Vemurafenib Synergizes with Nutlin-3 to Deplete Survivin and Suppresses Melanoma Viability and Tumor Growth

Abstract

Background For patients with advanced melanoma, primary and secondary resistance to selective BRAF inhibition remains one of the most critically compelling challenges. One rationale argues that novel biologically-informed strategies are needed to maximally cripple melanoma cells up front before compensatory mechanisms emerge. Since p53 is uncommonly mutated in melanoma, restoration of its function represents an attractive adjunct to selective BRAF inhibition. Experimental Design Thirty-seven BRAF(V600E)-mutated melanoma lines were subjected to synergy studies in vitro using a combination of vemurafenib and nutlin-3 (Nt-3). In addition, cellular responses and in vivo efficacy were also determined. We also analyzed changes in the levels of canonical apoptotic/survival factors in response to vemurafenib. Results Dual targeting of BRAF(V600E) and HDM2 with vemurafenib and Nt-3, respectively, synergistically induced apoptosis and suppressed melanoma viability in vitro and tumor growth in vivo. Suppression of p53 in melanoma cells abrogated Nt-3′s effects fully and vemurafenib’s effects partially. A survey of canonical survival factors revealed that both vemurafenib and Nt-3 independently attenuated levels of the anti-apoptotic protein, survivin. Genetic depletion of survivin reproduces the cytotoxic effects of the combination strategy. Conclusion These results demonstrate preclinical feasibility for overcoming primary vemurafenib resistance by restoring p53 function. Moreover, it identifies survivin as one downstream mediator of the observed synergism and a potential secondary target.

New Drug Combination Strategies in Melanoma: Current Status and Future DirectionsNew Drug Combination Strategies in Melanoma: Current Status and Future Directions

“…Combining the antagonist of the MDM2-p53 interaction, Nutlin3, with MAPK inhibitors suppresses melanoma growth and potentiates MAPK inhibition (47,48). Restoring the apoptotic function of p53 by inhibiting MDM2 and iASPP cooperates with V600E BRAF inhibition to suppress human melanoma cell growth both in Najem et al: Drug Combinations in Melanoma (Review) 5943 (49).…”

A novel multi-CDK inhibitor P1446A-05 restricts melanoma growth and produces synergistic effects in combination with MAPK pathway inhibitors

“…The A375 shTP53 and shGFP lines, as well as vemurafenib-resistant lines, were previously generated and described by our laboratory. [19][20][21] All cells were maintained in incubators at 37 C with an atmosphere of 95% room air and 5% CO 2 .…”

“…[19][20][21] In brief, equal amounts of protein lysate (10-20 mg) were run on pre-cast 4-20% SDSpolyacrylamide gels and transferred to PVDF membranes (BioRad, Hercules, CA). The membranes were blocked in 5% milk in TBS-Tween for 1 hour, and incubated with primary and secondary antibodies for 2 hours and 1 hour, respectively; blocking and antibody incubation were performed at room temperature.…”

Oncogene-Directed Small Molecule Inhibitors for the Treatment of Cutaneous Melanoma

“…In two preclinical studies, Ji et al demonstrated that the MDM2 inhibitor nutlin-3 was able to suppress melanoma viability across a large panel of cell lines, and synergize with vemurafenib to decrease melanoma growth in vitro and in vivo; the growth suppressive effect of MDM2 antagonism was additionally linked to restored p53 activity [97,98]. Antagonizing MDM4 with SAH-p53-8 has also been shown to suppress melanoma cell viability and xenograft tumor growth by inducing growth arrest and apoptosis [99].…”