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As many people with a family history of cancer know all too well, inheriting a mutation in the BRCA1 or BRCA2 genes dramatically increases cancer risk. People with these mutations can be 75% more likely to develop breast cancer during their lifetime and also face a higher risk of ovarian and prostate cancer. In hopes of catching cancer early, people with BRCA mutations typically undergo extra mammograms and screening procedures and some even elect to have risk-reducing surgeries such as mastectomies.

With V Foundation funding, Karlene A. Cimprich, PhD, from Stanford Cancer Institute is making new discoveries about how BRCA mutations lead to cancer. Her work is revealing potential new therapeutic targets for BRCA-related tumors and laying the groundwork for a new blood test to catch cancer sooner.

“Individuals with BRCA mutations frequently get cancers earlier in life,” said Cimprich. “We want to understand what drives this process, including what the BRCA genes do normally that isn’t taking place when mutations are present, and how this leads to early development of cancer.”

Escaping the nucleus

It all began years ago when Cimprich and her colleagues discovered something odd that occasionally happens inside the cell nucleus, the place where cells extract information from DNA to make proteins that keep the cell running. Normally, cells make RNA in the nucleus and then shuttle it out to the cytoplasm to serve as a template for making proteins. However, Cimprich found that under certain conditions RNA gets tangled with DNA in the nucleus, creating a structure called an R-loop.

If the cell doesn’t catch and correct this mistake, it can damage the DNA. After Cimprich’s discovery, another research group found that the BRCA1 and BRCA2 genes help stop R-loops from forming. This suggests BRCA mutations could allow R-loops to accumulate, leading to DNA damage.

Studying cells that lack normal BRCA1 and BRCA genes, Cimprich and her colleagues made another surprising discovery: in these cells, R-loops were not only found in the nucleus but also in the cell’s cytoplasm. The researchers gave these errant sections of DNA and RNA floating around the cytoplasm a name that sounds like science fiction: “cytoplasmic hybrids.”

Understanding harmful hybrids

The V Foundation awarded the team funding to find out where cytoplasmic hybrids come from, why they form and how they affect cell function. In the process, the researchers developed new imaging tools and new methods for isolating and genetically sequencing the hybrids.

“These cytoplasmic hybrids have never been reported before,” Cimprich said. “We not only showed they exist but developed ways to observe and characterize them.”

The researchers found that cytoplasmic hybrids activate an immune response that eventually leads to cell death. “This was a surprising finding and means that it might be possible to develop therapies that help boost this immune response in patients with BRCA1 or BRCA2 mutations,” Cimprich said. “We’re now working to better understand this immune response and how cancer cells have figured out how to evade this cell death.”

Easier screening

The researchers are also working on developing a way to isolate these cytoplasmic hybrids in blood. This could make it possible to develop a blood test that would offer a less invasive, more cost-efficient way of screening for BRCA-linked cancer. Toward this goal, they are now beginning to analyze blood samples from volunteers to see how well they can detect the cytoplasmic hybrids in blood and whether their levels correlate with cancer progression and initiation. They suspect that levels of the hybrids increase as cancer starts to form.

Cimprich says that the V Foundation support was critical in helping her bring together a group of Stanford scientists from different fields, which inspired new ideas and approaches. For example, Jim Ford, a geneticist and medical oncologist, encouraged the group to look for the cytoplasmic hybrids in blood. Also, team member Aaron Straight’s expertise in imaging was instrumental in figuring out how to visualize the cytoplasmic hybrids.

She also credits the V Foundation for being willing to take on a project that was high risk because it was different and new. “Our results were not exactly what we expected, but what we found out was still very new and exciting,” Cimprich said. “These findings are now driving much of the new research in my lab.”

About a third of all cancers diagnosed in women each year are breast cancers. The vast majority — 70% to 80% — of these tumors are estrogen receptor positive (ER+), which means they use the hormone estrogen to grow. Although ER+ breast cancer is often treatable with estrogen-blocking tamoxifen or other endocrine therapies, many patients eventually develop endocrine resistance that can lead to recurrence.

With funding from the V Foundation, Kexin Xu, PhD, from Mays Cancer Center at the University of Texas Health in San Antonio, is working to find new therapies that could circumvent this common form of treatment resistance and stop breast cancer from coming back. Her work may even uncover ways to make ER+ tumors more responsive to existing cancer treatments.

