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CORRESPONDENCE I read with great interest the recent article by Daskalakis et
al1 on the potential use of the DNA methyltransferase
inhibitor 5-Aza-2'-deoxycytidine (5adC) in patients with
myelodysplastic syndromes (MDSs). The authors demonstrate that
methylation-mediated silencing of the tumor suppressor gene
p15/INK4B can be partially overcome using 5adC. Bisulphite
sequencing of the p15 promoter shows significant CpG
demethylation after several courses of 5adC. Of the 23 patients
studied, 15 were scored with p15 methylation status above
15%. Twelve patients showed p15 hypermethylation before
5adC treatment, with reversal in 9 patients. But questions underlying
the precise consequence of methylation changes outside of the
p15 allele warrants attention. In an attempt to generate discussion over the topic, I am compelled to raise the issue of methylation-dependent silencing of the multidrug resistance gene 1 (MDR1) in myeloproliferative disorders.2 Recent
studies reveal MDR1 gene expression is controlled by
epigenetic silencing mechanisms such as histone
deacetylation3 and CpG methylation4,5 in
various hematologic malignancies. Methylation of the MDR1
promoter is inversely correlated with gene expression.6
Given that MDR1 expression is a negative prognostic factor
for response and survival in acute myeloid leukemia
(AML),7 this would suggest that epigenetic modification by
a DNA methyltransferase inhibitor may inadvertently aggravate the
MDR1 phenotype. The article presented by Daskalakis et al1 convincingly
demonstrates that 5adC has profound epigenetic effects on
p15 gene silencing. It is believed that incorporation of the
cytidine analog is substituted into newly synthesized DNA strands, thus
blocking methylation by covalently trapping the DNA methyltransferase
enzyme while attempting methyl-group transfer.8,9
Experimental evidence at present suggests that 5adC exposure invariably
results in a more "global" demethylation profile instead of
gene-specific demethylation, and microarray expression analysis reveals
a number of different genes are activated.10 The
methyl-CpG signal on MDR1 chromatin targets the
methylation-specific transcriptional repressor MeCP2 for gene
silencing.11 Methyl-CpG binding domain (MBD)
proteins physically associate with histone deacetylase to remodel
chromatin, and methylated DNA provides a dominant mechanism of
repression. Consistent with this epigenetic model, azacytidine-induced
demethylation causes the release of the transcriptional repressor and
primes the promoter for gene activation. The results to these
experiments suggest silent MDR1 chromatin can be efficiently
remodeled through the use of DNA methylation inhibitors. Given the evidence, it is likely that 5adC may activate other silent
genes. The authors indeed appreciate that MBD proteins recognize methylated CpG sites to repress gene activity, and this may
represent a pathway of transcriptional loss for p15. In a similar fashion, the methyl-CpG binding protein MBD2 cooperates with
histone deacetylase to silence p14 and p16. The
hypermethylated alleles can be reactivated by 5adC, with corresponding
release of MBD2 from the promoter sequence.12 Therefore, it is worthwhile remembering that the benefit of activating
proliferation-associated genes by DNA demethylation may also run the
risk of activating a drug resistance pathway believed to be a major
cause of treatment failure in patients with myeloproliferative disorders.
Assam El-Osta
References
1.
Daskalakis M, Nguyen TT, Nguyen C, et al.
Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2'-deoxycytidine (decitabine) treatment.
Blood.
2002;100:2957-2964
2.
Nakayama M, Wada M, Harada T, et al.
Hypomethylation status of CpG sites at the promoter region and overexpression of the human MDR1 gene in acute myeloid leukemias.
Blood.
1998;92:4296-4307
3.
Jin S, Scotto KW.
Transcriptional regulation of the MDR1 gene by histone acetyltransferase and deacetylase is mediated by NF-Y.
Mol Cell Biol.
1998;18:4377-4384
4.
Kantharidis P, El-Osta A, deSilva M.
Altered methylation of the human MDR1 promoter is associated with acquired multidrug resistance.
Clin Cancer Res.
1997;3:2025-2032[Abstract].
5.
Kusaba H, Nakayama M, Harada T, et al.
Association of 5' CpG demethylation and altered chromatin structure in the promoter region with transcriptional activation of the multidrug resistance 1 gene in human cancer cells.
Eur J Biochem.
1999;262:924-932[Medline]
[Order article via Infotrieve].
6.
Garcia-Manero G, Bueso-Ramos C, Daniel J, Williamson J, Kantarjian HM, Issa JP.
DNA methylation patterns at relapse in adult acute lymphocytic leukemia.
Clin Cancer Res.
2002;8:1897-1903
7.
Leith CP, Kopecky KJ, Chen IM, et al.
Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P-glycoprotein, MRP1, and LRP in acute myeloid leukemia: a Southwest Oncology Group Study.
Blood.
1999;94:1086-1099
8.
Juttermann R, Li E, Jaenisch R.
Toxicity of 5-aza-2'-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation.
