312 Epigenetically-enhanced PDT (ePDT) induces significantly higher levels of multiple extrinsic pathway apoptotic factors than conventional PDT, resulting in greater extrinsic and overall apoptosis of CTCL

Abstract

Conventional photodynamic therapy with aminolevulinate (ALA-PDT) selectively induces apoptosis in diseased cells primarily through the caspase 9-mediated intrinsic (mitochondrial) pathway and is highly effective for treating actinic keratoses. However, it is less effective for cutaneous T-cell lymphoma (CTCL). This failure may be related to the fact that although conventional PDT increases FASL expression via the JNK/c-Jun pathway, this same pathway can inhibit FAS expression. Our prior work showed that the apoptotic resistance of CTCL correlates with low expression of death receptors like FAS, and that methotrexate can upregulate FAS by inhibiting methylation of its promoter, acting as an epigenetic derepressor that restores the susceptibility of FAS-low CTCL to caspase 8-mediated extrinsic pathway apoptosis. We hypothesized that the limited efficacy of conventional PDT in CTCL is due to dysfunctional extrinsic apoptosis, and showed that epigenetic regulators like MTX will significantly increase the efficacy of PDT by sensitizing the targeted cells to apoptosis via up-regulation of death receptors such as FAS, a concept we refer to as epigenetically-enhanced PDT (ePDT) (Salva KA, Wood GS. Photochem Photobiol 91: 1444-51, 2015). Here, in CTCL lines, leukemic CTCL cells and normal blood T cells, we analyzed multiple components of the FAS, TRAIL and TNF families in vitro and ex vivo with multispectral imaging of immunostained cells, a quantitative method 5x more sensitive than standard immunocytology. Relative to conventional PDT, ePDT induced significantly greater FAS, FASL, TRAIL-R1 & -R2, and TNFa. This correlated with significantly greater induction of markers of extrinsic pathway apoptosis (cleaved caspase 8) and overall apoptosis (cleaved caspase 3) in all CTCL samples. There was no appreciable effect on normal T cells. These findings provide a rationale for clinical trials of ePDT for local therapy of CTCL.