Abstract 3072: A clinical pharmacodynamic biomarker assay that distinguishes potentially repairable, cytotoxic drug-induced DNA double strand breaks (DSBs) from DSBs associated with apoptotic cell death
Authors: Dull Angie B. , Wilsker Deborah , Kinders Robert J. , Parchment Ralph E. , Evans David , Teicher Beverly A. , Doroshow James H. 
Published: July 2017

Abstract
The effectiveness of certain classes of cytotoxic cancer therapeutics likely depends on whether drug-induced DNA damage is successfully repaired or not, with the latter situation leading to mutations and strand breaks. However, double strand breaks (DSBs) also occur independently of genotoxic insults during apoptotic cell death caused by many drug classes, as well as natural biological processes. We have developed an immunofluorescent confocal microscopy assay that uses a biomarker profile suitable for individual cell analysis designed to distinguish between DSBs caused by apoptosis and those caused by direct DNA damage from cytotoxic drug action. γ-H2AX is an established biomarker for DSBs and activated cleaved caspase 3 is an executioner caspase important for apoptosis, which leads to nuclear condensation, DNA fragmentation, plasma membrane blebbing, and subsequent cell death. Our assay defines the DSBs of apoptosis by co-localized γ-H2AX and cleaved caspase 3 in individual cells, while defining the DSBs from early drug effects of DNA damaging chemotherapeutics by γ-H2AX induction in the absence of cleaved caspase 3. Building on our published findings that topotecan strongly induces γ-H2AX and DSBs within 1-4 hours in vitro and in vivo, we observed exposure-dependent increases in γ-H2AX /cleaved caspase 3 double positive cells at later time points, both in an HT29 in vitro spheroid model and an MDA-MB-231 xenograft model. Fit-for-purpose studies in the MDA-MB-231 xenograft model treated with birinipant, a SMAC mimetic and IAP deregulator that does not directly produce lethal DSBs, demonstrated a dose-dependent increase in cellular co-localization of γ-H2AX/cleaved caspase 3 consistent with birinipant induced apoptosis and the established mechanism of action of this compound. Clinical feasibility was established in a canine clinical trial using formalin-fixed paraffin-embedded (FFPE) 18-gauge needle biopsies: two novel indenoisoquinolines, indotecan (LMP400) and indimitecan (LMP776), increased tumor cell co-localization of γ-H2AX/cleaved caspase 3 in tumor samples obtained on day 5 of qdx5 treatment. This PD biomarker assay of early and late DSB response to drug exposure could have important applications for elucidation of mechanisms of action of anticancer drugs and the development of investigational agents. Funded by NCI Contract No. HHSN261200800001E.
Citation Format: Angie B. Dull, Deborah Wilsker, Robert J. Kinders, Ralph E. Parchment, David Evans, Beverly A. Teicher, James H. Doroshow. A clinical pharmacodynamic biomarker assay that distinguishes potentially repairable, cytotoxic drug-induced DNA double strand breaks (DSBs) from DSBs associated with apoptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2017-3072