160 Mechanisms governing promoter methylation and apoptosis in cutaneous T-cell lymphoma: implications for therapy with methotrexate and JAK-inhibitors

Abstract

Cutaneous T-cell lymphoma (CTCL) is characterized by resistance to apoptosis involving dysregulation of the FAS ligand (FASL) - FAS death receptor pathway. CTCL cells also exhibit constitutively activated STAT proteins, which are known to upregulate and bind DNA methyltransferases (DNMTs). We hypothesized that, like many other tumor suppressor genes, FAS and FASL expression is at least partially regulated by promoter methylation. Earlier, we showed that methotrexate (MTX), a known folate antagonist, acts as a DNA methylation inhibitor via depleting the methyl donor, S-adenosylmethionine (SAM), in CTCL cells. Pyrosequencing analysis of FAS and FASL promoters showed that MTX inhibited methylation of certain CpG sites in these genes, which resulted in increased FAS/FASL expression and subsequent apoptosis via activated caspase 8. We found that shRNA mediated genetic knockdown of DNMT -1 and -3A also resulted in upregulation of FASL mRNA and protein in CTCL cell lines. However, the reduction in FASL promoter methylation induced by MTX was generally superior to that induced by shRNA knockdown and was not improved by a combination of MTX plus knockdown. Because STAT proteins help regulate DNMTs, we also evaluated the effect of multiple JAK inhibitors (fedratinib, tofacitinib, and ruxolitinib) in CTCL cells and Sezary patient’s blood. Among these, fedratinib treatment was most effective in decreasing viability, spheroid formation, and inducing apoptosis. These responses were accompanied by decreased phosphorylation of STAT3 at tyrosine-705. Taken together, our data demonstrate that enhanced apoptosis of CTCL can be induced by MTX, DNMT knockdown and JAK inhibitors. This is consistent with a mechanistic cascade in which activated STATs enhance DNMT activities that in turn suppress the expression of apoptotic factors and promote CTCL survival. Our findings suggest a therapeutic role for MTX and JAK inhibitors in the management of CTCL.