Downregulation of SAMHD1 Expression Correlates with Promoter DNA Methylation in Sézary Syndrome Patients
2013; Elsevier BV; Volume: 134; Issue: 2 Linguagem: Inglês
10.1038/jid.2013.311
ISSN1523-1747
AutoresSuresh de Silva, Fangfang Wang, Timothy S. Hake, Pierluigi Porcu, Henry K. Wong, Li Wu,
Tópico(s)Lymphoma Diagnosis and Treatment
Resumocutaneous T-cell lymphoma peripheral blood mononuclear cells sterile alpha motif and hemidesmosome domain containing protein 1 Sézary syndrome TO THE EDITOR Although only about 15% of non-Hodgkin lymphomas are T-cell lymphomas, the treatment of this subset of neoplasms remains a significant challenge in the field of hematological cancers owing to unclear mechanisms regulating malignant T-cell growth. Cutaneous T-cell lymphoma (CTCL) identifies a group of extranodal T-cell lymphomas characterized by the infiltration of malignant CD4+ T cells in the skin (Wong et al., 2011Wong H.K. Mishra A. Hake T. et al.Evolving insights in the pathogenesis and therapy of cutaneous T-cell lymphoma (mycosis fungoides and Sezary syndrome).Br J Haematol. 2011; 155: 150-166Crossref PubMed Scopus (119) Google Scholar). Sézary syndrome (SS) is an aggressive subtype of CTCL defined by diffuse pruritic rash, lymphadenopathy, and malignant T cells in the peripheral blood. The mechanisms underlying the proliferation of neoplastic CD4+ T cells in SS are not fully understood, but abnormal epigenetic regulation of gene expression including silencing of tumor suppressor genes likely has an important role (van Doorn et al., 2005van Doorn R. Zoutman W.H. Dijkman R. et al.Epigenetic profiling of cutaneous T-cell lymphoma: promoter hypermethylation of multiple tumor suppressor genes including BCL7a, PTPRG, and p73.J Clin Oncol. 2005; 23: 3886-3896Crossref PubMed Scopus (202) Google Scholar). Epigenetic mechanisms act as critical contributors in cancer initiation and progression through modulation of gene expression, and transcriptional repression of tumor suppressor genes via epigenetic mechanisms occurs in many cancers (Baylin and Jones, 2011Baylin S.B. Jones P.A. A decade of exploring the cancer epigenome - biological and translational implications.Nat Rev Cancer. 2011; 11: 726-734Crossref PubMed Scopus (2057) Google Scholar). Sterile alpha motif and hemidesmosome domain containing protein 1 (SAMHD1) is the first identified mammalian triphosphohydrolase that hydrolyzes deoxynucleoside triphosphates, implicating a role in nucleic-acid metabolism (Goldstone et al., 2011Goldstone D.C. Ennis-Adeniran V. Hedden J.J. et al.HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase.Nature. 2011; 480: 379-382Crossref PubMed Scopus (610) Google Scholar). SAMHD1 acts as an HIV-1 restriction factor in myeloid cells and in quiescent CD4+ T cells by diminishing the intracellular deoxynucleoside triphosphate pool to a level that is insufficient for viral replication (Baldauf et al., 2012Baldauf H.M. Pan X. Erikson E. et al.SAMHD1 restricts HIV-1 infection in resting CD4(+) T cells.Nat Med. 2012; 18: 1682-1689Crossref PubMed Scopus (452) Google Scholar; Lahouassa et al., 2012Lahouassa H. Daddacha W. Hofmann H. et al.SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates.Nat Immunol. 2012; 13: 223-228Crossref PubMed Scopus (620) Google Scholar). Non-dividing CD4+ T cells, monocytes, macrophages, and dendritic cells from healthy individuals express high levels of SAMHD1 protein and have significantly lower levels of intracellular deoxynucleoside triphosphates compared to activated CD4+ T cells, while several leukemia/lymphoma CD4+ T-cell lines lack SAMHD1 protein expression and have increased deoxynucleoside triphosphate levels necessary for cell division (Hrecka et al., 2011Hrecka K. Hao C. Gierszewska M. et al.Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein.Nature. 2011; 474: 658-661Crossref PubMed Scopus (913) Google Scholar; Laguette et al., 2011Laguette N. Sobhian B. Casartelli N. et al.SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.Nature. 2011; 474: 654-657Crossref PubMed Scopus (1123) Google Scholar; Baldauf et al., 2012Baldauf H.M. Pan X. Erikson E. et al.SAMHD1 restricts HIV-1 infection in resting CD4(+) T cells.Nat Med. 2012; 18: 1682-1689Crossref PubMed Scopus (452) Google Scholar). We found that promoter methylation represses SAMHD1 expression in human leukemia/lymphoma CD4+ T-cell lines, while the SAMHD1 promoter is unmethylated in primary CD4+ T-lymphocytes from healthy donors that express high levels of SAMHD1 protein (de Silva et al., 2013de Silva S. Hoy H. Hake T.S. et al.Promoter methylation regulates SAMHD1 gene expression in human CD4+ T cells.J Biol Chem. 2013; 288: 9284-9292Crossref PubMed Scopus (47) Google Scholar). The role of SAMHD1 in cancer remains unknown. To explore the potential