The Rho family of small GTP-binding proteins control a variety of signaling pathways regulating cytoskeleton organization, proliferation, adhesion and migration in eukaryotic cells. Aberrant signaling of these molecules has been implicated in promoting transformation and metastasis in human cancers. RhoH, a hematopoietic- specific member of the RhoE subfamily, was identified as a hypermutable gene locus in human B cell derived in malignancies. Importantly, RhoH, is GTPase-deficient and therefore remains in an active, GTP-bound state. Thus activity of RhoH is likely dependent, at least in part, on expression levels of the protein in the cell. In the previous funding period we have demonstrated that RhoH is critical for T cell development and T cell receptor signaling. We have identified a unique ITAM-like motif in RhoH that also plays a role in the regulation of RhoH function, via interaction with ZAP-70 and facilitation of its localization to the T cell immunological synapse. Since ZAP-70 is a prognostic indicator in chronic lymphocytic leukemia (CLL), we hypothesized that RhoH function may be critical in CLL cell behavior. In preliminary studies we demonstrate a significant correlation between RhoH and ZAP-70 expression in a large number of primary CLL samples. In addition, in a transgenic murine model of CLL, genetic knock-out of RhoH significantly delays the development of abnormal CD5+IgM+ cells in the bone marrow, spleen and peritoneal cavity. Using gene-targeting approaches, we have previously also demonstrated that Rac1 and Rac2 are required for hematopoietic stem cell proliferation, survival and adhesion, and therefore are critical for stem/progenitor cell localization within the hematopoietic microenvironment and trafficking of mature blood cell elements. Previous work by us and others have demonstrated that RhoH negatively regulates Rac activity in hematopoietic cells. Therefore, we hypothesize that RhoH and Rac crosstalk is critical for regulation of signaling pathways mediating normal and malignant lymphocyte functions. This crosstalk likely affects the activation of downstream targets of Rac. In myeloid cells, we have shown that RhoH functions via inhibition of activated Rac translocation to the membrane and this function is dependent on the conserved CaaX prenylation sequence in the carboxy-terminus of the RhoH protein. Thus, overall, we hypothesize that functions of RhoH vary with respect to cell lineage and are dependent on distinct motifs within the RhoH protein. Our goals are to elucidate the role(s) of RhoH and mechanism(s) by which RhoH controls growth, adhesion/migration and transformation and related intracellular signaling pathways in normal and leukemic hematopoietic cells.

Public Health Relevance

Recurrent chromosomal rearrangements and somatic hypermutation in the RhoH gene locus have been observed in NHLs, PCNSLs and DLBCLs, all B cell derived maligancies. RhoH is the first member of the Rho GTPase family to be reported with a high frequency of somatic mutations (up to 46%) in human cancers. However, the role(s) that RhoH plays in malignant processes remain unknown, although our studies and those of others have implicated RhoH as a negative regulator of Rac GTPase. Our recent studies have also implicated RhoH interaction with ZAP-70 in normal T cells and in CLL cells. In this grant, we propose to elucidate and compare the role and mechanism of RhoH in controlling cell growth, survival, transformation and adhesion/migration, and identify motifs determining functional interactions and interacting proteins of RhoH. Also, we will assess RhoH expression levels quantitatively and their correlations with ZAP-70 expression and cell function in primary human CLL cells and a murine model of CLL. These studies may provide critical clues to the molecular basis of RhoH function in hematologic malignancies and possibly establish an animal model for Rho GTPases in tumorigenesis. Given the lineage-restricted expression of RhoH such an observation could have important therapeutic implication. c: documents and settings destouche local settings temporary internet files olk149 project narrative.doc

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA113969-10
Application #
8629703
Study Section
Hematopoiesis Study Section (HP)
Program Officer
Mufson, R Allan
Project Start
2005-05-20
Project End
2015-03-31
Budget Start
2014-05-09
Budget End
2015-03-31
Support Year
10
Fiscal Year
2014
Total Cost
$315,782
Indirect Cost
$134,298
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
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Chae, Hee-Don; Siefring, Jamie E; Hildeman, David A et al. (2010) RhoH regulates subcellular localization of ZAP-70 and Lck in T cell receptor signaling. PLoS One 5:e13970
Sanchez-Aguilera, A; Rattmann, I; Drew, D Z et al. (2010) Involvement of RhoH GTPase in the development of B-cell chronic lymphocytic leukemia. Leukemia 24:97-104
Cancelas, Jose A; Williams, David A (2009) Rho GTPases in hematopoietic stem cell functions. Curr Opin Hematol 16:249-54
Chae, Hee-Don; Lee, Katherine E; Williams, David A et al. (2008) Cross-talk between RhoH and Rac1 in regulation of actin cytoskeleton and chemotaxis of hematopoietic progenitor cells. Blood 111:2597-605
Gu, Yi; Chae, Hee-Don; Siefring, Jamie E et al. (2006) RhoH GTPase recruits and activates Zap70 required for T cell receptor signaling and thymocyte development. Nat Immunol 7:1182-90