RAF265 inhibits the growth of advanced human melanoma tumors

Abstract

Purpose: The purpose of this preclinical study was to determine the effectiveness of RAF265, a multikinase inhibitor, for treatment of human metastatic melanoma and to characterize traits associated with drug response.

Experimental design: Advanced metastatic melanoma tumors from 34 patients were orthotopically implanted to nude mice. Tumors that grew in mice (17 of 34) were evaluated for response to RAF265 (40 mg/kg, every day) over 30 days. The relation between patient characteristics, gene mutation profile, global gene expression profile, and RAF265 effects on tumor growth, mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation, proliferation, and apoptosis markers was evaluated.

Results: Nine of the 17 tumors that successfully implanted (53%) were mutant BRAF (BRAF(V600E/K)), whereas eight of 17 (47%) tumors were BRAF wild type (BRAF(WT)). Tumor implants from 7 of 17 patients (41%) responded to RAF265 treatment with more than 50% reduction in tumor growth. Five of the 7 (71%) responders were BRAF(WT), of which 1 carried c-KIT(L576P) and another N-RAS(Q61R) mutation, while only 2 (29%) of the responding tumors were BRAF(V600E/K). Gene expression microarray data from nonimplanted tumors revealed that responders exhibited enriched expression of genes involved in cell growth, proliferation, development, cell signaling, gene expression, and cancer pathways. Although response to RAF265 did not correlate with pERK1/2 reduction, RAF265 responders did exhibit reduced pMEK1, reduced proliferation based upon reduced Ki-67, cyclin D1 and polo-like kinase1 levels, and induction of the apoptosis mediator BCL2-like 11.

Conclusions: Orthotopic implants of patient tumors in mice may predict prognosis and treatment response for melanoma patients. A subpopulation of human melanoma tumors responds to RAF265 and can be characterized by gene mutation and gene expression profiles.

Figure 1

A, orthotopic transplant tumor response to RAF265. The 17 human melanoma biopsy tissues were successfully grown on nude mice and the mean ± SD (biological repeats, n = 5) of measured tumor volumes of RAF265 treatment and control groups were shown. Seven melanoma biopsy tumor-derived orthotopic implants showed significant growth inhibition illustrated by unpaired Student t test analysis. The definition of treatment response in this study: When compared with control, more than 50% reduction of tumor volume is achieved by RAF265 at the end of 30-day treatment period. Mutational status (BRAF, cKIT, and NRAS) is noted in the right hand column. Annotations are as follows: *, mice carrying xenografts of patient V09 had only 1 mouse in the RAF265 treatment group and 4 in the control group, making statistical analysis of response not applicable (N/A); **, patient V12 received 800mg of the old formulation of PLX4032 which was inadequate and the patient progressed; ***, patient V29 was initially treated with 1600 mg of an older formulation of PLX4032, then switched to the newer formula and allowed to escalate to 360 and then to 720 mg/kg. After an initial response, patient V29 progressed at 15 months, and the PLX4032-resistant tumor was biopsied and used in the tumor implant study. B, graphical comparison of orthotopic transplant tumor size after 15- to 30-day treatment with RAF265. The tumor sizes on RAF265-treated nude mice were measured, and the difference of tumor volume between treatment and control groups was analyzed by unpaired Student t test (P <0.05). The figure shows normalized tumor sizes (mean ± SD, n = 5) in RAF265 treatment and control groups.

