Low-dose decitabine was safe and showed encouraging clinical and biologic activity in AML, but the addition of VA led to encephalopathy at relatively low doses. On the basis of these results, additional studies of decitabine (20 mg/m(2)/d for 10 days) alone or with an alternative deacetylating agent are warranted.
IntroductionMethylation of CpG islands in promoter region of genes is due to enzymatic addition of a methyl (CH 3 ) group at the carbon 5 position of cytosine and has been shown to inhibit gene transcription. 1 This enzymatic reaction is mediated by DNA methyltransferases (DNMTs: DNMT1, 3a, and 3b) that use s-adenosylmethionine (SAM) as a methyl donor. While DNMT3a and 3b are important to establish novel methylation sites on nascent DNA, DNMT1 plays a critical housekeeping role in maintaining established patterns of DNA methylation in dividing cells. 2 DNMTs have recently been found to be overexpressed in human acute myeloid leukemia (AML) and solid tumors, thereby supporting a role of these enzymes in the development and maintenance of the neoplastic phenotype. 3,4 Inhibition of DNMT1 by antisense or shRNA oligonucleotides or nucleoside analogs (eg, 5-aza-2Ј-deoxycytidine [decitabine]) induces DNA hypomethylation and reactivation of hypermethylated tumor suppressor genes in leukemia cells. [5][6][7][8][9][10][11] This ultimately restores normal patterns of cell proliferation, differentiation, and apoptosis, which in turn leads to a significant antitumor activity.To date, 2 hypomethylating nucleoside analogs (decitabine and 5-azacitidine, referred to hereafter as azanucleosides) have been approved by the FDA for the treatment of myelodysplastic syndromes (MDSs) and are currently in clinical trials for other types of cancers. 12,13 An accepted mechanism for the antitumor activity of these agents is their incorporation into newly synthesized DNA strands followed by covalent binding, sequestration, and depletion of the DNMT enzymes. 12,13 Clinical responses to azanucleosides, however, appear to be restricted to a minority of hematopoietic malignancies, which are characterized by a relatively high proliferative cell fraction. 14 Thus, development of novel hypomethylating compounds with mechanisms of action distinct from azanucleosides may broaden the therapeutic toolbox targeting epigenetic aberrations in human cancer.Recent studies suggest that DNMT1 expression is tightly regulated during normal cell growth, and its transcription is modulated by the Sp1 protein in mice. 15 Sp1 is a ubiquitous zinc finger transcription factor that binds GC-rich cis-acting elements ((G/ A)(G/A)GGCC(G/T)(G/A)(G/A)) in the promoter region of inducible genes. 16 The complexity of the regulatory functions mediated by Sp1 can be explained by a variety of posttranslational modifications (ie, ubiquitination, glycosylation, phosphorylation) of this protein 16,17 and/or its physical interaction with other transcription factors, such as those of the NF-B family 18,19 that are constitutively activated in AML and controlled by the proteasomal degradation. 20,21 Submitted August 31, 2007; accepted December 9, 2007. Prepublished online as Blood First Edition paper, December 14, 2007; DOI 10.1182 DOI 10. /blood-2007 S.L. and Z.L. contributed equally to this work.The publication costs of this article were defrayed in part by page charge payment. The...
