Abstract
Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 μM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.
Highlights
Inhibitors of BRAFV600E have changed the clinical management of patients with BRAF-mutant advanced melanoma, since significant improvements in progressionfree survival (PFS) and in overall survival (OS) have been reported in Phase 3 trials [1,2]
We used 49 BRAF-mutant melanoma cell lines isolated from surgical specimens of patients not previously treated with BRAFV600E inhibitors, nor with any other target-specific inhibitor, to test responsiveness to BRAFV600E (PLX4720), MEK1/2 (AZD6244), dual PI3K/mTOR (BEZ235) and dual mTORC1/2 (AZD8055) inhibitors (Figure 1A)
Enhanced inhibition of p-ERK and p-AKT by AZD6244BEZ235 compared to PLX4720-BEZ235, and to control animals treated with vehicle, was observed in-vivo, as indicated by a reduced staining for p-ERK and p-AKT in melanoma cells from neoplastic nodules removed after the last administration of inhibitors (Figure 6A, 6B and Supplementary Figure 8 for quantitative analysis). These results indicated that the asso ciation of MEK1/2 and PI3K/mTOR inhibitors induces a more effective inhibition of p-ERK at both early and late time points compared to BRAF and PI3K/mTOR dual blockade, in PLX4720-resistant melanoma cells and even in cell lines with a cross-resistant phenotype
Summary
Inhibitors of BRAFV600E have changed the clinical management of patients with BRAF-mutant advanced melanoma, since significant improvements in progressionfree survival (PFS) and in overall survival (OS) have been reported in Phase 3 trials [1,2]. These inhibitors can induce objective responses or stabilization of disease in a high fraction of patients [2,3], relapse occurs due to adaptive [4] or acquired [5] resistance mechanisms. Primary cross-resistance to BRAF and MEK inhibitors has been documented in a subset of melanomas, where it is related to the MITF profile [11,12,13], and in cell lines [14]
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