Newly Identified Pathway Helps Cancer Cells Survive DNA Damage

Robert Silverman, PhD

George Stark, PhD

Key Points

• A newly discovered biochemical pathway enables prostate cancer cells to survive DNA damage that normally would trigger cell death.
• In normal cells, poly-ADP-ribose polymerase-1 (PARP-1) modulates DNA repair and cellular survival by recognizing DNA strand breaks, binding to the DNA, inducing a conformational change and synthesizing poly-ADP ribose (PAR), the essential first step in DNA repair.
• Excessive DNA damage in a normal cell leads to PARP-1 overactivation, and oversynthesis of PAR, which kills the cell — a process called parthantos.
• In cancer cells, heightened concentrations of reactive oxygen species lead to DNA damage and ultimately upregulation of oligoadenylate synthetase enzymes (OASs), which greatly reduce PAR production. Decreased PAR synthesis enables cancer cells to escape parthanatos and survive an otherwise lethal amount of DNA damage.
• Inhibiting OAS synthesis could increase PAR production and sensitize many cancer cells to DNA damage while sparing normal cells.Cleveland Clinic researchers have discovered a novel biochemical pathway that enables prostate cancer cells to survive extensive DNA damage that normally would trigger cell death.

Blocking this modulatory pathway could increase cancer cells’ vulnerability to genotoxic therapies while sparing normal cells. The researchers are developing potential pathway inhibitors and will test their effectiveness paired with existing and new DNA-damaging treatments.

Identifying the pathway “is fundamentally interesting biochemistry, and also presents a clear opportunity to investigate a novel way to inhibit the ability of cancer cells to deal with excessive DNA damage, whether endogenous or exogenous,” said George Stark, PhD, of Cleveland Clinic Lerner Research Institute’s Department of Cancer Biology, who with colleague Robert Silverman, PhD, made the discovery. They are staff members of Cleveland Clinic’s Research Center of Excellence in Prostate Cancer.

The scientists, along with their collaborators at Cleveland Clinic, the University of Chicago and Thomas Jefferson University, are seeking a two-year, $1 million, Prostate Cancer Foundation to fund translational research.

Understanding DNA damage repair

Cellular DNA is regularly exposed to harmful factors and forces that can cause a variety of disruptions, including single- and double-strand breaks and base mismatches, insertions and deletions. To prevent accumulation of DNA damage and resultant genomic instability, as well as replication of DNA lesions, normal cells are equipped with numerous mechanisms to detect damage and effect repair. If damage exceeds repair capacity, programmed cell death occurs.

Cancer cells possess multiple defects in DNA repair mechanisms, but DNA damage caused by endogenous and/or exogenous means often is not cytotoxic, although it results in genomic instability, a hallmark of metastatic disease and a condition associated with poor clinical outcomes in prostate cancer patients.

Current treatments for metastatic castration-resistant prostate cancer only modestly improve survival, with a median response duration of 2.4 to 5.2 months. There is a critical need for new approaches.

One possibility, which takes advantage of recent advances in understanding the processes of DNA damage response in prostate cancer, is to manipulate one or more elements of those processes to boost cancer cells’ sensitivity to DNA-damaging treatments.

PAR’s role in repair

In normal cells, the nuclear enzyme poly-ADP-ribose polymerase-1 (PARP-1) plays an important modulatory role in DNA repair and cellular survival. PARP-1 recognizes DNA strand breaks, binds to the DNA, induces a conformational change and synthesizes the polymer poly-ADP ribose (PAR), the essential first step in DNA repair.

“PAR serves as an organizer for the DNA repair machinery, but in a normal cell it’s a transient response, after which continuing cellular processes remove PAR,” Dr. Stark says.

Excessive DNA damage in a normal cell leads to PARP-1 overactivation, massive consumption of the essential metabolite NAD (the precursor of PAR), and over-synthesis of PAR, which kills the cell by releasing the cell death effector apoptosis-inducing factor (AIF) from mitochondria into the nucleus — a process molecularly distinct from apoptosis and necrosis that Johns Hopkins University researchers dubbed parthantos.

In cancer cells, recent research has demonstrated that heightened concentrations of reactive oxygen species lead to DNA damage and formation of double-stranded RNA, which causes interferon synthesis. Interferon, in turn, triggers the upregulation of a subset of proteins, including the family of 2’,5’ oligoadenylate synthetase enzymes (OASs).

Making the OAS/PAR connection

From their previous research, Drs. Stark and Silverman were familiar with the role of OASs in interferon-induced antiviral response, where the enzymes catalyze synthesis of 2’,5’-linked oligoadenylates (2-5A). 2-5A’s function is to activate RNase L, which cleaves viral and cellular RNA and restricts replication.

