A Comparison of Azacitidine and Decitabine Activities in Acute Myeloid Leukemia Cell Lines

Background: The cytidine nucleoside analogs azacitidine (AZA) and decitabine (DAC) are used for the treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Few non-clinical studies have directly compared the mechanisms of action of these agents in a head-to-head fashion, and the agents are often viewed as mechanistically similar DNA hypomethylating agents. To better understand the similarities and differences in mechanisms of these drugs, we compared their in vitro effects on several end points in human AML cell lines. Methodology/Principal Findings: Both drugs effected DNA methyltransferase 1 depletion, DNA hypomethylation, and DNA damage induction, with DAC showing equivalent activity at concentrations 2- to 10-fold lower than AZA. At concentrations above 1 mM, AZA had a greater effect than DAC on reducing cell viability. Both drugs increased the sub-G1 fraction and apoptosis markers, with AZA decreasing all cell cycle phases and DAC causing an increase in G2-M. Total protein synthesis was reduced only by AZA, and drug-modulated gene expression profiles were largely non-overlapping. Conclusions/Significance: These data demonstrate shared mechanisms of action of AZA and DAC on DNA-mediated markers of activity, but distinctly different effects in their actions on cell viability, protein synthesis, cell cycle, and gene expression. The differential effects of AZA may be mediated by RNA incorporation, as the distribution of AZA in nucleic aci

Rapamycin (Sirolimus) and Rap-536 Increase Red Blood Cell Parameters through Distinct Mechanisms in Wild-Type and Thalassemic Mice

Luspatercept, a fusion protein composed of a modified activin receptor type-2B (ActRIIB) and IgG1 Fc, is a novel erythroid maturation agent that has been shown to increase red blood cell (RBC) and hemoglobin (Hb) levels. Luspatercept binds to and sequesters several endogenous transforming growth factor-beta superfamily ligands, thereby diminishing Smad2/3 signaling. RAP-536, the murine analog of luspatercept, has been shown to induce erythroblast maturation and increase RBC, Hb, and hematocrit levels in wild-type (WT) mice and in murine disease models of beta-thalassemia and myelodysplastic syndromes. Previous studies have shown that rapamycin (sirolimus), an mTOR inhibitor, improved anemia in a mouse model of beta-thalassemia by activating the autophagy pathway. In this study, rapamycin was tested in combination with RAP-536 by treating WT mice and th3/+ mice (a beta-thalassemia disease model; B6.129P2-Hbb-b1tm1Unc Hbb-b2tm1Unc/J) with vehicle, RAP-536 10 mg/kg twice weekly, rapamycin 4 mg/kg daily, or RAP-536 plus rapamycin for 2 weeks. Single-agent dosing with either RAP-536 or rapamycin increased RBC, Hb, and hematocrit levels significantly compared with vehicle treatment in both WT and th3/+ mice (Figure). The percentage increase in RBC, Hb, and hematocrit levels upon RAP-536 or rapamycin administration was substantially higher in th3/+ mice than in WT mice. Interestingly, much higher and significant increases in RBC and Hb levels in both WT and th3/+ mice, and in hematocrit levels in th3/+ mice, were observed with RAP-536 plus rapamycin, when compared with the single agents. For example, in th3/+ mice, the increase in Hb levels compared with vehicle control was 19.2% with RAP-536 alone, 13.0% with rapamycin alone, and 44.7% with RAP-536 plus rapamycin (Figure). In th3/+ mice, RAP-536 plus rapamycin treatment also significantly reduced spleen enlargement compared with vehicle (P &amp;lt; 0.001). RAP-536 increased and rapamycin decreased overall reticulocyte levels and immature reticulocyte relative percentages in whole reticulocyte populations in the blood of WT mice, suggesting that RAP-536 and rapamycin have different mechanisms of action through which they increase RBC and Hb levels. Altogether, these preclinical results provide a rationale for combining rapamycin, and potentially other mTOR inhibitors, with luspatercept to treat anemia associated with beta-thalassemia. Disclosures Acar: Bristol Myers Squibb: Ended employment in the past 24 months. Jupelli:Bristol Myers Squibb: Current Employment. MacBeth:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Schwickart:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. </jats:sec

Abstract 4457: The HDAC inhibitor romidepsin prevents the emergence of drug-tolerant cancer cells.

