Melanoma, cancer of the melanin-producing cells typically found in skin, is very difficult to treat and often fatal. Patients tend to respond to therapies that shut down a pro-cell growth pathway by activating a bypass pathway.
PLX4032 is a promising experimental new cancer drug in phase III clinical trials for treatment of melanoma. PLX4032 selectively targets tumor cells with activating mutations in the protein, B-RAF (V600E). The drug has shown an exceptional anti-tumor response rate of 80% in patients positive for the mutated B-RAF, however resistance often develops after 7 months of treatment, even for patients whose tumors seem to have disappeared. A recent news article discussed the findings of two separate groups of scientists who have independently discovered mechanisms behind the resistance to PLX4032. These mechanisms include the activation of other kinases to bypass the inhibited B-RAF, thus reactivating the downstream pathway. Also, cells resistant to the B-RAF inhibitor compensate by up-regulation of B-RAF independent growth factor receptor pathways. These findings provide starting points for development of combined therapies to overcome the resistance to PLX4032.
B-RAF mutations are found in about 60% of malignant melanomas, in high frequencies in thyroid and colon cancers, and in approximately 8% of all cancers. B-RAF, a serine-threonine protein kinase, normally functions in B-RAF/B-RAF homodimers or in heterodimers with a related protein, RAF1 kinase, generally in response to upstream signaling molecules, including the activation of the protein RAS. B-RAF signals through the MAPK/ERK signaling pathway, and when mutated, functions as a monomer rather then a dimer, thus activating downstream signals in the absence of upstream input. This signaling drives tumor growth through the activation of the MAPK pathway. PLX4032 inhibits the mutated B-RAF by occupying the pocket containing the mutation. This alters the mutated B-RAF’s 3-dimensional structure and thereby disrupts the monomer activity. The drug is exquisitely specific for B-RAF mutant tumors, which is important because it is effective only in high doses and such specificity should mean fewer undesirable side-effects.
The mechanisms of resistance to PLX4032 follow the common cancer drug resistance theme of bypassing the oncogenic pathway that has been shutdown. However, the expectation was to see compensating mutations in B-RAF leading to resistance, rather than activation of alternative signaling pathways.
One group of scientists sequenced the entire genome of 16 tumor samples from 12 patients who acquired resistance to PLX4032 during their treatment in phase I or II clinical trials. They found one patient acquired resistance via an additional mutation in N-RAS which reactivated the MAPK pathway. In 5 other patients samples, the scientists found that a receptor-tyrosine kinase (RTK)- survival pathway was activated as a result of over-expression of a platelet-derived growth factor receptor (PDGFRβ). They could not find mechanisms of resistance in the remaining patients’ samples.
The other group of researchers expressed roughly 600 different kinase protein open reading frames (ORFs) to identify which ones allowed the cells to grow in the presence of PLX4032. They found that the protein C-RAF, known to form dimers with normal B-RAF, and the protein COT, encoded by the gene MAP3K8, conferred resistance to the drug. Both kinases do this by independently activating the MAPK/ERK cell-growth pathway, similar to B-RAF and to N-RAS.
The findings are promising in that recognition of mechanisms of resistance suggests additional drug targets and could lead to therapies with drug combinations such as PLX4032 paired with another drug that inhibits a protein further downstream in the cell growth signaling pathway, such as ERK or MEK. In experimental models the RAF and MEK inhibitor combination was able to thwart resistance to a single RAF inhibitor. Some patients whose cancer relapsed after treatment with PLX4032 are enrolled in a phase II clinical trial of an MEK inhibitor. Additionally, PLX4032 will likely be tested in combination with this MEK inhibitor in clinical trials in the near future.
The finding that PDGF receptor overexpression can lead to resistance is analogous to the finding that insulin-like growth factor receptor (IGF-1R) overexpression results in resistance to a different B-RAF(V600E) inhibitor. Both IGF-1R and the PDGF receptor activate the same pathway, involving PI3 kinase and AKT. Apparently, this pathway could be another drug target.
The above, recent two studies included only a total of 17 patient samples. With the heterogeneity exhibited in the resistance mechanisms in such a small number of patients, larger studies will likely be needed to determine all possible resistance mechanisms.
In summary, these studies support the idea that combinatorial drug treatments, such as those that have revolutionized HIV treatment, will be important in cancer therapies. Further, screening techniques used by the researchers could show ways to stratify patients into distinct treatment groups, as is currently done for breast cancer. However, several questions will have to be answered in the process of developing drug combinations. For example, how many drugs are needed simultaneously and which can be tolerated in combination?
Abstract of article describing resistance mechanisms to PLX4032
Abstract from another article describing other resistance mechanisms to PLX4032
