Researchers Identify Genetic Basis for Cancer Cells’ Susceptibility to DNA Damage

A team including Cleveland Clinic researchers has identified a variety of genetic determinants that enable cancer cells to survive after exposure to radiation.

The groundbreaking research, which utilized a collection of 533 genetically profiled human tumor-derived cell lines, found that the cells’ sensitivity to ionizing radiation results from significant underlying biological diversity within and across lineages.

“Our team’s research helps explain why individual tumors may vary in their susceptibility to DNA-damaging radiation and drugs,” says Cleveland Clinic radiation oncologist Mohamed Abazeed, MD, PhD, the lead author of the study published in Nature Communications.

Mohamed Abazeed, MD, PhD.

The researchers showed that overall and individual somatic copy number alterations, gene mutations, and the expression of individual genes and gene sets correlated with cancer cells’ ability to survive radiation exposure.

Characterizing genetic factors that dictate cellular response to radiation is a fundamental step toward using biomarkers to predict individual cancer patients’ radiotherapy outcomes, and to tailoring radiation treatments to exploit the genetic alterations present in a patient’s tumor.

Although cancer genomes can be diverse, the researchers found that relevant alterations were frequent and spanned multiple cancer types. This suggests that combinations of only a limited number of functionally relevant alterations can confer resistance to therapeutic radiation within and across cancer types. Accordingly, this makes the task of personalization substantially less daunting since patients can be binned into more homogenous groups.

“The findings suggest that the promising strategies of personalized, genetically targeted cancer therapies can be extended to radiation therapy, and that we can develop predictive tools to guide clinical decision-making with improved patient selection and more precise drug-radiation combinations,” Dr. Abazeed says.

Seeking to optimize radiotherapy

Recent advances in genomic sequencing and analysis have enabled researchers to identify specific genetic alterations that contribute to tumor development and progression. These biomarkers have begun to serve as predictive tools that can indicate which patients may benefit from drugs targeting specific mutations and affected molecular pathways.

Clinical radiotherapy has not experienced comparable progress from these genomic insights. While radiotherapy contributes significantly to the curative and palliative cancer treatments, it is presently targeted based on the origin of the cancer and does not take into account the genetic complexity that may influence therapeutic response. The effectiveness of radiotherapy is tempered by a lack of biomarkers that can reliably predict tumors’ varying sensitivity to ionizing radiation and inform treatment decisions.

Dr. Abazeed and his collaborators studied the genetic determinants of cancer cells’ survival after irradiation using large-scale, high-throughput profiling of 533 genetically annotated human tumor cell lines encompassing 26 cancer types. The research was conducted by investigators at Cleveland Clinic, Case Western Reserve University, Korea’s Seoul National University College of Medicine, the Broad Institute at MIT and Harvard, the Dana-Farber Cancer Institute, Harvard University, and the Howard Hughes Medical Institute.

The researchers assessed clonogenic survival in the cell lines as a function of radiation dose and found significant survival variation across and within lineages — as large as five- to sevenfold in the latter (Figure 1).

“We found that the responses of cancers to DNA-damaging therapy are incredibly diverse,” Dr. Abazeed said. “Although oncologists have anecdotally appreciated the diversity of cancer’s response to radiation treatments, our study formalizes this diversity across 26 cancer types.”

The researchers next investigated the ability of several genetic factors to impact post-radiation cellular survival.

Probing the SCNA/radiation relationship

Somatic copy number alterations (SCNAs) are common in cancer and promote oncogenesis, but their relationship to cellular irradiation response has been unclear. Dr. Abazeed and his colleagues measured the fraction of each tumor genome that contained an SCNA (ƒSCNA) and found a positive correlation between ƒSCNA and radiation survival.

They surmised that this could be due to either:

• Tumor cells having increased capacity to repair radiation-induced DNA double-strand breaks using the same mechanisms that create SCNAs, or
• Individual SCNAs changing the expression of specific genes within structurally altered chromosomal segments to regulate survival

By correlating radiation survival with gene expression within altered segments, the researchers determined that SCNAs regulate cellular radiation-damage response in part through direct gene expression changes.

SCNA frequency and distribution varied across tumor lineages, the investigators found, with colorectal, uterine and ovarian cancers showing a positive correlation between ƒSCNA values and cellular survival.

Left Image: Distribution of cancer types profiled by lineage. Right Image: Integral survival is displayed by column scatter plot separated by lineage and histology where appropriate.

