We report that BRAFV600E melanomas chronically treated with a specific BRAF inhibitor acquire cross-resistance to several selective BRAF inhibitors through a RAF kinase switch. Chronic BRAF inhibition is associated with enhanced IGF-1R and PI3K/AKT activity in melanoma cells resistant to BRAF inhibitors. We propose that drug combinations co-targeting MEK and IGF-1R/PI3K may offer valid therapeutic approaches to overcome resistance to BRAF inhibitors.
Acquired resistance to anticancer agents is frequently encountered in clinical practice. Resistance to kinase inhibitors is often associated with secondary mutations in the target gene, which render the kinase insensitive to the inhibitor (Engelman and Settleman, 2008
). However, in our in vitro
system, we did not find secondary mutations in BRAF
that could explain resistance to BRAF inhibitors. We also did not identify de novo
mutations or changes in copy number in NRAS
, or PTEN
, three oncogenes commonly associated with melanoma. BRAFV600E
promotes persistent MAPK activity, leading to increased proliferation and survival. Acute BRAFV600E
inhibition by genetic depletion or kinase inhibitors can lead to cell cycle arrest and, in some instances, apoptosis in melanomas addicted to this oncogene (Bollag et al., 2010
; Hingorani et al., 2003
; Lee et al., 2010
; King et al., 2006
; Tsai et al., 2008
). Our studies demonstrate that upon chronic BRAF inhibition, melanomas rewire their signaling circuitries in order to utilize one of the other two RAF isoforms, ARAF or CRAF, to overcome the effect of BRAF inhibition.
Our data are consistent with a model whereby melanomas are initially addicted to the BRAF/MAPK pathway. If BRAF is repressed, melanomas trigger an alternative signaling program, involving a kinase switch, which allows the addicted tumor to continue to rely on MAPK for maintenance of the neoplastic phenotype. Our findings have important therapeutic implications as they highlight the relevance of MAPK signaling in melanoma and argue that targeting the MAPK pathway constitutes a valid therapeutic strategy.
Recent studies demonstrated that in the context of mutant RAS, acute inhibition of BRAF kinase activity promotes altered scaffolding and activation of CRAF, phosphorylation of ERK, and oncogenesis (Hatzivassiliou et al., 2010
; Heidorn S et al., 2010
; Poulikakos, 2010
). While Hatzivassiliou et al.
and Heidorn et al.
suggested that BRAF inhibition does not activate CRAF in V600E-mutant cells, our studies indicate that BRAFV600E
melanomas can flexibly switch among the three different RAF isoforms by a yet unidentified mechanism to overcome the effect of chronic BRAF inhibition and activate the MAPK pathway.
Montagut and colleagues described a model of resistance to the RAF inhibitor AZ628 through increased levels of CRAF protein (Montagut et al., 2008
). We also observed increased CRAF levels in cells chronically treated with the BRAF inhibitor 885. However, in our system, shRNA-mediated inhibition of CRAF did not affect ERK activation or proliferation, as resistant cells can also switch to ARAF. The differences between the two studies may be due to the distinct molecular and genetic profiles of the cell lines used, the mechanism of action of the drug used to target the tumor cells, and/or the duration of treatment among other factors.
Our data demonstrate that under conditions of chronic BRAF inhibition, melanomas rely on IR/IGF-1R -mediated survival pathways in order to circumvent adverse conditions favoring cell death. IGF-1R, which is expressed in all cells of melanocytic origin, has been implicated in resistance to therapy in other neoplasia, including lung and breast cancer (Casa et al., 2008
). Recently, Sharma et al.
have reported the existence of a subpopulation of drug-tolerant cells that survive acute drug treatment via engagement of IGF-1R signaling (Sharma et al., 2010
). The enhanced activity of PI3K/AKT associated with chronic BRAF inhibition suggests the possible existence of a negative crosstalk between the two pathways. Crosstalk between MAPK and PI3K has been reported in several cancer systems (Carracedo et al., 2008
; Cheung et al., 2008
; Mirzoeva et al., 2009
), but not much is known in melanoma; this issue deserves further exploration.
BRAFV600E/PTEN+ melanomas, which are sensitive to BRAF inhibitors, have low levels of pAKT (our unpublished data). In contrast, melanoma cells that acquire resistance to BRAF inhibitors have enhanced levels of pAKT associated with increased IGF-1R signaling. These observations raise the possibility that IGF-1R/PI3K-mediated signaling in the context of chronic BRAF inhibition promotes survival of BRAF inhibitor-resistant melanomas, and cooperates with the MAPK pathway to support drug resistance. Consistent with this notion, inhibitors of MEK and IGF-1R or PI3K in combination were more effective inducing cell death of BRAF-inhibitor resistant cells than when used as single agents.
Although results from recent clinical trials with PLX4032 are encouraging, responding tumors eventually develop resistance. Increased expression of IGF-1R in post-relapse tumor biopsies of two patients who developed resistance to PLX4032, one of whom also had increased levels of phospho-AKT, constitute proof-of-principle that IGF-1R/PI3K/AKT-mediated signaling may be associated with resistance to BRAF inhibitors, and provide insight into future therapies for the treatment of patients who become refractory to these drugs. The absence of changes in BRAF, NRAS, and PTEN mutation status in patient 1 supports the idea that a non-genetic mechanism can be underlying resistance to BRAF inhibitors in some patients. Our findings suggest that melanomas can respond to chronic BRAF inhibition through dynamic changes by re-wiring their signaling circuitries, allowing the tumor cells to adapt to pharmacological challenges. Given the high degree of heterogeneity and plasticity of melanoma, it is likely that several mechanisms of resistance will arise in response to chronic BRAF inhibition, raising challenges to our quest in search of effective therapies for this malignancy. Of note, homozygous loss of PTEN and increased phospho-AKT were identified in post-relapse samples in one patient, suggesting that alternative mechanisms leading to PI3K/AKT activation may also be associated with acquired resistance to BRAF inhibitors.
Our studies and others’ demonstrate that targeting solely one pathway is not sufficient to eradicate melanoma (Lasithiotakis et al., 2008
; Smalley et al., 2006
). This study provides further evidence that combination strategies targeting key oncogenic pathways are required for successful therapy. Furthermore, our findings provide a molecular rationale for combining MEK and IGF-1R/PI3K inhibitors as we demonstrate that: 1) melanomas are addicted to the MAPK pathway - thus, shutting off this pathway results in oncogenic shock, rendering cells susceptible to apoptosis; 2) chronic BRAF inhibition is associated with enhanced IGF-1R/PI3K-dependent survival pathways as a protective cellular mechanism, and 3) concomitant MEK and IGF-1R/PI3K inhibition shifts the balance towards induction/activation of pro-apoptotic molecules and inhibition of pro-survival factors in melanomas resistant to BRAF inhibitors.
Combining MEK and IGF-1R/PI3K inhibitors constitutes a promising approach, as these two signaling pathways cooperate to drive tumor growth, survival, and resistance to therapy. Thus, combination strategies targeting these two pathways merit further evaluation as a potential approach to treat melanomas refractory to BRAF inhibitors.