Tumor and Stem Cell Biology Protein Kinase D3 Sensitizes RAF Inhibitor RAF265 in Melanoma Cells by Preventing Reactivation of MAPK Signaling

RAS mutations occur in more than 30% of all human cancers but efforts to directly target mutant RAS signaling as a cancer therapy have yet to succeed. As alternative strategies, RAF and MEK inhibitors have been developed to block oncogenic signaling downstream of RAS. As might be expected, studies of these inhibitors have indicated that tumors with RAS or BRAF mutations display resistance RAF or MEK inhibitors. In order to better understand the mechanistic basis for this resistance, we conducted a RNAi-based screen to identify genes that mediated chemoresistance to the RAF kinase inhibitor RAF265 in a BRAF (V600E) mutant melanoma cell line that is resistant to this drug. In this way, we found that knockdown of protein kinase D3 (PRKD3) could enhance cell killing of RAF and MEK inhibitors across multiple melanoma cell lines of various genotypes and sensitivities to RAF265. PRKD3 blockade cooperated with RAF265 to prevent reactivation of the MAPK signaling pathway, interrupt cell cycle progression, trigger apoptosis, and inhibit colony formation growth. Our findings offer initial proof-of-concept that PRKD3 is a valid target to overcome drug resistance being encountered widely in the clinic with RAF or MEK inhibitors. Cancer Res; 71(12); 4280–91. 2011 AACR. Introduction RAS–RAF mitogen-activated protein kinase (MAPK) signaling cascade plays a central role in the regulation of cell proliferation and survival, whereas the deregulation of this pathway frequently occurs in human cancers (1–3). As mutations in RAS or BRAF occur in more than 30% of human cancers, these proteins are very attractive therapeutic targets in many cancer types. Among them, BRAF mutations occur in about 7% of human cancers, with highest prevalence in melanomas (66%) and thyroid (35%–70%) tumors (4, 5). Interestingly, 80% of all BRAF mutations are concentrated on a single substitution of glutamic acid for valine (V600E) within the kinase domain (4). Compared with BRAF, mutations in the 2 RAF isoforms, ARAF and CRAF, are rarely found in human cancers, which is likely due to lower basal kinase activities (6, 7). All 3 RAS isoforms (KRAS, NRAS, and HRAS) are found mutationally activated in 30% of all human cancers, with highest prevalence in pancreas (90%), colon (50%), thyroid (50%), and lung (30%) cancers (1, 2). Although the RAS oncogene has been studied for more than 3 decades, there is no drug on the market that sufficiently inhibits RAS, despite extensive efforts to inhibit activated RAS with low molecular weight inhibitors (3, 8). As an alternative therapeutic strategy, RAF and MAP/ERK kinase (MEK) inhibitors have been developed to inhibit the pathway downstream of RAS, and only 1 RAF inhibitor Sorafineb has been approved by Food and Drug Administration and several inhibitors are still undergoing clinical trials (8). However, clinical responses from these reagents are not as effective or durable as expected, drug resistance frequently occurs in tumors treated with RAF or MEK inhibitors (2, 8). PLX4032 seems to be an effective RAF inhibitor in malignant melanoma with an overall response rate of 81%, but responsive time from patients ranged from 2 to more than 18 months and this could limit the long-term efficacy of the drug (9). A recent report suggested that upregulation of N-RAS and other RTK signals such as platelet-derived growth factor receptor b (PDGFRb) are responsible for the acquired resistance to PLX4032 (10). Potential mechanisms of resistant to RAF or MEK inhibitors in RAS or BRAF mutant cancers can be attributed to either coactivation of parallel or downstream survival pathways prior to drug treatments or compensatory activation of alternative survival pathways upon drug administration (11–22). In either situation, combinatorial inhibition of multiple survival pathways is required to achieve potent antitumor effects. RAS signaling pathway is more complex than a linear RAS–RAF–MEK signaling cascade (1). Activated RAS protein Authors' Affiliations: Novartis Institute for BioMedical Research, Inc., Cambridge, Massachusetts Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Authors: Jian Chen, Department of Developmental and Molecular Pathways, Novartis Institute for Biomedical Research, Inc., 500 Technology Square, Cambridge, MA 02139. Phone: 617-871-7411; Fax: 617-871-5783; E-mail: [email protected] or L. Alex Gaither, Department of Developmental and Molecular Pathways, Novartis Institute for Biomedical Research, Inc., 500 Technology Square, Cambridge, MA 02139. Phone: 617-871-7209; Fax: 617-871-5783; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-10-3761 2011 American Association for Cancer Research. Cancer Research Cancer Res; 71(12) June 15, 2011 4280 on April 19, 2017. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst April 28, 2011; DOI: 10.1158/0008-5472.CAN-10-3761