Overall PPG Objectives and Points of Integration for Project 3: The central theme of this program project grant application is to clarify the molecular basis ofthe Fanconi anemia phenotype and to exploit this knowledge to identify strategies that will reduce morbidity and mortality in children and adults with this disease. Project 3 focuses largely on the hematopoiefic phenotype seeking define roles of FA proteins in three signal transduction pathways and to test (directly in hematopoietic cells) the notion that FA defects lead to dysfunction of these pathways consequently to bone marrow failure.(14-16) Since this project was last reviewed, we have developed a nano-immunoassay that will permit us to test the validity of our model of STAT5 dysfunction in FA stem cells. We have also developed and validated two short-term in vitro assays suitable for use in our shared small molecular screening efforts one of which is derived from a method that we had already published(l) and the second of which has been submitted for review to the journal Blood (Appendix). In fact, using the latter assay we have used the primary screening method to screen 50 small molecules and have identified agents that both enhance FANCC-/- hematopoietic stem cell (HSC) survival and reduce the release of TNFa by FANCC deficient mononuclear phagocytes stimulated with toll-like receptor 8 (TLRS) agonists. Because these assays are robust and quantitative, they have supplanted the assay (based upon defective STAT5 activation) we had proposed in the last application. All ofthe projects and cores share two objectives;to identify small molecules that might be of value in the clinic to reduce the complications of FA and to identify mechanisms by which agents with favorable in vitro profiles exert their effects on FA cells.
Each aim of this project (Project 3) has integrated a novel assay applicable to Fancd2 mice. Project 3 will conduct such assays as they become relevant in the light of new findings in Projects 1 and 2. For example, project 2 has confirmed that HSC of Faned2 deficient mice are significantly less quiescent than those of wild type mice. Their flow cytometry based assay will be conducted by Project 2 using HSC from mice studied in project three, adding depth to the functional and biochemical studies being conducted on primary HSCs in project 3. Likewise, the leader of project 2 will create Fancc mutant knock-in mice suggested by one of the reviewers. Goals for Project 3: Bone marrow failure and acute myelogenous leukemia are major causes of morbidity and mortality in FA. For this reason our project has focused on the defining the molecular pathogenesis of these cardinal hematopoietic phenotypes (17-20). We have defined three key signaling functions of some FA proteins that are distinct from their nuclear core complex functions (18) (also see preliminary studies and progress report) and during the past year have demonstrated that at least two of these mechanisms (TNFa hypersensitivity in HSC and hematopoietic progenitor cells (HPCs) increase the coefficient of selection for neoplastic stem cell clones.(1) Reasoning that prevention of marrow failure in FA will reduce the risk of AML, our long term objectives are to: (A) Clarify the precise molecular mechanisms that underlie the survival signaling functions of FA proteins, (B) demonstrate that marrow failure arises from defective survival signaling and HSC senescence but not as a result of the aberrant DNA damage response, (C) to identify survival signaling defects in cells from all of the most common FA complementation groups, and (D) to identify therapeutic agents that will reduce stem cell apoptosis and senescence and reduce over-production of TNFa, a cytokine we have shown to be of significance in both the bone marrow failure and acute leukemia phenotype.(1;21) Each of the three aims below focuses on one of the three signaling defects we have identified in FA hematopoietic cells.
|Garbati, Michael R; Hays, Laura E; Rathbun, R Keaney et al. (2016) Cytokine overproduction and crosslinker hypersensitivity are unlinked in Fanconi anemia macrophages. J Leukoc Biol 99:455-65|
|Zhang, Haojian; Kozono, David E; O'Connor, Kevin W et al. (2016) TGF-Î² Inhibition Rescues Hematopoietic Stem Cell Defects and Bone Marrow Failure in Fanconi Anemia. Cell Stem Cell 18:668-81|
|Lombardi, Anne J; Hoskins, Elizabeth E; Foglesong, Grant D et al. (2015) Acquisition of Relative Interstrand Crosslinker Resistance and PARP Inhibitor Sensitivity in Fanconi Anemia Head and Neck Cancers. Clin Cancer Res 21:1962-72|
|Xie, Jenny; Kim, Hyungjin; Moreau, Lisa A et al. (2015) RNF4-mediated polyubiquitination regulates the Fanconi anemia/BRCA pathway. J Clin Invest 125:1523-32|
|Zhang, Qing-Shuo; Deater, Matthew; Schubert, Kathryn et al. (2015) The Sirt1 activator SRT3025 expands hematopoietic stem and progenitor cells and improves hematopoiesis in Fanconi anemia mice. Stem Cell Res 15:130-40|
|Zhang, Qing-Shuo; Benedetti, Eric; Deater, Matthew et al. (2015) Oxymetholone therapy of fanconi anemia suppresses osteopontin transcription and induces hematopoietic stem cell cycling. Stem Cell Reports 4:90-102|
|Zhang, Qing-Shuo; Marquez-Loza, Laura; Sheehan, Andrea M et al. (2014) Evaluation of resveratrol and N-acetylcysteine for cancer chemoprevention in a Fanconi anemia murine model. Pediatr Blood Cancer 61:740-2|
|Kim, Hyungjin; Dejsuphong, Donniphat; Adelmant, Guillaume et al. (2014) Transcriptional repressor ZBTB1 promotes chromatin remodeling and translesion DNA synthesis. Mol Cell 54:107-18|
|Fargo, John H; Rochowski, Andrzej; Giri, Neelam et al. (2014) Comparison of chromosome breakage in non-mosaic and mosaic patients with Fanconi anemia, relatives, and patients with other inherited bone marrow failure syndromes. Cytogenet Genome Res 144:15-27|
|Owen, Nichole; Hejna, James; Rennie, Scott et al. (2014) Bloom syndrome radials are predominantly non-homologous and are suppressed by phosphorylated BLM. Cytogenet Genome Res 144:255-63|
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