By Nicole Davis, PhD
Just like humans, cells get stressed, too — especially cancer cells. For decades, Dana-Farber researchers have been characterizing a complex molecular condition in cancer cells known as DNA replication stress. Now, fueled in part by that pioneering research, a new generation of treatments is emerging that takes aim at the vulnerabilities created by this "stressed out" state. These advances could open up new therapeutic options for patients, especially those with ovarian and uterine cancers.
Alan D'Andrea, MD
"There's a lot of interest now in targeting DNA replication stress," said Alan D'Andrea, MD, who directs Dana-Farber's Susan F. Smith Center for Women's Cancers and the Center for DNA Damage and Repair. "It's become clear over the last several years that certain tumors, including high-grade serous ovarian cancer, have naturally high levels of replication stress. And how these cells cope with such stress leads to molecular vulnerabilities that can be exploited from a therapeutic standpoint."
A hallmark of cancer cells is their ability to rapidly — and continuously — divide. Yet before any cell, malignant or not, can divide in two, it must copy (or "replicate") its DNA, providing each future daughter cell with a complete set of genetic instructions. DNA replication is a highly choreographed molecular dance. It involves six known methods for fixing mistakes that can arise in the genetic code, which cells must resolve or risk transmitting to their progeny. In addition, there are also multiple checkpoints when the cellular machinery pauses to ensure that DNA replication is unfolding in an orderly way.
But in cancer cells, which are wired to grow quickly, replication often does not proceed in an efficient and coordinated fashion, and as a result, the process slows down. That's replication stress. And as Dr. D'Andrea and his Dana-Farber colleagues are learning, when cancer cells become stressed, they also become vulnerable. Moreover, the researchers now recognize that if they block the cells' coping mechanisms — the molecular signals and pathways that help repair DNA and get DNA replication back on track — the cancer cells will die.
PARP Blockers and Repurposed Drugs
Dr. D'Andrea didn't set out to study ovarian cancer. Trained as a hematologist, he initially became interested in a rare childhood disease known as Fanconi anemia (FA) and spent his early career unlocking its biology, which led him and his colleagues to discover a key DNA repair pathway.
"I was looking for a niche where DNA repair and replication stress mattered and that drew me to ovarian cancer," says Dr. D'Andrea. "It is very difficult to treat the disease and new, targeted approaches are needed."
One class of therapies that has been developed for ovarian cancer is PARP inhibitors. These drugs block the activity of a key protein called poly (ADP-ribose) polymerase or PARP, which is involved in a type of DNA repair that mends single-strand breaks in the DNA double helix. PARP inhibitors can also enhance replication stress, making those cells vulnerable. While PARP inhibitors have spurred a paradigm shift in ovarian cancer treatment, some patients do not respond to them and most who do, unfortunately, go on to develop resistance.
Several years ago, Dr. D'Andrea and his team identified a new molecular target that is now showing early promise in overcoming PARP inhibitor resistance. The researchers were studying tumor cells that carry mutations in the BRCA1 or BRCA2 gene, scouring molecular readouts to glean clues about their potential vulnerabilities. The cells harbor defects in a form of DNA repair that mends double strand breaks and, as a result, are hyper-dependent on other forms of DNA repair — such as the one controlled by PARP. (That's why PARP inhibitors are most effective in killing BRCA-deficient tumors; they amount to a one-two punch, known in scientific parlance as a synthetic lethal hit.)
The researchers noticed that these BRCA-deficient cells harbored very high activity levels for a gene called DNA polymerase theta. And when they blocked the activity of polymerase theta, the cells died.
"So, polymerase theta became a very interesting target," said Dr. D'Andrea. "It was as strong of a synthetic lethal hit as a PARP inhibitor — if you inhibit PARP, you kill a BRCA-deficient cell; and if you inhibit polymerase theta, you also kill a BRCA-deficient cell."
Geoffrey Shapiro, MD, PhD
There was another remarkable finding: when the scientists blocked polymerase theta activity in BRCA-deficient cells that had grown resistant to PARP inhibitors, those cells died, too. Based on these results, Dr. D'Andrea and his team conducted a screen for chemicals that could inhibit polymerase theta and they pulled out novobiocin, a drug that had already been tested in humans decades ago. Originally developed as an antibiotic, it is no longer prescribed in humans as it has been replaced by more effective drugs. But now, novobiocin is being resurrected. Dr. D'Andrea and Dana-Farber's Geoffrey Shapiro, MD, PhD, with support from the National Cancer Institute's Experimental Therapeutics (NeXT) program, are launching a phase 1 clinical trial testing novobiocin as a single agent therapy in patients with mutations in BRCA genes and other related DNA repair genes.
"This is a true drug development project that began here at Dana-Farber," said Dr. Shapiro, who serves as Dana-Farber's senior vice president for Developmental Therapeutics and clinical director for the Center for DNA Damage and Repair. "The phase 1 trial is an important step because it will give us preliminary information on the safety of novobiocin when given chronically as a cancer therapy. As an antibiotic, which is the purpose for which it was originally approved, the drug was administered only for short periods of time."
Meanwhile, Drs. D'Andrea and Shapiro and their colleagues continue to pursue translational studies that help bridge what researchers are learning about the underpinnings of cancer and the drugs designed and developed to treat it. In Dana-Farber's Center for DNA Damage and Repair, they have amassed a variety of cell and animal models of DNA replication stress and can test candidate drugs to characterize their effects. The approach can also help unearth molecular biomarkers that signal not only whether cells have replication stress, but also if they are likely to respond to a particular drug.
