Leukemia (1997) 11, 175–180  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

REVIEW Pharmacological approach for optimization of the dose schedule of 5-Aza-2’- deoxycytidine (Decitabine) for the therapy of leukemia

RL Momparler, S Coˆte´ and N Eliopoulos

De´partement de Pharmacologie, Universite´ de Montre´al; and Centre de Recherche Pe´diatrique, Hoˆpital Ste-Justine, Montre´al, Que´bec, Canada

5-Aza-2Ј-deoxycytidine (5-Aza-CdR; Decitabine) is an active drome (MDS).8 One problem in evaluating the clinical effi- antineoplastic agent in patients with leukemia. Since 5-Aza- cacy of 5-Aza-CdR in these studies is that most of the leu- CdR is an S phase specific agent and has a short plasma half- life, its antileukemic activity is dose schedule-dependent. Leu- kemic patients received prior therapy with Ara-C. Since both kemia patients who are candidates for 5-Aza-CdR therapy fol- 5-Aza-CdR and Ara-C are metabolized by the same enzymes, lowing relapse after therapy with cytosine arabinoside are at the possibility exists that subpopulations of drug-resistant leu- greater risk for the problem of drug resistance since these cyto- kemic cells were present at the time of administration of the sine nucleoside analogues are metabolized by the same first course of 5-Aza-CdR therapy. enzymes. Due to its unique mechanism of action of demethylat- ing DNA, 5-Aza-CdR has the potential to activate tumor (growth) suppressor and differentiation genes that have been accidentally silenced by DNA methylation in leukemic cells. All Drug resistance to cytosine nucleoside analogues these factors should be taken into account in the design of the optimal dose schedule of this analogue. The optimal dose The major mechanisms of resistance to these cytosine nucleo- schedule of 5-Aza-CdR should be based on the kinetic para- side analogues are summarized in Table 1. Both 5-Aza-CdR meters of deoxycytidine kinase, its pharmacokinetics, its and Ara-C must first be phosphorylated by deoxycytidine kin- effects on DNA methylation and the cell cycle parameters of ase to be active inhibitors. Cells lacking this enzyme are com- the leukemic cells and the normal hematopoietic stem cells. 9,10 Since granulocytopenia is the major toxic effect produced by pletely resistant to these analogues. Cells with an increased 5-Aza-CdR, the use of hematopoietic growth factors to shorten pool of dCTP also show drug resistance to these analogues the duration of leukopenia should be investigated. Another since dCTP is a feedback inhibitor of deoxycytidine kinase.9 approach which we are investigating is to use the methods of In addition, dCTP reduces the incorporation of 5-Aza-CdR and gene therapy to insert the cytidine deaminase gene into normal Ara-C into DNA by competition for DNA polymerase with the hematopoietic progenitor cells so as to make them drug resist- 9,11 ant to 5-Aza-CdR. The use of other agents that can induce the triphosphate forms of these analogues. Finally, cells with differentiation of leukemic cells in combination with 5-Aza-CdR increased cytidine deaminase show drug resistance since may have the potential to increase the clinical effectiveness of deamination of these analogues results in a loss of antineo- this analogue for the therapy of leukemia. plastic activity.12–14 Keywords: 5-Aza-2Ј-deoxycytidine; DNA methylation; chemo- therapy Activation of tumor (growth) suppressor genes by 5-AZA- CdR Introduction The methylation in DNA of cytosine to 5-methylcytosine in 5-Aza-2Ј-deoxycytidine (5-Aza-CdR; Decitabine) was first the promoter region of a gene can suppress its expression.15 shown to be an active antileukemic agent in a mouse model As shown in Figure 1 the incorporation of 5-Aza-CdR into by Sorm and Vesely.1 In the mouse L1210 leukemia model, DNA can block DNA methylation11,16 which can result in 5-Aza-CdR was demonstrated to be much more effective than gene activation and the induction of differentiation.16–18 cytosine arabinoside (Ara-C),2 one of the most effective agents Recent investigations have shown that the lack of for the treatment of acute myeloid leukemia.3 In vitro studies expression of several types of tumor (growth) suppressor genes showed that 5-Aza-CdR is also a more potent antineoplastic is due to aberrant DNA methylation.19 As summarized in agent against human myeloid leukemic cells than Ara-C.4 Table 2, 5-Aza-CdR has been demonstrated to activate the Phase I–II studies in Canada and Europe on 5-Aza-CdR expression of tumor suppressor genes: p16,20,21 p15,22 VHL23 showed that this cytosine analogue could induce complete and retinoic acid receptor ␤ (RAR␤)24 that have been silenced remission in patients with acute leukemia who had relapsed by DNA methylation. Recent reports support the hypothesis 5–7 on conventional chemotherapy. In addition, 5-Aza-CdR has that RAR␤ is a tumor suppressor gene.25,26 shown clinical activity in patients with myelodysplastic syn- The role of most tumor suppressor genes in the pathogenesis