The translocation t(8;21)(q22;q22) in acute myeloid leukemia (AML) results in the expression of the fusion protein RUNX1/ MTG8, which in turn recruits histone deacetylases (HDAC) to silence RUNX1 target genes [e.g., interleukin-3 (IL-3)]. We previously reported that expression of the RUNX1/MTG8 target gene IL-3 is synergistically restored by the combination of inhibitors of HDACs (i.e., depsipeptide) and DNA methyltransferases (DNMT; i.e., decitabine) in RUNX1/MTG8-positive Kasumi-1 cells. Thus, we hypothesized that DNMT1 is also part of the transcriptional repressor complex recruited by RUNX1/ MTG8. By a chromatin immunoprecipitation assay, we identified a RUNX1/MTG8-DNMT1 complex on the IL-3 promoter in Kasumi-1 cells and in primary RUNX1/MTG8-positive AML blasts. The physical association of RUNX1/MTG8 with DNMT1 was shown by coimmunoprecipitation experiments. Furthermore, RUNX1/MTG8 and DNMT1 were concurrently released from the IL-3 promoter by exposure to depsipeptide or stabilized on the promoter by decitabine treatment. Finally, we proved that RUNX1/MTG8 and DNMT1 were functionally interrelated by showing an enhanced repression of IL-3 after coexpression in 293T cells. These results suggest a novel mechanism for gene silencing mediated by RUNX1/MTG8 and support the combination of HDAC and DNMT inhibitors as a novel therapeutic approach for t(8;21) AML. (Cancer Res 2005; 65(4): 1277-84)
In t(8;21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemogenesis by recruiting class I histone deacetylase (HDAC)-containing repressor complex to the promoter of AML1 target genes. Valproic acid (VPA), a commonly used antiseizure and mood stabilizer drug, has been shown to cause growth arrest and induce differentiation of malignant cells via HDAC inhibition. VPA causes selective proteasomal degradation of HDAC2 but not other class I HDACs (i.e., HDAC 1, 3, and 8). Therefore, we raised the question of whether this drug can effectively target the leukemogenic activity of the AML1/ETO fusion protein that also recruits HDAC1, a key regulator of normal and aberrant histone acetylation. We report here that VPA treatment disrupts the AML1/ETO-HDAC1 physical interaction, stimulates the global dissociation of AML1/ ETO-HDAC1 complex from the promoter of AML1/ETO target genes, and induces relocation of both AML1/ETO and HDAC1 protein from nuclear to perinuclear region. Furthermore, we show that mechanistically these effects associate with a significant inhibition of HDAC activity, histone H3 and H4 hyperacetylation, and recruitment of RNA polymerase II, leading to transcriptional reactivation of target genes (i.e., IL-3) otherwise silenced by AML1/ETO fusion protein. Ultimately, these pharmacological effects resulted in significant antileukemic activity mediated by partial cell differentiation and caspase-dependent apoptosis. Taken together, these data support the notion that VPA might effectively target AML1/ETO-driven leukemogenesis through disruption of aberrant HDAC1 function and that VPA should be integrated in novel therapeutic approaches for AML1/ETO-positive AML.
Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death concurrent with down-modulation of phosphoAkt and cyclin D1 and subsequent cellcycle arrest. However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. In the present study, we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes and increased LC3-II and p62 levels. We also show that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagiclysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. This finding provided rationale for examining combination therapy with FTY720 and milatuzumab, an anti- IntroductionMantle cell lymphoma (MCL) is a B-cell malignancy that comprises 3%-8% of non-Hodgkin lymphoma cases diagnosed each year. 1 Whereas the current treatment approach of using combination chemotherapeutic regimens can lead to complete remission, virtually all MCL patients relapse and outcome remains poor, with a median survival of only 3 years. 2 The aggressive clinical behavior of MCL may be because of the complex pathophysiology of the disease, which includes cell-cycle dysregulation driven by cyclin D1 overexpression, alteration in the DNA-damage response, and constitutive activation of key antiapoptotic pathways such as PI3K/Akt and NF-B. [3][4][5][6] Given the absence of curative therapy and the limited number of options for patients with relapsed/refractory MCL, it will be essential to improve our understanding of the complex biology of this disease so that novel treatment approaches can be developed. FTY720 (fingolimod), is a synthetic analog of sphingosine that was developed as an immunosuppressive agent. 7,8 Based on the results of a recent phase 3 clinical trial, FTY720 has been approved by the US Food and Drug Administration (FDA) to treat relapsed multiple sclerosis. 9 We have recently reported that FTY720 has in vitro and in vivo activity in MCL. 10 FTY720 promotes death of MCL cell lines and primary MCL tumor cells via caspaseindependent radical oxygen species (ROS) generation, downmodulation of phospho-Akt and cyclin D1, with accumulation of cells in G 0 /G 1 and G 2 /M phases of the cell cycle. Whereas these data provided information explaining the antitumor activity of FTY720, the effects of this drug on the pathophysiology of MCL required further characterization.In the present study, we show that FTY720 inhibits autophagic flux and induces MCL cell death through lysosomal membrane permeabilization and subsequent translocation of lysosomal hydrolases in the cytosol. Because the autophagy-lysosomal pathway represents an important regulatory mechanism governing the cellular proteome, we hypo...
