Scientists say additional changes to the sequence of BRAF(V600E) that arise in melanoma tumors on repeated exposure to anticancer therapy may be responsible for this type of cancer acquiring resistance to treatments such as the RAF inhibitor vemurafenib. Studies in melanoma cell lines and patient-derived tumors by a Memorial Sloan-Kettering Cancer Center-led team have found that acquired resistance to vemurafenib is associated with the loss of key exons in BRAF(V600E) that encompass the RAS-binding domain.
As a result, the alternatively spliced proteins show enhanced dimerization in cells, leading to an inability of drug therapy to inhibit ERK signaling. David B. Solit, M.D., and colleagues, report their findings in Nature, in a paper titled “RAF inhibitor resistance ismediated by dimerization of aberrantly spliced BRAF(V600E).”
ATP-competitive RAF inhibitors activate ERK signaling by transactivating RAF dimers. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and the drugs can bind to BRAF(V600E) monomers and inhibit their activity. RAF inhibitors have thus been of great clinical utility in patients with melanomas that harbor mutant BRAF(V600E). However, the cancers invariably become resistant.
To identify mechanisms behind this drug resistance, the Memorial Sloan-Kettering Cancer Center rendered an initially vemurafenib-sensitive melanoma cell line (SKMEL-239) resistant to the drug by exposing the cells to continuous drug treatment for two months. Development of resistance was associated with reduced sensitivity of ERK signaling to vemurafenib therapy. All five resistant clones retained sensitivity to the MEK inhibitor PD0325901 (MEK inhibitors act at points downstream of RAF inhibitors), and all retained expression of BRAF(V600E).
Analysis of BRAF protein expression showed that each of the resistant clones expressed a 90 kDa band that co-migrated with the band observed in parental cells. Interestingly, however, an approximately 61 kDa band was also identified in the C1, C3, and C4 clones. Designated p61BRAF(V600E), this band size wasn’t detected in parental SKMEL-239 cells or in a panel of 22 other melanoma cell lines. cDNA analysis also showed that the 2.3 kb transcript representing full-length BRAF was present in parental cells, but two transcripts, of 2.3 kb and 1.7 kb, were found in C3 cells.
The 1.7 kb sequence was identified as a BRAF transcript containing the V600E mutation and an in-frame deletion of exons 4–8. The resulting sequence was predicted to encode a 554 amino acid protein of 61 kDa, consistent with the BRAF band previously detected by immunoblotting. Importantly, exons 4–8 encode the RAS-binding domain critical for RAF activation.
The researchers cloned the 1.7 kb transcript into an expression vector and expressed it in 293H cells, either on its own or together with full-length wild-type BRAF. Expression of p61BRAF(V600E) in these cells and in parental SKMEL-239 cells and HT-29cells, resulted in failure of vemurafenib to inhibit ERK signaling effectively.
Then, to test whether ERK signaling in C3 cells was also dependent on p61BRAF(V600E), the researchers used siRNAS to selectively knock down either p61BRAF(V600E) or full length BRAF. What they found, was that in parental cells, ERK signaling was inhibited by knockdown of full-length BRAF(V600E). In C3 cells, phosphorylation of MEK, cyclin D1 expression and cell growth were inhibited upon knockdown of p61BRAF(V600E) but not of full length wild-type BRAF, ARAF, or CRAF. Moreover, the team adds, in C3 cells in which the expression of full-length BRAF or CRAF was knocked down, ERK signaling remained resistant to Vemurafenib.
To determine whether deletion of exons 4–8 has an effect on dimerization of p61BRAF(V600E), constructs encoding either p61BRAF(V600E) or full-length BRAF(V600E) were tagged with Flag or V5 and then immunoblotted for V5 after immunoprecipitating Flag. The results indicated that levels of p61BRAF(V600E) dimerization were markedly higher than those of full-length BRAF(V600E).
Mutation of the R509 residue within the BRAF dimerization interface significantly diminishes dimerization of wild-type BRAF and results in loss of its catalytic activity in cells. To test whether resistance mediated by p61BRAF(V600E) was the result of elevated dimer formation, the researchers introduced the R509H dimerization-deficient mutation into p61BRAF(V600E).
Phosphorylation of ERK was elevated and insensitive to vemurafenib in 293H cells expressing p61BRAF(V600E). ERK activity was also raised (though to a slightly lesser degree) in cells expressing 61BRAF(V600E/R509H). Notably, though “p61BRAF(V600E/R509H) showed impaired dimerization, confirming that the R509H mutation located within the dimerization interface disrupts the formation of p61RAF(V600E) dimers,” they write.
In addition, monomeric p61BRAF(V600E/R509H) was sensitive to RAF inhibitors and, in cells ectopically expressing this mutant ERK signaling, was inhibited by vemurafenib. “Thus, the R509H mutation restores sensitivity to the RAF inhibitor by impairing dimerization of p61BRAF(V600E).”
The researchers moved on to analyze tumors from 19 melanoma patients with acquired resistance to vemurafenib. PCR analysis of cDNA from pretreatment tumor samples identified the expected, 2.3 kb sequence that was confirmed to include both BRAF(V600E) and wild-type BRAF transcripts. Conversely two PCR products were identified in six of the post-treatment samples. The shorter of these in each sample was found to encode BRAF(V600E) transcripts that lacked either exons 4–10, exons 4–8 (identical to the variant identified in the resistant cell lines), exons 2–8, or exons 2–10. In contrast, BRAF splicing variants weren’t found in two samples from patients with de novo resistance to vemurafenib or in another 27 melanomas from patients that hadn’t been treated using the drug.
“The identification of BRAF variants lacking the RAS-binding domain in six of nineteen patients with acquired resistance indicates that this mechanism is clinically important,” the authors conclude. “Expression of BRAF(V600E) splicing variants is the first resistance mechanism identified that involves a structural change in BRAF
"These data support the model that inhibition of ERK signaling by RAF inhibitors is dependent on levels of RAS–GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.
The authors note that tumors carrying the BRAF(V600E) splicing variants should still be sensitive to inhibitors of downstream components of the same pathway such as MEK. “Therefore, MEK inhibitors used in combination with vemurafenib could delay or prevent resistance by this mechanism.”