Table 1 Drug resistance to 5-Aza-CdR and Ara-C Correspondence: RL Momparler, Centre de Recherche Pe´diatrique, Hoˆpital Ste-Justine, 3175 Chemin Coˆte Ste-Catherine, Montre´al, Que´- bec H3T 1C5 Canada 1 Deficiency in deoxycytidine kinase This paper will also appear in a supplementary monograph of the 2 Increase intracellular pool of dCTP journal Leukemia, Workshop on Clinical Results with Decitabine (5- (a) Inhibition of deoxycytidine kinase AZA-2Ј-deoxycytidine) in Hematological Malignancies, Guest Editor (b) Competition for DNA polymerase Dr Hagop M Kantarjian, 1997; 11 3 Increase in cytidine deaminase Received 1 July 1996; accepted 16 October 1996 Review RL Momparler et al 176 action is also schedule-dependent. At diagnosis or relapse, the leukemic patient may have 109 to 1010 leukemic stem cells that should be eradicated by treatment in order to produce curative therapy. To accomplish this difficult task, every para- meter that can influence the response to the 5-Aza-CdR should be evaluated. In Table 3 are summarized the major parameters that should be taken into account in the design of the optimal dose schedule for 5-Aza-CdR. Chemotherapy studies in mice with leukemia showed that 5-Aza-CdR treatment produces ‘cures’ only at the higher dose levels.31 Extrapolation to the clinical therapy of leukemia, the higher dose ranges of 5-Aza-CdR should be given a priority. The steady state plasma concentration of 5-Aza-CdR during a Figure 1 Inhibition of methylation of cytosine by the incorporation continuous i.v. infusion should be above its minimal cytotoxic of 5-Aza-CdR (AZA) into DNA. After DNA replication, methylation of the newly synthesized DNA strand takes place at complementary sites concentration as determined by in vitro colony assays on determined by presence of methyl groups (CH ) in parental DNA human leukemic cells. Since the minimal cytotoxic concen- 3 − strand in order to maintain a specific pattern of DNA methylation. The tration for 5-Aza-CdR is estimated to be about 10 7 M,32 the incorporation of AZA in place of cytosine at a specific methylation steady state plasma concentration of this analogue should be − site produces an inactivation of DNA methylase resulting in a region above 10 7 M. This would ensure that adequate cytotoxic con- of hypomethylation. centrations of 5-Aza-CdR will be obtained in all anatomical compartments. In certain anatomical sites the concentration of 5-Aza-CdR will be less than that of the plasma concen- Table 2 Activation of tumor suppressor genes by 5-Aza-CdR tration. For example, the concentration of 5-Aza-CdR in the Tumor suppressor gene Ref. cerebral spinal fluid is about 50% lower than the concen- tration in the plasma.33 Due to the high level of cytidine

INK4B1 22 deaminase, the enzyme that inactivates 5-Aza-CdR, in liver p15 Herman et al 34 p16INK4 Otterson et al20 and spleen, it is possible that the concentration of this ana- Merlo et al21 logue could fall below the minimal cytotoxic concentration Retinoic acid receptor ␤ Coˆte´ and Momparler24 creating a biochemical sanctuary for leukemic cells in these von Hippel-Lindau (VHL) Herman et al23 organs. Serious consideration should be given to the use of cytidine deaminase inhibitors to overcome this problem.35 What is the optimal plasma concentration of 5-Aza-CdR? Clinical studies on Ara-C have shown that it is possible to use of leukemia is unclear at this time since DNA methylation very high doses with cytosine nucleoside analogues for the studies have been performed primarily on tumor cells. The therapy of leukemia.36 However, very high doses can produce tumor suppressor gene p16 does not appear to be modified unexpected side-effects such as the neurological toxicity that by methylation in leukemia.22 However, the p15 tumor sup- sometimes is produced by high dose Ara-C therapy.37 Since pressor gene was demonstrated to be hypermethylated in leu- intermediate dose Ara-C therapy can produce similar kemic cells in six of seven patients with AML and four of eight responses as high-dose therapy,3 it is reasonable to also favor patients with ALL.22 The p15 gene is a cyclin-dependent kin- the use of intermediate dose 5-Aza-CdR for the therapy of leu- ase inhibitor.27 Cyclin-dependent kinases play an important kemia. The optimal plasma concentration for 5-Aza-CdR is

