Gary S. Wood, MD-UWHealth
BA Northwestern University
MD University of Illinois
Cutaneous T-cell lymphoma (CTCL) consists of mycosis fungoides (MF) and its leukemic variant, Sezary syndrome (SS). It accounts for approximately 2/3 of all primary cutaneous lymphomas. Less than 10% of cases are cured by available therapy. Advanced CTCL is usually fatal within a few years. A major impediment to the development of more effective treatment is our lack of understanding of the pathogenesis of CTCL and associated disorders. Our ongoing research supports the hypothesis that the FAS (CD95) apoptotic pathway is defective in many cases. We showed that the death receptor-ligand partners, FAS and FASL, are frequently expressed only weakly in CTCL cell lines, leukemic blood and lesional skin. We have observed an inverse correlation between FAS expression and promoter methylation in CTCL lines and leukemic cells. Using laser capture microscopy and pyrosequencing, we have developed a method to assess promoter methylation in cells microdissected from lesional skin. Initial results indicate the same inverse relationship in-situ. We found methylation correlates directly with DNA methyltransferase (DNMT)-1 and STAT3 levels. Our data involving EMSA, supershift, ChIP and luciferase reporters also show that interaction of transcription factors with the FAS promoter can be decreased by methylation (e.g. NFKB p50/p65) and by germline SNPs (e.g. STAT1) resulting in less FAS expression. In addition, our data suggest that transcription factors such as STAT3 and c-Jun may also regulate FAS expression in CTCL. Currently, we are delving deeper into FAS regulation and extending our focus to other FAS pathway members that we find to be under-expressed (e.g. FASL) or over-expressed (e.g. cFLIP). Our discovery that MTX can reverse FAS promoter methylation and restore FAS expression, combined with our observation that the FAS response to IFNa is attenuated by the -671 GG promoter SNP, form the basis for determining the in-vivo effects of these therapies in the specific context of these novel findings. To perform these highly translational studies, we take advantage of our Cutaneous Lymphoma Clinic where we treat CTCL patients with these agents, either as monotherapy or in combination. Turning to mechanism of action, our studies suggest that MTX functions as a demethylator by depleting cellular S-adenosyl methionine, the principal methyl donor for DNMTs. This is an entirely novel approach to DNMT inhibition, suggesting broad potential for MTX as a general demethylator with relevance far beyond CTCL. We plan to further clarify MTX’s mechanism of action and determine if related compounds might be superior as demethylators.
In related studies, we showed that the M phase cell cycle regulator, polo-like kinase 1 (Plk1), is selectively up-regulated in advanced stage CTCL and that its inhibition results in apoptosis and cell cycle arrest. In addition, we showed that the histone deacetylase, Sirt-1, is also over-expressed in CTCL. These findings provide insight into CTCL pathogenesis and suggest novel therapeutic targets.
Current therapies for unresectable, metastatic melanoma have very limited impact on patient outcome. Interferon alpha 2b is the FDA-approved adjuvant therapy for patients with resected stage III melanoma. While this adjuvant therapy has a confirmed impact on relapse-free survival, its impact on overall survival is controversial. Interferon alpha 2b has demonstrated some single-agent activity in metastatic melanoma. Epigallocatechin-3-gallate (EGCG) is a non-toxic natural product that is the major antioxidant in green tea. We showed that it has anti-melanoma effects in vitro and in vivo in mouse models, and that this anti-melanoma activity can be enhanced when EGCG is combined with interferon. We hypothesize that EGCG in combination with interferon alpha-2b will be well tolerated and demonstrate significant anti-melanoma effects in patients with advanced, unresectable melanoma. We also hypothesize that the subset of patients most likely to respond to interferon can be identified by analysis of a germline SNP in the FAS gene promoter. In collaboration with Dr. Mark Albertini (Medical Oncology), we plan to test these hypotheses in a phase I study of EGCG and interferon alpha-2b in patients with advanced, unresectable melanoma.