Purpose: Inhibition of ribonucleotide reductase reduces the availability of the endogenous pool of deoxycytidine and may increase cytarabine (AraC) cytotoxicity. We performed a phase I dose escalation trial of AraC combined with GTI-2040, a 20-mer antisense oligonucleotide shown in preclinical studies to decrease levels of the R2 subunit of ribonucleotide reductase, to determine the maximum tolerated dose in adults with relapsed/refractory acute myeloid leukemia. Experimental Design: Twenty-three adults (ages 18-59 years) were enrolled in this dose escalation phase I trial, receiving high-dose AraC twice daily combined with infusional GTI-2040. An ELISA-based assay measured plasma and intracellular concentrations of GTI-2040. R2 protein changes were evaluated by immunoblotting in pretreatment and posttreatment bone marrow samples. Results: The maximum tolerated dose was 5 mg/kg/d GTI-2040 (days 1-6) and 3 g/m 2 /dose AraC every 12 hours for 8 doses. Neurotoxicity was dose limiting. Eight patients (35%) achieved complete remission. Mean bone marrow intracellular concentration of GTI-2040 were higher at 120 hours than at 24 hours from the start of GTI-2040 (P = 0.002), suggesting intracellular drug accumulation over time. Reductions in bone marrow levels of R2 protein (>50%) were observed at 24 and 120 hours. Higher baseline R2 protein expression (P = 0.03) and reductions after 24 hours of GTI-2040 (P = 0.04) were associated with complete remission. Conclusions: GTI-2040 and high-dose AraC were coadministered safely with successful reduction of the intended R2 target and encouraging clinical results. The clinical efficacy of this combination will be tested in an upcoming phase II study.Nucleoside analogues constitute the backbone of several primary and salvage chemotherapy regimens for acute myeloid leukemia (AML; refs. 1, 2). By mimicking endogenous nucleosides, these compounds are incorporated into newly synthesized DNA and induce inhibition and chain termination of newly synthesized nucleic acid, thereby leading to apoptosis (3). Among pyrimidine analogues, cytarabine (AraC) differs from the endogenous cytidine counterpart by the presence of an arabinoside rather than a ribose sugar. This compound undergoes enzymatic phosphorylation into the active metabolite Ara-CTP before being incorporated in newly synthesized DNA. A direct correlation between intracellular levels of Ara-CTP and antileukemic effect has been identified, and different strategies to increase levels of the active metabolite have been investigated (4 -7).Inhibition of the ribonucleotide reductase (RNR) has the potential to increase AraC cytotoxicity. Composed of two subunits, R1 and R2, RNR is required for the reductive conversion of ribonucleotides to deoxynucleotides, a crucial rate-limiting step during DNA synthesis and repair (8, 9). Overexpression of RNR, commonly found in malignant cells, increases the endogenous pool of deoxynucleoside triphosphates, a potential mechanism of chemoresistance to nucleoside analogues competing for DN...
Therapeutic use of oligodeoxynucleotides (ODNs) that hybridize to and downregulate target messenger RNAs (mRNAs) encoding proteins that contribute to malignant transformation has a sound rationale, but has had an overall limited clinical success in cancer due to insufficient intracellular delivery. Here we report a development of formulations capable of promoting targeted delivery and enhanced pharmacologic activity of ODNs in acute myeloid leukemia (AML) cell lines and patient primary cells. In this study, transferrin (Tf) conjugated pH-sensitive lipopolyplex nanoparticles (LPs) were prepared to deliver GTI-2040, an antisense ODN against the R2 subunit of ribonucleotide reductase that has been shown to contribute to chemoresistance in AML. LPs had an average particle size around 110 nm and a moderately positive zeta potential at ~ 10 mV. The ODN encapsulation efficiency of LPs was > 90%. These nanoparticles could release ODNs at acidic endosomal pH and facilitate the cytoplasmic delivery of ODNs after endocytosis. In addition, Tf-mediated targeted delivery of GTI-2040 was achieved. R2 downregulation at both mRNA and protein levels was improved by 8-fold in Kasumi-1 cells and 2 to 20-fold in AML patient primary cells treated with GTI-2040-Tf-LPs, compared to free GTI-2040 treatment. Moreover, Tf-LPs were more effective than non-targeted LPs, with 10 to 100% improvement at various concentrations in Kasumi-1 cells and an average of 45% improvement at 3 µM concentration in AML patient primary cells. Treatment with 1 µM GTI-2040-Tf-LPs sensitized AML cells to the chemotherapy agent cytarabine, by decreasing its IC50 value from 47.69 nM to 9.05 nM. This study suggests that the combination of pH sensitive LP formulation and Tf mediated targeting is a promising strategy for antisense ODN delivery in leukemia therapy.
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