role in the regulation of cellular progression into the S phase probably in the range of the Km value of deoxycytidine kinase, 32 and the control of cell proliferation. the rate limiting enzyme that activates this analogue. The Km In order for tumor suppressor genes to participate in cellular is the concentration of drug at which the enzyme reaction transformation, both alleles must be inactivated. Inactivation takes place at 50% of its maximum velocity. For deoxycytid-

of tumor suppressor genes can occur by chromosomal ine kinase the Km of 5-Aza-CdR is in the range of approxi- − deletion or rearrangement, mutation and DNA methylation. In mately 10 5 M.32 In order to obtain a plasma concentration of evaluation of the clinical response of leukemic patients to 5- this range, 5-Aza-CdR should be infused at a rate of about 1– Aza-CdR, the prior chemotherapy should be examined. As 2 mg/kg/h or 30–60 mg/m2/h.5,6 The pharmacokinetics of 5- illustrated in Figure 2, prior treatment with mutagenic or DNA Aza-CdR are also important. Since 5-Aza-CdR has a short damaging anticancer drugs have the potential to produce plasma half life of 15–20 min,6 a constant continuous i.v. mutations and chromosomal translocations28 that can inacti- infusion of this analogue is the best way to maintain a steady − vate tumor suppressor genes and possibly decrease the state level of approximately 10 5 M in the plasma. response to subsequent therapy with 5-Aza-CdR. The duration of the 5-Aza-CdR therapy will depend on the cell cycle parameters of the leukemic cells and the normal hematopoietic stem cells. Recent investigations on the cell Optimization of dose schedule of 5-Aza-CdR kinetics of myeloid leukemic cells in man gave an estimate of the length of the cell cycle with a mean value of 59.8 h (range The classical investigation by Skipper et al29 demonstrated 18–211 h).38 From these data the estimated optimal duration that the antileukemic activity of the S phase specific agent, of 5-Aza-CdR therapy is probably in the range of 90 h (only Ara-C, was markedly schedule-dependent in murine leukemia six of 45 patients showed a cell cycle time Ͼ90 h). models. These data plus additional data on the pharmacology The 5-Aza-CdR therapy will also induce terminal differen- of Ara-C were used by Momparler30 to propose a model for tiation of the dividing hematopoietic progenitor cells. There- the therapy of leukemia with this analogue. Since 5-Aza-CdR fore, the success of the therapy depends on the survival of the is also an S phase specific agent, its in vivo antileukemic resting hematopoietic stem cells. If prior therapy with other Review RL Momparler et al 177

Figure 2 Tumor (growth) suppressor gene can be inactivated by either mutation (M), chromosomal deletion or DNA methylation (CH3). Both alleles of the tumor suppressor gene must be inactivated to result in a complete loss of expression. Prior treatment with mutagenic or DNA damaging drugs by inactivation of both alleles can reduce the clinical response to 5-Aza-CdR.

Table 3 Parameters to consider in the design of optimal dose- gation in our laboratory is to use gene therapy to protect the schedule of 5-Aza-CdR for the therapy of leukemia normal hematopoietic stem cells from drug-induced toxicity. The enzyme cytidine deaminase, as discussed above, inacti- 1 Curative therapy of murine leukemia occurs at high vates cytosine nucleoside analogues such as 5-Aza-CdR and dose. 2 Minimal cytotoxic concentration for human leukemic Ara-C by deamination. We have cloned the human cytidine 40 cells. deaminase cDNA and used a retroviral vector to insert and 3 Inactivation by cytidine deaminase in liver and spleen. express this gene in murine fibroblast cells. We have observed 4 Pharmacokinetics. that these transduced cells are drug resistant to both 5-Aza- 14,41,42 5 Km value of deoxycytidine kinase. CdR and Ara-C. Recently, we have inserted the cytidine 6 Cell cycle parameters of leukemic stem cells. deaminase gene into normal murine hematopoietic cells and 7 Cell cycle parameters of normal hematopoietic stem 42 cells. demonstrated that they are drug resistant to Ara-C. We are 8 Stage of leukemic disease. currently investigating if these hematopoietic cells are also cross-resistant to 5-Aza-CdR. One problem to overcome before this type of gene therapy can be used clinically is the accidental insertion of the cytid- drugs or disease progression produces a marked reduction in ine deaminase gene into a few leukemic cells that may con- the number of these resting stem cells, the 5-Aza-CdR will taminate a normal marrow aspirate. Future technological produce a prolonged granulocytopenia. Under these con- advances in the cloning of normal hematopoietic stem cells ditions a more conservative approach to therapy with 5-Aza- can overcome this problem. In addition, if the leukemic cells CdR should be used. have an identifiable genetic marker that can be detected by the polymerase chain reaction, the possible contamination by leukemic cells can be verified prior to transplantation of the Approaches to overcome the problem of hematopoietic normal stem cells. toxicity