role of SAMHD1 in CTCL, we measured SAMHD1 mRNA levels in peripheral blood mononuclear cells (PBMCs) taken from 14 healthy donors and 9 CTCL patients using a real-time quantitative-PCR assay (de Silva et al., 2013de Silva S. Hoy H. Hake T.S. et al.Promoter methylation regulates SAMHD1 gene expression in human CD4+ T cells.J Biol Chem. 2013; 288: 9284-9292Crossref PubMed Scopus (47) Google Scholar). The patients comprised eight with SS and one with advanced-stage mycosis fungoides (Table 1), which are two related subtypes of CTCL originating from CD4+ skin-homing T cells (Wong et al., 2011Wong H.K. Mishra A. Hake T. et al.Evolving insights in the pathogenesis and therapy of cutaneous T-cell lymphoma (mycosis fungoides and Sezary syndrome).Br J Haematol. 2011; 155: 150-166Crossref PubMed Scopus (119) Google Scholar). Interestingly, quantitative-PCR results revealed that PBMCs from the 8 SS patients and an advanced-stage mycosis fungoides patient expressed on average threefold lower SAMHD1 mRNA levels (P=0.0013, two-sample t-test) compared with those from 14 healthy donors (Figure 1a and Table 1). These results indicate that SAMHD1 expression is significantly downregulated in PBMCs from SS patients relative to healthy individuals.Table 1Clinical information of CTCL patients and relative levels of SAMHD1 mRNA and promoter methylation in PBMCs from the patientsPatient no.CTCL subtype1No patient in this group was treated with methotrexate. These patients have been on bexarotene and IFN at some time during their treatments.Disease stageCD4+ cells (%)2CD4+ cells (%) indicate percentage of CD4-positive cells in patient PBMCs by flow cytometry analysis.Absolute CD4 countCD4:CD8 ratioSézary cell count (%)Relative SAMHD1 mRNA levels3Relative levels of SAMHD1 mRNA in PBMCs from 9 CTCL patients (mean±SD is 0.372±0.285) compared with the average level of 14 healthy donors (1.113±0.553).Relative SAMHD1 promoter methylation levels4Relative levels of SAMHD1 promoter methylation in PBMCs from 9 CTCL patients (mean±SD is 51.27±43.32) compared with the average level of 8 healthy donors (1.000±1.703).#1SSIVA98N/AN/A290.07930.74#2SSIVA93N/AN/A450.99539.31#3SSIVA280N/AN/A310.2170.000#4SSIVA65N/AN/AN/A0.38719.43#5SSIVA278N/AN/A610.469135.79#6MFIIB301631.8N/A0.04892.27#7SSIVA85178528N/A0.48319.34#8SSIVA288334682N/A0.22943.29#9SSIVA82146613N/A0.44181.32Abbreviations: CTCL, cutaneous T-cell lymphoma; MF, mycosis fungoides; N/A, not available; SAMHD1, sterile alpha motif and hemidesmosome domain containing protein 1; SS, Sézary syndrome.1 No patient in this group was treated with methotrexate. These patients have been on bexarotene and IFN at some time during their treatments.2 CD4+ cells (%) indicate percentage of CD4-positive cells in patient PBMCs by flow cytometry analysis.3 Relative levels of SAMHD1 mRNA in PBMCs from 9 CTCL patients (mean±SD is 0.372±0.285) compared with the average level of 14 healthy donors (1.113±0.553).4 Relative levels of SAMHD1 promoter methylation in PBMCs from 9 CTCL patients (mean±SD is 51.27±43.32) compared with the average level of 8 healthy donors (1.000±1.703). Open table in a new tab Abbreviations: CTCL, cutaneous T-cell lymphoma; MF, mycosis fungoides; N/A, not available; SAMHD1, sterile alpha motif and hemidesmosome domain containing protein 1; SS, Sézary syndrome. Next, we examined whether the downregulation of SAMHD1 mRNA levels would translate to reduced SAMHD1 protein levels in PBMCs of CTCL patients. To this end, we measured expression levels of surface CD4 and intracellular SAMHD1 proteins in PBMCs from three CTCL patients with high circulating neoplastic T cells (patient 3, 4, and 5 in Figure 1b and Table 1) and four healthy donors using immunostaining and flow cytometry (Figure 1b) (Descours et al., 2012Descours B. Cribier A. Chable-Bessia C. et al.SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4+ T-cells.Retrovirology. 2012; 9: 87Crossref PubMed Scopus (263) Google Scholar). The limited sample size was because of thePBMCs being available only from three CTCL patients. The percentage of total SAMHD1-positive cells in PBMCs from CTCL patients (37±9%) was significantly lower (P=0.0024) than that from healthy donors (71±6%) (Figure 1c). Furthermore, the percentage of SAMHD1-expressing cells in CD4-negative PBMCs from CTCL patients (3±2%) was significantly lower than that from healthy donors (19±8%) (P=0.023). Analysis of SAMHD1 and CD4 double-positive cells in PBMCs from CTCL patients (34±9%) and healthy donors (52±8%) showed a statistical difference (P=0.042), suggesting significant downregulation of SAMHD1 protein expression in the neoplastic CD4+ cell subset from CTCL patients. Given that PBMCs from CTCL patients comprise mainly CD4+ cells, we also compared the percentage of SAMHD1-expressing