MAPK Pathway Inhibitors Sensitize BRAF-Mutant Melanoma to an Antibody-Drug Conjugate Targeting GPNMB

Abstract

Purpose: To determine if BRAF and/or MEK inhibitor-induced GPNMB expression renders melanomas sensitive to CDX-011, an antibody-drug conjugate targeting GPNMB.

Experimental design: The Cancer Genome Atlas melanoma dataset was interrogated for a panel of MITF-regulated melanosomal differentiation antigens, including GPNMB. BRAF-mutant melanoma cell lines treated with BRAF or MEK inhibitors were assessed for GPNMB expression by RT-qPCR, immunoblot, and FACS analyses. Transient siRNA-mediated knockdown approaches were used to determine if MITF is requirement for treatment-induced GPNMB upregulation. GPNMB expression was analyzed in serial biopsies and serum samples from patients with melanoma taken before, during, and after disease progression on MAPK inhibitor treatment. Subcutaneous injections were performed to test the efficacy of MAPK inhibitors alone, CDX-011 alone, or their combination in suppressing melanoma growth.

Results: A MITF-dependent melanosomal differentiation signature is associated with poor prognosis in patients with this disease. MITF is increased following BRAF and MEK inhibitor treatment and induces the expression of melanosomal differentiation genes, including GPNMB. GPNMB is expressed at the cell surface in MAPK inhibitor-treated melanoma cells and is also elevated in on-treatment versus pretreatment biopsies from melanoma patients receiving MAPK pathway inhibitors. Combining BRAF and/or MEK inhibitors with CDX-011, an antibody-drug conjugate targeting GPNMB, is effective in causing melanoma regression in preclinical animal models and delays the recurrent melanoma growth observed with MEK or BRAF/MEK inhibitor treatment alone.

Conclusions: The combination of MAPK pathway inhibitors with an antibody-drug conjugate targeting GPNMB is an effective therapeutic option for patients with melanoma. Clin Cancer Res; 22(24); 6088-98. ©2016 AACR.

Figure 1.

A MITF transcriptional signature comprised of melanosomal differentiation antigens correlates with poor outcome in melanoma patients. A, Structural/enzymatic proteins and the stages during melanosomal differentiation in which they function: PMEL: premelanosome protein 17; GPR143: G protein-coupled receptor 143; MART1: melanoma antigen recognized by T cells 1; TYR: tyrosinase; TYRP1, tyrosinase-related protein 1; GPNMB: glycoprotein (transmembrane) Nmb; DCT: dopachrome tautomerase; OCA2: oculocutaneous albinism II. B, The mRNA expression levels of selected melanosomal differentiation antigens following 48hr treatment of melanoma cells (A375, WM2664, MDA-MB-435) with a BRAF (Vemurafenib) or MEK (trametinib) inhibitor. Error bars represent SEM from 3 independent experiments. C, Gross images of sub-cutaneous WM2664 melanomas from mice treated for 12 days with vehicle (DMSO), trametinib (2mg/kg/day), or vemurafenib (60mg/kg/day) plus trametinib (2mg/kg/day). D, Heat map depicting mRNA expression of individual melanosomal differentiation antigens within the MITF-melanosomal differentiation signature (MITF Sig.) in the TCGA dataset. E, Kaplan-Meier analysis of melanoma patients separated into high, medium, or low expression of a MITF-melanosomal differentiation signature.

Figure 2.

GPNMB expression is upregulated in melanoma cells following MAPK pathway inhibition in a MITF-dependent manner. A, Immunoblot analysis of GPNMB and MITF expression in melanoma cells (A375, WM2664) following 48hr treatment with vehicle (DMSO) or increasing concentrations of BRAF (Vemu, Dabr) or MEK (Tram, Selu) inhibitors. Inhibitor efficacy was assessed by pERK/ERK immunoblot analysis. Vemu: vemurafenib; Dabr: dabrafenib; Tram: trametinib; Selu: selumetinib. B, Quantification of the percentage MITF nuclear positivity and immunofluorescence intensity of nuclear localized MITF following treatment with vehicle (DMSO), Dabrafenib (Dabr; 100nM) or Trametinib (Tram; 10nM). The number of cells analyzed from 3 independent experiments for each condition: n = 645 for DMSO; n = 351 for Dabrafenib; n = 361 for Trametinib. Error bars represent SEM. *; P < 0.001, **; P < 0.01, ***; P < 0.0001. C, Immunoblot analysis of GPNMB expression in response to vehicle (DMSO), vemurafenib (Vemu) or trametinib (Tram) treatment in melanoma cells with normal (Control siRNA) or reduced (MITF siRNA) MITF levels. Inhibitor efficacy was assessed by pERK/ERK immunoblot analysis. D, Fluorescence activated cell-sorting analysis of cell surface GPNMB expression in DMSO (black lines), vemurafenib (green lines) or trametinib (blue lines) treated melanoma cells. The geometric mean ratio (GMR) indicates the fluorescence intensity (solid lines)/fluorescence intensity measured with the secondary antibodies alone (dotted lines).

Figure 3.

