Reduction of Fas/CD95 Promoter Methylation, Upregulation of Fas Protein, and Enhancement of Sensitivity to Apoptosis in Cutaneous T-Cell Lymphoma FREE

Cutaneous T-cell lymphoma (CTCL) is a hematologic malignant disease whose most common variant, mycosis fungoides, is characterized by flat cutaneous patches that become progressively infiltrated to form plaques and tumors.¹ Extracutaneous tissues can become involved in the later stages of disease. Sézary syndrome is the term given to the leukemic variant of CTCL when accompanied by erythroderma. Considerable evidence suggests that early CTCL has apoptotic defects that render it more of a lymphoaccumulative disorder than a lymphoproliferative disorder.^2- 10 This includes a low apoptotic rate, low proliferative rate, relatively indolent clinical course, and no enhanced survival following conventional chemotherapy.

There are several potential apoptotic pathways in lymphoid cells, including those involving extrinsic death receptors like Fas, tumor necrosis factor (TNF), and TNF-related apoptosis-inducing ligand receptor (TRAIL), as well as intrinsic ones including the mitochondrial pathway. The Fas pathway is well established as a major apoptotic mechanism for T cells. Previously, we reported that CTCL cells are frequently deficient in Fas protein expression relative to normal and inflammatory T cells (Wu et al²). In addition, we showed that CTCL cells exhibit a direct correlation among expression of Fas transcript, the level of cell surface Fas protein, and sensitivity to apoptosis mediated via the Fas pathway. Furthermore, Fas upregulation by transfection restored apoptotic sensitivity to CTCL cells that were otherwise resistant to Fas ligand. In related work, we showed that some CTCL lines have lost 1 or both Fas alleles (Wu et al¹¹). Loss of heterozygosity by tumor cells has been demonstrated by others in patients with leukemic CTCL.^7- 8 Nevertheless, we and others have shown that the Fas gene sequences detectable in samples from patients with CTCL rarely exhibit functionally significant somatic mutations in promoter or exon coding regions that might account for the low Fas protein levels observed in many cases.^9- 11

Given its key effects on regulating programmed cell death, Fas can be considered a tumor suppressor gene. A common characteristic among many malignant diseases is the epigenetic silencing of tumor suppressors through methylation of their promoter regions. This concept and our prior data set the stage for the current studies in which we explored the methylation status of the Fas promoter region and the consequences of its manipulation.

Human CTCL lines HH, SZ4, MyLa, Hut 78, SeAx, and human T-cell acute lymphoblastic leukemia cell lines Jurkat and JFL (Fas-low variant of Jurkat) have been described previously.² “SS1” represents circulating tumor cells from an African American woman with Sézary syndrome. She had 60 000 Sézary cells/mm³ that were CD4⁺, CD7⁻, and CD26⁻ with a CD4 to CD8 ratio of more than 100:1. Cells were treated with 5-azacytidine (aza) (Sigma-Aldrich, St Louis, Missouri) for 3 days or with methotrexate (Fisher Scientific, Fair Lawn, New Jersey), interferon alfa (Schering, Kenilworth, New Jersey), or methotrexate plus interferon alfa for 5 days. The reagents were dissolved in dimethyl sulfoxide (DMSO) as stock solution, and the final concentrations in the culture were 5μM of 5-aza, 10μM of methotrexate, and 100 U/mL of interferon alfa. Control cells were treated with DMSO. The reagents were added every 2 days when media were changed.

The eTable displays pyrosequencing primer sets. The reagents, primer sequences, and procedures for real-time polymerase chain reaction (RT-PCR) detection of Fas messenger RNA (mRNA), flow cytometric detection of cell surface Fas, and the aggregated Fas ligand–induced apoptosis assay were described in our previous publication.²

Statistical analysis was performed by t test. A 2-tailed P <.05 was considered statistically significant for the treatment sample relevant to control sample (as shown by an asterisk in the figures).

