The hENT1 and DCK genes underlie the decitabine response in patients with myelodysplastic syndrome

doi:10.1016/j.leukres.2014.08.016

Leukemia Research

Volume 39, Issue 2, February 2015, Pages 216-220

https://doi.org/10.1016/j.leukres.2014.08.016 Get rights and content

Highlights

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The decitabine metabolic pathway in vivo may affect its demethylated efficacy for MDS patients.
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Patients with higher expression of hENT1 mRNA relates to a good response to decitabine.
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DCK mRNA expression was significantly reduced when the patients relapsed during decitabine treatment.

Abstract

Decitabine is approved for the treatment of MDS, but resistance to this agent is common. To determine the mechanisms underlying decitabine resistance, we measured the mRNA expression of metabolism (hENT1, DCK, CDA) and apoptosis (BCL2L10) genes and found that the hENT1 mRNA level was significantly higher in response compared with non-response patients (P = 0.004). Furthermore, the DCK level was significantly reduced for relapse (P = 0.012) compared with those with continued marrow CR (P = 0.222). These findings indicate that the decitabine metabolic pathway affects its therapeutic effects, lower hENT1 expression may induce primary resistance and down-regulated DCK expression may be related to secondary resistance.

Introduction

Myelodysplastic syndrome (MDS) comprises a group of biologically and clinically heterogeneous clonal hematopoietic neoplasms characterized by aberrant myeloid differentiation, dysplastic changes, ineffective hematopoiesis and increased genomic instability, which clinically manifest as peripheral blood (PB) cytopenias and variably increased rates of leukemia progression [1]. Epigenetic deregulation (particularly gene hypermethylation and histone deacetylation) plays an important role in the pathogenesis of MDS [2]. The inhibitor of DNA methyltransferases (DNMTs) 2-deoxy-5-azacitidine/decitabine has been approved by the US Food and Drug Administration (FDA) for the treatment of MDS [3], [4].

A number of clinical trails have shown that decitabine provides significant clinical benefit for patients with MDS [5], [6], [7], [8], but a number of patients do not initially respond (primary resistance), and a majority of patients lose response or progress despite continued therapy (secondary resistance) [9]. Furthermore, the mechanisms underlying decitabine resistance remain unknown. Previous studies indicate that a metabolic pathway is related to primary and secondary resistance [10], and genes related to decitabine metabolism include human equilibrative nucleoside transporter 1 (hENT1), hENT2, deoxycytidine kinase (DCK) and cytidine deaminase (CDA) [11], [12], [13]. Moreover, other studies have demonstrated that BCL2L10 is a predictive factor for resistance to the DNA methyltransferase inhibitor azacitidine for MDS and AML patients [14]. In view of this information, we aimed to investigate whether the expression level of these genes could predict the response to decitabine for MDS patients.

Section snippets

Patients

Adults with a diagnosis of MDS who were referred to Guangdong General Hospital & Guangdong Academy of Medical Science were enrolled in this study according to FAB criteria. 28 newly diagnosed patients with MDS who received decitabine more than 2 cycles of standard treatment (2–23 cycles, median number cycles: 7). A total of 14 patients had an overall response after treatment. 14 patents showed no response. Among the responders, 5 patients achieved more than 8 courses of mCR (continued mCR), but

Statistical analyses

Statistical analyses were performed using SPSS version 11.5 for Windows. Statistical differences between different groups were analyzed by nonparametric tests. The Mann–Whitney U test was used to compare the difference of genes expression between two groups. The difference of gene expression at diagnosis vs relapse or mCR was compared by Wilcoxon Signed Ranks Test. P values less than or equal to 0.05 were considered statistically significant.

Results

In this study, we investigated the mechanisms of primary and secondary resistance to decitabine. Thus, we examined the expression level of different metabolism genes in MDS patients who received decitabine and compared that of responders and non-responders for primary resistance. For secondary resistance, we analyzed the expression level of different genes in patients who lost response or progressed and continued mCR while on hypomethylating agent-based therapy. The patient characteristics are

Discussion

Decitabine (5-aza-2′-deoxycytidine) is a nucleoside analog of cytidine in which the 5-carbon of the pyrimidine ring is replaced by nitrogen. The preceding decade has seen the approval of frontline therapies, such as decitabine, for all myelodysplastic syndrome (MDS) subtypes [16]. Studies have shown ORRs of 28–48% with CR rates of 6–34% [17]. In responding patients, the median duration of response was between 8 and 10 months with improved OS and decreased transfusion requirements. In patients

Conflict of interest statement

The authors declare no competing financial interests.

