Our group and others have been intensively investigating the molecular mechanisms underlying resistance to BRAF inhibitors using a variety of approaches (12
). In our studies, we modeled the emergence of resistance to BRAF inhibitors by selecting a panel of BRAFV600E/PTEN+ melanoma cells which are highly sensitive to BRAF inhibition and chronically exposing them to increasing doses of SB-590885 (GlaxoSmithKline), a BRAF-selective inhibitor (15
). Drug-resistant cells emerged approximately 6 months after persistent drug exposure and were able to proliferate and survive in the continuous presence of 1 μM SB-590885, unlike their parental counterparts. Importantly, chronic BRAF inhibition led to cross-resistance to several BRAF-selective inhibitors, including PLX4032, indicating that resistance is not likely to be easily overcome by switching to a new RAF inhibitor. All resistant clones were able to proliferate at normal rates, retained their anchorage independent growth, and were able to grow in a 3D-tumor-like microenvironment even in the presence of high doses of BRAF inhibitors.
Although a frequent mechanism of anti-cancer drug resistance is the development of secondary mutations in the target gene, we did not identify secondary mutations in BRAF in any of our resistant cell lines, all of which retained the BRAFV600E mutation. Biochemically, our resistant melanoma cells were able to reactivate the MAPK pathway in a BRAF-independent manner. While the parental (BRAF inhibitor-sensitive) cells rely on BRAF for MAPK activation, the BRAF-inhibitor resistant cells had elevated expression of CRAF and ARAF, and were able to dynamically use either of these two RAF isoforms to sustain MAPK activity and promote proliferation; nevertheless, the resistant cells were still sensitive to MEK inhibitors which target downstream of RAF (). Treatment of BRAF-inhibitor resistant cells with various structurally different MEK inhibitors had mostly cytostatic effects, suggesting that additional bypass mechanisms could be promoting survival. Indeed, our resistant cells displayed differential activation of several RTKs, in particular IGF-1R. Although the parental melanoma cells, like all cells of melanocytic origin, express the IGF-1R receptor, some of our BRAF-resistant melanomas expressed higher surface levels of IGF-1R. We observed that treatment of parental cells with BRAF inhibitors led to a decrease in phospho-IGF-1R levels; however, phosphorylation of IGF-1R was sustained in our BRAF-inhibitor resistant cells. We further noted that enhanced IGF-1 mediated signaling was not due to amplification or mutations of the IGF-1R gene. Even though the precise mechanisms of enhanced IGF-1R expression and signaling in the context of chronic BRAF inhibition are not yet completely understood, our results suggest possible cross-talk between BRAF and RTKs, particularly IGF-1R-dependent networks, that requires further investigation.
Figure 1 Simplified schematic of signaling pathways driving resistance to BRAF inhibitors. In BRAF V600E mutant melanoma cells (left), BRAF inhibition causes growth arrest and apoptosis by blocking the MAPK pathway. These BRAF-inhibitor (BRAFi-) sensitive cells (more ...)
IGF-1R plays an important role in survival and resistance to anti-cancer therapies (16
) that could be mediated through activation of MAPK and PI3K signaling. In the context of resistance to BRAF inhibition, we found that IGF-1R promotes activation of PI3K and phosphorylation of AKT, but has no effect on the MAPK pathway. Pharmacological or genetic inhibition of IGF-1R inhibited only downstream PI3K/AKT signaling; exogenous IGF-1 increased PI3K-mediated signaling, but was not sufficient to induce resistance. Additionally, the MAPK and PI3K pathways appeared to jointly regulate the levels of the anti-apoptotic factor Mcl-1 to promote survival. Clearly, the MAPK and IGF-1R/PI3K/AKT signaling pathways cooperate to promote survival and expansion of the BRAF-inhibitor resistant cells. Validating this conclusion, we found that co-inhibition of these two pathways was more efficient in inducing apoptosis of BRAF-inhibitor resistant cells than inhibition of each individual pathway. Combination of MEK inhibitors, including GSK1120212 or AZD6244 with PI3K inhibitors such as GSK 2126458 or IGF-1R inhibitors led to striking cytotoxic effects in 3D BRAF-inhibitor resistant melanoma spheroids. Our findings strongly advocate the potential use of these combinatorial approaches to treat patients refractory to BRAF inhibitors.
To assess the clinical relevance of our studies, we compared paired biopsies (pre-treatment and post-relapse) in five patients with metastatic melanoma treated with PLX4032. All five patients initially responded to PLX4032 but relapsed after 4-15 months of treatment. Sequencing of all five paired tumor biopsies indicated that the mutation encoding BRAFV600E was present in all samples (pre-treatment and post-relapse), but no secondary mutations in BRAF, CRAF or RAS were identified. Through immunohistochemical analysis of these biopsies, we found increased levels of IGF-1R in the post-relapse samples of two patients, one of which also had increased levels of phosphorylated AKT. These findings are consistent with our in vitro data suggesting that enhanced IGF-1R expression and PI3K/AKT activity are associated with resistance to BRAF inhibitors in some patients. We also noted a homozygous loss of PTEN and increased pAKT levels in the post-relapse biopsy of one patient, suggesting that PTEN loss could also be linked to resistance to BRAF inhibitors in some patients; however, it is not likely to be the sole player. These findings need to be further investigated both in vitro and in clinical samples.