Inside Salk; Salk Institute

Cancers in the Crosshairs

Nothing stopped Dan Lewis—until that day in 2006.

The hardworking president of a global management and strategy consulting firm, as well as a commercial aviation industry consultant, Lewis was always on the go. But that day, in a New York doctor's office after a busy weekend in Chicago, he was brought up short.

"I'll bet you think you're going somewhere tomorrow on business," the doctor told him. "But you're not going anywhere. Not until I tell you to."

As president of Booz Allen Hamilton Global Management Consulting, Lewis was required to get frequent checkups, which included blood tests. A test he'd taken before his Chicago trip had sounded an alarm. The doctor explained that Lewis had chronic myelogenous leukemia, or CML, a cancer of the white blood cells that starts in the bone marrow and causes uncontrolled growth of white blood cells called myeloid cells. Left untreated, the diseased cells overrun the bone marrow and circulatory system, leading inevitably to death.

But there was also good news. Had he contracted the disease a decade earlier, his prognosis would have been bleak: the average survival time with standard chemotherapy back then was just three to five years. By 2006, when he received his diagnosis, an entirely new kind of drug, a targeted cancer therapy called Gleevec, was rewriting the rules for how doctors treated CML—and by extension other forms of cancer. For most patients, Gleevec had transformed CML from a fatal disease into a chronic disease— with nearly 90 percent of patients surviving at least five years, many with no symptoms.

Tony Hunter

Tony Hunter

Lewis later learned that the breakthrough that made Gleevec possible, a discovery by a scientist named Tony Hunter of a form of protein regulation in cells, had taken place at the Salk Institute, where Lewis happened to be a member of a far-flung group of supporters known as the International Council. "I've been on Gleevec for six or seven years now, which is a testament to the importance of Dr. Hunter's discovery," says Lewis, who has since joined Salk's Board of Trustees. "Before, people were thinking about trying to kill cancers with chemotherapy, but Tony's work helped launch a whole new paradigm."

Indeed, that new paradigm, the development of precisely targeted cancer therapies, owes much to discoveries made by Salk scientists. Just a few decades ago, cancer was a black box. Little was known about the biological processes underlying its development and progression, and finding diagnostics and treatments for cancers was very much a hit-or-miss process. Scientists of the Salk Cancer Center, which was established in 1970 and designated in 1973 by the National Cancer Institute (NCI) as the first of seven basic cancer research centers in the United States, have played a central role in prying open that black box. Salk researchers were well ahead of the curve, for instance, in approaching cancer as a disease of normal cells gone rogue due to genetic mutations—a discovery for which the late Salk scientist Renato Dulbecco received the Nobel Prize.

And Salk isn't resting on its laurels. Using new technologies, including powerful imaging techniques, stem cell-based studies and genome sequencing, the Institute's scientists are speeding their exploration of the molecular mechanisms underlying cancers. Even as they continue to aggressively root out the molecular bad actors, they are building on their expertise in cellular mechanics to identify and test promising therapies against tumors. By bridging the gap between basic biological research and clinical trials, Salk researchers are ensuring that laboratory discoveries become medical breakthroughs.

"These are complex diseases at the molecular level, which means sophisticated science is needed to develop the tools for diagnosing and treating them," says Hunter, who directs the Salk Cancer Center and holds the Renato Dulbecco Chair in Cancer Research. "Thanks to Gleevec and other targeted therapies, the prognosis for patients with certain cancers has improved dramatically. We think that can be true for all cancers, that the mutations leading to cancer can be tamed."

Inder Verma

Inder Verma

AN UNLIKELY ALLY

Odd as it seems, viruses have proven one of the most valuable tools for Salk's cancer researchers. Much maligned for their pathological tendencies, viruses are whizzes at infiltrating other organisms' cells and altering their DNA, an ability that scientists have appropriated for studying cancer. In fact, it was studies of a tumor virus that led to Hunter's discovery of the new type of enzyme that is targeted by Gleevec. And researchers in the laboratory of Salk professor Inder Verma have made great progress using defanged viruses to study a particularly aggressive form of brain cancer, glioblastoma multiforme (GBM).

Every year more than 14,000 Americans succumb to malignant brain tumors, and the average life expectancy after the disease is detected is just over a year. Verma's team developed a mouse model to study the pathophysiology of these tumors. To do this, they harnessed the power of modified viruses, of a viral type called lentiviruses, to disable tumor suppressor genes that regulate the growth of cells and inhibit the development of tumors.

Scientists had long believed that GBM begins in glial cells that make up supportive tissue in the brain or in neural stem cells. But through studies of their mouse model, Verma's team found that the tumors can originate from other types of differentiated cells in the nervous system, including cortical neurons. Their findings, reported recently in Science, also offer an explanation for the recurrence of GBM following treatment and suggest potential new targets to treat these deadly brain tumors.

