The signal transduction protein BRAF is an important member of the MAPK/ERK pathway, which transmits a signal from a growth factor binding to its receptor at the cell surface into the cell nucleus, stimulating protein expression. In many cancers, BRAF is mutated so that it no longer requires signal transduction from its upstream protein, RAF, active. Several BRAF inhibitors are in development as anti-tumour drugs, and one of these, vemurafenib, has recently been approved by the FDA as a treatment for melanoma. This drug, and other compounds in its series, has remarkable activity in melanomas in which BRAF carries the common mutation V600E. However, this activity is always compromised by the development of resistance.

Inhibitors of BRAF are known to act by binding to protein monomers and preventing them from associating to form the active dimers, but the mechanism through which resistance to these drugs develops is not yet fully understood. A large group of researchers led by David Solit at Memorial Sloan-Kettering Cancer Center, New York, USA has now elucidated a novel mechanism for the development of this resistance.

Solit and co-workers first generated a vemurafenib-resistant BRAF(V600E) mutant melanoma cell line by exposing the SKMEL-239 cell line, which bears this mutation, to a high concentration of the drug for approximately two months, approximately mimicking the exposure given in a course of chemotherapy. The researchers found ERK signalling, which is downstream of BRAF in the same pathway, to be less sensitive to vemurafenib in cells that had become resistant to this drug. Two distinct clones were identified in these cells: one with a dramatic (100-fold) increase in the IC₅₀ for vemurafenib-mediated pERK inhibition, and the other with only a modest (4.5-fold) IC₅₀ increase. Both clones retained sensitivity to inhibitors of the downstream signalling protein MEK.

Analysis of the expression of BRAF(V600E) showed that all vemurafenib-resistant cells expressed the full length 90kDa protein, but the clone with the greatest IC50 increase also expressed a truncated, 61kDa protein. PCR analysis of cDNA in these cells showed that exons 4-8 of wild type BRAF were not transcribed in the truncated form. These missing exons contain the RAS-binding and cysteine-rich domains of BRAF, and signalling in analogous deletions in wild-type proteins of this family is independent of RAS binding. The truncated protein does, however, include the kinase domain bearing the mutated residue 600.

The transcript encoding the truncated BRAF protein was cloned and expressed in melanoma cells with or without the full-length mutant protein. Expression of the truncated form prevented vemurafenib from inhibiting ERK signalling, and knock-down of the truncated but not the full-length protein using siRNA inhibited both signalling and cell growth. In cells with reduced RAS signalling (and so reduced "normal" signals for BRAF dimerisation), the truncated protein but not the full-length one formed dimers. Introducing a further mutation that prevents dimerisation (R509H) into the truncated protein restored the inhibitory activity of vemurafenib.

These results imply that resistance to vemurafenib and, by implication, other BRAF inhibitors can arise by the formation of truncated mutants that can dimerise, and therefore become active, in the absence of RAF activation. In order to investigate their clinical significance, Solit and his co-workers analysed vemurafenib-resistant tumours from nineteen melanoma patients for wild-type and mutant BRAF transcripts. Six of these patients' tumours contained truncated splice variants of this protein that lacked the RAS binding domain and would therefore be expected to be able to dimerise in a RAS-independent manner. The tumours, however, should retain sensitivity to inhibition of signalling proteins downstream of BRAF in the cascade, and combinations of drugs targeting several proteins in this pathway might be a useful therapeutic strategy in these patients.

Reference

Poulikakos, P.I., Persaud, Y., Janakiraman, M. and 21 others, RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E), Nature, published online ahead of print 24 November 2011, doi:10.1038/nature10662