OASs are present only in small amounts in normal cells, but are commonly much more abundant in cancer cells, including about 30 percent of prostate cancers. Extensive PAR-mediated DNA-damage repair (or, conversely, parthanatos) is not occurring in cancer cells. Could there be an OAS/PAR connection affecting DNA repair?

“We knew that a DNA virus can cause activation of OASs and the production of 2-5A,” Dr. Stark explains. “In thinking about how cancer cells respond to DNA damage, we began to wonder whether DNA damage could similarly activate OASs and the 2-5A system to affect PAR synthesis. If you look at the structure of PAR, the polymer chain ends in an adenosine diphosphate ribose, which can serve perfectly well as a primer for 2-5A addition.”

Using an antibody to the 2’,5’-linked core structure of 2-5A that Dr. Stark had developed more than three decades ago, he and Dr. Silverman confirmed that OASs do greatly reduce PAR production in cancer cells. The overabundant OASs (activated by DNA damage) cap and terminate PAR by adding 2’,5’-linked AMP residues to the ends of PAR chains, preventing their propagation.

By significantly reducing PAR synthesis in this way, cancer cells are able to escape parthanatos and survive an amount of DNA damage that typically would be lethal to cells.

Since interferon is an inhibitor of cell growth and is used to treat cancer, it seems paradoxical that cancer cells’ interaction with interferon would result in a survival assist from OAS overexpression and PAR attenuation. The degree of interferon exposure may account for the difference.

“There is a small subset of genes expressed when cells are exposed constitutively rather than acutely to interferon, which is present in the microenvironment all of the time, and that subset includes the OASs,” Dr. Stark says. “Cancer cells manage to deal with exposure to interferon in a way that helps them. We’re striving to understand that. This discovery of the PAR modulation pathway is a part of the story.”

Drs. Stark and Silverman believe that inhibiting OAS synthesis will increase PAR production and sensitize many cancer cells to DNA damage, with little probable effect on normal cells since they have very low OAS levels.

Pairing OAS inhibition with enhanced DNA damage

There are no known OAS inhibitors, so the researchers are working with Babal Kant Jha, PhD, a structural biologist in Cleveland Clinic’s Department of Translational Hematology and Oncology Research, to develop novel compounds as well as screen libraries of existing compounds for candidate inhibitors that can be tested in cell culture and patient-derived xenografts.

“There are two contexts in which you could imagine using an OAS inhibitor,” Dr. Stark says. “One is in order to kill cancer cells outright. Cancer cells are damaging their DNA, particularly by oxidative stress, so it may be that blocking OAS activity will cause them to make too much PAR, triggering programmed cell death by parthanatos as occurs in normal cells.

“The other scenario is to enhance the cytotoxic effect in cancer cells by combining an OAS inhibitor with exogenously delivered DNA damage generated by ionizing radiation, cisplatin, or by reactive oxygen species that form in cancer cells in response to Vitamin C,” he says.

The researchers plan to explore the efficacy of this OAS-inhibition/enhanced-DNA-damage combination in both primary and metastatic prostate cancer.

They also will compare the efficacy of OAS inhibitors with PARP inhibitors such as olaparib, which have beneficial therapeutic effects by blocking PAR synthesis and synergizing with cancer cells’ intrinsic DNA repair defects.

“OAS inhibitors and PARP inhibitors both modulate PAR synthesis, but in very dissimilar ways, so we’ll assess them objectively,” Dr. Stark says. “PARP inhibitors are pretty toxic. The attraction of an OAS inhibitor is that it’s not going to inhibit PARP, which has important roles in other biological processes. OAS inhibition is a different route to get at PAR’s role in helping cancer cells avoid termination from extensive DNA damage. I suspect it’s going to be a lot less toxic.”

Lastly, in a related approach, the researchers will evaluate combining the new drug CBL0137 with cisplatin to enhance DNA damage in cancer cells in response to ionizing radiation.

CBL0137 is a novel small molecule that inhibits FACT (facilitates chromatin transcription), a histone chaperone complex predominantly expressed in undifferentiated cells. Dr. Stark and collaborators recently determined that FACT preferentially kills cancer stem cells and synergizes with cisplatin and other DNA-damaging treatments to kill prostate cancer organoid cells and enhance the killing of multiple types of cancer cells. Other researchers have found that FACT inhibition by CBL0137 also is necessary for DNA repair, since histones must be rearranged at DNA damage sites.

Because of the low toxicity potential of OAS inhibitors, CBL0137 and vitamin C and the well-established use of cisplatin and radiotherapy, Drs. Stark and Silverman believe that their research into the PAR modulation pathway could lead to clinical trials relatively soon. Although they are focusing on prostate cancer, their approach has broader implications. “If we prove the principle in prostate cancer and develop effective OAS inhibitors, I think there would be significant interest in translating this into other cancers,” Dr. Stark says.