Proc Natl Acad Sci U S A.
1994;91:11797-11801
9.
Ferguson AT, Vertino PM, Spitzner JR, Baylin SB, Muller MT, Davidson NE.
Role of estrogen receptor gene demethylation and DNA methyltransferase.DNA adduct formation in 5-aza-2'-deoxycytidine-induced cytotoxicity in human breast cancer cells.
J Biol Chem.
1997;272:32260-32266
10.
Liang G, Gonzales FA, Jones PA, Orntoft TF, Thykjaer T.
Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2'-deoxycytidine.
Cancer Res.
2002;62:961-966
11.
El-Osta A, Kantharidis P, Zalcberg JR, Wolffe AP.
Precipitous release of methyl-CpG binding protein 2 and histone deacetylase 1 from the methylated human multidrug resistance gene (MDR1) on activation.
Mol Cell Biol.
2002;22:1844-1857
12.
Magdinier F, Billard LM, Wittmann G, et al.
Regional methylation of the 5' end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.
Faseb J.
2000;14:1585-1594
We have read with interest the thoughtful comments of Dr El-Osta
regarding our observation of in vivo demethylating activity of
5-aza-2'-deoxycytidine (decitabine; DAC) upon the p15/INK4B gene in myelodysplastic syndrome (MDS) patients.1
Since many genes can be inactivated by DNA methylation,2
reactivation of additional genes bearing a pathologic hypermethylation
pattern in these patients is not unlikely and warrants sequential
analyses of other putative target genes before and during treatment
with demethylating agents. Additionally, many genes up-regulated by azanucleosides do not have CpG islands in their promoters or have not
been shown to be methylated before treatment.3,4 Based on
the concern that treatment with demethylating agents may also reactivate genes that could potentially interfere with anticancer treatment, studies have addressed the possibility that the multidrug resistance pump P-glycoprotein (Pgp), the product of the MDR1 gene, may be up-regulated via demethylation. Indeed, this gene was
shown to be up-regulated by both 5-azacytidine and
5-Aza-2'-deoxycytidine (as described by Dr El-Osta) in several cell
line models. Other studies have challenged this view: fine-mapping of
its CpG-rich promoter has suggested that the methylation status of this
domain does not act as a switch to regulate MDR1
expression,5 and a study using an anthracycline-resistant
subclone of the myeloid leukemia cell line K562 in fact demonstrated
down-regulation of MDR1 by a demethylating agent (possibly
mediated by demethylation of a silencer element in the MDR1
promoter.6 The concern that in vivo responsiveness of malignant cells to
chemotherapy is mitigated by pretreatment with a demethylating agent
can first be addressed by the fact that Pgp confers in vitro resistance
to some but clearly not all cytostatic drugs in common use. Cytosine
arabinoside, which is considered to be the most active drug against
AML, is no substrate of P-glycoprotein. In fact, pretreatment with a
demethylating agent may even sensitize previously drug-resistant
xenograft tumors to both MDR1-dependent and -independent
cytostatic drugs.7. Second, while it is well established
that MDR1 expression is a negative prognostic factor in
AML,8 it is also associated with an immature, stem
cell-like phenotype (CD34+ blast cells) in AML and MDS,
which is also a poor prognostic factor in AML.9
Correspondingly, MDR1 as well as other drug transporters,
such as the breast cancer resistance protein (BCRP, also known as
ABCG2) are highly expressed in normal hematopoietic stem cells, and
their expression is down-regulated during myeloid differentiation.10,11 Taken together, in some cases
MDR1 expression may be an indicator of an early phenotype,
rather than the primary cause for failure to antileukemic induction chemotherapy. Among 101 MDS patients treated with DAC at 2 centers, 15 patients
(median age, 63 years; range, 50-77 years) received standard induction chemotherapy at time of progression to AML. Eight of 15 had
shown a previous response to DAC during a median of 7 treatment courses
(range, 3-9 courses, with or without a trial of retreatment at
relapse), whereas 7 of 15 had not responded to this drug (median, 1 course; range, 1-2 courses). Nine (60%) of 15 patients receiving induction treatment of secondary AML achieved a response, including 6 (40%) of 15 achieving complete remissions (Table
1).
