Flap Structure-Specific Endonuclease 1 Coordinates with a Helicase (ASCC3) to Drive Genomic Instability, Proliferation and Poor Clinical Outcome in Myeloma

Genomic instability leads to an increased frequency of aberrations at the single nucleotide and chromosomal levels, thus contributing to cancer progression and poor clinical outcomes. We have previously reported FEN1 as an important driver of genomic evolution in MM. Suppression of FEN1 in cancer cells inhibits DNA breaks, HR activity and genomic instability, whereas its overexpression in non-cancerous cells increases those activities. In addition, FEN1 overexpression also increased DNA replication (as assessed by BrdU labeling) and the growth rate of non-cancerous cells.

Here, we further demonstrate that overexpression of FEN1 in normal cells increases genomic instability and cell growth, whereas its suppression in a cancer cell line inhibits these functional activities. During FEN1 overexpression in non-cancerous cell types, the pathways of DNA repair (especially HR) and DNA replication were upregulated, whereas several metabolic and apoptosis pathways were downregulated. Mass spectrometry in two MM cell lines (MM1S, RPMI8226) revealed that the FEN1 interactome was mainly comprised of DNA damage response and repair proteins, including several RNA and DNA helicases. Since helicase activity is required for HR as well as DNA replication, we hypothesized that interaction with a helicase may enable FEN1 to participate in both these processes.

Based on analysis of RNA-seq data and the mass spectrometry with FEN1 immuno-precipitate, we investigated the functional significance of a DNA helicase (ASCC3) and the importance of its interaction with FEN1. As observed for FEN1, the overexpression of ASCC3 in normal human fibroblast cells increased DNA replication by ~2-fold, whereas its knockdown in MM cell lines (H929 and JJN3) reduced it to a similar extent. ASCC3 knockdown also inhibited spontaneous and FEN1-induced HR activity and genomic instability in MM as well as other solid tumor cell types. Consistently, whereas the overexpression of FEN1 increased the growth rate of MM cell lines (MM1S, H929), ASCC3 knockdown reduced it. Importantly, the knockdown of ASCC3 in FEN1-overexpressing MM cell lines prevented the FEN1-mediated increase in growth rate. For example, at day 5 after selection, FEN1-overexpression in H929 cells was associated with a ∼50% increase in the viable cell fraction, whereas ASCC3-knockdown was associated with a complete loss of the viable cell fraction in the control and FEN1-overexpressing cells. Consistent with these observations, the high expression of both FEN1 and ASCC3 correlated with poor overall survival in myeloma patients (in MMRF patient dataset; p < 0.008). Furthermore, high ASCC3 expression further separated patients with high FEN1 expression into high- and low-risk categories (p = 0.025). Similarly, high FEN1 expression further separated patients with high ASCC3 expression into high- and low-risk categories (p = 0.00025). These data suggest that interaction between a helicase, ASCC3, and a nuclease, FEN1, drives genomic instability and cell proliferation thus contributing to poor clinical outcome. These data provide the basis to further study molecular characteristics of the high-risk MM cells and molecular targets for therapeutic consideration, especially in high-risk patients.

Munshi:Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; GSK: Consultancy; Takeda Oncology: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Legend: Consultancy; Adaptive Biotechnology: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy; Abbvie: Consultancy; Pfizer: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties.