Background: Recent advances with B-raf and Mek targeted therapies have transformed the therapeutic landscape for metastatic melanoma and improved survival times for patients. Despite this progress, resistance to targeted therapies remains a universal problem that limits treatment efficacy. Combination therapy strategies represent a viable option to overcome the problem of therapeutic resistance.
Glycoprotein Non-Metastatic B (GPNMB) is a cell surface protein that is highly expressed in a wide variety of cancer types, and regulates: tumor growth, cancer cell migration, invasion and metastasis. Glembatumumab vedotin (CDX-011) is a GPNMB-targeted antibody-drug conjugate that is in clinical trials for the treatment of melanoma and breast cancer. Interestingly, a wide spectrum of kinase inhibitors (KI) has been reported to induce GPNMB expression in a variety of cell types. Here we investigate the mechanism(s) responsible for elevated GPNMB levels in melanoma cells treated with clinically relevant KI, determine whether KI-mediated induction of GPNMB expression synergizes with CDX-011 in the treatment of melanoma.
Methods: B-raf mutant (A375, WM-2664) and N-ras mutant (SkMel2) melanoma cells were exposed to inhibitors of B-raf (vemurafenib, dabrafenib), or Mek (trametinib, selumetinib). Immunoblot analysis or fluorescence-activated cell sorting was used to assess GPNMB expression. B-raf mutant cells were passaged in the presence of vemurafenib over the course of several weeks to months to generate vemurafenib-resistant cells. Transient siRNA knockdown studies were used to assess the requirement for the transcription factor, MiTF, in KI-mediated induction of GPNMB. To assess the interaction between individual KI and CDX011, dose response curves for the KI and CDX-011 alone or in combination in melanoma cells in vitro were generated. Melanoma cells were either 1) pretreated or 2) pre and co-treated with KI for 48 hours prior to treatment with CDX011 for 96 hours. Cell viability was assessed by XTT assay.
Results: B-raf inhibition led to an induction of total and cell surface GPNMB protein in B-raf mutant, but not in B-raf WT melanoma cells. Mek inhibition induced GPNMB expression in all three melanoma cell lines. In addition to acute inhibitor treatment, Vemurafenib-resistant cells also expressed higher levels of GPNMB when compared to parental cells (inhibitor sensitive). Knock-down of the MiTF transcription factor abrogated the vemurafenib-mediated induction of GPNMB. Pretreatment of melanoma cells with Mek inhibitors led to a reduction in cell viability, compared to untreated cells, that was further exacerbated by exposure to CDX-011, in a dose-dependent manner. Interestingly, sub-optimal doses with B-raf inhibitors did not significantly affect cell viability of WM-2664 cells when used in isolation, but did enhance the sensitivity of melanoma cells to CDX-011.
Conclusions: Induction of GPNMB in response to B-raf/Mek inhibition is a MiTF-dependent process that occurs in both B-raf mutant and WT cells. In this context, induction of GPNMB expression sensitizes cells to CDX-011 mediated cell killing. As such, the combination of B-raf/Mek inihibitors with CDX-011 represents an intriguing new combination therapy strategy for the treatment of melanoma, and warrants further validation in in vivo mouse models.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C232.
Citation Format: April A.N. Rose, Peter M. Siegel. B-raf/Mek/Erk Pathway inhibition induces GPNMB expression and sensitizes melanoma cells to the antibody-drug conjugate glembatumumab vedotin. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C232.