Analysis of the approximately 1.8-kb Fas promoter region immediately upstream of the first exon (National Center for Biotechnology Information Human Genome Database, location: 10q24.1) revealed 37 CpG islands. Using pyrosequencing, we determined the level of methylation present at each of these islands in 4 CTCL lines: MyLa, Hut-78, HH, and SZ4. A fifth CTCL line (SeAx) could not be studied because it lacks Fas genes. We reported previously that MyLa and Hut-78 express high levels of Fas mRNA and cell surface protein, whereas HH and SZ4 are Fas-low.² As shown in Figure 1 and Figure 2, there was an inverse relationship between Fas promoter methylation and expression of both Fas transcript and cell surface protein. Fas-low SZ4 and HH had high levels of methylation involving 6 CpG islands relative to Fas-high MyLa and Hut-78.

These findings suggested that promoter methylation regulates Fas expression in CTCL. To confirm this, we used 5-aza to demethylate the Fas promoter. As shown in Figure 1 and Figure 2, there was little effect on CTCL lines MyLa and Hut-78, which have very low baseline promoter methylation. In contrast, Sz4 and HH exhibited a major reduction in promoter methylation accompanied by a statistically significant increase in expression of both Fas mRNA and protein (P <.05).

To determine the functional consequences of these relationships, we used an aggregated Fas ligand assay to study their effect on the sensitivity of CTCL cells to Fas pathway apoptosis. As shown in Figure 3, Fas-high lines (MyLa and Hut-78) had high baseline sensitivity to Fas ligand, whereas Fas-low lines (SZ4 and HH) were resistant. However, demethylation with 5-aza upregulates Fas expression (Figure 2) to an extent that causes these cells to become sensitive to apoptosis mediated through the Fas pathway.

To explore the mechanisms by which epigenetic alterations in Fas promoter methylation affect Fas expression, we studied the impact of methylation on the interaction of the Fas promoter with transcription factors known to upregulate Fas expression. One example is the NFkB (nuclear factor kappa–light chain enhancer of activated B cells) family. Prior reports indicate that canonical NFkB mediators NFkB1/p105 (cleavage product p50) and RelA/p65 are the major family members active in CTCL.¹²

Analysis of the Fas promoter using the Transcription Element Search System (TESS) (free, downloadable software available at http://www.cbil.upenn.edu/cgi-bin/tess/tess) revealed 3 NFkB binding elements containing CpG islands that are situated between CpGs 13 and 24. As shown in Figure 4, chromatin immunoprecipitation assays demonstrated that the baseline level of NFkB1 and RelA transcription factor binding to this region of the Fas promoter was low in highly methylated SZ4 and HH compared with sparsely methylated MyLa and Hut-78. Following demethylation of SZ4 and HH with 5-aza, there was a significant increase in NFkB binding to their Fas promoters, whereas the largely unmethylated MyLa and Hut-78 generally showed only minimal changes in NFkB binding. When combined with our earlier data concerning the effects of methylation on expression of Fas mRNA and protein, these results indicate that DNA methylation plays a major role in the regulation of Fas promoter function by NFkB family members.

Because many patients with CTCL exhibit low Fas expression² and there is a correlation with high promoter methylation in CTCL lines (current data) and leukemic CTCL blood samples (current data),¹³ our findings suggested that demethylation might be a useful treatment strategy for enhancing the apoptotic sensitivity of tumor cells in patients with CTCL. The dihydrofolate reductase inhibitor, methotrexate, also blocks the synthesis of S-adenosyl-methionine (SAM), which is the major methyl group donor used by DNA methyltransferases; therefore, we hypothesized that by depleting SAM, methotrexate might indirectly block DNA methylation and lead to Fas promoter demethylation.

To test this theory, we used methotrexate to treat the highly methylated CTCL lines (SZ4 and HH) and circulating tumor cells from a patient with leukemic CTCL (SS1). Figure 5 shows that methotrexate treatment significantly reduced SAM levels in each CTCL cell type. As shown in Figure 6, there was a reduction in promoter methylation at all relevant CpG islands in each case. This was accompanied by an increase in Fas cell surface protein expression (Figure 7) and functional sensitivity to apoptosis mediated through the Fas pathway (Figure 8).

