Overall PPG Obiectives and Points of Integration for Project 3: The central theme of this program project grant applicafion 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 dysfuncfion 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 idenfified agents that both enhance FANCC'''hematopoiefic stem cell (HSC) survival and reduce the release of TNFa by FANCC deficient mononuclear phagocytes sfimulated with toll-like receptor 8 (TLRS) agonists. Because these assays are robust and quanfitafive, they have supplanted the assay (based upon defective STAT5 acfivation) we had proposed in the last application. All ofthe projects and cores share two objecfives;to identify small molecules that might be of value in the clinic to reduce the complicafions 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 funcfional 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 hematopoiefic phenotypes (17-20). We have defined three key signaling functions of some FA proteins that are disfinct from their nuclear core complex funcfions (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 hematopoiefic progenitor cells (HPCs) increase the coefficient of selecfion for neoplasfic stem cell clones.(1) Reasoning that prevention of marrow failure in FA will reduce the risk of AML, our long term obiectives are to: (A) Clarify the precise molecular mechanisms that underiie the survival signaling funcfions of FA proteins, (B) demonstrate that marrow failure arises from defecfive 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 complementafion groups, and (D) to identify therapeufic 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 hematopoiefic cells.
|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|
|Park, Eunmi; Kim, Hyungjin; Kim, Jung Min et al. (2013) FANCD2 activates transcription of TAp63 and suppresses tumorigenesis. Mol Cell 50:908-18|
|Zhang, Qing-Shuo; Watanabe-Smith, Kevin; Schubert, Kathryn et al. (2013) Fancd2 and p21 function independently in maintaining the size of hematopoietic stem and progenitor cell pool in mice. Stem Cell Res 11:687-92|
|Dao, Kim-Hien T; Rotelli, Michael D; Brown, Brieanna R et al. (2013) The PI3K/Akt1 pathway enhances steady-state levels of FANCL. Mol Biol Cell 24:2582-92|
|Mistry, Helena; Hsieh, Grace; Buhrlage, Sara J et al. (2013) Small-molecule inhibitors of USP1 target ID1 degradation in leukemic cells. Mol Cancer Ther 12:2651-62|
|Park, Eunmi; Kim, Jung Min; Primack, Benjamin et al. (2013) Inactivation of Uaf1 causes defective homologous recombination and early embryonic lethality in mice. Mol Cell Biol 33:4360-70|
|Garbati, Michael R; Hays, Laura E; Keeble, Winifred et al. (2013) FANCA and FANCC modulate TLR and p38 MAPK-dependent expression of IL-1* in macrophages. Blood 122:3197-205|
|Anur, Praveen; Yates, Jane; Garbati, Michael R et al. (2012) p38 MAPK inhibition suppresses the TLR-hypersensitive phenotype in FANCC- and FANCA-deficient mononuclear phagocytes. Blood 119:1992-2002|
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