"Every time we study one of these drugs, we learn more about the basic biology," said Dr. D'Andrea. "For example, we have found 15 different mechanisms of PARP inhibitor resistance, and each one has distinct biology that helps us better understand how cells work and how they can outwit cancer drugs. Our work is really able to move back and forth between the laboratory bench and the bedside."
Targeting Replication Stress for Cancer Therapy
Other tumor types have high levels of DNA replication stress, too, including some of the more aggressive types of uterine cancers, which are sometimes referred to as high-grade endometrial cancer. While these tumors can share certain molecular features with high-grade serous ovarian cancers, they typically lack the defects in DNA repair that render many ovarian tumors vulnerable to the cancer-killing effects of PARP inhibitors. And for tumors with that molecular profile, which also includes a particular type of uterine cancer called uterine serous cancer, there are currently few treatment options beyond conventional chemotherapy.
Joyce Liu, MD, MPH
"Replication stress as a clinical target in cancer has really emerged over the past few years," said Joyce Liu, MD, MPH, associate chief and director of clinical research for Gynecologic Oncology at Dana-Farber.
Many cells with replication stress depend heavily on the checkpoints that are built into the process of cell division (or "mitosis"). During these checkpoints, the molecular machinery is instructed to pause, allowing cells to verify that their DNA has been fully and faithfully copied. One of these checkpoints, known as the G2-M checkpoint, is controlled by a protein called WEE1.
"We hypothesized that these cells are highly reliant on the G2-M checkpoint to prevent them from going into mitosis with under-replicated or misreplicated DNA," said Dr. Liu. "If you bring in a WEE1 inhibitor, you basically remove that checkpoint, and as a result, the cell is forced to enter mitosis before it is ready to do so and dies."
About five years ago, Dr. Liu and her colleagues launched a clinical trial that examined the use of a WEE1 inhibitor, called adavosertib, as a single-agent therapy for patients with uterine serous cancer. The results were noteworthy: nearly 30% of patients saw their tumors shrink, and 50% showed no signs of significant growth in their cancer for at least six months. "If we think about what standard chemotherapy offers these patients, this is a significant signal of activity," said Dr. Liu.
She and her colleagues have since devoted their efforts to dissecting the biology that underlies tumors' response to adavosertib. Together with Dr. D'Andrea, Dr. Shapiro, Dipanjan Chowdhury, PhD, and colleagues at Dana-Farber's Belfer Center for Applied Cancer Science, the researchers developed a method that harnesses biopsies of patients' tumors and propagates the tumors cells in the laboratory as "organoids", enabling the team to understand what happens to the cells and their levels of replication stress when exposed to adavosertib.
"The question we really want to answer here is, 'What are the signals that we can see in these biopsy samples that allow them to respond to WEE1 inhibition?'" said Dr. Liu. "As we understand mechanistically how this works, it will help us learn what other drugs we might add to enable better, more durable responses or to allow patients whose tumors are not currently sensitive to WEE1 inhibitors to become sensitive to them."
Panagiotis Konstantinopoulos, MD, PhD
Dana-Farber's Panagiotis Konstantinopoulos, MD, PhD, director of translational research in Gynecologic Oncology and co-director of the Center for BRCA and Related Genes has also been pioneering new approaches to targeting replication stress. He and his colleagues have been testing molecular inhibitors of key proteins that help cancer cells cope with DNA replication stress. These include an inhibitor of the ATR protein, called berzosertib, as well as a drug, prexasertib, which blocks the activity of two related proteins, CHK1 and CHK2.
In a multi-center, randomized phase 2 clinical trial, Dr. Konstantinopoulos and his colleagues showed that adding berzosertib to standard gemcitabine chemotherapy improved outcomes in platinum resistant ovarian cancer; they also identified a replication stress biomarker associated with patients' response to treatment. In addition, his initial study of prexasertib, a multi-center clinical trial of patients with various forms of ovarian cancer, including platinum-resistant tumors, revealed that the drug was active in some patients. However, the response rate was low — only 12%. Now, Dr. Konstantinopoulos is co-leading a clinical trial that incorporates the use of a molecular signature that can help define which patients are likely to respond to prexasertib. The trial is enrolling patients with ovarian cancer as well as those with endometrial cancer.
In addition, Dr. Konstantinopoulos is leading an investigator-initiated clinical trial that combines an ATR inhibitor with an immunotherapy drug, called avelumab. The trial is exploring the effectiveness of this drug combination in a specific cohort of patients with endometrial cancer. These are patients whose tumors carry mutations in a gene called ARID1A and have grown resistant to prior immunotherapy treatment.
"We know from our work in preclinical models that both drugs work better together — and we believe this synergy could help re-sensitize tumors to immunotherapy," said Dr. Konstantinopoulos.
He is also teaming up with Dr. Liu and Ursula Matulonis, MD, chief of Gynecologic Oncology and the Brock-Wilson Family Chair at Dana-Farber, to pursue a major research program aimed at targeting replication stress in uterine cancer. The researchers recently received a prestigious, five-year grant to carry out this work, which includes three projects. Dr. Konstantinopoulos will lead a project that investigates the combination of two drugs — an ATR inhibitor as well as a drug that blocks a key signaling pathway, called the PI3 kinase pathway. The two other projects will include studies of adavosertib as well as drug combinations that simultaneously target both replication stress and tumors' immune environment.
"Replication stress represents a very important field of study for cancer therapeutics," said Dr. Konstantinopoulos. "We believe the work we're doing now will yield insights down the road that can improve the lives of our patients."
