P-057: A helicase “ASCC3” is coupled to FEN1-mediated genomic instability and cancer cell proliferation

Abstract

<h3>Background</h3> We have previously reported the identification of FEN1 a driver of genomic instability in myeloma (MM) as well as esophageal adenocarcinoma (EAC) cells. We demonstrated that FEN1 is overexpressed in MM cell lines and in clinical datasets of MM and several other cancers including EAC, and FEN1-knockdown inhibited spontaneous DNA breaks, homologous recombination (HR) activity as well as genomic instability in MM cells. We now show that FEN1-overexpression in non-cancerous (normal fibroblasts and bone marrow/stroma HS5) cells increases DNA breaks and genomic instability, as assessed by micronucleus assay. <h3>Methods</h3> In order to further evaluate the impact on genomic instability, control and FEN1-overexpressing cells were cultured for three weeks and new genomic changes acquired in cultured relative to "day 0" cells (representing baseline genome), were identified using single nucleotide polymorphism (SNP) arrays. <h3>Results</h3> Overall, the acquisition of amplification and deletion events were increased by ~ 3-fold in FEN1-overexpressing relative to control cells. Evaluation by RNA sequencing in two different non-cancerous cell types showed that FEN1-overexpression was associated with upregulation of several interconnected pathways including DNA double strand break repair, cell cycle, mitotic G2 M phases, TP53, homologous DNA pairing and strand exchange. Top downregulated pathways included several metabolic pathways and an apoptosis pathway. These data demonstrate a significant role and the impact of FEN1 on DNA repair and genome maintenance, especially HR. We next identified FEN1-interacting proteins from two different MM cell lines (MM1S, RPMI8226) by mass spectrometry. Forty-one proteins interacted with FEN1 in both MM cell lines including two helicases (ASCC3, RUVBL2) and several proteins involved in DNA damage response and recruitment. To investigate the functional relevance of FEN1-ASCC3 interaction, FEN1 was overexpressed in non-cancerous (stromal HS5) cells and ASCC3 was suppressed in control as well as FEN1-overexpressed cells, and impact on different parameters of genome stability (HR activity, micronuclei) and growth (cell viability and DNA replication) monitored. FEN1-overexpression increased HR activity, whereas ASCC3-knockdown inhibited spontaneous as well as FEN1-induced HR activity. Consistent with these data, FEN1-overexpression also increased genomic instability, whereas ASCC3-knockdown inhibited spontaneous as well as FEN1-induced genomic instability as assessed by micronucleus assay. Importantly, FEN1-overexpression also increased DNA replication (as assessed by BrdU-labelling), whereas ASCC3-knockdown inhibited spontaneous as well as FEN1-induced DNA replication in these cells. <h3>Conclusion</h3> These data suggest that helicase activity of ASCC3 is coupled to endonuclease activity of FEN1 to cause genomic instability and cancer cell proliferation, and is currently being investigated in MM and other cancer models to develop translational application.