Further, in the uterine and colorectal cell lineages they discovered a correlation between radiation survival and SCNA, an anti-correlation between radiation survival and mutation frequency, and correlations between mutations in individual genes and radiation sensitivity. Collectively, these indicate a relationship between low SCNA, high mutation frequency, gene disruption of DNA repair and radiation sensitivity in uterine and colorectal cancer cell lines.

Looking at genes and gene pathways

The researchers then identified gene mutations associated with radiation sensitivity. A majority of the 19 most strongly correlated genes had not previously been implicated in radiation-induced damage response. Further analysis revealed that certain mutations directly regulated cellular radiation response rather than merely having an association with radiation sensitivity. For example, mutations in KEAP1 confer radiation resistance by regulating oxidative damage response.

The investigators also found that genetic pathways are differentially correlated with radiation response. Top pathways correlated with radiation sensitivity included DNA damage response, cell cycle, chromatin organization and RNA metabolism, while pathways correlated with radiation resistance included cellular signaling, lipid metabolism and transport, stem-cell state, cellular stress, and inflammation.

This diversity of pathways across tumor lineages suggests that extensive cellular processes play a role in survival after irradiation, and reveals several cellular receptors that may be targetable for radiosensitization.

An in vivo testbed for radiosensitivity

Breast cancer provides a good example of the potential therapeutic benefits of identifying predictive biomarkers for radiation therapy.

Although breast-conserving surgery and mastectomy can eliminate detectable macroscopic cancer, tumor foci can remain in local and regional tissue. Radiotherapy significantly reduces the odds of recurrence and mortality, but some patients are more likely than others to fail treatment. Determining who is at risk is a priority.

Analyzing breast cancer cell lines, Dr. Abazeed and colleagues looked for genetic determinants of cellular radiation survival in breast cancer. They found multiple genetic pathways associated with radiation resistance. One of the most strongly correlated gene sets was linked to androgen signaling.

Androgen receptor (AR) expression has been found to promote radiation resistance in prostate cancer, and the combination of androgen blockage and radiotherapy is the standard of care for locally advanced disease. Abnormally high AR expression has been detected in most breast cancers but its role in breast oncogenesis is unclear.

Using various testing methods, the researchers demonstrated for the first time that AR expression plays a pivotal role in protecting breast cancer cells from DNA damage, and that the suppression of androgen signaling in breast cancer cells that express AR results in increased DNA damage.

To test AR’s radioprotective role in vivo, the researchers created orthotopic xenografts by injecting AR-positive breast cancer-derived cells into the inguinal mammary glands of female immunodeficient mice. When tumors developed, the mice were randomized to receive one of four treatments: mock; the potent AR agonist enzalutamide (ENZ); ionizing radiation; or ENZ and radiation. The combined ENZ/radiation therapy more effectively suppressed tumor growth than either modality separately.

Using findings to develop predictive tests and guide treatment

The researchers’ determination that there is a genetic basis for cancer cells’ variable vulnerability to radiation has both diagnostic and therapeutic implications. Genetic alterations that dictate cellular response to DNA damage could be used predictively to assess individual patients’ likely response to radiotherapy, to suggest possible combinatorial therapy strategies, and to indicate opportunities for precision targeting of molecular pathways that confer radioresistance.

“We can potentially bin patients into more homogeneous categories that are likely to maximize their ability to respond to our therapies,” Dr. Abazeed said. “Our initial work has spawned several confirmatory studies in multiple cancer types. We are actively working on developing certified genetic tests that are destined to be incorporated into clinical practice. These tests are designed to assist the oncologist in identifying patients who are more or less likely to respond to these treatments.”

The findings may help shift the use of radiotherapy and DNA-damaging drugs from the current generic, population-based approach to a much more personalized application guided by the genetic alterations present in an individual patient’s tumor.

“Cancer treatments are currently guided by population studies of genetically heterogeneous patients,” Dr. Abazeed said. “We and others believe that grouping patients in this manner is a suboptimal strategy, namely because it does not reflect the uniqueness of individual patients. Our work to date represents the construction of a critical scaffold that will serve as a basis for future studies.”

Reference: Yard BD, Adams DJ, Chie EK, Tamayo P, Battaglia JS, Gopal P, Rogacki K, Pearson BE, Phillips J, Raymond DP, Pennell NA, Almeida F, Cheah JH, Clemons PA, Shamji A, Peacock CD, Schreiber SL, Hammerman PS, Abazeed ME. A genetic basis for the variation in the vulnerability of cancer to DNA damage. Nat Commun. 2016 Apr 25;7:11428.

Photo: Russell Lee