimulating factor (GM‐CSF) receptor, having differential signaling and leukemogenic activity. This model is suited to large‐scale gene‐profiling, and we have used a factorial time‐course design to generate a substantial and powerful data set. Linear modeling was used to identify gene‐expression changes associated with continued proliferation, differentiation, or leukemic receptor signaling. We focused on the changing transcription factor profile, defined a set of novel genes with potential to regulate myeloid growth and differentiation, and demonstrated that the FDB1 cell line model is responsive to forced expression of oncogenes identified in this study. We also identified gene‐expression changes associated specifically with the leukemic GM‐CSF receptor mutant, V449E. Signaling from this receptor mutant down‐regulates CCAAT/enhancer‐binding protein α (C/EBPα) target genes and generates changes characteristic of a specific acute myeloid leukemia signature, defined previously by gene‐expression profiling and associated with C/EBPα mutations.

The granulocyte-associated transcription factor Krüppel-like factor 5 is silenced by hypermethylation in acute myeloid leukemia

Krüppel-like factor 5 (KLF5) has been implicated as a tumor suppressor in various solid tumors such as breast and prostate, and recent studies have demonstrated a role for this protein in neutrophil differentiation of acute promyelocytic leukemia cells in response to ATRA. Here, we show that KLF5 expression increases during primary granulocyte differentiation and that expression of KLF5 is a requirement for granulocyte differentiation of 32D cells. In AML, we show that KLF5 mRNA expression levels are reduced in multiple French-American-British subtypes compared to normal controls, and also in leukemic stem cells relative to normal hematopoietic stem cells. We demonstrate that in selected AML cases, reduced expression is associated with hypermethylation of the KLF5 locus in the proximal promoter and/or intron 1, suggesting that this may represent a Class II genetic lesion in the development of AML.

The GM-CSF receptor utilizes β-catenin and Tcf4 to specify macrophage lineage differentiation

Granulocyte-macrophage colony stimulating factor (GM-CSF) promotes the growth, survival, differentiation and activation of normal myeloid cells and is essential for fully functional macrophage differentiation in vivo. To better understand the mechanisms by which growth factors control the balance between proliferation and self-renewal versus growth-suppression and differentiation we have used the bi-potent FDB1 myeloid cell line, which proliferates in IL-3 and differentiates to granulocytes and macrophages in response to GM-CSF. This provides a manipulable model in which to dissect the switch between growth and differentiation. We show that, in the context of signaling from an activating mutant of the GM-CSF receptor β subunit, a single intracellular tyrosine residue (Y577) mediates the granulocyte fate decision. Loss of granulocyte differentiation in a Y577F second-site mutant is accompanied by enhanced macrophage differentiation and accumulation of β-catenin together with activation of Tcf4 and other Wnt target genes. These include the known macrophage lineage inducer, Egr1. We show that forced expression of Tcf4 or a stabilised β-catenin mutant is sufficient to promote macrophage differentiation in response to GM-CSF and that GM-CSF can regulate β-catenin stability, most likely via GSK3β. Consistent with this pathway being active in primary cells we show that inhibition of GSK3β activity promotes the formation of macrophage colonies at the expense of granulocyte colonies in response to GM-CSF. This study therefore identifies a novel pathway through which growth factor receptor signaling can interact with transcriptional regulators to influence lineage choice during myeloid differentiation.

Interleukin‐3‐mediated regulation of β‐catenin in myeloid transformation and acute myeloid leukemia

Aberrant activation of β‐catenin is a common event in AML and is an independent predictor of poor prognosis. Although increased β‐catenin signaling in AML has been associated with oncogenic translocation products and activating mutations in the FLT3R, the mechanisms that activate β‐catenin in AML more broadly are still unclear. Here, we describe a novel link between IL‐3 signaling and the regulation of β‐catenin in myeloid transformation and AML. In a murine model of HoxB8 and IL‐3 cooperation, we show that β‐catenin protein levels are modulated by IL‐3 and that Cre‐induced deletion of β‐catenin abolishes IL‐3‐dependent growth and colony formation. In IL‐3‐dependent leukemic TF‐1.8 cells, we observed increased β‐catenin protein levels and nuclear localization in response to IL‐3, and this correlated with transcriptional induction of β‐catenin target genes. Furthermore, IL‐3 promoted β‐catenin accumulation in a subset of AML patient samples, and gene‐expression profiling of these cells revealed induction of WNT/β‐catenin and TCF4 gene signatures in an IL‐3‐dependent manner. This study is the first to link β‐catenin activation to IL‐3 and suggests that targeting IL‐3 signaling may be an effective approach for the inhibition of β‐catenin activity in some patients with AML.

Methylation of KLF5 contributes to reduced expression in acute myeloid leukaemia and is associated with poor overall survival.

Sonya M. Diakiw, Michelle Perugini, Chung H. Kok, Grant A. Engler, Nik Cummings, Luen Bik To, Andrew H. Wei, Ian D. Lewis, Anna L. Brown, Richard J. D’Andrea

p53-Dependent transcriptional responses to interleukin-3 signaling.

p53 is critical in the normal response to a variety of cellular stresses including DNA damage and loss of p53 function is a common feature of many cancers. In hematological malignancies, p53 deletion is less common than in solid malignancies but is associated with poor prognosis and resistance to chemotherapy. Compared to their wild-type (WT) counterparts, hematopoietic progenitor cells lacking p53 have a greater propensity to survive cytokine loss, in part, due to the failure to transcribe Puma, a proapoptotic Bcl-2 family member. Using expression arrays, we have further characterized the differences that distinguish p53−/− cells from WT myeloid cells in the presence of Interleukin-3 (IL-3) to determine if such differences contribute to the increased clonogenicity and survival responses observed in p53−/− cells. We show that p53−/− cells have a deregulated intracellular signaling environment and display a more rapid and sustained response to IL-3. This was accompanied by an increase in active ERK1/2 and a dependence on an intact MAP kinase signaling pathway. Contrastingly, we find that p53−/− cells are independent on AKT for their survival. Thus, loss of p53 in myeloid cells results in an altered transcriptional and kinase signaling environment that favors enhanced cytokine signaling.

The preferential occurrence of FLT3-TKD mutations in inv(16) AML and impact on survival outcome: a combined analysis of 1053 core-binding factor AML patients

Chung H. Kok, Anna L. Brown, Michelle Perugini, Diana G. Iarossi, Ian D. Lewis, Richard J. D’Andrea

High prevalence of relapse in children with Philadelphia-like acute lymphoblastic leukemia despite risk-adapted treatment

Acute lymphoblastic leukemia (ALL) remains a leading cause of cancer-related death in children and young adults. Since the 1960s, improvements in the treatment of children with ALL have led to 10-year survival rates now exceeding 85%.[1][1] Philadelphia-like (Ph-like) ALL is characterized by a gene