Abstract Background: Drug resistance represents a major obstacle to successful cancer treatment. Various resistance mechanisms have been described (e.g., drug efflux pumps). Recent data points to the pre-existence of a subpopulation of cancer cells termed drug-tolerant persisters (DTPs) that exhibit an epigenetically-mediated tolerance to high concentrations of chemotherapeutic drugs (Sharma, 2010). Although DTPs were largely quiescent, a small fraction resumed growth even in the presence of high drug concentrations, giving rise to drug-tolerant expanded persisters (DTEPs). Levels of histone H3 lysine 4 (H3K4) methylation and H3K14 acetylation were decreased in DTPs and DTEPs, and co-treatment (Q3D) of cancer cells with the anticancer drug in combination with histone deacetylase inhibitors (HDACi) inhibited DTP/DTEP growth, implicating an epigenetic mechanism. Purpose: To assess whether the HDACi romidepsin (ROMI) can prevent the emergence of drug-tolerant cancer cells, to determine the stage at which ROMI acts (killing DTPs or preventing DTEP expansion), and to establish the dose-schedule requirements for the effect. Methods: The M14 melanoma and HCC827 NSCLC cell lines were treated in 24-well plates with AZ628 (B-Raf inhibitor) or Erlotinib (ERL; EGFR inhibitor), respectively, alone or in combination with ROMI, TSA, or azacitidine (AZA). ROMI, TSA, AZ628, and ERL were added every 3 days, while AZA was added daily. Cell viability was monitored periodically over 45 days using CellTiter-Glo. In subsequent experiments, reduced schedules from the “continuous” (Q3D for 45 days) schedule were tested, including Q3D schedules given for 1-8 times. To evaluate minimal daily exposure, cancer cells were exposed to ROMI for only 6 hours. Results: Massive cell death was observed after 9 days of treatment of M14 and HCC827 cells with 100X IC50 concentrations of AZ628 and ERL, respectively. With continued AZ628/ERL treatments, however, the few remaining cells (DTPs) began to proliferate, ultimately yielding a large population of AZ628/ERL-resistant cells (DTEPs). “Continuous” (Q3D for 45 days) co-treatment of cancer cells with AZ628/ERL in combination with sub-lethal concentrations of ROMI and TSA, but not AZA, prevented the emergence of DTEPs. Short (6-hour) exposures to higher ROMI and TSA concentrations on the “continuous” schedule were also sufficient to prevent DTEP growth. Interestingly, “continuous” dosing of HDACi was not required to prevent DTEPs from forming, as reducing the schedule to 2 weeks Q3D had the same effect, suggesting that there is a period of susceptibility to the DTP or early DTEP population. Conclusions: ROMI prevents DTEP emergence. Dosing ROMI for 2 weeks Q3D and 6-hour exposures are sufficient for this effect. These results show that co-treatment with ROMI can prevent drug resistance and provide guidance to the dose and schedule requirements to consider for clinical evaluation in this setting. Citation Format: Manith Norng, Aaron N. Nguyen, Jorge F. DiMartino, Kyle J. MacBeth. The HDAC inhibitor romidepsin prevents the emergence of drug-tolerant cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4457. doi:10.1158/1538-7445.AM2013-4457</jats:p

Abstract 4084: Exploring predictive biomarkers of in vitro sensitivity to azacitidine in breast cancer

Abstract The cytidine nucleoside analog azacitidine (AZA) is approved for the treatment of patients with myelodysplastic syndromes and acute myeloid leukemia. In contrast to its activity in patients with these blood cancers, AZA has demonstrated low response rates in clinical trials on unselected patient populations with solid tumors. To realize the potential of AZA in solid tumor patients, we must understand the mechanisms of response and resistance to AZA and develop predictive biomarkers for patient selection. In this study, we sought to identify molecular markers of in vitro sensitivity to azacitidine in breast cancer, using in vitro sensitivity screening of a panel of breast cancer cell lines and gene expression profiling. In vitro sensitivity (EC50) to AZA was determined in 4-day cell viability assays for 21 luminal breast cancer cell lines, while baseline gene expression profiles of cell lines were generated from cells cultured in drug-free growth media. The panel of 21 cell lines displayed markedly differential sensitivity (varied EC50 values) to AZA, revealing both AZA-sensitive and AZA-resistant cells. Analysis of pharmacodynamic (PD) markers for AZA activity revealed substantial reduction of DNA methylation and induction of DNA damage and apoptosis upon AZA treatment of sensitive cell lines in comparison to resistant cell lines. Genes over or under-expressed in association with sensitivity or resistance were identified using two approaches: 1) PAMR analysis, based on fold change across sensitive versus resistant groups (defined by the panel's median EC50) and 2) correlation analysis of baseline gene expression versus AZA EC50. Comparative gene expression analysis of sensitive versus resistant cell lines revealed differential baseline expression of a set of genes involved in multi-drug resistance, cell proliferation and apoptosis. Differential baseline expression of candidate genes in sensitive versus resistant cell lines, as well as their expression after AZA treatment, was validated using QuantiGene mRNA quantification assay. Multi-drug resistance genes were expressed at higher baseline levels in resistant cell lines and showed sustained high expression even after AZA treatment, consistent with a role in AZA resistance. A subset of genes, previously reported as putative tumor suppressors that are silenced by DNA methylation in breast cancer, were found to be under-expressed in sensitive cell lines. AZA treatment resulted in DNA hypomethylation and subsequent re-expression of such genes indicating their predictive value and plausible role in mediating AZA-sensitivity. Taken together, our data provide insights into possible molecular mechanisms and corresponding predictive biomarkers mediating sensitivity to AZA. Further validation of these candidate predictive biomarkers may provide the basis for a hypothesis driven clinical study of AZA in breast cancer or other solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4084. doi:1538-7445.AM2012-4084</jats:p

Lenalidomide Promotes Degradation of Casein Kinase 1a (CK1a) through Cereblon: Implications for the Efficacy of Lenalidomide in MDS and AML

Abstract Background: Lenalidomide has broad clinical activity in hematologic malignancies, including lymphoid malignancies (multiple myeloma (MM), non-Hodgkin’s lymphomas, B cell chronic lymphocytic leukemia) and myeloid malignancies (myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML)). Lenalidomide’s molecular mechanism involves modulation of the cullin 4 RING E3 ubiquitin ligase complex (CRL4-CRBN), with downstream effects on protein homeostasis. The binding of lenalidomide to CRL4-CRBN promotes ubiquitination and degradation of Aiolos and Ikaros in B cell lineages (MM, lymphoma), and regulation of Ikaros has been shown to be a key effector of lenalidomide’s anti-myeloma tumoricidal and T cell immunomodulatory activities. Multiple mechanisms of clinical efficacy have been hypothesized for lenalidomide activity in del(5q) MDS; however, lenalidomide-regulated substrates of therapeutic relevance to myeloid malignancies have not been defined. We undertook a proteomics approach to identify such substrates. Methods: Changes in global cellular protein levels were measured by tandem-mass-tagged proteomics in a del(5q) MDS cell line (MDS-L) and an AML cell line (HNT-34), following treatment with vehicle or 10 uM lenalidomide for 8, 24 and 72 hours. Western blot analysis was used to subsequently validate proteins that were differentially regulated in these lenalidomide-sensitive cell lines. Sensitivity to lenalidomide treatment in a panel of myeloid cancer cell lines was evaluated by tritiated thymidine and/or BrdU assays. Results: Across a panel of myeloid cancer cell lines evaluated for sensitivity to lenalidomide, HNT-34 and MDS-L cells were the most sensitive to lenalidomide, with EC50’s of 0.6 and 1.5 µM, respectively, while other cell lines were predominantly insensitive (EC50 &gt; 10 µM). Proteomic analysis of lenalidomide-regulated proteins in MDS-L cells revealed Ikaros as the most down-regulated protein at 72 hrs (5.3 fold), and CK1α was the second most down-regulated protein (2.8 fold). The decrease of Ikaros and CK1α proteins by lenalidomide was confirmed by Western blot analysis in MDS-L and HNT-34 cells. Ikaros protein levels were reduced by lenalidomide in cell lines that were sensitive or insensitive to lenalidomide, suggesting that modulation of other proteins may be responsible for lenalidomide sensitivity of myeloid cancer cells. Ikaros levels were also decreased by a CRBN-binding glutarimide analog in HNT-34 cells, which were insensitive to the analog, further suggesting that Ikaros was not a substrate of consequence to sensitivity. In contrast, an initial study of five cell lines showed that lenalidomide promoted the greatest degradation of CK1a in the most sensitive lines (HNT-34, MDS-L), but did not degrade CK1a in the most insensitive line (MOLM-13). Mechanistic studies addressing CK1α regulation in HNT-34 cells revealed that CK1α protein levels were reduced by lenalidomide treatment in a time- and dose-dependent manner, with maximal reduction of 3.3 fold observed at 4 hrs using 10 µM lenalidomide, and CK1α degradation observed with lenalidomide at a dose as low as 0.1 µM. Pre-treating HNT-34 cells with the proteasome inhibitor MG-132 stabilized CK1α protein levels in the presence of lenalidomide, demonstrating proteasome-dependent degradation. A competition experiment performed by pre-treating HNT-34 cells with the glutarimide analog that binds CRBN, but does not result in the degradation of CK1a, resulted in CK1α protein stabilization in the presence of lenalidomide, demonstrating CRBN-dependence of the lenalidomide-induced degradation. Degradation of CK1α was also observed in primary peripheral blood mononuclear cells of AML patients treated with lenalidomide. Conclusions: CK1α is a lenalidomide-induced substrate of CRL4-CRBN, with initial links to myeloid cancer cell line sensitivity to lenalidomide. As the CSNK1A1 gene is located at 5q32, a commonly deleted region in MDS, further reduction of haplo-insufficient expression of CK1α is a potential mechanism of sensitivity to lenalidomide in del(5q) MDS. The therapeutic relevance of CK1α regulation by lenalidomide in AML requires further exploration. CK1α gene silencing and additional correlative studies of lenalidomide-induced degradation of CK1α are ongoing to define mechanistic links to myeloid cancer cell sensitivity to lenalidomide. Disclosures Hollenbach: Celgene: Employment, Equity Ownership. Lu:Celgene Corp: Employment. Gandhi:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership. MacBeth:Celgene: Employment, Equity Ownership. </jats:sec

