Combination Targets For Cancer: Some Drugs Work Well Together, Studies Suggest

Reported at the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, multiple-target applications of new and existing drugs are offering new hope in the fight against cancer and drug resistance, from lung and breast cancer to rare tumors of the bile duct.

Multi-level targeting in breast cancer cells

The chemical signaling pathways that control the life cycle of cells offer many important targets for cancer researchers hoping to stop the growth of tumors, yet the complex nature of these pathways may make it impossible to kill a cell with a single therapeutic bullet, as researchers from the University of California, San Francisco, (UCSF) have discovered in a study of breast cancer cells. Their findings suggest that molecules used to inhibit the MEK protein, similar to those being studied to control breast cancer growth, can "switch on" another pathway that keeps cancer cells from dying.

Their solution is to look elsewhere to see where these pathways intersect, which has allowed them to uncover two targets that, when chemically inhibited at the same time, caused apoptosis --cell suicide. This combination therapy approach might make it difficult for cancer cells to resist the disruption of a single protein along a particular signaling pathway.

"The chemical networks involved in cell cycle control have a tendency to be much more complicated than we initially expect, with intersecting pathways and feedback loops," said Michael Korn, M.D., an associate professor in residence at the UCSF's Comprehensive Cancer Center. "To understand the connections between these pathways we have taken a systems biology approach, one that may uncover new anti-cancer drug targets on different levels within connecting pathways."

As part of a multi-institutional research effort funded by the National Cancer Institute's Integrative Cancer Biology Program, Dr. Korn and his colleagues at UCSF, Lawrence Berkeley National Laboratories and The University of Texas M. D. Anderson Cancer Center are creating computer models informed by their systemic approach that will enable them to identify important proteins within the signaling networks. "There so many different components involved, that modeling these pathways is as complicated as creating computer models that accurately predict the weather," Dr. Korn said.

The MEK protein is an enzyme involved in the well-studied RAS pathway, which plays a central role in promoting cell growth and multiplication. When not functioning properly, this pathway has been implicated in a number of cancers, Dr. Korn says, including breast cancer. When the researchers treated breast cancer cells with molecules designed to bind to and inhibit MEK, named U0126 and CII040, they observed that instead of dying, the cells simply froze at a single point in their life cycle.

While U0126 and CII040 prevented breast cancer cells from growing, they did not kill the cells implying that some breast cancer cells would survive treatment with MEK inhibitors, Dr. Korn says. In analyzing the performance of the MEK inhibitors, researchers noticed that a similar pathway, identified by the activity of an enzyme called PI3-kinase, had been activated. "So, of course, we decided that we would try blocking the PI3-kinase pathway as well, assuming that simply blocking both pathways would work," Dr. Korn said.

However, combining the MEK inhibitors with a PI3-kinase inhibitor resulted in cells even more steadfastly resolved to stay alive.

"That is when we decided to look elsewhere along the MEK and PI3-kinase pathways, eventually trying MEK inhibitors in combination with a number of inhibitors along the PI3-kinase pathway," Dr. Korn said. "The resulting studies show that, with the select combination of targets, we can elicit a synergistic effect between anti-cancer drugs that are much stronger than either drug's effect alone." Dr. Korn concludes the key to ultimate MEK inhibition may be to bypass the MEK pathway altogether. The researchers found that targeting PI3-kinase and a protein farther down the pathway, called TOR, caused apoptosis in the breast cancer cells. One molecule known to inhibit TOR is rapamycin, an immunosuppressant whose anti-cancer effects are under study. Dr. Korn and his colleagues are currently studying the combination in other breast cancer cell lines in anticipation of later clinical trials.

Therapeutic response in a lung tumor xenograft model

Researchers at the University of Manchester have found that a therapeutic currently in Phase II clinical trials is more effective at shrinking tumors when combined with radiotherapy. In a mouse model for human lung cancer, AZD6244 may increase the potency of radiotherapy by shutting down the cellular machinery that allows cancer cells to cope with a lack of oxygen, a state called hypoxia, they report.

AZD6244 is a small molecule inhibitor developed by AstraZeneca to bind to and inhibit MEK1 and MEK2 proteins, which together form a critical point on the pathway that controls the ongoing survival of cells. Defects along the pathway can lead to increased cell proliferation and cancer, and the drug was developed to halt this important route toward tumor progression. "The MEK proteins are also part of the pathway that controls the production of HIF-1, the hypoxia-related protein involved in stabilizing cells in the absence of oxygen," said Aoife M. Shannon, Ph.D., a postdoctoral fellow at the University of Manchester. "Furthermore, HIF is associated with protecting tumors from the effects of radiation."

As a cancer treatment, radiotherapy, which works by damaging DNA with ionizing radiation, is relatively less effective against tumors that are deprived of oxygen, or hypoxic, the researchers say. As tumors grow into large masses, they strain the available supply of oxygen and exist in a hypoxic state.

Shannon and her colleagues studied the combined effects of radiation and AZD6244 on human lung cancer cell cultures and a mouse model of human lung cancer, in studies funded by AstraZeneca. In mice they found that the combination of radiotherapy and drug was more effective than either therapy alone. While the drug alone and the combination therapy caused the tumors to shrink to nearly a third of their original volume within 10 days, regrowth took significantly longer in the combination group than in the drug- or radiation-only groups.

