Supplemental methods, figures, captions, and references. Supplemental Figure S1. Isotype controls. Supplemental Figure S2. ATM deficiency attenuates pS343-Nbs1 signal induction. Supplemental Figure S3. Comparison of the percent nuclear area positive for γH2AX versus the proportion of cells positive for γH2AX signal. Supplemental Figure S4. pS343-Nbs1 is a sensitive marker of genotoxic agents across different classes. Supplemental Figure S5. Development of nuclear masks for quantitation of DAPI area, nuclear marker expression, and nuclear foci. Supplemental Fig. S6. pS343-Nbs1 and γH2AX calibrators. Supplemental Figure S7. DDR activation in the context of homologous recombination defects. Supplemental Figure S8. Induction of Rad51 and colocalization with cell cycle marker geminin following gemcitabine exposure.
ARTICLE ABSTRACT
We sought to examine the pharmacodynamic activation of the DNA damage response (DDR) pathway in tumors following anticancer treatment for confirmation of target engagement.
We evaluated the time course and spatial activation of 3 protein biomarkers of DNA damage recognition and repair (γH2AX, pS343-Nbs1, and Rad51) simultaneously in a quantitative multiplex immunofluorescence assay (IFA) to assess DDR pathway activation in tumor tissues following exposure to DNA-damaging agents.
Because of inherent biological variability, baseline DDR biomarker levels were evaluated in a colorectal cancer microarray to establish clinically relevant thresholds for pharmacodynamic activation. Xenograft-bearing mice and clinical colorectal tumor biopsies obtained from subjects exposed to DNA-damaging therapeutic regimens demonstrated marked intratumor heterogeneity in the timing and extent of DDR biomarker activation due, in part, to the cell-cycle dependency of DNA damage biomarker expression.
We have demonstrated the clinical utility of this DDR multiplex IFA in preclinical models and clinical specimens following exposure to multiple classes of cytotoxic agents, DNA repair protein inhibitors, and molecularly targeted agents, in both homologous recombination–proficient and -deficient contexts. Levels exceeding 4% nuclear area positive (NAP) γH2AX, 4% NAP pS343-Nbs1, and 5% cells with ≥5 Rad51 nuclear foci indicate a DDR activation response to treatment in human colorectal cancer tissue. Determination of effect-level cutoffs allows for robust interpretation of biomarkers with significant interpatient and intratumor heterogeneity; simultaneous assessment of biomarkers induced at different phases of the DDR guards against the risk of false negatives due to an ill-timed biopsy.