This included both DAC responders and nonresponders. Thus, pretreatment
with DAC (independent of its effectiveness) did not result in a
leukemia phenotype with complete in vivo resistance to
AraC/anthracycline-based induction treatment. In fact, the response
rates are comparable to other published series of patients older than
55 to 60 years with AML from MDS receiving AraC-based induction
treatment.9 But since we did not experimentally address
the question of P glycoprotein (Pgp) up-regulation during
decitabine treatment in MDS patients, we cannot formally exclude that
in the cells belonging to the MDS clone, MDR1 was
up-regulated during an early treatment phase in the responding
patients. Whether or not a demethylation-induced up-regulation of
MDR1 may translate into clinical treatment failure is
currently unknown and requires further study. In addition, one should
keep in mind that in several tumor entities, including breast and
ovarian cancer, overexpression of the MDR1 gene is not
necessarily correlated with clinical drug resistance.13,14 Finally, recent studies suggest that the AML subgroup of elderly patients with frequent myelodysplastic features, a higher rate of
poor-risk cytogenetics, and p15 hypermethylation may already have
increased MDR1 expression prior to any
treatment.15-17 It will be of interest to see whether in
comparative studies of normal hematopoietic progenitor cells versus
leukemic myeloid cells treated with demethylating agents, preferential
up-regulation of MDR1 might occur in the normal cells. This
could be hypothesized based on the observation that untransformed
murine fibroblasts were more susceptible to the differentiating
activity of a demethylating agent than their transformed
counterpart.18
Michael Lübbert, Pierre W. Wijermans, Thomas Licht, and Peter A. Jones Supported by Wilhelm Sander-Stiftung (grant 99.032.1) References
1.
Daskalakis M, Nguyen TT, Nguyen C, et al.
Demethylation of a hypermethylated p15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2'-deoxycytidine treatment.
Blood.
2002;100:2957-2964
2.
Melki JR, Vincent PC, Clark SJ.
Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia.
Cancer Res.
1999;59:3730-3740
3.
Karpf AR, Peterson PW, Rawlins JT, et al.
Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells.
Proc Nat Acad Sci U S A.
1999;96:14007-14012
4.
Liang G, Gonzales FA, Jones PA, Orntoft TF, Thykjaer T.
Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2'-deoxycytidine.
Cancer Res.
2002;62:961-966 5. Fryxell KB, McGee SB, Simoneaux DK, Willman CL, Cornwell MM. Methylation analysis of the human multidrug resistance 1 gene in normal and leukemic hematopoietic cells. Leukemia. 1999;13:910-917[CrossRef][Medline] [Order article via Infotrieve]. 6. Ando T, Nishimura M, Oka Y. Decitabine (5-Aza-2'-deoxycytidine) decreased DNA methylation and expression of MDR-1 gene in K562/ADM cells. Leukemia. 2000;14:1915-1920[CrossRef][Medline] [Order article via Infotrieve].
7.
Plumb JA, Strathdee G, Sludden J, Kaye SB, Brown R.
Reversal of drug resistance in human tumor xenografts by 2'-deoxy-5-azacytidine-induced demethylation of the hMLH1 gene promoter.
Cancer Res.
2000;60:6039-6044
8.
Pirker R, Wallner J, Geissler K, et al.
MDR1 gene expression and treatment outcome in acute myeloid leukemia.
J Nat Cancer Inst.
1991;83:708-712
9.
Hiddeman W, Kern W, Schoch C, et al.
Management of acute myeloid leukemia in elderly patients.
J Clin Oncol.
1999;17:3569-3576 10. Chaudhary PM, Roninson IB. Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell. 1991;66:85-94[CrossRef][Medline] [Order article via Infotrieve]. 11. Zhou S, Schuetz JD, Bunting KD, et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med. 2001;7:1028-1034[CrossRef][Medline] [Order article via Infotrieve]. 12. Willemze R, Suciu S, Archimbaud E, et al. A randomized phase II study on the effects of 5-Aza-2'-deoxycytidine combined with either amsacrine or idarubicin in patients with relapsed acute leukemia: an EORTC Leukemia Cooperative Group phase II study (06893). Leukemia. 1997;11(suppl 1):24-27[Medline] [Order article via Infotrieve]. 13. Wang CS, LaRue H, Fortin A, Gariepy G, Tetu B. mdr1 mRNA expression by RT-PCR in patients with primary breast cancer submitted to neoadjuvant therapy. Breast Cancer Res Treat. 1997;45:63-67[CrossRef][Medline] [Order article via Infotrieve].
14.
Izquierdo MA, van der Zee AG, Vermorken JB, et al.
Drug resistance-associated marker Lrp for prediction of response to chemotherapy and prognoses in advanced ovarian carcinoma.
J Nat Cancer Inst.
1995;87:1230-1237
15.
Wong IH, Ng MH, Huang DP, Lee JC.
Aberrant p15 promoter methylation in adult and childhood acute leukemias of nearly all morphologic subtypes: potential prognostic implications.
Blood.
2000;95:1942-1949
16.
Leith CP, Kopecky KJ, Godwin J, et al.
Acute myeloid leukemia in the elderly: assessment of multidrug resistance (MDR1) and cytogenetics distinguishes biologic subgroups with remarkably distinct responses to standard chemotherapy: a Southwest Oncology Group study.
Blood.
1997;89:3323-3329
17.
Pedersen-Bjergaard J, Andersen MK, Christiansen DH, Nerlov C.
Genetic pathways in therapy-related myelodysplasia and acute myeloid leukemia.
Blood.
2002;99:1909-1912 18. Taylor SM, Jones PA. Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell. 1979;17:771-779[CrossRef][Medline] [Order article via Infotrieve].   CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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