"One of the reasons for the lack of clinical advances in GBMs has been the insufficient understanding of the underlying mechanisms by which these tumors originate and progress," says Verma, holder of the Irwin and Joan Jacobs Chair in Exemplary Life Science. "The cancer-causing insults to neurons or glial cells reprogram them into stem cells which can continue to proliferate and induce tumor formation, thereby perpetuating the cycle of continuous cell replication to form malignant gliomas."

Verma has also used his viral vector technique to create a mouse model for non-small cell lung cancer, a type that accounts for as much as 80 percent of all lung cancer cases in humans. In one study with these mice, Verma's team found that blocking the activity of the enzyme IKK2, which helps activate the body's inflammation response, slowed the growth of tumors in mice with lung cancer and increased their lifespan. The findings suggested that drugs targeting this inflammation pathway may present a new avenue for treating certain lung cancers.

"These models offer us a way to study cancer in a living organism that faithfully follows the pathology, physiology and molecular signatures of human cancers," says Verma. "It not only lets us explore the mechanics behind the various forms of cancer, but also provides us with a platform for testing therapeutics against tumors. What we're working on now is developing models for other cancers, which is important because different cancers operate in different ways."

Clodagh O'Shea, an associate professor at Salk, is exploring another highly promising avenue for using viruses in the development of cancer therapeutics, based on modified adenovirus, a type of cold virus. Adenovirus has developed molecular tools that allow it to hijack a cell's operations, including large cellular machines involved in growth, replication and cancer suppression.

Clodagh O'Shea

Clodagh O'Shea

O'Shea studies E4-ORF3, a cancer-causing protein encoded by adenovirus, which prevents the p53 tumor suppressor protein from binding to its target genes. Normally, p53 suppresses tumors by causing cells with DNA damage—a hallmark of cancer—to self-destruct. This tumor suppressor pathway is inactivated in almost every human cancer, allowing cancer cells to escape normal growth controls and acquire new cancer-causing mutations. Similarly, by inactivating p53, the E4- ORF3 protein enables adenovirus replication in infected human cells to go unchecked.

O'Shea's team revealed the ultrastructure of the remarkable polymer that E4-ORF3 assembles in the nucleus—something that previously had proven difficult since the polymer is effectively invisible using conventional electron microscopy. "What you see is the E4-ORF3 polymer bending and weaving and twisting its way through the nucleus," she says. "It does appear to have a single repeating pattern and creates a matrix that captures several different tumor suppressors and silences p53 target genes."

O'Shea's findings may help scientists develop drugs capable of destroying tumors by binding and disrupting large and complex cellular components that allow cancer cells to grow and spread. Understanding how viruses overcome healthy cells may also help scientists engineer tumor-busting viruses. Such modified viruses would destroy only cancer cells because they could only replicate in cells in which the p53 tumor suppressor has been deactivated. When a cancer cell is destroyed, it would release additional copies of the engineered viruses, which in turn would seek out and kill remaining cancer cells that have spread throughout the body.

GAME-CHANGING TECHNOLOGIES

The evolution of cancer research has always relied on the progression of technology, and now, more than ever, Salk scientists benefit from a critical mass of cutting-edge research technologies. Verma's virus-based gene delivery systems and mouse models are just one example.

Stem cell techniques provide another powerful method of charting important growth and development pathways involved in cancers. Salk's Waitt Advanced Biophotonics Center allows Salk researchers to visualize the inner workings of cancers in stunning visual detail. And new high-throughput sequencing devices provide a tool for intercellular cartography, letting scientists map out the entire genetic code of a patient or a tumor. In combination, these technologies prove particularly valuable, helping researchers tackle longstanding questions that were difficult or impossible to answer in the past. In one case, they've even allowed a Salk lab to breathe new life into a theory that was first proposed 150 years ago but was never fully explored due to technological limitations.

While reading the writings of two 19th-century scientists, Francesco Durante and Julius Cohnheim, Salk professor Geoff Wahl realized that their theories about where cancers originate might still have merit for modern science. In the 1870s, Durante and Cohnheim proposed that cancers come from cells in adults that persist in an immature, embryonic-like state.

Geoff Wahl

Geoff Wahl

"What Durante and Cohnheim were saying makes a lot of sense," says Wahl, who holds Salk's Daniel and Martina Lewis Chair. "Tumors and embryonic cells share many similarities. They divide rapidly, can generate different cell types, have similar metabolism that enables them to grow in limited oxygen, and in some tissues must develop the capacity to move and burrow into adjacent tissues. Given these similarities, we wondered whether the genomic programs active in developing embryos might be reactivated in certain types of cancers, but to the detriment, not benefit, of the individual."

Thanks to advances in stem cell research and genomic sequencing, Wahl's team was able to explore this question with tools unavailable until very recently. Using sophisticated methods of stem cell and developmental biology, genetic manipulation of mice, microscopy and microfluidics, the Salk researchers were able to isolate stem cells found in the developing mammary glands of mice. Using genomic sequencing techniques, they compared the genetic activity of these mammary stem cells with stem-like cells found in breast cancers.

