Sunday, May 18, 2014

The Biochemical Mechanism Behind Paradoxical Activation in BRAF Wild-type Cells by BRAFV600E inhibitors

In my last two blog posts I wrote about a unique case in cancer therapeutics where the successful treatment for a BRAFV600E metastatic melanoma with the vemurafenib BRAF inhibitor drug caused the development of chronic myelomoncytic leukemia in the same patient with a preexisting KRASG12D mutation.  It was shown through in vitro assays that the Map Kinase pathway was being turned on in KRAS mutant and BRAF wild-type cells causing uncontrollable cellular proliferation.

This diagram is showing that in mutant melanocytes (that cause melaonma) the higher the drug concentration the lower the ERK activation (pERK) and theoretically the patient has been cured (not taking into account resistance to the drug later on). In the wild type cells it is showing that at a certain drug concentration ERK is activated causing cellular proliferation and in some cases new primary melanomas. This is the paradoxical activation theory.

In an attempt to understand the biochemical mechanisms behind the paradoxical activation I read a mini review paper that summed up all of the possible theories behind this inconvenient side effect of an otherwise successful melanoma therapy (ignoring chemo resistance). There have been many proposed mechanisms of BRAF activity over the last 15-20 years but the one I will focus on is the most recent discovery of BRAF dimerization and auto inhibitory phosphorylation. It is important to remember that this paradoxical activation is occurring in wild type BRAF cells not the melanoma cells.

A) When the cell is not actively proliferating the RAF Kinase has a check to make sure there is no down stream signaling. The RAF isomers (ARAF, BRAF, and CRAF) autophosphorylate each other as an inhibitory mechanism so that MEK cannot be phosphorylated. Notice that this is when there is little to no drug present.

RAF is a kinase, which means it is an enzyme that transfers a phosphate from ATP to its substrate (MEK) to activate it in a process known as phosphorylation. The BRAF inhibitors are ATP competitive, which means they occupy the active site and inhibit the terminal phosphate group of ATP from being transferred to the substrate.

B) When low concentrations of a BRAF inhibitor are present the drug will bind in the active site of one of the RAF molecules and prevent it from phosphorylating the other RAF molecule so the auto-inhibitory phosphorylation check is lost. Now when triggered by upstream signaling from RAS the RAF Kinase is active and can phosphorylate MEK, which phosphorylates ERK, which then turns on many transcription factors for cellular proliferation.

This mechanism predicts that all catalytic RAF inhibitors are likely to cause some paradoxical activation of the MAP Kinase pathway. When there is the addition of a RAS mutation upstream the activation and signaling to MEK is much stronger and that is why we see excessive proliferation (paradoxical activation) and development of different types of cancer in the RAS mutated and BRAF wild-type cells.

C) When the drug is present at higher concentrations the RAF inhibitor binds in the active site of both RAF molecules and signaling downstream to MEK is blocked so there is decreased cellular proliferation.

Although this is only a theory it is widely accepted and to solve this problem the drug companies have been developing “paradox breaker” drugs that are showing to be more potent and not cause paradoxical activation. The paradox breakers will bind to RAF and cause a different conformational change in the enzyme that does not inhibit the auto phosphoryalation of the other RAF molecule in the autoinhibitory complex.

Holderfield, M., T. E. Nagel, and D. D. Stuart. "Mechanism and Consequences of RAF Kinase Activation

by Small-molecule Inhibitors." British Journal of Cancer 10.1038 (2014): 1-6. Web.