“About 30% to 40% of patients with localized ER+ tumors, and nearly all patients with ER+ metastatic breast cancer, will develop resistance to hormone therapies like tamoxifen,” said Xu. “This is why we’re very interested in finding new therapeutic options for patients with therapeutic-resistant breast cancer.”

Epigenetics could be key

At the heart of Xu’s strategy is an area of science called epigenetics that has gained substantial attention in cancer research. Epigenetic changes are chemical modifications that alter how genes are expressed without changing the genetic code. Because these changes are reversible, it is possible to use drugs to change cancer-linked epigenetic patterns back into normal patterns. In fact, drugs that work this way are already being used to treat other types of cancers.

“A lot of studies have suggested that epigenetic abnormalities play a very important role in the development of endocrine resistance,” said Xu. “Thus, we think epigenetics may represent a new direction for developing drugs for endocrine resistant ER+ breast cancer.”

In earlier work, Xu’s team identified an epigenetic enzyme called EZH2 as a very promising therapeutic target for treatment-resistant metastatic breast cancer. Experiments using an FDA-approved drug that inhibits EZH2-mediated epigenetic abberations also showed encouraging initial results.

For their V Foundation project, the researchers are digging deeper into how this drug works to put the brakes on breast cancer. They also developed a new breast cancer mouse model in which tissue from a patient with ER+ breast cancer is implanted into the mouse and moved from an endocrine sensitive to resistant status. Although they can be difficult to establish, this type of mouse model can be very useful for discovering and testing new therapies because the tumors are so similar to those found in patients.

Reversing treatment resistance

Using their new mouse model, Xu and colleagues found that the FDA-approved EZH2 inhibitor slowed the growth of ER+ breast tumors, especially tumors with endocrine resistance. They also observed that an EZH2-targeting drug can change the tumor microenvironment in a way that may make ER+ breast cancer more responsive to immunotherapy, a type of treatment that stimulates the body’s own immune system to attack cancer.

“This is an exciting finding because ER+ cancers usually don’t respond to immunotherapy,” said Xu. “We are now studying how these EZH2 inhibitors might synergize with immune checkpoint inhibitors to block the endocrine resistant breast cancer. Since both therapies are already FDA-approved, this combination therapy could move quickly into clinical testing.”

The researchers also developed new EZH2 inhibitors that show stronger effects against endocrine resistant breast cancer than the currently approved ones. They plan to use their new mouse models to test these promising drugs further.

“The support from V Foundation was key for developing the new models, which are an invaluable system for not only testing EZH2 inhibitors but also some other promising treatments,” said Xu. “This gives our lab a very unique tool that puts us in a priority position to carry out certain types of research.”

Xu says that the V Foundation support also allowed her team to collect the preliminary data necessary to expand into new research areas and gain new funding resources. This data has already helped them secure funding from the NIH, the Susan G. Komen Breast Cancer Foundation and the Mays Cancer Center.

To help fund important breast cancer research through the V Foundation, give through our Clea Shearer Breast Cancer Research Fund.

Thanks to new treatments, survival rates for children with cancer are high. However, the treatments they receive put them at higher risk for a variety of chronic health conditions later in life — a risk that is unfortunately amplified for those with socioeconomic disadvantages.

With V Foundation funding, Zhaoming Wang, Ph.D., and I-Chan Huang, Ph.D., from St. Jude Children’s Research Hospital are working to better understand the role of socioeconomic status in health problems among adult survivors of childhood cancer. This work could lead to new ways to help these survivors stay healthy throughout their lives.

“Most cancer survivorship research focuses on documenting the long-term toxicity of cancer treatment,” explained Wang. “We are among the first to look at the biological causes of health disparities in childhood cancer survivors. This group is vulnerable because they age faster, which means that chronic health conditions affect them even earlier. Someone who had cancer treatment as a child might have a heart attack in their thirties, for example.”

In pioneering new work, the researchers are using the concept of social epigenetics to study how social factors and cancer treatment work together to change the way a person’s genes are expressed. By helping to reveal the biological mechanisms behind these disparities, researchers hope to find ways to identify those at highest risk and intervene to reduce or prevent the chronic health conditions they face.

