Theodora S. Ross - US grants

DESCRIPTION (Applicant's Description): The TEL-PDGFBR fusion protein was identified as the protein product of a t(5;12) translocation in a patient with chronic myelomonocytic leukemia (CMML). The protein fuses the amino portion of TEL with the transmembrane and cytoplasmic domains of the PDGFBR. TEL, a member of the ETS family of transcription factors, has subsequently been described as a common site of rearrangement in multiple forms of leukemia. This is not yet the case for the PDGFBR. The applicants find, however, in the Preliminary Results, that another patient with CMML has a novel t(5;7) translocation. Southern blotting analysis has identified a breakpoint in this patient at the same genomic localization in the PDGFBR as the t(5;12) translocation. Their hypothesis is, that in this patient, as for the t(5;12) TEL-PDGFR patients, PDGFBR is constitutively activated by fusion with a 7q24 partner. Although rare (as for identification of TEL), the PDGFBR fusion partner at 7q24 may identify a gene involved in a broader group of malignancies. Also, in light of the facts that other patients with CMML have PDGFR containing fusions and the region of 7q24 is frequently deleted in MDS, the cloning, characterization and manipulation of this fusion protein is paramount. Hence, in Specific Aim 1, they will use anchored PCR to clone the breakpoint. Their specific oligos will come from the known PDGFBR sequence. In Specific Aim 2, they will obtain a full length cDNA and determine the relevance of this by performing ribonuclease protection assays and mapping back to chromosome 7. Finally, for Specific Aim 3 they propose to characterize the fusion protein by determining its transforming, activity(s) and biological properties using mutational and biochemical analyses.

chimeric proteins; oncoproteins; cell transformation; protein tyrosine kinase; neoplastic transformation; disease /disorder model; chromosome translocation; biological signal transduction; bone marrow transplantation; platelet derived growth factor; chronic myelogenous leukemia; gene targeting; laboratory mouse; immunofluorescence technique; cell line;

Huntingtin Interacting Protein 1 (HIP1) is a clathrin and inositol lipid binding protein that may be involved in neurodegeneration by virtue of its interaction with huntingtin, the protein mutated in Huntington's disease. It is also associated with leukemia by discovery of the oncogenic HIP1/PDGF_R fusion protein that resulted from a t(5;7) chromosomal translocation in a patient with chronic myelomonocytic leukemia (CMML). We hypothesize that HIP1 is involved in tumorigenesis for three additional reasons. First, the HIP1 portion of the HIP1/PDGFI3R fusion protein is necessary for cellular transformation. Second, HIP1 is over-expressed in multiple tumors (preliminary data section). Third, expression of a dominant negative mutant of HIP1 (preliminary data section) or genetic deletion of HIP1 leads to apoptosis. The first hypothesis we propose to test is that HIP 1 is tumorigenic and its over-expression in vivo leads to cancer. As a corollary, we predict that when HIP1 is not expressed, there will be a diminished susceptibility to the development of cancer. Second, we propose to test the hypothesis that HIP lr complements HIP 1 function(s) in endocytosis, cell growth and carcinogenesis. Finally, we propose to test the hypothesis that regulation of clathrin mediated trafficking by HIP1 and HIPlr results in an increase in growth factor receptor (GFR) signaling. We suggest that this maybe accomplished by increasing the number of the cell surface receptors to increase sensitivity to growth factor and thereby promote cellular survival and/or growth.

Huntingtin Interacting Protein 1 (HIP1) is a clathrin, actin and inositol lipid binding protein that has been implicated in neurodegeneration by virtue of its interaction with huntingtin, the protein mutated in Huntington's disease. It is also associated with leukemia by our discovery of the oncogenic HIP1/PDGF¿R fusion protein that resulted from a t(5;7) chromosomal translocation in a patient with chronic myelomonocytic leukemia (Ross et al., 1998). We hypothesize that HIP1 is involved in tumorigenesis for several additional reasons. First, the HIP1 portion of the HIP1/PDGF¿R fusion protein is necessary for cellular transformation (Ross and Gilliland, 1999). Second, HIP1 is upregulated in multiple tumors (Rao et al., 2002). Third, expression of a dominant negative mutant of HIP1 or genetic deletion of HIP1 leads to apoptosis in several cell types including tumor cells (Rao et al., 2002 and 2003). Fourth, HIP1 deficiency inhibits prostate tumorigenesis (Bradley et al., 2005) and finally, overexpression of HIP1 in fibroblasts transforms them (Rao et al., 2003). The first hypothesis we propose to test is that different types of HIP1 mutations (coding, splicing or over-expression) transform primary cells in vivo. As a corollary, we predict that when HIP1 is not expressed, there will be a diminished susceptibility to the development of cancer in vivo. Second, will determine if the deficiency of the only known mammalian homologue of HIP1, HIP1-related (HIP1r), modifys HIP1's role in tumorigenesis. Using mice with targeted mutations in HIP1 and HIP1r, we have found that HIP1 and HIP1r compensate for one another (preliminary data section). We therefore predict that loss of HIP1r expression would inhibit HIP1 mediated transformation and gain of HIP1r expression would promote HIP1 mediated transformation. Third, we propose to investigate how HIP1 and its mutant forms change endocytic, actin and signal transduction pathways to promote neoplastic proliferation.

PROJECT SUMMARY Role of BRCA1 in normal and neoplastic bone marrow cells We have recently discovered that Brca1 is necessary for normal hematopoiesis, but mutation of this gene in hematopoietic cells rarely predispose carriers to leukemia. We have collected a number of convergent data sets that serve as the foundation for the studies proposed here. First, we have found that humans heterozygous for BRCA1 mutations may have an increased risk for chemotherapy-associated febrile neutropenia. Second, mice deficient for Brca1 in the hematopoietic system experience bone marrow failure associated with severe hematopoietic stem cell and progenitor defects, and third, mice heterozygous for Brca1 deficiency have slight defects in bone marrow reconstitution due to problematic functional hematopoietic stem cell activity. From these data, we have developed two hypotheses that we propose to test: BRCA1 is required for normal hematopoiesis and BRCA1 plays a role in emergency granulopoiesis, which is the acute response of hematopoietic progenitors to infection or other stressors. We propose that BRCA1 is regulated by interferon regulatory factor 8 (IRF8) expression, which is induced by infections or other stresses that lead to emergency granulopoiesis. Interestingly, an absolute requirement of BRCA1 for hematopoiesis may explain why people with BRCA1 mutations do not have an increased risk for leukemia: their bone marrow stem and progenitor cells die without BRCA1 before these cells have a chance to transform. Here, we will first identify the cellular and molecular events required for Brca1 to maintain normal hematopoiesis and examine the phenotypes of different human BRCA1 mutations using a humanized Brca1 mouse model. We will also conduct a study with additional patients from our cancer genetics clinic to examine the relationship between BRCA1 mutations and hematopoietic toxicity. Finally, we will examine the increases in BRCA1 levels during stress granulopoiesis and BRCA1 function in this pathway. This area of investigation is unexplored, and our results will facilitate a better understanding of the requirements for Brca1 in normal and neoplastic hematopoiesis and the chemotherapy toxicities in patients, ultimately leading to improved treatment of hereditary breast and ovarian cancer syndrome patients as well as insight into the tissue specificity of BRCA1 mutation-associated tumorigenesis.