Abstract 4656: Development of a robust preclinical model for studying the mechanism of azacitidine priming for platin-induced cytotoxicity.

Abstract Background: Chemosensitization by azacitidine (AZA) priming has been demonstrated preclinically and is now supported by clinical data, but the underlying mechanism for the priming effect is not well understood. Previous in vitro studies of priming have focused on individual cell lines and specific epigenetically-silenced genes; however, we show that sensitization is not a universal effect of AZA pretreatment in all cancer cell lines or with all combination agents. Studying the molecular differences between cell lines that are sensitized and those that are not may yield insights into the mechanisms of priming. Purpose: To identify solid cancer cell lines that consistently show AZA sensitization to platin-induced cytotoxicity, for future molecular characterization of the priming mechanism. Methods: Ninety-two solid cancer cell lines, from seven indications, were treated with vehicle or 1μM AZA for 24h before being treated with carboplatin (CARB), cisplatin (CIS), or Abraxane (ABX) for 72h. CellTiter-Glo was used to measure cell viability. The fractional product method and normalized dose-response curves were used to characterize the drug combination interaction. In subsequent experiments, various AZA pretreatment durations (4-72h) were tested. DNA was prepared for DNA methylation (LINE-1) analysis, and cell lysates were harvested for DNMT1 Western blotting. Results: In a subset of cancer cell lines, AZA pretreatment resulted in enhanced sensitivity of cells to CARB and CIS. Seven cell lines across 5 cancer indications consistently showed a sensitization effect with AZA priming, decreasing the CARB IC50 by a mean of 59.6% (range 52.9-68.4%) and CIS IC50 by a mean of 44.6% (range 36.2-54.6%). Priming for CARB was concordant with priming for CIS, but not for ABX, indicating that the AZA pretreatment does not globally increase the susceptibility of cells to cytotoxic agents through non-specific toxic effects; rather, these results suggest that priming by AZA has specific pharmacodynamic effects unique to the combination agent. Follow-on experiments demonstrated that AZA pretreatment of cells for 18-24h was required for priming, whereas the magnitude of priming was not increased with longer (48-72h) AZA pretreatment durations. Effects of AZA on DNMT1 depletion and DNA hypomethylation were detected in all cell lines tested. The magnitude of these proximal PD effects did not correlate with priming, suggesting that differences in sensitization cannot be attributed to differential drug uptake and DNA incorporation. Conclusions: We identified a panel of solid cancer cell lines that consistently demonstrate sensitization to CARB and CIS with AZA priming. By comparing genetic and epigenetic profiles between these lines and a closely matched set of cell lines that does not show priming, we hope to generate clinically testable hypotheses for predictors of sensitization in patients. Citation Format: Aaron N. Nguyen, Manith Norng, Antonio Luna-Moran, Kyle J. MacBeth, Jorge F. DiMartino. Development of a robust preclinical model for studying the mechanism of azacitidine priming for platin-induced cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4656. doi:10.1158/1538-7445.AM2013-4656</jats:p