A unique clinical resource

The new research draws on the St. Jude Lifetime Cohort, a clinical cohort St. Jude Children’s Research Hospital established to follow its pediatric cancer patients over their lifetimes. The approximately 6,000 patients and survivors currently enrolled in the cohort visit the clinic every few years for comprehensive medical assessments that include blood draws, physical performance tests, neuro-psychological assessments and more.

“Our study wouldn’t be possible without the data from this group of patients, which is one of the largest cohorts of this type in the country,” said Huang. “Thanks to the extensive assessments, we are able to examine more than 175 chronic health conditions, assess various socioeconomic factors and also conduct epigenetics analyses on stored blood samples.”

The researchers are studying a particular type of epigenetic change — a change to the genome that affects how genes are expressed — known as DNA methylation. For the first part of their project, they examined how cancer treatments affect DNA methylation and, subsequently, chronic health conditions. As published in a Genome Medicine paper, they identified thousands of DNA methylation sites that were affected by cancer treatment, especially radiation.

“This shows that the treatment leaves a permanent signature on the epigenome,” said Wang. “We also found that some of these changes are linked to cardiometabolic conditions, which helps explain why the treatment increases the risk of chronic health conditions.”

Next, they examined how each person’s education level, personal and household income, and neighborhood disadvantaged socioeconomic status correlated with DNA methylation patterns. For this study, published in an Epigenetics paper, they measured DNA methylation at about 1 million sites and found around 130 DNA methylation sites were correlated with these socioeconomic factors. For example, people with higher education had very different methylation signatures than those with less education. The researchers then began to take a closer look at these 130 socioeconomically linked markers to see if any were linked with chronic health conditions. This ongoing research suggests that some of these markers may mediate the association between socioeconomic factors and impaired lung function.

Closing the disparity gap

The research could eventually be used to help identify people at high risk of certain conditions, to develop interventions aimed at modifying socioeconomic exposures, and possibly even develop drugs that target the genes involved in these disparities.

“These methylation signatures can also be used to measure how well interventions are working,” said Wang. “Changes in epigenetics are immediately accessible, which means we don’t have to wait and see if patients go on to develop a chronic disease to find out if an intervention is working.”

Next, the researchers plan to use RNA sequencing to study how methylation levels relate to expression levels for individual genes, uncovering more information about the biological mechanisms at work. They are also applying for NIH grants to extend their work and develop interventions that could help improve the unmet social needs of childhood cancer survivors with the aim of counteracting certain health disparities in this vulnerable population.

Although immunotherapy has saved many lives, it can also come with serious side effects.

“As a clinician, the worst-case scenario is when I have a patient who not only doesn’t respond to a treatment but who also experiences severe toxicity because of it,” said V Scholar Aadel Chaudhuri, MD, Ph.D., from Washington University School of Medicine in St Louis. “Unfortunately, this can happen with immunotherapy because it can inadvertently cause the immune system to attack healthy tissues. Predicting who will experience this type of severe toxicity is very challenging.”

With funding from the V foundation and other sources, Chaudhuri has made significant progress toward generating a way to identify patients with the highest risk for severe toxicity from immunotherapy. He is also working to find ways to predict treatment response so that one day a simple blood test might provide clinicians the information necessary to select a treatment that will not only get rid of the cancer but also be the least harmful for a specific patient.

Predicting toxicity

To figure out who is most likely to experience toxicity from immunotherapies, Chaudhuri and his colleagues at Washington University, Yale University and Stanford University, used RNA sequencing and mass cytometry to analyze immune cells in blood samples from patients with melanoma. The samples were taken from blood circulating throughout the body during routine laboratory blood draws taken before patients started an immunotherapy known as immune checkpoint blockade.

As described in a recently published Nature Medicine paper, the researchers found that patients who experienced severe toxicity had a higher abundance of certain immune cells – activated CD4 memory T cells – and also a greater diversity of receptors on the T-cells in their blood. Based on these characteristics, the researchers created a composite model for predicting who will develop severe or life-threatening toxicity.

