PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibits V600E-positive melanomas

Abstract

Targeted intervention of the B-Raf V600E gene product that is prominent in melanoma has been met with modest success. Here, we characterize the pharmacological properties of PLX4032, a next-generation inhibitor with exquisite specificity against the V600E oncogene and striking anti-melanoma activity. PLX4032 induces potent cell cycle arrest, inhibits proliferation, and initiates apoptosis exclusively in V600E-positive cells in a variety of in vitro experimental systems; follow-up xenograft studies demonstrate extreme selectivity and efficacy against melanoma tumors bearing the V600E oncoproduct. The collective data support further exploration of PLX4032 as a candidate drug for patients with metastatic melanoma; accordingly, validation of PLX4032 as a therapeutic tool for patients with melanoma is now underway in advanced human (Phase III) clinical trials.

Figure 1. PLX4032 Inhibits Raf Signaling in a V600E-dependent Manner

A panel of melanoma cell lines with either mutant (left panel) or wild-type (right panel) B-Raf was treated with increasing doses of PLX4032 for 2 hours before lysates were collected for immunoblotting. pERK levels were measured to determine activity of the Raf/MEK/ERK signaling cascade both pre- and post-treatment. β-actin serves as a loading control.

Figure 2. In vitro activity of PLX4032 in Melanoma

A) Proliferation of V600E+ (left panel) or wild-type B-Raf (right panel) cells in increasing concentrations of PLX4032. B) Colony formation assay; cells were seeded in 6-well dishes and exposed to 100 nM and 1 μM PLX4032 for 14 days. Surviving cells were stained in methylene blue and photographed. C) Representative cell cycle analysis of mutant (1205Lu) and wild-type (C8161) melanoma cells in response to PLX4032 treatment. D) Annexin V/PI staining of mutant and wild-type B-Raf cells at 24-hour intervals after treatment with 1 μM PLX4032.

Figure 3. PLX4032 Exhibits Anti-melanoma Activity in 3D-based Cellular Models

A) Collagen-embedded melanoma spheroids from established lines were treated with indicated doses of PLX4032 for 72 hours and stained for viability with calcein-AM (green) and ethidium bromide (red). B) Primary spheroids from freshly-isolated human melanomas were embedded in collagen and exposed to PLX4032 for 72 hours, followed by staining as described in 3A. C&D) Artificial skin reconstructs were generated with either mutant or wild-type B-Raf cells and treated with 1 μM PLX4032 for 72 hours before harvesting and immunostaining for the indicated protein markers; Ki67 indicates proliferation, TUNEL represents apoptosis.

Figure 4. PLX4032 Initiates Potent Tumor Regression in V600E-positive Xenografts

A,B) Xenograft tumors established from either B-Raf wild-type (A) or mutant (B) cell lines were administered 100 mg/ml PLX4032 BID and tumor volume was recorded every 72 hours for 15 days. C) 1205Lu tumors from control and treated mice were harvested, paraffin-embedded, and stained with pERK to measure target efficacy. Photos are depicted at 40x magnification. D) 1205Lu tumors control and treated mice were harvested, paraffin-embedded, and stained with Ki67 to indicate relative proliferation within respective tumors. E) Pharmocokinetic analysis of plasma drug levels from xenografts on the first and last day of treatment at 2- and 6-hours post treatment.