Synergy Of Small-Molecule Inhibitors In Cutaneous T-Cell Lymphoma Cells: A Discovery Tool To Define New Therapeutic Targets In T-Cell Receptor (TCR) Signaling Pathways

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) and patient cells. Further investigations have shown combined treatment with Enz and ARA increased β-catenin protein levels and that β-catenin downstream transcription activation negatively impacts CTCL viability. However, β-catenin alone is not sufficient to induce CTCL apoptosis. These data indicate there are additional mechanisms of cell death stimulated by the combined inhibition of PKC and GSK3.

To identify the β-catenin independent targets of combined PKC and GSK3 inhibition, we have assayed drug-treatment induced changes in global gene expression through a microarray approach. We have identified transcripts that are significantly regulated after combined inhibition of PKC and GSK3 with Enz, ARA, and a combination of both small molecules. We are focusing our efforts on those targets and signaling pathways that are minimally regulated by each drug alone (or no change at all), but are dramatically impacted by the combination treatment to identify mechanisms of synergy. Using Gene Set Enrichment Analysis (GSEA), we noted enrichment in two pathways that are key to T-cell viability, namely the T-cell receptor (TCR) pathway and interferon response pathway. To elucidate candidate genes that may have therapeutic importance and to further explore the clinical relevance of our data set, in a pilot study, we interrogated the gene expression profile of a set of CTCL patients normalized to normal CD4+ T-cells. We then selected those genes whose expression is negatively correlated between CTCL patients and drug treated CTCL cell lines. From this pilot study, we have identified members of the TCR pathway that are over-expressed in CTCL patients and strikingly down-regulated by combined inhibition of PKC and GSK3. Taken together, these observations prompted us to further explore genes of the TCR signaling cascade as potential therapeutic targets. These data will provide the basis for downstream target identification for further drug development.