Thursday, November 21, 2024
11/21/2024
SUMMARY. Kinase inhibitors targeting the catalytic subunit of the DNA-dependent protein kinase (DNAPKcs) are being developed to sensitize tumors to clastogenic (chromosomal-breaking) agents, such as radiotherapy. The DNAPKcs kinase inhibitor M3814 (EMD Serono) is in clinical trials, and other new potent inhibitors have been reported. Elucidating the role of DNAPKcs in the repair of chromosomal breaks is significant because it will enable the identification of the contexts (e.g. tumor genetic background and treatment regimens) in which DNAPKcs kinase inhibitors are most effective, and thereby develop biomarkers to predict tumor response. DNAPKcs has long been recognized as important for promoting radioresistance, but its role in chromosomal break repair has remained elusive. Namely, DNAPKcs associates with factors in the canonical non-homologous end joining (C-NHEJ) pathway for DNA double-strand break (DSB) repair but is not required for all C-NHEJ events (e.g. is largely dispensable for V(D)J recombination signal EJ). Furthermore, until recently, specific assays to detect C-NHEJ of chromosomal DSBs, apart from V(D)J recombination, have not been available, due to partial redundancy with the alternative EJ (ALT-EJ) pathway. To address this gap in technology, our laboratory recently identified a specific hallmark of C-NHEJ. Namely, we reported that several C-NHEJ factors (i.e. XLF, KU70, and XRCC4) are required for EJ between blunt-ended DSBs (induced by the Cas9 nuclease) that are joined without causing insertion/deletion mutations (indels), i.e. for accurate/No Indel EJ. In preliminary data, we show that DNAPKcs is partially required for No Indel EJ, but its role is substantially magnified in cells with XLF reduced-function mutations (hypomorphs), including mutations in an XLF motif important for binding the KU heterodimer and DNA. Building on these findings, we will define the role of DNAPKcs in chromosomal EJ and clastogen resistance. In Aim 1, we hypothesize that XLF and DNAPKcs function synergistically to ensure No Indel EJ, and this synergy is critical for clastogen resistance. We will also examine the interplay between these two factors in DNA replication, and define the key domains of XLF and DNAPKcs important for each of these functions. In Aim 2, we investigate the interplay between DNAPKcs and a set of KU-binding factors, as well as ALT-EJ, on clastogen resistance and genome stability. We test whether DNAPKcs functions synergistically not only with XLF, but also with each of four KU-binding factors (CYREN/MRI, PAXX, APLF, and WRN), for No Indel EJ, clastogen resistance, and robust recruitment of KU to DNA damage. These studies will fill a major gap in our understanding of C-NHEJ mechanisms. Finally, we posit that combined disruption of DNAPKcs and ALT-EJ causes aberrant DSB end processing, leading to large deletions, and/or persistent, unrepaired DSBs, thereby causing reduced clonogenic survival and/or clastogen sensitization. In summary, we will test the overall hypothesis that DNAPKcs promotes accurate EJ (i.e. No Indel EJ), that its role in such EJ is magnified by defects in a set of KU-binding factors, and that it is critical for genome stability in cells deficient in ALT-EJ.
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