Fanconi Anemia (FA) is an autosomal or X-linked recessive disorder caused by mutation in any of the 22 genes involved in Interstrand cross link repair (ICL). During DNA replication due to cross linking of bases the replication fork gets stalled at the site of damage; FA pathway proteins work coordinately with other DNA damage repair pathways to rectify the halt and precede replication. Non-functional FA pathway causes chromosomal aberrations and genomic instability hence FA mutant cells are highly sensitive to DNA cross linking agents. Although the core function of FA pathway remains to be ICL repair, alongside it is involved in non canonical functions in regulating cellular mechanisms like cell proliferation, immune response, and apoptosis. In our study we evaluated the effects of non-functional FA pathway and the interconnection between other pathways in regulating cellular processes like cell division, DNA repair regulation and maintenance of redox potential in FA clinical samples and fibroblast cells. Firstly, we studied the interconnection between autophagy and Notch signaling pathway in rendering proliferation advantage to FA A mutant cells. We had inspected the gene and protein levels of the above mentioned pathway targets in both cell lines and in FA clinical samples and found the interplay between the molecules. Later the effect of these dysregulated pathways in cell proliferation was studied by the addition of an autophagy inducer Rapamycin, in FA cells as we observed impaired autophagy in FA. We had identified that inefficient autophagic mechanism can be modified on addition of an activator and regulate the expression of Notch signaling genes; harmoniously these pathways alter the cell cycle genes expression pattern in FA cells. Secondly, regulation of ubiquitination process involved in protein degradation and DNA repair are analyzed by gene expression studies in FA cells and bone marrow samples. Importantly, Skp2 were found low at both gene and protein level in FA cells and bone marrow samples. Interconnection between ubiquitinating enzymes and cell cyclins was predicted by networking map and found that cyclins were tightly regulated by the ligases. Finally, the involvement of intracellular stress in altering mitochondrial properties and cellular death was studied in FA cells. The proinflammatory IL6 was found high in FA causing cancer progression. In our study we had knocked down the expression of IL6 in FA cells and the after effects were analyzed. We found that lower levels of the proinflammatory cytokine, IL6 subsides the inflammatory response and rescues FA mutant cells from cellular stress thereby lesser percentage of apoptotic cells were observed. Conclusively, in this research work we aimed to spatially screen for biological processes that are dysregulated in FA and more specifically we looked for significant targets in each processes that are consistently varied under FA condition.

Publication:

[1] Binita Zipporah E, Patra B, Govarthanan K, Yadav R, Mohan S, Shyamsunder P, Verma RS. Defective cell proliferation is an attribute of overexpressed Notch1 receptor and impaired autophagy in Fanconi Anemia. Genomics. 2020