GPNMB mRNA and protein expression is increased in melanoma patients undergoing treatment with MAPK pathway inhibitors. A, RT-qPCR of MITF and GPNMB expression performed on melanoma biopsy specimens taken from patients prior to treatment (pre-therapy) and while receiving therapy (on-treatment). The data is expressed as the on-treatment/pre-treatment ratio for MITF (blue) and GPNMB (red). B, Immunohistochemical (IHC) staining for GPNMB in select patients prior to initiation of therapy and while receiving MAPK inhibitors. Scale bar represents 20 μm and applies to all panels. C–D, RT-qPCR analysis of GPNMB (C) and MITF (D) expression performed on RNA extracted from pre-treatment, on-treatment and progression melanoma biopsy specimens from melanoma patients undergoing MAPKi therapy. The data is presented as on-therapy/pre-therapy or progression/pre-treatment ratios. E, ELISA analysis of soluble GPNMB extracellular domain in pre-treatment, on-treatment and progression serum samples from melanoma patients undergoing MAPK pathway inhibitor therapy. The data is presented as on-therapy/pre-therapy or progression/pre-treatment ratios.

Figure 4.

Combination of intermittent MAPK pathway inhibition and CDX-011 effectively impairs melanoma growth. A–B, Tumor lysates derived from the sub-cutaneous injection of A375 and WM2664 melanoma cells were subjected to immunoblot analysis for GPNMB, pERK and ERK. Tumor lysates were prepared following 7 days of treatment with trametinib or vehicle alone. Immunoblots for α-Tubulin served as a loading control. C–D, Sub-cutaneous tumor growth of A375 and WM2664 melanoma cells in mice treated with vehicle (black line), CDX-011 alone (green line), discontinuous trametinib alone (blue line), continuous daily trametinib alone (red line – panel D) and a combination of discontinuous trametinib plus CDX-011 (purple line). All treatments were started when sub-cutaneous tumors reached 250 mm³ and were administered as follows: CDX-011 (green arrows below x-axis) was administered as a single agent once every three-weeks (panel C) or once every two-weeks (panel D); continuous daily trametinib treatment (panel D, red line); discontinuous trametinib treatment (blue line) was administered daily for one week on and two weeks off inhibitor (panel C) or one week on and one week off inhibitor (panel D); discontinuous trametinib treatment with CDX-011 administered during the mid-point of the on treatment cycle (purple line). In all cases, tumor growth is plotted as the percentage change in tumor volume relative to the size of the tumors when treatment was initiated.

Figure 5.

Continuous treatment with BRAF/MEK inhibitors and CDX-011 significantly impairs melanoma growth. A, Sub-cutaneous tumor growth of WM2664 melanoma cells in mice treated with vehicle (black line), CDX-011 alone (green line), continuous dabrafenib + trametinib (red line) and a combination of continuous dabrafenib/trametinib plus CDX-011 (purple line). Treatments were started when sub-cutaneous tumors reached 400 mm³ and were administered as follows: CDX-011 was administered once weekly as a single agent (green arrows); daily dabrafenib/trametinib treatment (red line); daily dabrafenib/trametinib treatment with CDX-011 administered once weekly (purple line). In all cases, tumor growth is plotted as the percentage change in tumor volume relative to the size of the tumors when treatment was initiated. B, Tumor lysates from WM2664 melanomas were subjected to immunoblot analysis for GPNMB, MITF, pERK and ERK and β-actin following 2 weeks of treatment with vehicle alone, dabrafenib and trametinib or at experimental end-point (9 weeks) from mice treated with dabrafenib and trametinib +/− CDX-011. C, Gross images of sub-cutaneous WM2664 melanomas from mice treated with vehicle, CDX-011, dabrafenib/trametinib (Dab/Tram), or dabrafenib/trametinib/CDX-011 (Dab/Tram/CDX-011). Fontana-Masson stained sections are shown and quantification of the degree of pigmentation is indicated below each image. Scale bar = 100μm and applies to all sections.

Figure 6.

Combination therapy targeting the MAPK pathway and GPNMB in BRAF mutant melanoma. BRAF mutations (*) are present in approximately 50% of melanomas, which constitutively activate the MAPK pathway. Downstream ERK activation leads to MITF phosphorylation, resulting in decreased MITF protein stability and cytoplasmic retention. Clinically approved MAPK pathway inhibitors targeting BRAF (vemurafenib, dabrafenib) or MEK (trametinib, selumetinib, cobimetinib), stabilize and increase MITF nuclear localization. Nuclear MITF transcriptionally regulates many melanosomal genes, including GPNMB, which causes an increase in melanosome biogenesis and hyperpigmentation. MITF also induces PGC-1α expression, a master regulator of mitochondrial biogenesis. Thus, MAPKi treated melanoma cells undergo a metabolic shift from anerobic glycolytic metabolism to aerobic oxidative metabolism, leading to increased oxygen consumption and ROS production. Elevated melanin associated with increased melanosome biogenesis can serve as a scavenger for excess ROS produced during a MAPKi-induced switch to oxidative phosphorylation. GPNMB is a transmembrane glycoprotein present in endosomes, late stage melanosomes and at the plasma membrane. Cell surface GPNMB is the target of the antibody-drug-conjugate, CDX-011 (Glembatumumab vedotin). The GPNMB:CDX-011 complex is internalized and traffics to lysosomes, at which point the linked cytotoxin, monomethyl auristatin E (MMAE), is liberated from the antibody by proteolytic cleavage of an amino-acid linker. MMAE is a potent inhibitor of tubulin dynamics and causes apoptotic cell death.