Another agent with proven efficacy against CTCL is interferon alfa. In fact, very high response rates have been obtained using the combination of methotrexate and interferon alfa in advanced CTCL.¹⁴ The potential for synergy between these 2 agents derives in part from their different mechanisms of Fas upregulation. In contrast to methotrexate, interferon alfa acts through the Janus kinase–signal transducer and activator of transcription (JAK-STAT) signal transduction pathway.¹⁵ As shown in Figure 7 and Figure 8, treatment of CTCL cells with interferon alfa also enhanced Fas protein expression and sensitivity to Fas pathway apoptosis. Furthermore, the combination of methotrexate and interferon alfa resulted in a greater effect than either agent alone. In fact, Figure 8 shows that combination therapy induced more apoptotic cells than the sum of individual therapies in each sample: HH (72% vs 69%), SZ4 (72% vs 58%), and SS1 (90% vs 65%).

We conducted the current study to explore the status and functional consequences of epigenetic modification of the Fas promoter in CTCL, specifically, the role of DNA methylation. We found that the Fas promoter has multiple CpG islands and that the methylation status of 6 of them correlated inversely with the expression of both Fas transcript and cell surface protein. The tightly linked parallelism between the levels of Fas mRNA and protein is not surprising given the fact that Fas is regulated largely at the transcriptional level in T cells and other cells.^16- 20 Using the demethylating agent 5-aza, we showed that Fas promoter methylation can be reversed, that reversal is accompanied by increased Fas expression, and that the magnitude of this change is sufficient to restore functional sensitivity of tumor cells to apoptosis triggered by Fas ligand. Furthermore, these alterations are associated with an increase in the binding of NFkB family transcription factors to a Fas promoter region containing 3 binding sites involving CpG islands. These data are consistent with the view that alterations in Fas promoter methylation affect Fas protein expression, at least in part through their impact on NFkB transcription factor binding.

Because many patients with CTCL exhibit low Fas expression and there is a correlation with high promoter methylation, our findings thus far suggest that demethylation might be a useful treatment strategy for enhancing the apoptotic sensitivity of tumor cells in patients with CTCL. The toxic effects of 5-aza render it undesirable for clinical use. Its nucleoside analog, 5-aza-2′-deoxycytidine (decitabine), is approved by the US Food and Drug Administration for the treatment of certain hematologic disorders²¹; however, its profile of adverse effects makes it less than ideal as a clinical demethylator. DNA methylation is mediated by DNA methyltransferases (DNMTs). This is a dynamic process involving initial methylation, demethylation, and remethylation to maintain promoter silencing.

The dihydrofolate reductase inhibitor, methotrexate, is widely known to block cell division in the S phase by preventing the conversion of dihydrofolate to tetrahydrofolate, which is required for the synthesis of thymidylate and purine nucleotides needed for DNA and RNA synthesis. A lesser known property of methotrexate is that it also blocks the synthesis of SAM and increases adenosine by inhibiting methionine synthetase and aminoimidocaboxyamido-ribonucleotide transformylase. SAM is the major methyl group donor used by DNMTs; therefore, we hypothesized that by depleting SAM, methotrexate might indirectly block DNMT function and lead to Fas promoter demethylation. Our current results support this view. Methotrexate treatment resulted in decreased SAM levels, decreased Fas promoter methylation, increased Fas expression, and enhanced sensitivity to Fas pathway apoptosis.

Methotrexate has a long history as a drug with proven efficacy against CTCL when used as monotherapy or in combination with other agents.²² In fact, one of the most impressive treatment responses on record for CTCL involved a series of 158 patients with refractory, advanced stage (stages IIB-IVB) CTCL treated with methotrexate and interferon alfa.¹⁴ Methotrexate was administered at a dose of 10 mg/m² biweekly together with 9 million U of interferon alfa 3 times per week. If a complete response was achieved, then patients were maintained on interferon alone. At 1 year, there was a 74% complete response. Overall survival was 68% with a 13-year median follow-up.

