Dynamics of DNA Double-strand Break Repair Pathways and Unlocking New Therapeutic Opportunities by Exploiting DSB Repair in Head and Neck Cancer

Abstract

Among different types of DNA damage, DNA double-strand breaks (DSBs) are the most lethal, disrupting genome integrity and potentially causing cancer via faulty repair. Major DSB repair pathways in humans include homology-directed repair (HDR), nonhomologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ). Head and neck cancer (HNC), with over 450,000 annual global deaths and the most common cancer in India, was investigated for its ability and mechanism to repair DSBs. Assays in four HNC lines (SCC084, SCC131, Cal27, RPMI2650) showed proficient NHEJ and MMEJ. RPMI2650 exhibited the highest NHEJ but the lowest MMEJ. MMEJ needed ≥8 nt microhomology, efficiency increasing with length. Consistently, upregulation of several genes associated with NHEJ, MMEJ, and HR was observed in RNA-Seq data from >500 HNC patients in the TCGA dataset. Immunofluorescence using 53BP1, a marker of DSBs, showed high repair efficiency at the chromosomal level following IR in HNC patients and cell lines. Furthermore, knockdown of DSB repair proteins led to a significant delay in repair response following IR exposure, demonstrating involvement of both NHEJ and MMEJ at the genome level. Thus, we establish that HNC cells possess efficient DSB repair via NHEJ and MMEJ, and the efficacy of repair is directly linked to the expression of key DSB repair genes. Previously, SCR7 was identified as a potent inhibitor of NHEJ, targeting DNA Ligase IV. Recently, we derivatized SCR7 by synthesizing a series of mercaptopyrimidine-based molecules called SCR116 and SCR132, as third-generation NHEJ inhibitors. SCR116 and SCR132 exhibited cytotoxicity in different HNC cell lines, including their resistant counterparts. SCR116 and SCR132 resulted in the accumulation of 53BP1 foci, an increase in the sub-G1 population, and induction of cell death via the intrinsic pathway of apoptosis. Both compounds demonstrated significant antitumor activity in multiple xenograft models induced by HNC sensitive as well as resistant cell lines. Further, assessment of the safety profile in mice and rats showed no toxicity in terms of mortality, abnormal appearance, body weight, kidney and liver function tests. Collectively, these findings highlight SCR116 and SCR132 as promising candidates for novel cancer therapeutics against head and neck cancer.