Acknowledgements

Contributions: PW and XD participated in the design of the study, analysis of data and draft the manuscript. SXG performed the RNA extraction, cDNA synthesis, and RT-PCR experiments. JYW, CXD, ZSL and CWL were responsible for the patients’ diagnosis and participated in the clinical data acquisition. All authors read and approved the final manuscript. This work was supported by grants from National Public Health Grand Research Foundation (No. 201202017), the National Natural Science Foundation

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Myeloid malignancies such as myelodysplastic syndrome (MDS) & acute myeloid leukemia (AML) are clonal diseases that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells, that are not eliminated by conventional cytotoxic therapies. Hypomethylating agents(HMA), azacytidine and decitabine are the first line agents for treatment of MDS and a combination with BCL-2 inhibitor, venetoclax, is approved for AML induction in patients above 75 years and is also actively being investigated for use in high risk MDS. Resistance to these drugs has become a significant clinical challenge in treatment of myeloid malignancies. In this review, we discuss molecular mechanisms underlying the development of resistance to HMA and venetoclax. Insights into these mechanisms can help identify potential biomarkers for resistance prediction, aid in the development of combination therapies and strategies to prevent resistance and advance the field of cancer therapeutics.
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Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds.
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Resistance to the hypomethylating agents (HMAs) 5-azacytidine (AZA) and 5-aza-2′-deoxycytidine (DAC) represents a major obstacle in the treatment of elderly patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) which are not suitable for hematopoietic stem cells transplantation. Approximately 50 % of patients do not respond to HMA treatment because of intrinsic (primary) resistance, while others could acquire drug resistance during the repeated cycles of the treatment. To prevent, delay or surmount resistance development, the molecular mechanisms underlying drug resistance must be first identified. This is crucial as no further standard therapeutic opportunities are available for these patients who failed hypomethylating agents-based treatment. The current review provides an updated information about the different mechanisms that may contribute to the development of resistance to HMAs. Despite the similar structure and mechanism of action of HMA, several studies did not report the expected development of cross-resistance. It is clear that in addition to the common modalities of chemoresistance, there must be some specific mechanisms of drug resistance. Changes in transport and metabolism of HMAs are among the most studied mechanisms of resistance. Drug uptake provided by two solute carrier (SLC) families: SLC28 and SLC29 (also known as the concentrative and equilibrative nucleoside transporter families, respectively), could represent one of the mechanisms of cross-resistance. Changes in the metabolism of these drugs are the most likely mechanism responsible for the unique mode of resistance to AZA and DAC. Deoxycytidine kinase and uridine-cytidine kinase due to their necessity for drug activation, each could represent one of the response markers to treatment with DAC and AZA, respectively. Other mechanisms involved in the development of resistance common for both drugs involved: i. increased DNA repair (caused for example by constitutive activation of the ATM/BRCA1 pathway and inhibition of p53-dependent apoptosis); ii. changes in the regulation of apoptosis/disrupted apoptotic pathways (specifically increased levels of the anti-apoptotic protein BCL2) and iii. increased resilience of leukemic stem cells to multiple drugs including HMAs. Despite intense research on the resistance of MDS and AML patients to HMAs, the mechanisms that may reduce the response of these cells to HMAs are not known in detail. We herein highlight the most important directions that future research should take.
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Drug resistance during DAC treatments represents a major limitation in chemotherapy. Wu et al. showed that the weak expression of equilibrative nucleotide transporter 1 (ENT1) may induce natural resistance, while the down-regulated expression of dCK may be related to acquired resistance [3]. We previously reported the resistance to DAC by long-term treatment with DAC in colorectal cancer cell line HCT116 [4].
Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor is a novel anti-cancer drug regulating epigenetic mechanisms. Similar to conventional anti-cancer drugs, drug resistance to DAC also has been reported, resulting in tumor recurrence. Our previous study using colorectal cancer HCT116 cells found the decrease in deoxycytidine kinase (dCK) (activation enzyme of DAC) and the increase in cytidine deaminase (inactivation enzyme of DAC) in acquired DAC-resistant HCT116 (HCT116/DAC) cells. The aim of our study was to clarify the involvement of dCK and CDA in DAC resistance. In order to tackle DAC resistance, it was also examined whether other DNMT inhibitors such as azacytidine (AC) and polyphenols are effective in DAC-resistant cancer cells. When dCK siRNA was transfected into HCT116 cells, IC₅₀ value of DAC increased by about 74-fold and reached that of HCT116/DAC cells with attenuated dCK. dCK siRNA to HCT116 cells also abolished DNA demethylation effects of DAC. In contrast, CDA siRNA to HCT116 cells did not influence the efficacy of DAC. In addition, CDA siRNA to HCT116/DAC cells with increased CDA did not restore the compromised effects of DAC. These results suggested that attenuated dCK but not increased CDA mainly contributed to DAC resistance. Regarding dCK in HCT116/DAC cells, a point mutation with amino acid substitution was observed while the product size and expression of mRNA coding region did not change, suggesting that dCK protein was decreased by post-transcriptional regulation. AC and polyphenols showed no cross-resistance in HCT116/DAC cells. AC but not polyphenols exerted DNA demethylation effect. Among polyphenols, curcumin (Cur) showed the most synergistic cytotoxicity in combination with AC while DNA demethylation effect of AC was partly maintained. Taken together, combination of AC and Cur would be a promising alternative to tackle DAC resistance mainly due to attenuated dCK.
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The present study aimed to compare the efficacy and safety of azacitidine and decitabine in patients with myelodysplastic syndrome (MDS). A total of 88 patients diagnosed with refractory anemia with excess blast (RAEB) treated with azacitidine (n = 57) or decitabine (n = 31) were evaluated. Comparisons between azacitidine and decitabine groups were performed in the whole cohort, and in a 1:1 propensity score-matched cohort in order to reduce the simple selection bias. Patients who received azacitidine or decitabine had comparable overall response rates in both the unmatched (49.1% vs. 64.5%, p = 0.166) and the propensity-matched cohorts (52% vs. 68%, p = 0.248). The cumulative incidence of AML transformation at one year was comparable between azacitidine and decitabine in the unmatched (24.0% vs. 31.3%, p = 0.26) and in the propensity-matched cohorts (18.7% vs. 31.5%, p = 0.11). There was no difference in terms of transfusion requirement, febrile neutropenia episodes or the need for antifungal use during the treatment cycles in the propensity-matched cohort. The median overall survival was 20.4 months for azacitidine and 16.8 months for decitabine (p = 0.59). Finally, we found that at least a four-cycle treatment with any HMA was a favorable factor. In conclusion, both azacitidine and decitabine have similar efficacy and toxicity profiles in the treatment of MDS-RAEB.

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¹: These authors contributed equally to this work.

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The hENT1 and DCK genes underlie the decitabine response in patients with myelodysplastic syndrome

Highlights

Abstract

Introduction

Section snippets

Patients

Statistical analyses

Results

Discussion

Conflict of interest statement

Acknowledgements

Blood Rev

Best Pract Res Clin Haematol

Semin. Hematol

Blood

Semin Oncol

Clin Lymphoma Myeloma Leuk

Blood

DacogenTM (decitabine) for injection

An epigenetic approach to the treatment of advanced MDS; the experience with the DNA demethylating agent 5-aza-2’-deoxycytidine (decitabine) in 177 patients

Ann Hematol

Low-Dose Decitabine Versus Best Supportive Care in Elderly Patients With Intermediate- or High-Risk Myelodysplastic Syndrome (MDS) Ineligible for Intensive Chemotherapy Final Results of the Randomized Phase III Study of the European Organis

J Clin Oncol.

Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study

Cancer

Outcome of patients with myelodysplastic syndrome after failure of decitabine therapy

Cancer

Nucleoside analogues: mechanisms of drug resistance and reversal strategies

Leukemia