Therapeutic research in fanconi anemia
- Autores: Helena Montanuy Escribano
- Directores de la Tesis: Jordi Surrallés Calonge (dir. tes.), Jordi Minguillón Pedreño (codir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2017
- Idioma: español
- Tribunal Calificador de la Tesis: Jordi Carreras (presid.), Massimo Bogliolo (secret.), María Castella Castella (voc.)
- Programa de doctorado: Programa de Doctorado en Genética por la Universidad Autónoma de Barcelona
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
Much effort is being made on Fanconi anemia therapeutics to treat bone marrow failure and cancer, the most life threatening signs of the disease. There are also studies searching for prevention, with chronic treatment proposals. As mortality rate by bone marrow failure has been dramatically reduced in the last years, thanks to improved hematopoietic stem cell transplantation protocols, and with the promise of new approaches such as gene therapy, the next step in the horizon of Fanconi anemia treatment may be focused on malignancies. Patients have very low survival rate after solid tumors appear, and no effective therapy currently exists beyond surgical resection. That is the reason why we developed two drug screening assays to measure different Fanconi biomarkers with the purpose of finding new drugs that ameliorate or cure the Fanconi anemia disorder and could be investigated in preclinical studies as a new therapy. We adapted the flow cytometric in vitro micronucleus test to screen drugs for their ability to reduce chromosome fragility and cell cycle arr est in FANCA-deficient cells. Screening of 10 candidate drugs based on literature search and a redox library of 84 antioxidants/antiinflammatory compounds identified several drugs that consistently restored chromosome fragility and G2/M cell cycle arrest in lymphoblast cell lines from 3 independent FANCA-deficient patients. We selected two drugs, CRD3 and CRD8, widely used in clinics, to test in Fanca-deficient mice in vivo. One year later 60 % of placebo-treated mice died. Only 20 % mice of CRD3 treated group were sacrificed due to excess weight loss while all CRD8 treated mice were alive and healthy at the end of the experiment. In a second assay, we set up a cell system based on FANCD2-Ub foci detection in FANCA-deficient cells and we screened 3802 drugs, which included all FDA-approved, kinase inhibitors and chemical diversity compounds in search of a drug to cure Fanconi anemia by re-activating the FA/BRCA pathway. We selected 7 hits and none of them could be validated on phenotypic assays. Taking advantage of this screening, we searched for drugs that killed cells by a non-genotoxic mode of action. Selected drugs were tested in Fanconi anemia patient-derived head and neck squamous cell carcinoma (HNSCC) cell lines and primary fibroblasts. Two potential drugs, CAT2 and CAT29, were especially relevant as they are highly selective for cancer cells. They are already approved for cancer treatment and their mechanism of action does not involve DNA damage. Neither CAT2 nor CAT29 induced chromosome fragility in a FANCA-deficient lymphoblastoid cell line or activate FANCD2 ubiquitination, suggesting that the Fanconi anemia pathway is not involved in the cellular response to these drugs. In vivo experiments of tumor xenografts in mice showed that CAT2 and CAT29 inhibited the growth of Fanconi HNSCC VU1131-T2.8 in a highly significant manner. Given the results presented in this PhD thesis, we propose to continue the preclinical studies in order to finally repurpose CRD3 and CRD8 for Fanconi anemia prevention and CAT2 and CAT29 to treat Fanconi HNSCC.
