Drug to block double-strand break repair in breast cancer

Institution: Stanford University
Investigator(s): Gilbert Chu, M.D. -
Award Cycle: 2014 (Cycle 20) Grant #: 20IB-0126 Award: $160,500
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure



Initial Award Abstract (2014)

This project was supported in part by a generous donation from the Katie Ann Buzbee Trust.

DNA double-strand breaks leave a chromosome broken into two pieces. These breaks are so dangerous that cells use two overlapping methods for repairing the damage: homologous recombination (HR) and non-homologous end joining (NHEJ). HR uses the intact partner (or "homologous") chromosome as a blueprint for repair, restoring the damaged chromosome to its original state. By contrast, NHEJ does not need an intact "homologous" chromosome, but instead simply joins the broken ends directly.

Many breast cancers have defects in HR, such as mutations in the BRCA1 or BRCA2 genes. The cancer cells are defective in HR because both copies of the BRCA1 or BRCA2 gene are mutated. By contrast, the patient's normal cells are intact for HR because only one copy of the gene is mutated. The aim is to find an oral drug that disrupts NHEJ in HR-defective breast cancers, crippling the ability of the cancer cells to repair DNA double-strand breaks.

The question(s) or central hypotheses of the research: The first hypothesis is that the drug will cure HR-defective breast cancer, without side effects for the patient. The drug will work either alone, or in combination with agents that generate DNA double-strand breaks, such as ionizing radiation or doxorubicin. These agents have proven benefit against breast cancer, but the drug will allow them to be used at doses that are low enough to eliminate their side effects.

A second hypothesis is that the new era of sequencing whole cancer genomes will soon enable physicians to identify breast cancers with defects in any of the HR genes. As a result, the drug will be used to treat a growing proportion of breast cancers.

The general methodology: We will exploit powerful technologies to find the drug. The Stanford High-Throughput Bioscience Center (HTBC) has collected 130,000 compounds, which were selected because their chemical structure suggests that oral administration will be feasible. Robotic machinery at the HTBC will first dispense the compounds into wells containing purified proteins that perform the NHEJ reaction, and then dispense reagents for identifying the compounds that disrupt the NHEJ reaction.

Compounds that disrupt the NHEJ reaction will be tested for their ability to kill BRCA-mutated breast cancer cell lines, while sparing cell lines with intact BRCA function. We will test the compounds alone, or in combination with ionizing radiation or doxorubicin.

Innovative elements of the project: The first innovative element is the strategy of treating HR-defective breast cancers with a drug that disrupts NHEJ. Currently, no FDA-approved drug exists for disrupting NHEJ, so this would be the first of a new class of drugs. Because both HR and NHEJ will be disrupted in the cancer cells, they will be exquisitely sensitive to DNA double-strand breaks. Thus, the drug has the potential for achieving the holy grail of curing HR-defective breast cancer without side effects.

The second innovative element is our "test tube" NHEJ reaction to find the drug. We have successfully reproduced NHEJ in the "test tube" using purified proteins that will join the ends of a linear DNA molecule to form a DNA circle. We discovered that these proteins bring DNA ends together by forming a large complex on the DNA with many sites of potential vulnerability.

The third innovative element is our low-cost method for measuring NHEJ. The method uses an enzyme that digests inward from the free ends of linear DNA that has not been joined. The enzyme digests only DNA ends, thus sparing DNA that has been joined into a circle. A fluorescent dye stains the DNA circles, which are detected by laser scanners at the Stanford HTBC. This new method costs 2 cents per reaction, thus permitting us to screen a large number of compounds at very low cost.




Progress Report 1 (2015)

Cells use two major pathways for repairing DNA double-strand breaks: homologous recombination (HR), and non-homologous end joining (NHEJ). This proposal will find a small molecule drug that inhibits NHEJ. The goal is to kill HR-defective breast cancer cells, while sparing HR-competent normal cells, thus curing patients without toxicity.

We performed a systematic optimization of the assay for finding the drug. We showed that the signal for successful joining was accurate and reproducible. In the course of our optimization, we discovered an alternative assay for finding the drug. We then tested a drug already known to inhibit the NHEJ reaction and showed that application of both the original assay and the alternative assay would have led to the proper identification of the known drug as an inhibitor of NHEJ.

These experiments demonstrated that our assay system is feasible and can be used to screen a large number of compounds in search of a potential drug for treating breast cancer. Our next step is to undertake a screen using compounds that have already received FDA approval for other purposes.