Etoposide-induced dna damage is increased in p53 mutants: identification of atr and other genes that influence effects of p53 mutations on top2-induced cytotoxicity

Abstract

// Daniel Menendez 1 , 4 , * , Jay R. Anand 2 , 5 , * , Carri C. Murphy 1 , Whitney J. Bell 1 , Jiaqi Fu 3 , Nadia Slepushkina 3 , Eugen Buehler 3 , Scott E. Martin 3 , Madhu Lal-Nag 3 , John L. Nitiss 2 and Michael A. Resnick 1 1 Chromosomal Stability Group, Genome Integrity and Structural Biology Laboratory, NIEHS, NIH, Durham, NC 27709, USA 2 Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois, Rockford, IL 61107, USA 3 Functional Genomics Laboratory, National Center for Advancing Translational Sciences, NIH, Bethesda, MD 20850, USA 4 Environmental Cardiopulmonary Disease Group, Immunity, Inflammation and Disease Laboratory, NIEHS, NIH, Durham, NC 27709, USA 5 Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA * These authors contributed equally to this work Correspondence to: Daniel Menendez, email: menendez@niehs.nih.gov Michael A. Resnick, email: resnick@niehs.nih.gov Keywords: tumor suppressor; siRNA screen; synthetic lethality; ICE assay; Top2 covalent complexes Received: December 15, 2021 Accepted: January 28, 2022 Published: February 14, 2022 Copyright: © 2022 Menendez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT The functional status of the tumor suppressor p53 is a critical component in determining the sensitivity of cancer cells to many chemotherapeutic agents. DNA topoisomerase II (Top2) plays essential roles in DNA metabolism and is the target of FDA approved chemotherapeutic agents. Topoisomerase targeting drugs convert the enzyme into a DNA damaging agent and p53 influences cellular responses to these agents. We assessed the impact of the loss of p53 function on the formation of DNA damage induced by the Top2 poison etoposide. Using human HCT116 cells, we found resistance to etoposide in cell growth assays upon the functional loss of p53. Nonetheless, cells lacking fully functional p53 were etoposide hypersensitive in clonogenic survival assays. This complex role of p53 led us to directly examine the effects of p53 status on topoisomerase-induced DNA damage. A deficiency in functional p53 resulted in elevated levels of the Top2 covalent complexes (Top2cc) in multiple cell lines. Employing genome-wide siRNA screens, we identified a set of genes for which reduced expression resulted in enhanced synthetic lethality upon etoposide treatment of p53 defective cells. We focused on one hit from this screen, ATR, and showed that decreased expression sensitized the p53-defective cells to etoposide in all assays and generated elevated levels of Top2cc in both p53 proficient and deficient cells. Our findings suggest that a combination of etoposide treatment with functional inactivation of DNA repair in p53 defective cells could be used to enhance the therapeutic efficacy of Top2 targeting agents.