Hematopoietic growth factors, such as G-CSF or GM-CSF, are In vitro assays to evaluate clinical response currently used to reduce the duration of leukopenia following therapy with cytotoxic anticancer agents.39 We have observed Since a conventional clinical trial on an experimental therapy a gradual reduction in the white blood cell count (WBC) fol- for leukemia can take up to 5 years and a significant number lowing therapy with 5-Aza-CdR,5,6 which one would expect of patients for complete evaluation, in practice only a very for an agent that induces cellular differentiation. The terminal small number of trials can be performed. This poses a serious differentiation of the dividing hematopoietic progenitor cells problem for an agent such as 5-Aza-CdR whose clinical effi- induced by 5-Aza-CdR is a slow process requiring many days cacy is dose-schedule dependent. One approach to overcome and thus the feedback mechanism to activate the recruitment this problem is to perform a small series of pilot studies in of resting hematopoietic stem cells into the proliferation com- which in vitro tests before and after therapy are used to evalu- partment may take place much later in time than produced ate efficacy in conjunction with the conventional clinical by cytolytic drugs. If these conditions exist, treatment with response end points. For example, we have used an in vitro G-CSF or GM-CSF after 5-Aza-CdR therapy may reduce the drug sensitivity test to evaluate the response of leukemic duration of granulocytopenia. patients to 5-Aza-CdR. We observed in some patients that the Another approach to overcome the problem of hematopo- leukemic cells that survived the 5-Aza-CdR treatment showed ietic toxicity produced by 5-Aza-CdR which is under investi- drug resistance.5,6,13 One possible interpretation of these data Review RL Momparler et al 178 is that the dose schedule of 5-Aza-CdR used was capable of kemic blasts without any sign of drug toxicity (Momparler RL, eradicating all of the drug-sensitive leukemic blasts, but not Rivard GE, unpublished observation). In accordance with this the small fraction of drug-resistant leukemic cells. One observation is our mouse study, using L1210 leukemic cells important conclusion in this type of analysis is that only an that are drug resistant due to the deficiency in deoxycytidine ‘optimal’ dose schedule of 5-Aza-CdR can eliminate all the kinase, in which we demonstrated that 3-DU markedly drug-sensitive leukemic blasts and permit the survival of the increased the effectiveness of therapy with 5-Aza-CdR.10 drug-resistant cells. Using this approach it should be possible to evaluate different dose schedules of 5-Aza-CdR in a period of months instead of years by the conventional method and it Differentiation therapy of leukemia will also require fewer patients. The spontaneous frequency of mutation in mammalian cells One important aspect of the action of 5-Aza-CdR is its for a single copy gene is about 10−5.9 Since a cell contains capacity to induce leukemic cell differentiation.17,18 Since a two copies of the autosomal gene, deoxycytidine kinase, the very large number of leukemic cells must be eradicated by theoretical predicted frequency of drug resistance due to a the 5-Aza-CdR therapy, the possibility exists that a very small complete loss of enzyme activity due to a mutation in each fraction of the leukemic cell population will not reach the allele of this gene is 10−10 (10−5 × 10−5). This means that theor- threshold level of gene expression so as to undergo irrevers- etically one cell in about 10 billion leukemic stem cells would ible terminal differentiation. This subpopulation of leukemic be drug resistant due to the complete loss of deoxycytidine cells may survive the 5-Aza-CdR treatment. One approach to kinase activity. Therefore, the 5-Aza-CdR therapy would have overcome this problem is to use other differentiation agents to produce about a 10-log reduction in leukemic stem cells in combination with 5-Aza-CdR. The remarkable observation in order to permit survival of a leukemic cell with this type of that retinoic acid can induce complete remission of patients drug resistance. Only the ‘optimal’ dose schedule of 5-Aza- with acute promyelocytic leukemia48,49 suggests that this vit- CdR can produce a reduction in the number of leukemic stem amin A metabolite may be a good agent to test in combination cells of such a magnitude. If prior to 5-Aza-CdR treatment, the with 5-Aza-CdR. In vitro