A first-in-class allosteric RAF inhibitor for breast cancer

Institution: University of California, San Diego
Investigator(s): David Cheresh, Ph.D. -
Award Cycle: 2013 (Cycle 19) Grant #: 19IB-0130 Award: $187,500
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (2013)

Non-technical overview of the research topic and relevance to breast cancer:
RAF kinases are a family of enzymes or substances that help cancer cells grow and spread to distant sites in the body. Recently, the hyper-activation of one RAF family member, CRAF, has been linked to poor outcome for patients with breast cancer. To target CRAF, a team of chemists and biologists in Dr. Cheresh's lab created Ď3G8í, an experimental drug that helps slow down the activity of the breast cancer cells and makes them more sensitive to the effects of chemotherapy. These scientists believe that a daily dose of '3G8' taken by mouth will improve the chances of fighting some types of breast cancer in human beings when combined with the current treatment options, yet with fewer negative consequences or side effects. Experiments are now underway to test this strategy in mice, but the goal of Dr. Cheresh's lab is to be able to offer this therapy to breast cancer patients in the near future.

The question(s) or central hypotheses of the research:
Before 3G8 therapy can move forward into clinical testing for breast cancer, there are a number of questions to answer. First, we will ask whether CRAF (and specifically its activation on a single site, Serine 338) plays an important role in all breast cancer cells, or if it is more or less important in certain breast cancers. Second, we will ask how CRAF drives cancer progression in order to identify biomarkers suitable for monitoring CRAF activity and the efficacy of 3G8 within a tumor. Third, we will ask which FDA-approved breast cancer drugs will best synergize with 3G8 to produce the most effective and least toxic new therapeutic strategy for patients with breast cancer.

The general methodology:
In Aim 1, we will examine tumor biopsy tissue obtained from a large cohort of breast cancer patients who enrolled in the Women's Healthy Eating Lifestyle (WHEL) Study between the years 1995 and 2000. This tissue represents a valuable and unique resource, as disease progression for each patient continues to be updated quarterly. Therefore, we can ask whether high CRAF activity predicts relapse or poor survival. In this Aim, we will also test the ability of 3G8 to suppress growth for a diverse panel of breast cancer cells we have assembled in our laboratory. Analyzing these genetically diverse patient samples and cell lines will allow us to determine whether certain breast cancer subtypes may be associated with high CRAF activity, suggesting these as the best candidates for 3G8 therapy.

In Aim 2, we will investigate how CRAF functions in breast cancer cells. We will determine what signaling pathways CRAF promotes, and how this impacts tumor cell behavior. Then, we will grow breast tumors in mice, and determine whether treatment with 3G8 is able to block the activation of these signals.

In Aim 3, we will combine 3G8 with different FDA-approved breast cancer chemotherapy drugs. Our preliminary data predicts that any drug that induces damage to the cellís DNA will work much better in the presence of 3G8, as our drug disables the tumor cellís ability to repair DNA. By removing DNA repair in the tumor, 3G8 can reduce the amount of chemotherapy drug required to eradicate that tumor. Thus, our goal is to provide the greatest suppression of tumor growth with the fewest side effects, and to identify particularly good combinations to advance to testing in clinical trials in the appropriate population of breast cancer patients.

Innovative elements of the project:
Although there is sufficient rationale to warrant CRAF as a target for breast cancer, this has not become a popular strategy due to a lack of suitable drugs. Because 3G8 locks CRAF into an inactive shape, it changes the way this protein can function inside a cell, while avoiding the pitfalls associated with traditional drugs that target RAF kinases. In our proposal, we will identify which breast cancer types are the most suited for 3G8 therapy, and will test how 3G8 could be best combined with existing breast cancer chemotherapy drugs. If successful, our work will provide the basis for opening a clinical trial to evaluate 3G8 in breast cancer patients at UCSD to maximize therapeutic benefit while minimizing side effects.

Final Report (2015)

CRAF is a signaling molecule that can promote a variety of physiological cellular functions in healthy cells. When activated by factors prevalent within the tumor environment, CRAF endows tumor cells with the ability to evade the effects of therapy, thereby driving tumor progression. We have recently developed a unique drug to block these functions of CRAF. The overall goal of our proposal is therefore to assess this approach for targeting CRAF as a new strategy for breast cancer therapy.

We first evaluated CRAF phosphorylated on Serine 338 (pS338) as a predictive biomarker for breast cancer progression. We established a new automated method to detect and measure this form of CRAF activity in biopsy specimen slides, but we found that CRAF pS338 levels detected in tumor biopsies taken at diagnosis are not predictive of disease recurrence for patients treated with chemotherapy or tamoxifen. In general, we saw no correlation between CRAF pS338 levels and tumor stage or grade. However, all breast cancer cell lines tested showed a strong sensitivity to 3G8, an allosteric inhibitor of CRAF pS338 activity. 3G8 provided significant cell killing even for lines that were highly resistant to radiation, suggesting CRAF as critical pathway common among genetically distinct breast tumor cell lines and subtypes.

Mechanistically, CRAF recruits and activates CHK2, an enzyme involved in DNA repair, following exposure to radiation or chemotherapy. Inactivating CRAF mutations or treating cells with CRAF inhibitors can prevent the activation of CHK2 and thereby sensitize cells to therapy. From a translational perspective, our findings suggest that regardless of baseline levels of CRAF pS338, most breast cancer cell lines should be uniformly and highly sensitive to CRAF inhibition, suggesting this as a potential replacement for other types of DNA damaging chemo or radio-therapies. In addition, for breast cancers that show moderate sensitivity to radiation, CRAF inhibition could be added in combination to lower the radiation dose required to achieve tumor killing.