Elucidating the role of DNAPKcs in chromosomal break end joining and clastogen

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, and is implicated in the canonical non-homologous end joining pathway (C-NHEJ), but nonetheless its role in chromosomal break repair has remained elusive. The goals of the parent grant, and the project in this supplement application, are

to define the role of DNAPKcs in chromosomal DSB repair and clastogen resistance, and thereby support development of DNAPKcs as a cancer therapeutic target. To this end, 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. We have been using this hallmark of C- NHEJ to define the role of DNAPKcs in this process. Regarding the specific project for this supplement

application, the candidate for this application, Ms. Kaela Makins, has found that the factor RIF1 is particularly important for No Indel EJ in cells treated with a DNAPKcs kinase inhibitor. She will develop these preliminary findings to 1) test the hypothesis that RIF1 and DNAPKcs synergistically promote No Indel EJ, which includes

development of a RIF1 knockout (KO) human cell line to test this hypothesis, 2) test the genetic interplay between RIF1 and DNAPKcs for radioresistance, 3) test for effects of RIF1 on DNAPKcs autophosphorylation, 4) examine the interplay of RIF1 and DNAPKcs using double-knockout cell lines, and 5) develop a genetic

complementation assay for RIF1. Her training plan is to develop 1) key research design and experimental skills, 2) manuscript and grant writing skills, 3) presentation and networking skills, 4) mentoring skills, and 5) a breadth of knowledge in Cancer Biology. Altogether, this supplement to enhance diversity in health-related

research will further the goals of the parent grant to define the contexts that influence the role of DNAPKcs in chromosomal break repair and clastogen response, as well as provide comprehensive training for the candidate Ms. Kaela Makins to develop into an independent and impactful cancer researcher.