High-throughput DNA damage analyses identify synergistic drug combinations to treat malignant brain tumours

Current methods to treat malignant brain tumors are highly intrusive, leading to poor quality of life with abysmal patient survival rates. Combined with radiation therapy, first/second-line DNA damaging chemotherapeutics marginally improve patient survival. However, high rates of drug-resistance and recurrence are due to development of intra/intertumoral heterogeneity due in part from the emergence of chemoradioresistant clones. The targeted use of DNA repair inhibitors to sensitize and augment tumour cell killing is an emerging tool in cancer therapy. However, the biochemical and functional readout of combining chemotherapeutic drugs with one or more DNA damaging inhibitors is limited by use of traditionally low-throughput analytical techniques that preclude their adequate interrogation and translational potential.

We have developed novel high-throughput DNA repair assay methods, which allow us to combine multiple DNA repair inhibitors and genotoxic agents in order to identify synergistic interactions that sensitize CNS tumours to chemoradiotherapeutics. This combinatorial analysis approach combines DNA damage, cell cycle and cell viability readouts for determining drug selection and dosing effectiveness.

Part 1 of my talk will detail our development of a high-throughput comet assay (HTCOMET) that enables simultaneous, unbiased multi-well/sample DNA damage analysis. Using this strategy, we have compared drug/repair inhibitor responses amongst patentderived glioblastoma multiforme (GBM) and medulloblastoma (MB) cell lines. Using this approach we have found differential sensitivities amongst tumor types and amongst tumor lines. Validation studies have identified key molecular differences that prescribe these differential sensitivities.

Part 2 of my talk will outline our development of the high-throughput γH2AX analysis (HT-γH2AX) whereby γH2AX imaging and foci quantification has enabled a detailed examination of poly(ADP-ribose) polymerase inhibitor (PARPi) use in GBM lines in conjunction with the first-line DNA alkylating agent, Temozolomide (TMZ), and secondline DNA Toposiomase-1 inhibitor, Topotecan (TPT). This analysis has enabled a new level of molecular insight into the properties of these drugs whereby γH2AX foci analysis combined with predictive markers of the DDR pathway and cell cycle analysis (in both dose-escalating and time-dependent manners) identify specific properties in select PARPis that can synergize with chemotherapeutics to better enable tumor cell killing.

Our novel high-throughput DNA damage assay methodology enables molecular dissection and understanding of the interplay of DNA repair pathways with broad implications to fundamental science and clinical treatment. Fast, efficient and reproducible analyses, drug-screening and validation can be achieved using patientderived tumor specimens and live cells to identify targeted and/or personalized therapies for improved patient outcomes.

Learning Objectives:

1. To understand the cellular DNA damage response and types of DNA repair pathways

2. To identify DNA damage-specific repair pathways that brain tumor cells use to resolve damage induced by chemotherapy which act to evade cell death.

3. To describe how conventional low-throughput DNA repair methodologies can be streamlined using unbiased semi-automated high-throughput techniques to enable drug combination synergy analysis and drug discovery through the lens of DNA damage repair – a fundamental outcome of most anti-cancer chemotherapeutics.