The broad goal of our research program is to understand how mutations of transcriptional regulators may set up patterns of aberrant gene expression that yield cancer. This requires a detailed understanding of the normal function of these transcription factors. Such information includes an understanding of the role of these factors in cell growth, differentiation and developmental, the normal DNA binding and transcriptional activity of these proteins, identification of the critical protein partners of the factors and elucidation of the downstream targets of these genes. The mutations that occur in cancer are tragic experiments of nature that may alter critical amino acid residues for the function of the proteins. By modeling the function of both the normal and mutated forms of these factors we hope to better understand the molecular basis of cancer and potentially identify new therapeutic targets and pathways in this disease.
The Molecular Basis of Wilms' Tumor The WT1 gene was one of the earliest tumor suppressor genes identified. WT1 is mutated in a significant proportion of Wilms' tumor and was recently implicated in the pathogenesis of leukemia. WT1 protein is a gene regulator with a controlling role in normal kidney development, normally up-regulated as the glomerulus develops from a primitive mesenchymal precursor. To date we found that WT1 can act as both a transcriptional repressor and an activator. We currently believe that transcriptional activation may be the most critical function of the protein as we identified three tumor-associated missense mutants of the protein which were competent for transcriptional repression but could not activate target promoters and could not suppress cell growth. We are continuing to identify Wt1 target genes relevant for kidney development and tumor suppression using Gain of function and loss of function models, Affymetrix arrays and chromatin immunoprecipitation assays
The Sprouty Proteins The sprouty1 gene is expressed in the developing kidney in a pattern overlapping with the WT1 gene. We found that sprouty inhibits the activation of MAP kinase and likely represents a counter-regulatory signaling molecule that limits the effects of signaling through receptor tyrosine kinases. We demonstrated that Sprouty1 and Sprouty2 inhibit RTK signaling at the level of Ras activation. Current studies focus on knockouts of the Spry genes and the effects of these genes on animal development and signal transduction.
The Molecular Biology of Acute Promyelocytic Leukemia Acute promyeloctyic leukemia (APL) is an unusual form of leukemia which can be treated with retinoic acid. All patient with this disease had a consistent molecular defect in the retinoic acid receptor, in which the protein is fused to another gene product known as PML. Our lab studies a variant form of APL, which was shown to be highly resistant to retinoic acid or conventional chemotherapy. In this syndrome the retinoic acid receptor is linked to a different protein known as PLZF, which in turn is a DNA-binding repressor of gene expression. We have shown that PLZF is high expressed in early hematopoietic cells, growth suppresses a number of different cell lines and can repress the expression of the regulator of cell division cyclin A. We have characterized the cognate DNA binding sequences of PLZF and found that the protein contains a conserved self-association and repression domain called the BTB/POZ domain. Working with Dr. Gil Prive of the Ontario Cancer Institute, who successfully crystallized this domain we have made a number of single and two amino acid mutations in the domain which can break up dimerization by the protein or preserve dimerization and destroy the ability of the protein to repress gene transcription. We are identifying the normal targets of the PLZF gene. We have created a cell line with inducible expression of PLZF and have used Affymetrix and glass slide arrays to reveal that c-myc and myc target genes are regulated by PLZF. Opther studies in Porgress include knockdowns of PLZF in hematopoietic cells, and chromatin IP assays.
The Molecular Basis of Multiple Myeloma Multiple myeloma (MM) one of the commonest hematological malignancies represents the malignant transformation of plasma cells. For many years the pathogenesis of MM was quite obscure, but over the past decade there has been progress based upon the characterization of consistent chromosomal translocations in MM involving the immunoglobulin heavy chain (IgH). These translocations implicate particular genes in the pathogenesis of myeloma. MMSET (MULTIPLE MYELOMA SET DOMAIN) gene was identified at the breakpoint of the t(4;14) translocation, present in 15-20% of multiple myeloma. MMSET has a SET domain previously identified in histone methyl transferases. We demonstrated that the MMSET protein is strikingly overexpressed in myeloma cells harboring the t(4;14) translocation. Our preliminary data indicate that MMSET has proprieties of a transcriptional co-factor, including nuclear localization, the ability to bind to sequence specific transcription factors 1, transcriptional co-factors and histone deacetylases. Furthermore we found that MMSET has histone methyl transferase activity, modifying histone H3 and H4. These data lead to our overarching hypothesis that aberrant overexpression of MMSET leads to deregulated gene expression in B cells, contributing to the pathogenesis of myeloma.
Molecular Basis of Myeloproliferative Disease Hematological malignancies display a spectrum of phenotypes. At one end is acute leukemia, characterized by proliferation of a population of cells blocked in their differentiation. Myelodysplasia is characterized by blocked differentiation and intramedullary cell death, leading to pancytopenia. In contrast, the myeloproliferative disorders (MPD) are characterized by an excess of well-differentiated cells. Chronic myelogenous leukemia, the archetypal MPD, is associated with the constitutively activated BCR-ABL tyrosine kinase that is specifically targeted by Imatimib. There are a spectrum of MPD represented by aberrant accumulation of each of the respective components of terminally differentiated myeloid lineage cells. These include CML, chronic myelomonocytic leukemia (CMML), agnogenic myeloid metaplasia (AMM) polycythemia vera (PV), essential thrombocythemia (ET), hypereosinophilic syndrome (HES), and systemic mast cell disease (SMCD). Polycythemia vera (PV), a MPD characterized by accumulation of erythrocytes is virtually always associated with a consitutive activating mutation of JAK2 tyrosine kinase. Currently we are 1) Comparing the gene expression profiles of CD34+ cells from PV patients with the profile generated by inserting of mutant JAK2 into human CD34+ cells in culture, 2) Searching for areas of chromosomal changes in PV, MM and ET patients using Affymetrix SNP Chips ; these may represent secondary changes in these diseas. 3) Comparing and contrasting gene expression changes mediated by normal erythropoetic/Jak2 signalign with mutant Jak2 dignalin in terms of proliferation and cell survival pathways. 4) Modeling the action of other activating kinase mutations in MPDs.
Defeating Transcriptional repression in Leukemia-Translational studies The overall hypothesis of this line of investigation is that aberrant transcriptional repression is a root cause of many leukemias and lymphomas. We have begun an investigator initiated clinical trial using the histone deacetylase inhibitor valproic acid in order to stimulate expression of differentiation-associated genes in leukemia in combination with retinoic acid. Target genes of this combination are being identified in a cell line system and will be validated in tissue specimens.