“Although research has shown that patients who experience toxicity also have higher rates of response to immunotherapy, our research suggests that response and toxicity can be immunologically decoupled,” said Chaudhuri. “We think we’ve identified immune underpinnings that are specific to severe toxicity and aren’t significantly associated with treatment response.”

Going in a new direction

Chaudhuri points out that when he started studying immunogenomics and toxicity, he didn’t have a track record in this area. “This would have made it difficult to get NIH funding for this project,” he said. “The V Foundation took a leap of faith that allowed me to pivot my research to an important and interesting area that has enormous potential to improve cancer treatment.”

Chaudhuri and his collaborators are now working with the Melanoma Research Alliance to design a prospective study that will, if successful, translate their published research findings into a precision diagnostic blood test for predicting toxicity before treatment begins.

“We started this line of research with metastatic melanoma because immune checkpoint blockade is a front-line therapy in this disease,” said Chaudhuri. “However, we aim to translate our findings to predict immunotherapy toxicity in other cancer types too.”

Farther down the road, these new insights into the mechanisms responsible for immunotherapy toxicity could help inform the development of drugs that selectively block toxicities from these otherwise highly effective medicines.

Head and neck cancers are particularly devastating because they often affect the way a patient looks, as well as crucial functions such as eating and speaking.

“For a long time, head and neck cancers were thought of as resulting from poor choices such as smoking or drinking, which unfortunately led to a dearth of funding in this area,” said V Foundation Scholar Sidharth Puram, M.D., Ph.D., from Washington University School of Medicine. “Now, there’s a growing recognition that studying head and neck cancers is important, and this is creating opportunities to make major strides in understanding these cancers so that better treatments can be developed.”

As a head and neck surgeon, Puram is acutely aware of the ostracization and stigmatization patients with these cancers can experience. This has motivated his quest to better understand how and why head and neck cancers sometimes spread, or metastasize. His work could eventually lead to new therapies or allow doctors to tailor treatments based on whether a tumor is likely to spread.

Looking at what makes cancer spread

The new project expands on findings from a previous study in which Puram and his colleagues analyzed gene expression in head and neck cancers in individual cells, one of the first single-cell analyses of its type to be performed on solid tumors.

“Our previous single-cell work revealed that a subset of cells appears to be particularly important for invasion and metastasis,” said Puram. “These cells undergo what is called a partial epithelial to mesenchymal transition (partial EMT), which means they change in a way that allows them to ultimately spread.”

With the V Foundation funding, the researchers studied these partial EMT cells in detail to find out how they work and what protein markers could be used to identify them. In collaboration with Shannon Stott, Ph.D., and her group at Massachusetts General Hospital, they used a new technology known as multi-spectral imaging that allows simultaneous, high-resolution imaging of up to 15 cell markers across a tissue section that may contain thousands of cells.

“We used this approach to analyze millions of cancer cells from around 10 different tumor samples,” said Puram. “We sought to answer important questions such as how do markers that relate to partial EMT drive metastasis?”

The spectral analysis helped the researchers pinpoint a group of partial EMT markers that appear to play a role in metastasis. It also showed that partial EMT cells might hide from the body’s immune system, perhaps allowing them to avoid destruction and most likely contributing to their ability to spread. A collaboration with Itay Tirosh, Ph.D., and his group at the Weizmann Institute of Science also revealed that cells undergo partial EMT in other types of tumors beyond head and neck cancers.

Working toward better treatment

“In terms of clinical translation, we think that the presence of partial EMT cells could be useful as a readout of cancer aggressiveness, which could allow doctors to dial in the treatment that appropriately matches that aggressiveness,” said Puram. “As we keep learning more about the partial EMT process, it might also be possible to develop a drug that targets partial EMT to make tumors less aggressive or prevent metastasis.”

Puram says that the V Foundation funding made it possible to generate the preliminary data that is critical for securing additional grants. It also helped set up his lab with the tools and personnel necessary for working toward translating their research into the clinic. The funding has already led to a new study in which the researchers will scale up the multi-spectral imaging analysis by examining 200 new tumor samples. This will help them find out whether patients with partial EMT cells are more likely to have poor outcomes or show resistance to treatment, for example.

“Funding for head and neck cancer research is very important because of how much catching up we have to do in our field compared to other tumors,” said Puram.