The experimental drug PLX4032, which is a selective inhibitor of the key enzyme B-RAF kinase, is showing significant promise in early clinical trials against malignant melanoma. However, some patients have been shown to develop resistance to this drug at an early stage in treatment. Discovering the mechanisms through which resistance to PLX4032 occurs is an important step towards the development of more effective therapies for this disease. Two groups of researchers have now published papers that highlight subtly different putative mechanisms for the development of resistance to this drug.

PLX4032 targets tumours in which the proto-oncogene B-RAF has been converted into its oncogenic form through the V600E mutation; this mutation is particularly common in melanoma, being found in 60% of these cancers. Roger Lo, Ramin Nazarian and colleagues from the Dana-Farber Cancer Institute in Boston, MA, in collaboration with co-workers in Los Angeles, Nashville, TN and Melbourne, Australia, investigated the mechanism through which resistance to PLX4032 is acquired by comparing PLX4032-sensitive melanoma cell lines with the same cell lines that have acquired resistance through chronic exposure to the drug [1].  Sanger and subsequent “deep” Illumina sequencing of the B-RAF gene from the resistant cell lines showed that in all cases the V600E mutation had been retained and no secondary missense mutations had been introduced. This lack of secondary mutations was confirmed by sequencing B-RAF in tumour biopsies from patients with clinically acquired resistance.

Lo and co-workers used phospho-specific antibodies to investigate the activation of the kinases MEK and ERK, which act downstream of B-RAF in the MAPK signal transduction pathway. Different PLX4032-resistant cell lines showed different patterns of ERK and MEK activation, indicating that more than one resistance mechanism was involved, and this was confirmed using gene expression profiling. Some cell lines showed significant increases in the activation of these genes, indicating that the MAPK pathway had been re-activated, with further investigations revealing mutations in another oncogene in the RAS family, N-RAS. A second subset of cell lines showed high induced levels of the growth factor receptor PDGFRb. These cells have low levels of MAPK pathway activity, indicating that tumour cell survival is mediated via an alternative signal transduction pathway.

In the second study, a large group of researchers led by Levi Garraway and Cory Johannessen at the Broad Institute of Harvard and MIT, MA, USA expressed a panel of about 600 human kinases in PLX4032 sensitive melanoma cell lines and treated these with the B-RAF directed kinase inhibitor to identify kinases that could confer resistance to the drug [2]. Nine of the kinases were found to confer resistance at statistically significant levels in at least one cell line. Two kinases, COT and C-RAF – both of which are members of the Ser/Thr MAP kinase kinase kinase family – were highlighted in particular as conferring the most significant resistance in multiple cell lines. These kinases are involved in the MAPK pathway, and their ability to confer resistance suggests that this is arising through re-activation of this pathway. Three of the other seven kinases identified seemed to confer resistance in a MAPK-independent manner.

In further experiments, Garraway and co-workers focused on details of the mechanism through which COT confers resistance. This protein, which is encoded by the gene MAP3K8, was expressed in human melanocytes, although at greatly decreased levels if they also expressed mutated B-RAF. In cells with this mutation, resistance to PLX4032 could be induced by COT expression. These cells showed higher expression levels of all kinases in the MAPK pathway compared to B-RAF mutated cell lines. COT expression was also recorded in tumour tissue obtained from patients whose melanomas had acquired resistance to PLX4032.

Taken together, these studies indicate that resistance to this promising drug can arise through a variety of mechanisms, some involving downstream kinases in the MAPK pathway but none involving secondary mutations in the B-RAF gene. Further analysis of these mechanisms suggests several kinases that might be good drug targets for melanomas that have acquired resistance to PLX4032 and other B-RAF targeted drugs.