cells in the CD4+ gated population between healthy donor samples (81±3%) and CTCL patient samples (46±6%) and found a significant reduction (P=0.0024) in SAMHD1-expressing cells (Supplementary Figure S1 online). Download .pdf (1.23 MB) Help with pdf files Supplementary Information We hypothesized that the SAMHD1 promoter in PBMCs from the CTCL patients is methylated and thereby inhibits SAMHD1 expression. To compare the methylation status of the SAMHD1 promoter in PBMCs from the CTCL patients selected with that from healthy donors, genomic DNA of PBMCs was treated with the methylation-sensitive HpaII endonuclease, or left untreated, and then subjected to PCR amplification using SAMHD1 promoter-specific primers as described (de Silva et al., 2013de Silva S. Hoy H. Hake T.S. et al.Promoter methylation regulates SAMHD1 gene expression in human CD4+ T cells.J Biol Chem. 2013; 288: 9284-9292Crossref PubMed Scopus (47) Google Scholar). The SAMHD1 promoter contains five HpaII sites, and methylation of these sites prevents digestion by HpaII. The intact undigested sequence can serve as a template for PCR amplification to yield a 1.2-kb product. As an input control of genomic DNA, a 0.25-kb region within the glyceraldehyde-3-phosphate dehydrogenase gene lacking HpaII sites was PCR amplified (Supplementary Figure S2 online). The SAMHD1 promoter in PBMCs from eight of nine CTCL patients tested was methylated (Supplementary Figure S2a online, 1.2-kb bands). Strikingly, PBMCs from eight healthy donors showed that the SAMHD1 promoter was unmethylated (Supplementary Figure S2b online). The purity of the genomic DNA for complete enzyme digestion as well as the intact nature of the HpaII sites in the SAMHD1 promoter sequence in healthy donor and CTCL patient genomic DNA was confirmed by restriction digestion with MspI (an isoschizomer of HpaII), which cleaves the HpaII site irrespective of its methylation status (Supplementary Figure S3 online). Densitometry analysis of the PCR products in Supplementary Figure S2 online was used to quantify the relative level of methylation of the SAMHD1 promoter in PBMCs from CTCL patients and healthy donors. Our analysis revealed 51-fold higher average levels of methylation of the SAMHD1 promoter in PBMCs from nine CTCL patients (P=0.0052) relative to eight healthy donors (Figure 1d and Table 1). These results suggest a positive correlation between downregulation of SAMHD1 expression and methylation of the SAMHD1 promoter in PBMCs from CTCL patients. However, we observed a lack of correlation between reduced SAMHD1 expression and promoter methylation in patient 3, which suggests that, besides promoter methylation, other transcriptional and epigenetic regulatory mechanisms, such as microRNA and histone modifications, may also contribute to the regulation of SAMHD1 expression in CTCL patients. SAMHD1 somatic mutations have been identified in patients with lung adenocarcinoma, medulloblastoma, glioblastoma, breast, pancreatic, and colorectal cancers, albeit at a very low frequency (Sjoblom et al., 2006Sjoblom T. Jones S. 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Hammerman P.S. et al.Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing.Cell. 2012; 150: 1107-1120Abstract Full Text Full Text PDF PubMed Scopus (1368) Google Scholar). Transcriptional repression of tumor suppressor genes through DNA methylation and histone modifications is a common mechanism of gene silencing in numerous types of cancer. Inhibition of epigenetic suppression in vitro using specific inhibitors to block DNA methyltransferase and/or histone deacetylase can reactivate the expression of tumor suppressor genes silenced in cancer. Downregulation of dNTP catabolic enzymes such as SAMHD1 may lead to imbalances in the intracellular dNTP pool, which can induce mutations and genomic instability as key features of CTCL. This study was approved by the Institutional Review Board of the Ohio State University. The study was conducted according to the Declaration of Helsinki guidelines and all participants gave their written informed consent. PBMCs from 14 healthy donors and 9 patients with CTCL were obtained from the clinics at the Ohio State University Medical Center with an approved Institutional Review Board protocol. We thank Dr Olivier Schwartz (Institut Pasteur) for the kind gift of SAMHD1 antibody (clone I19-18) and Heather Hoy for her excellent technical assistance. This work was supported in part by grants AI098524 and AI102822 to LW, and CA164911 to HKW and PP from the National Institutes of Health. LW is supported in part by the Public Health Preparedness for Infectious Diseases Program of The Ohio State University. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
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