Figure 2

A, Western blot analysis of lysates from 5 selected melanoma implants reveals that RAF265 treatment downregulates level of phosphorylated MEK without affecting ERK1/2 phosphorylation. It also reduces phospho-cyclin D1 and PLK1 levels, and elevates BIM1 and COT as compared with vehicle-treated controls in tumors responsive to the drug treatment. Top, tumor growth charts for 3 representative Raf265-responsive melanoma implants, 1 partial responder, and 1 nonresponder tumor during the 14-day period of daily treatment with Raf265 or vehicle control. Bottom, Western blot analysis of phospho-MEK, phospho-ERKand total ERK,Cot, phospho-Cyclin D1, PLK, BIM, BAX, BCL2, MCL1, and cleaved caspase-3 levels in lysates extracted from Raf265 and vehicle-treated melanoma tumors shown above. Western blot with β-actin–specific antibody was used as a loading control everywhere except for cases indicated with * in which GAPDH levels were analyzed to ensure equal loading. B, photomicrographs showing immunohistochemical localization of pMEK1 (green) and cyclin D1 (red) in tissue cores from TMAs assembled from tumors implanted into mice and treated with RAF265 or vehicle control. Nuclei are stained with DAPI and appear blue. a, pMEK1 staining of tumor core from V27 treated with vehicle control. Magnification, ×20. b, pMEK staining of tumor core from V27 treated with RAF265, ×20 magnification. c, pMEK1 staining of tumor core from V27 treated with vehicle control, ×40 magnification. d, pMEK staining of tumor core from V27 treated with RAF265, ×40 magnification. e, cyclin D1 staining of tumor core from V27 treated with vehicle control, ×20 magnification. f, cyclin D1 staining of tumor core from V27 treated with RAF265, ×20 magnification. g, cyclin D1 staining of tumor core from V27 treated with vehicle control, ×40 magnification. h, cyclin D1 staining of tumor core from V27 treated with RAF265, ×40 magnification. i, pMEK1 staining of tumor core from V05 treated with vehicle control, ×20 magnification. j, pMEK staining of tumor core from V05 treated with RAF265, ×20 magnification. k, pMEK1 staining of tumor core from V05 treated with vehicle control, ×40 magnification. l, pMEK staining of tumor core from V05 treated with RAF265, ×40 magnification. m, cyclin D1 staining of tumor core from V05 treated with vehicle control, ×20 magnification. n, cyclin D1 staining of tumor core from V05 treated with RAF265, ×20 magnification. o, cyclin D1 staining of tumor core from V27 treated with vehicle control, ×40 magnification. p, cyclin D1 staining of tumor core from V05 treated with RAF265, ×40 magnification.

Figure 3

Heatmap of differentially expressed genes and their patterns. Unsupervised Clustering categorized wild typeBRAFhuman melanoma into two groups which was in agreement of the separation based on RAF265-induced responsiveness. The lines on the left are used to group the similar gene expression pattern together. These lines separate into two large groups based on whether the gene expression is up-regulated or down-regulated. Further, into each group, there are subgroups, each having genes sharing even more similar patterns as identified by the unsupervised clustering software. There are seven levels of gene expression patterns shown on the left. The top lines of the figure are used to group samples of RNA based on the similarity of gene expression patterns shown on the right side of the figure. These RNA samples were from primary patient tumors so they were labeled P0, for example V28P0 means passage 0 primary melanoma tumor from patient V28. Gene expression of three RNA sample repeats from each patient, except V19, 25, and 24 which only have two repeats, were included. The pseudo-color red indicates the up-regulation while green color for down-regulation of gene expression in the tumor RNA samples.

Figure 4

Pathway analysis of differentially expressed genes in RAF265 responding versus nonresponding tumors. Ingenuity software was utilized to recognize pathways connecting genes that were differentially expressed in RAF265 responding versus nonresponding patients. A, Pathway analysis of genes involved in cell development and growth regulation that are differentially expressed in BRAFWT responding tumors to RAF265 versus those not responding to RAF265. B, Pathway analysis of genes involved in cell signaling, nucleotide metabolism, and small molecule biochemistry that are differentially expressed in BRAFWT tumors responding to RAF265 versus those not responding to RAF265. C, Pathway analysis of genes involved in cell signaling interactions that are differentially expressed in RAF265 responding versus nonresponding tumors, irrespective of BRAF mutation status.