studies have shown an additive anti- leukemic patient is treated with a mutagenic drug, one would neoplastic effect of retinoic acid in combination with 5-Aza- 50 predict an increase in the frequency of drug resistance. CdR on human myeloid leukemic cells. Vitamin D3 which As discussed earlier, lack of response to 5-Aza-CdR therapy can also induce differentiation of leukemic cells was demon- can also be due to deletions, mutations or DNA hypermethyl- strated to produce a synergistic antineoplastic effect in combi- ation in tumor suppressor genes. In vitro tests, such as by nation with 5-Aza-CdR on human myeloid leukemic cells.51 Southern blotting with specific DNA probes to the region of Vitamin D analogues which have reduced calcium toxicity the first exon after digestion of genomic DNA with methyl- may be interesting agents to use in combination with 5-Aza- ation-sensitive and insensitive restriction enzymes can be used CdR in phase I studies in leukemia.52 Interferon-␣ also has to detect tumor suppressor genes that have been silenced by some potential to induce leukemic cell differentiation and has DNA hypermethylation.20–23 In addition, novel techniques shown an interesting interaction with 5-Aza-CdR on human have been developed to identify the specific sites of cytosine myeloid leukemic cells.53 An interesting differentiating agent methylation by DNA sequencing.43–45 These in vitro tests to investigate for use in combination with 5-Aza-CdR is which can be performed on the leukemic cells before and hexamethylene bisacetamide which has been evaluated after treatment with 5-Aza-CdR will assist in the evaluation of in patients with myeloid leukemia and myeodysplastic the clinical response to 5-Aza-CdR. syndrome.54 The action of steroids on leukemic cells is also an interest- ing area of investigation. It should be pointed out that 5-Aza- Approaches to overcome the problem of drug resistance CdR treatment of a steroid-resistant T lymphoid leukemic cell line produced an increased sensitivity to the cytolytic effects The use of other antileukemic agents in combination with the of dexamethasone,55 suggesting that this cytosine analogue 5-Aza-CdR therapy is the conventional way to overcome the may be an effective therapeutic agent for the treatment of ster- problem of drug resistance to this cytosine analogue. The oid-resistant lymphoid leukemias. Remarkably, estrogen choice of drug and its time of administration should be care- receptor expression is silenced by DNA methylation in most fully chosen. As discussed above, the use of a mutagenic or hematological neoplasms suggesting that modification of its DNA damaging antileukemic drug prior to or simultaneously gene may be one of the early events in the pathogenesis of with the 5-Aza-CdR therapy is not recommended. leukemia and suggesting a possible therapeutic role of de- Interesting experimental agents that can be used in combi- methylating agents.56 In this regard, it is interesting to note nation with 5-Aza-CdR are 3-deazauridine (3-DU) and cyclo- that 5-Aza-CdR was demonstrated to activate estrogen recep- pentyl-cytosine (CPC). 3-DU and CPC are biochemical modu- tor (ER) expression in ER-negative breast cancer cells by de- lators which inhibit CTP synthetase resulting in a secondary methylation of its gene.57 reduction in the intracellular pool of dCTP.46,47 As discussed above, one mechanism of resistance to 5-Aza-CdR is an increased pool of dCPT. Using in vitro studies with leukemic Conclusions cells we demonstrated a synergistic antineoplastic action when either 3-DU or CPC were used in combination with 5- The novel mechanism of action of 5-Aza-CdR in the activation Aza-CdR. One very interesting aspect about 3-DU is that cells of tumor (growth) suppressor and differentiation genes and its that are lacking deoxycytidine kinase activity are very sensi- potent antileukemic activity in animal models make it a very tive to the cytotoxic action of this analogue. In one terminal interesting agent to investigate in the therapy of leukemia. leukemic patient that was treated with 5-Aza-CdR, we Since it is an S-phase specific agent and its antileukemic detected drug resistance due to a deficiency in deoxycytidine activity is dose-schedule dependent, a careful analysis of its kinase; 3-DU therapy completely eliminated the blood leu- pharmacology is essential in order to design its optimal dose Review RL Momparler et al 179 schedule. 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