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BRAF/MEK: The science of targeting one oncogenic pathway

About half of all melanomas are “addicted” to an activating mutation in BRAF, which fuels cancer growth by constituently activating the kinases MEK and ERK. To overcome drug resistance to the selective BRAF inhibitors (RG7204/PLX4032, Roche) (GSK2118436, GlaxoSmithKline), researchers are testing the addition of a MEK inhibitor and are eyeing other targets in the same pathway and in the PI3K pathway. Courtesy of Keith Flaherty/Annals of Internal Medicine

In theory, it sounds straightforward: Find genetic mutations essential to the growth and survival of the cancer, identify specific inhibitors in the lab, and validate them in human studies.

In practice, it is often a complicated path from mutation discovery to a viable targeted therapeutic agent, said Levi Garraway, MD, PhD (DFCI). Several years ago, for example, failure of the first drug in clinical trials designed to block molecular drivers of metastatic melanoma made the BRAF mutation seem a dubious target. And despite last year’s striking results in early phase trials, it seems clear that inhibiting mutant BRAF activity alone cannot eradicate melanoma tumors nor even hold them back for long.

“The good news is that the other kinases in the pathway remain a hot area for drug discovery,” said Garraway. Large-scale genetic studies in his lab are beginning to untangle the dynamics of this cancer-driving pathway to find new ways to boost the effectiveness of BRAF inhibitors and to thwart drug resistance. “We know how to discover drugs against kinases,” he said. “Even more exciting, the other kinases could also be points of vulnerability.”

BRAF in melanoma

The BRAF mutation was identified as an oncogene in melanoma in 2002. Scientists soon worked out the mechanics of the pathway and its key role in melanoma. BRAF is a version of RAF in the MAP kinase signaling pathway of RAS-RAF-MEK-ERK (see diagram).

The early growth and survival of about half of all melanomas seems to depend upon a BRAF mutation that dials up the activity of the protein, pumping up activity at each next step, MEK and then ERK, which directs cell proliferation and survival, among other things. About 90 percent of BRAF mutations are in one spot: V600E, a substitution of one amino acid for another that renders BRAF deaf to the molecules that normally turn down its volume.

In healthy cells, BRAF is found in the testes, some hematopoietic precursors, and some brain cells (which develop from the same embryonic tissue as melanocytes). In contrast, BRAF’s better-known cousin CRAF is essential to the daily function of most other cells. Researchers hope highly selective inhibition of BRAF will translate to fewer debilitating toxicities for patients.

Try and try again

The failures and triumphs in testing BRAF as a target have lessons for developing the next selective inhibitors, “It had looked like a drug development dream,” Garraway said, but “as recently as 2008, there was a fair bit of skepticism as to what extent we would be able to gain therapeutic traction against the pathway.”

The first apparent setback came with the first intensively tested drug developed as a RAF inhibitor. In test tubes, sorafenib had looked promising, However, in BRAF-mutant melanoma cell lines the effect was modest. In people, the drug showed minimal activity against BRAF-mutant tumors. It had some benefit with chemotherapy in early stage trials, but not in phase III randomized trials of melanoma patients. The sorafenib data will be published later this year by Keith Flaherty, MD (MGH) and his co-investigators. Last year, in a study that dug more deeply into the broad action of sorafenib, researchers found no evidence that it worked through the RAF pathway in practice.

Next came a much more selective BRAF inhibitor (RG7204/PLX4032, Roche) in clinical trials also led by Flaherty. A more thorough molecular workup established its targeting credentials in malignant cells and mice, but the second generation inhibitor showed no effects initially in the phase I testing against a variety of cancers. A reformulation boosted the drug levels in people’s bodies with better results, as did a trial extension that limited participants to those with BRAF-mutant metastatic melanoma. The short-term response rate rose to 81 percent when 32 more people were enrolled with BRAF mutations and given the recommended phase II dose.

The BRAF bypass

Now that BRAF has been validated as a single target, “the question of resistance has moved to center stage,” said Garraway. In December, Garraway’s group published an initial salvo in the journal Nature of what resistance to BRAF inhibitors might look like, based on a new technique for methodically screening kinase-coding genes for those that confer resistance to the Roche drug.

It was no surprise to Garraway that the two biggest hits both converged on MEK to turn the pathway back on: RAF1 (also known as CRAF, described above), which signals in healthy tissue and in cancers without BRAF mutations, and MAP3K8/COT, another kinase that can bypass the BRAF inhibition. Additionally, Garraway’s team found a mutation that increased MEK activity in a tumor sample from a patient with melanoma who developed resistance to the Roche BRAF inhibitor after an initial dramatic response, they reported in March in the Journal of Clinical Oncology.

“We’ve been advocating the combination of BRAF and MEK for years,” he said. A phase II study of combined BRAF and MEK inhibitors (GSK2118436 and GSK1120212, GlaxoSmithKline) for metastatic melanoma and selected other BRAF-mutant cancers is underway at DF/HCC, led by Flaherty and his co-investigators.

Garraway’s group is following up by characterizing tumor samples from patients in the trials. “That’s the gold standard of understanding resistance,” he said.

A different resistance mutation in response to BRAF inhibition, this time in upstream NRAS, also restored the downstream MEK activation, reported a California team in the same issue of Nature. (NRAS activates CRAF, and can also turn on another oncogenic pathway known as PI3K.) In tumor samples of five other relapsed patients, a different parallel signaling pathway kicked in, possibly triggering the same cell proliferation and survival. The resistance mechanisms in six other patients are a mystery.

What’s particularly notable so far is that the resistance-conferring mutations do not seem to appear in the original BRAF target, which is different from several other inhibitors that target key oncogenic mutations, such as imatinib (Gleevec) in chronic myelogenous leukemia and in gastrointestinal stromal tumor, as well as erlotinib (Tarceva) in non-small cell lung cancer, Flaherty said.

Researchers have only begun to understand the complex genetics of resistance, but thanks to the data so far, “we have a solid scientific basis on which to start testing two-drug combinations,” Garraway said. “We know that some type of combinations will be critical to achieve durable responses.” In addition to muffling MEK, ERK inhibitors have also entered clinical testing and ultimately may be tested with RAF inhibitors. Scientists have not yet identified selective inhibitors of NRAS or COT.

Powerful new sequencing technologies hold promise for building a more comprehensive catalogue of the genetics of cancer and of resistance. “Cancer genomes are riddled with mutations,” Garraway said. “Melanoma exemplifies the potential to identify and genetically stratify a spectrum of pathways. If we use a systematic approach to identify the molecular cartography in theory, it may tell us about new targets and suggest clever combinations that may take care of a lot of resistance. It might help us better characterize samples from patients so that we can treat them with the right therapeutic combinations.”

Garraway and his colleagues foresee an era when cancer researchers and physicians can know enough in advance about the genetics of a cancer and the mechanisms of resistance to anticipate and ward off resistance with smartly targeted combination therapies matched against key components of a tumor’s molecular and genetic profile.

“Meanwhile, we can be designing drugs that target those mechanisms, so we can have one or more ready for new trials of therapeutic combinations,” said Garraway. His lab has also developed a tumor profiling test for several hundred highly recurrent mutations in a patient’s cancer, known as OncoMap. Last year, he and several colleagues started Foundation Medicine, a company that aims to harness next-generation sequencing and other advanced technologies to capture a patient’s relevant tumor genetic and molecular information and connect it with clinical data to help guide treatment.

— Carol Cruzan Morton