Given these encouraging results, we tested the combined effect of methotrexate and interferon alfa on CTCL lines and leukemic blood. We demonstrated augmentation of Fas expression and enhanced sensitivity to apoptosis that was greater than either agent alone. Interferon alfa increases Fas expression^6,23- 24 by a JAK-STAT mechanism¹⁵ that does not involve Fas promoter methylation. It augments signal transducer and activator of transcription 1 (STAT1) binding to an unmethylated gamma activation site within the Fas promoter.^25- 26 Therefore, the benefits of methotrexate, which involve CTCL S-phase cell cycle arrest and DNA demethylation, are distinct from those of interferon alfa, which involve increased CTCL Fas levels mediated by STAT1, modulation of the host immune response, and other effects. This provides a compelling rationale for combination therapy with these particular agents, a rationale supported by the impressive clinical responses reported previously¹⁴ and the in vitro responses demonstrated by our current study.

In the clinical situation, one might question the relevance of increased CTCL Fas expression if exogenous Fas ligand is not added to trigger apoptosis as it was in our in vitro studies. However, there are multiple potential apoptotic triggers in vivo. First, tumor-infiltrating lymphocytes can express Fas ligand and could serve as a trigger. We and others have established that CTCL prognosis and response to therapy correlate positively with preservation of the host immune system.^27- 28 Second, CTCL tumor cells themselves may express Fas ligand that could potentially trigger apoptosis in an autocrine fashion when their own Fas levels are enhanced. In fact, we showed previously that overexpression of Fas via transfection also results in modest upregulation of Fas ligand, suggesting that the 2 may be coordinately regulated (Wu et al²). It is important to note that this coordinate expression of endogenous Fas and Fas ligand was associated with modest apoptosis in vitro without the addition of exogenous Fas ligand. In the current study, this effect can also be seen in response to drug treatment (Figure 3 and Figure 8). Third, there is evidence in in vivo systems that Fas can be activated independent of Fas ligand.^29- 30 It is also worth noting that there is cross-talk between extrinsic death receptor pathways like Fas and the intrinsic mitochondrial pathway. This can result in the amplification of Fas apoptotic signals by recruitment of additional apoptotic mechanisms.

Recent studies have demonstrated loss of heterozygosity for the 10q24 chromosomal region that contains the Fas gene in some cases of leukemic CTCL.^7- 8 This raises the possibility that such CTCL cases might not be able to restore Fas levels significantly in response to promoter demethylation because half their complement of Fas gene has been deleted. However, using cytogenetic analysis and fluorescence in situ hybridization, we have shown previously that the HH cell line is hemizygous for Fas (Wu et al¹¹). Despite this, our current findings show that demethylation of HH resulted in a major increase in Fas expression and apoptotic sensitivity. Therefore, CTCL cases harboring similar partial Fas deletions should still benefit from promoter demethylation.

In summary, we have shown that methylation of the Fas promoter in CTCL correlates inversely with Fas expression and sensitivity to apoptosis, that DNA demethylation can upregulate Fas, at least partially via increased NFkB transcription factor binding, and that in addition to its well-recognized function as an antimetabolite, methotrexate functions indirectly as a demethylating agent whose affects on Fas expression are enhanced by interferon alfa, which operates by a separate, methylation-independent mechanism. These findings provide a novel approach to CTCL therapy and help explain the impressive clinical response of advanced-stage CTCL patients to combination therapy with methotrexate and interferon alfa.

Correspondence: Gary S. Wood, MD, Department of Dermatology, University of Wisconsin, One South Park, Seventh floor, Madison, WI 53715 (gwood@dermatology.wisc.edu).

Accepted for Publication: October 18, 2010.

Published Online: December 20, 2010. doi:10.1001/archdermatol.2010.376. This article was corrected online on January 5, 2011, for an error in the key for Figure 5.

Author Contributions: Both authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Wood. Acquisition of data: Wu. Analysis and interpretation of data: Wood. Drafting of the manuscript: Wood. Critical revision of the manuscript for important intellectual content: Wu and Wood. Statistical analysis: Wu and Wood. Obtained funding: Wood. Administrative, technical, and material support: Wu and Wood. Study supervision: Wood.

Financial Disclosure: None reported.

Funding/Support: This study was supported by funding from the Department of Veterans Affairs and the University of Wisconsin.