Abstract B14: Synergy of small-molecule inhibitors in cutaneous T-cell lymphoma cells: A discovery tool to define new therapeutic targets

Abstract

Abstract Cutaneous T-Cell Lymphomas (CTCL) represent a heterogeneous group of hematopoietic malignancies that account for 5-10% of Non-Hodgkin Lymphomas. The initial disease presentation is characterized by the infiltration of the skin by malignant clonal CD4+ lymphocytes that possess a mature memory helper T-cell phenotype. CTCL patients present in a spectrum of defined disease phenotypes, Mycosis Fungoides (MF) and Sézary Syndrome (SS) being the most common. Thus, it is not surprising that no underlying molecular basis for disease has been identified. Standard treatment protocols are designed to provide palliation, as no chemotherapeutic compound(s) has demonstrated increased long-term or disease-free survival. Currently, no curative therapy for CTCL exists. Our long-term goal is to gain a better understanding of the pathways governing CTCL cell survival and proliferation while identifying druggable targets for the development of new and more effective therapeutics. The principles of chemical biology, to discover and elucidate molecular pathways fundamental in cellular, developmental, and disease biology through synthetic organic chemistry, are readily applied to the field drug discovery. Using these methods, we have previously observed that inhibition of protein kinase C (PKC) β with the small molecule Enzastaurin (Enz), combined with inhibition of glycogen synthase kinase 3 (GSK3) with AR-A014418 (ARA), causes synergistic apoptosis in CTCL cell lines (Hut78 and H9). Critically, this cell death is dependent on the increase in active β-catenin protein expression. Treatment of cells with ICAT, a protein that prevents the interaction of β-catenin and TCF, demonstrated that β-catenin-mediated transcriptional activity participates in the combined compound—induced cell death. In order to assess potential targets of Enz and ARA treatment, we recently screened downstream β-catenin-mediated targets with a commercially available signaling pathway PCR array and validated them with qRT-PCR. Several targets of β-catenin were differentially regulated by the combined small-molecule treatment. We decreased cellular β-catenin by either siRNA knockdown or shRNA in stable cell lines and determined that β-catenin is required for compound-induced cytotoxicity of CTCL cell lines. In contrast, overexpression of β-catenin in CTCL cell lines demonstrated β-catenin alone is not sufficient to induce apoptosis. Thus, while we have identified β-catenin as a key regulator of CTCL viability, there are likely additional undescribed mechanisms of cell death stimulated by the combined inhibition of PKC and GSK3. From these data, we conclude that the combined inhibition of PKC and GSK signaling in CTCL causes cell death by impacting β-catenin specific targets as well as by stimulating cell death independent of β-catenin. To further elucidate these mechanisms we are assaying drug-treatment induced changes in global gene expression through a microarray approach. We are identifying transcripts that are significantly regulated after combined inhibition of PKC and GSK3 with Enz and ARA respectively. These data will provide the basis for downstream target identification for further drug development. Citation Format: Meghan Bliss-Moreau, Weimin Xiao, Preethi Gunaratne, Nancy L. Krett, Steven T. Rosen. Synergy of small-molecule inhibitors in cutaneous T-cell lymphoma cells: A discovery tool to define new therapeutic targets. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B14.