They found that the genetic signatures of the mouse cells were remarkably similar to the stem-like cells found in aggressive breast cancers. This was true for a significant fraction of virulent cancers labeled "triple-negative," a name that indicates the tumors do not express the genes for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER2).

When a patient's tumor tests positive for HER2 or one of these other markers, the doctor's treatment approach is more precise and typically far more effective. By blocking the activation of the receptor, the drug Herceptin stops the growth of breast cancers. Because triple-negative tumors lack the HER2 receptor, however, there are fewer good options for treatment, so the outcomes are usually worse—something Wahl aims to change.

His team's findings suggest that triple-negative cancers rely on signaling through pathways similar to those that drive fetal breast stem cell growth. They also found that the fetal breast stem cells are sensitive to a class of targeted therapies that already exists, so these therapies might also work in the considerable subset of triple-negative breast cancers that resemble the fetal breast stem cells. Laboratory studies and clinical trials are currently underway to test this possibility.

"We've now got the tools to find the weaknesses of these cancers, to give them molecular names that reflect what they are, not what they aren't," says Wahl. "I don't want to call a tumor triple-negative. I want it to have a name that tells a doctor how to stop it from growing."

DISCOVERIES INTO CURES

Like Wahl, Salk professor Ron Evans sees translational research—the bridging of basic and clinical science—as the natural evolution of his foundational research on the physiology of cells.

An authority on hormones, both their normal activities and their roles in disease, Evans discovered a large family of molecules named nuclear receptors, which respond to various steroid hormones, vitamin A and thyroid hormones. In addition to impacting our daily health by controlling sugar, salt, calcium and fat metabolism, the receptors are primary targets in the treatment of breast cancer, prostate cancer and leukemia. Evans's discoveries have led to a remarkable number of clinically relevant discoveries, so much so that Nature Biotechnology named him the top translational researcher in the world in 2012, based on the number of patents resulting from his studies (114).

Ron Evans

Ron Evans

One of Evans's discoveries resulted in Entinostat, a new breast cancer drug that targets certain enzymes found in cancers that have become resistant to chemotherapy. Entinostat has proven so effective in early clinical trials that the U.S. Food and Drug Administration designated it a "Breakthrough Therapy," which will speed the drug's progress toward clinical use.

More recently, Evans's lab has studied the role of hormone receptors in pancreatic cancer, where the lifespan after diagnosis is typically counted in mere months. Inflammation of the pancreas, or pancreatitis, is a serious pathologic state associated with both acute and chronic inflammation that is linked to the development of pancreatic cancer. Stromal cells, supporting cells found in connective tissues of the pancreas, are known to release substances that stimulate tumor growth, tumor invasion and tumor resistance to therapy.

"These cells are a promising target for stopping pancreatic inflammation, pancreatitis and cancer," says Evans, a Howard Hughes Medical Institute investigator and holder of the March of Dimes Chair in Molecular and Developmental Biology. "We know the key molecular players in these signaling pathways, so we may be able to control their activity. Tackling inflammation may represent an entirely new therapeutic approach to controlling the disease process."

His team's preliminary studies identified the vitamin D receptor (VDR), a known potent anti-inflammatory regulator, in inactivated stromal cells. They hypothesize that drugs that modify VDR signaling may prove effective at suppressing the genomic events that lead to cancer development. The lab is currently collaborating on a human clinical trial that explores whether VDR activators, alone or in combination with the anticancer drug gemcitabine, can stop inflammation and the development of pancreatic cancer.

Like Evans, Salk professor Reuben Shaw has also discovered links between metabolism, inflammation and cancer. Shaw, a Howard Hughes Medical Institute early career scientist, studies a tumor suppressor gene called LKB1, which is lost in 30 percent of lung cancers. The gene's normal action is to turn on a metabolic enzyme called AMPK when energy levels run low in cells, suppressing their growth and proliferation. But in certain lung cancer tumors lacking the normal LKB1 gene, cells cannot sense their own energy levels, resulting in out-of-control growth. This led Shaw to speculate that drugs that lower cellular energy levels and slow cellular metabolism may kill lung tumors with defective LKB1.

Reuben Shaw

Reuben Shaw

Interestingly, the best-known drugs to lower cellular energy levels are used in diabetes treatment. This past year, Shaw and his team demonstrated that phenformin, a derivative of the widely-used type 2 diabetes drug metformin, decreased the size of lung tumors in mice and increased the animals' survival.

Like the Gleevec and other targeted medicines, the treatment is most effective in lung tumors carrying the specific genetic mutation that sensitizes them to this therapeutic option, in this case, the LKB1 gene.

The next step is to determine how phenformin would perform in combination with other existing standard-of-care lung cancer drugs. In addition, Shaw's team is further studying other ways to alter cellular metabolism and target this large proportion of lung cancer patients.

"Increasingly, this is where Salk's cancer research is headed," says Shaw. "Cancer is complicated, which is part of the reason clinical progress has been slow compared to other diseases. But we now have the technologies to marry laboratory science and clinical studies—and that's going to give doctors the targeted therapies they need to turn these deadly acute diseases into manageable chronic conditions."