CO-TARGETING OF ONCOGENIC AND DEATH RECEPTORS PATHWAYS IN HUMAN MELANOMA:PRE-CLINICAL RATIONALE FOR A PRO-APOPTOTIC AND ANTI-ANGIOGENIC STRATEGY

INTRODUCTION: Resistance to cell death is one of the well-known hallmarks of cancer. Metastatic melanoma is an aggressive disease whose treatment has significantly improved thanks to the recent development of either target-specific or immune-modulating agents. Since the FDA approval of the BRAFV600E inhibitor Vemurafenib in 2011, several other inhibitors, targeting relevant oncogenic signaling pathways (i.e. the MEK/ERK or the PI3K/Akt/mTOR pathways) have been used to treat melanoma patients. However intrinsic or acquired resistance limits the efficacy of these compounds, with relapses in most of the patients within 6 months. Therefore, there is an immediate necessity for new therapeutic strategies to face this issue, and one option could be represented by combinatorial treatments associating different anti-tumor agents able to target not only tumor cells but also to counteract pro-tumoral mechanisms in melanoma microenvironment. HYPOTHESIS: Our goal was to obtain pre-clinical evidence for the efficacy of an anti-tumor approach based on the combination of MEK, PI3K inhibitors and TRAIL; building upon the hypothesis that these agents should: 1) be able to overcome melanoma intrinsic resistance to programmed cell death by the concomitant targeting of both the extrinsic (mainly through TRAIL activity) and the intrinsic (mainly due to the activity of MEK and PI3K pathway inhibitors) apoptosis pathways; 2) be able to promote an anti-angiogenic effect combining the well-known vascular disrupting activity of TRAIL and the effects of inhibition of pro-angiogenic pathways in tumor and in tumor-associated vasculature due to targeting of ERK and AKT cascades. METHODS: A large panel of patient-derived melanoma cell lines was used to test the in vitro efficacy of the association between AZD6244/Selumetinib (a MEK1/2 inhibitor), the dual PI3K/mTOR inhibitor BEZ235, and soluble TRAIL. Chou-Talalay drug interaction analysis was used to determine Combination Indexes and Fraction Affected values for all the possible combinations of anti-tumor agents. Whole-genome gene expression profiling, flow cytometry experiments, western blot analysis, proteomic arrays and ELISAs were used to clarify the mechanism behind the synergy shown for the AZD6244+TRAIL and AZD6244+BEZ235+TRAIL associations. Moreover, xenografts in SCID mice were used to confirm the in vivo efficacy and mechanism of action of the combination between the MEK inhibitor and TRAIL, using tumor growth rates and immunohistochemistry on tumor nodules to evaluate effects of the association. Human Umbilical Vein Endothelial Cells (HUVEC) were chosen to model the endothelial-melanoma cell interaction in order to analyze its effects on response to the combinatorial treatment, both in terms of apoptosis induction and endothelial differentiation/activation status RESULTS: While half of the melanoma cell lines we tested were resistant to the death receptor ligand TRAIL, several were susceptible either to AZD6244 or to BEZ235, evidencing independent susceptibility profiles to these drugs and setting the rationale for their association. The combination of the MEK inhibitor, with or without the PI3K/mTOR inhibitor, and TRAIL achieved synergistic anti-tumor activity in 20/21 melanoma cell lines tested, including tumors resistant to either one of the agents. Mechanistically, an increment in induction of caspase-dependent cell death and of mitochondrial depolarization was evidenced for the association, and a significant modulation of key regulators of extrinsic and intrinsic apoptosis pathways including c-FLIP, BIM, BAX, clusterin, Mcl-1 and several IAP family members was confirmed. Moreover, silencing experiments defined Apollon downmodulation as a central event for the promotion of the melanoma apoptotic response to our combinatorial treatments. SCID mice bearing melanoma xenografts were treated with the MEK inhibitor and TRAIL, alone or in combination, obtaining a more significant tumor growth inhibition by the combinatorial treatment, with no detectable adverse events on mice body weight and tissue histology. TUNEL staining on tissues sections indicated also in vivo an increased promotion of tumor apoptosis, which was associated with suppression of several pro-angiogenic molecules like HIF1, VEGF, IL-8 and TGF1as well as a marked reduction in CD31 positive cells. Furthermore, initial results on HUVECs pointed at a possible effect of the interaction between endothelial and melanoma cells, affecting responsiveness of HUVECs to combinatorial treatments, as documented by increased endothelial cell apoptosis, in response to MEK inhibitor and TRAIL treatment, after co-culture with melanoma cells. The modulatory effect of melanoma on endothelial cells was evidenced not only by “activation” markers (upregulation of ICAM-1/CD54) but also the “differentiation” status of endothelial cells, indicated by increased alpha-SMA levels, reduction in the expression of vascular cadherin CD144 and downmodulation of the endothelial marker CD31. CONCLUSIONS: Results of this work suggest that concomitant targeting of melanoma oncogenic signaling pathways and the TRAIL receptor cascade can not only overcome in vitro tumor resistance to different anti-tumor agents, but can also have in vivo effects on tumor microenvironment, promoting pro-apoptotic effects and inhibition of tumor angiogenesis. Moreover, this could be associated to a modulation of endothelium responsiveness to anti tumor agents by direct interaction with melanoma cells.