According to Shannon, their findings support further development of AZD6244 and radiotherapy as combined regimen, and indicate a novel role for AZD6244 in inhibiting the tumor hypoxia response.

"It is possible that the effect of AZD6244 on HIF as well as tumor blood vessel growth induced by HIF contributed towards the remarkable effects of the combination," Shannon said. "It is a double-pronged approach that will likely be relevant to ongoing trials of MEK inhibitors and inform future studies."

Enhanced responses induced by EGFR kinase inhibitors

Researchers at Memorial Sloan-Kettering Cancer Center have developed a strategy to enhance tumor response in some lung cancer patients treated with the tyrosine kinase inhibitor (TKI), erlotinib, by combining it with ABT-737, a small molecule drug that primes tumor cells to undergo apoptosis, or programmed cell death.

"About ten percent of tumors from lung cancer patients harbor mutations in the kinase domain of the epidermal growth factor receptor (EGFR) gene, and these mutations are associated with increased sensitivity to EGFR TKIs." said Yixuan Gong, Ph.D., a post-doctoral researcher at Memorial Sloan-Kettering. "EGFR TKIs, while certainly beneficial, unfortunately, do not cure patients whose tumors have these mutations, and resistance develops after long time use. We're attempting to enhance the effectiveness of EGFR TKIs by combining TKI therapy with ABT-737, a BCL-2 inhibitor."

According to Gong, mutated EGFRs produce growth signals that drive uncontrolled lung tumor cell division. Inhibition of mutated EGFR is a very effective treatment for these types of lung cancers, she says.

Gong and her colleagues at Memorial Sloan-Kettering demonstrated how lung cancer cell lines with EGFR mutations undergo apoptosis when exposed to erlotinib. In particular, they found that such cells die by a mechanism called the intrinsic apoptotic pathway. Erlotinib, the researchers say, induces dramatic changes in the pro-apoptotic protein, BIM, and this protein is required for erlotinib-triggered cell death.

"The improved understanding of how EGFR mutant cells actually respond to EGFR TKIs will help us to identify strategies to enhance tumor cell death and will also help us to look for possible reasons why tumor cells persist despite treatment," Gong said.

In studies of EGFR TKI-sensitive cancer cells, the researchers found that ABT-737 significantly enhanced erlotinib-induced cell death. "It is proof of principle that you can enhance tumor response to EGFR TKIs by priming tumor cells to a more death--prone state with small molecules that manipulate the intrinsic apoptotic pathway," Gong said. "We hope to design clinical trials based on this strategy, in order to achieve better and longer clinical responses for patients who benefit from EGFR TKIs."

Gong is part of the laboratory of William Pao, M.D., Ph.D. Their studies were funded by the Doris Duke Charitable Foundation and the Labrecque Foundation. ABT-737 is under development by Abbott Laboratories, Inc.

Enhanced anti-tumor activity in cholangiocarcinoma

A common chemotherapy regimen, when given before either one of two engineered human antibodies, greatly increases the effectiveness of the antibodies against cholangiocarcinoma (CCA), a rare and deadly cancer of the ducts that drain bile from the liver, say researchers from Human Genome Sciences, Inc., a biopharmaceutical research company based in Rockville, Maryland. If their findings in cell culture and animal models of CCA can be applied to humans, the combination of cisplatin, 5-fluorouracil or gemcitabine with a TRAIL-receptor antibody would be the first effective treatment for this disease, they say.

The two antibodies react with different, yet related, cell surface proteins called TRAIL-R1 and TRAIL-R2, which are common to many human cancers. While they are distinct receptors, these two receptors both receive signals that activate biochemical pathways leading to programmed cell death, the researchers say. The TRAIL-R1 antibody (known as mapatumumab) and TRAIL-R2 antibody (lexatumumab) are both being evaluated in clinical trials by Human Genome Sciences.

"Many cancers use the TRAIL pathway as a preferred way to die -- a dominant biochemical pathway -- and we are in the process of exploring how we can better activate the TRAIL pathways to enhance cell death in the tumor cell," said Robin C. Humphreys, Ph.D., associate director in the Oncology Research Department at Human Genome Sciences. "When we pre-treat CCA cell lines with conventional chemotherapies, the drugs seem to increase the cytotoxicity of the TRAIL-receptor antibodies, as if conventional chemotherapy can lower the cell death threshold of cancer cells enough for the TRAIL pathway to be more effective."

Humphreys and his colleagues experimented with combinations of cisplatin, 5-fluorouracil and gemcitabine given with or prior to treatment with the human TRAIL-receptor antibodies in cholangiocarcinoma cell cultures. Their findings indicated that treating CCA cell lines with a single chemotherapeutic agent 24 hours before administering either antibody increased cytotoxicity, killing nearly 90 percent of cells. According to Humphreys, mouse xenograft studies showed that co- or pre-treatment of a CCA xenograft tumor with cisplatin or gemcitabine before administering mapatumumab significantly inhibited tumor growth and displayed tumor regression over the 40-day period of the trials.

Since human biliary cancer remains a relatively rare cancer, with an annual incidence of about one or two cases per 100,000 people in the Western world, there has been little research toward a specific treatment for this disease, the researchers contend. This pre-clinical data supports potential clinical trials of a TRAIL-R mAb and chemotherapy combination in CCA, Humphreys says.