Abstract 2280: Functional characterization of combining epigenetic modifiers azacitidine and AG-221 in the TF-1:IDH2R140Q AML model

Abstract Approximately 15% of AML patients have an IDH2 mutation which leads to production of the oncometabolite 2-hydroxyglutarate (2HG). Accumulation of 2HG inhibits aKG-dependent DNA and histone demethylases, resulting in epigenetic disregulation, which in turn leads to a block in cellular differentiation, promoting AML. AG-221 is a selective inhibitor of IDH2 mutant enzyme and is in development for AML patients carrying IDH2 mutation. Clinical responses to AG-221 therapy have been observed, including decreases in blast percentages and evidence of differentiated functional blood cells; however, preliminary analysis indicated that the mutant allelic burden was not reduced in a majority of subjects treated with AG-221. Azacitidine (AZA) is another epigenetic modifying agent with clinical activity in AML. We hypothesized that combining AG-221 with AZA could synergize in releasing the differentiation block in IDH2-mutant AML cells and enhance cell killing. TF-1:IDH2R140Q are human erythroleukemia cells engineered to express R140Q-mutant IDH2 and model the differentiation block conferred by 2HG accumulation. TF-1:IDH2R140Q cells were treated with AG-221, AZA or the combination of AG-221 + AZA, and measures of cell differentiation (hemoglobinization, KLF1 and HBG qRT-PCR, CD34/CD38 flow cytometry) and death (IncuCyte Zoom caspase 3/7) were evaluated. Measures of cell differentiation were evaluated in TF-1:IDH2R140Q cells, using an in vitro erythropoietin differentiation assay. Single agent AG-221 and AZA increased heme production in a dose-dependent manner, as evidenced by increased red color of cells. With AZA + AG-221 combination, hemoglobinization was greater than with single agents. Dose-dependent increases in RNA expression of differentiation markers KLF1 and HBG were observed with single agents, and the combination resulted in additive, or greater than additive, increases. Quantification of hematopoietic stem /progenitor cell populations demonstrated that as single agents, both AG-221 and AZA reduced CD34+/CD38+ and CD34+/CD38- cell populations, and the combination resulted in additive or greater than additive decreases. Real-time quantification of cell death showed that single agent AG-221 had no effect, while single agent AZA increased apoptosis. Concurrent combination of AZA + AG-221 increased cell death beyond that of single agents. Whole genome expression data (RNA-seq) showed enrichment of differentiation and cell death gene signatures with the combination when compared to single agents alone. We have demonstrated the beneficial effects of combining AG-221 + AZA in the TF-1:IDH2R140Q AML cell line, including greater than additive increases in hemoglobinization and expression of differentiation markers, reduced stem /progenitor cell populations, and potentiation of death. Further exploration of the molecular mechanism of the combination is ongoing. Citation Format: Vivek S. Chopra, Brian Avanzino, Konstantinos Mavrommatis, Adam Olshen, Jorge DiMartino, Kyle J. MacBeth. Functional characterization of combining epigenetic modifiers azacitidine and AG-221 in the TF-1:IDH2R140Q AML model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2280.</jats:p

Lenalidomide as a disease-modifying agent in patients with del(5q) myelodysplastic syndromes:linking mechanism of action to clinical outcomes

Deletion of the long arm of chromosome 5, del(5q), is the most prevalent cytogenetic abnormality in patients with myelodysplastic syndromes (MDS). In isolation, it is traditionally associated with favorable prognosis compared with other subtypes of MDS. However, owing to the inherent heterogeneity of the disease, prognosis for patients with del(5q) MDS is highly variable depending on the presence of factors such as additional chromosomal abnormalities, &gt; 5 % blasts in the bone marrow (BM), or transfusion dependence. Over recent years, the immunomodulatory drug lenalidomide has demonstrated remarkable efficacy in patients with del(5q) MDS. Advances in the understanding of the pathogenesis of the disease have suggested that lenalidomide targets aberrant signaling pathways caused by haplosufficiency of specific genes in a commonly deleted region on chromosome 5 (e.g., SPARC, RPS14, Cdc25C, and PP2A). As a result, the agent specifically targets del(5q) clones while also promoting erythropoiesis and repopulation of the bone marrow in normal cells. This review discusses recent developments in the understanding of the mechanism of action of lenalidomide, and how this underlies favorable outcomes in patients with del(5q) MDS. In addition, we discuss how improved understanding of the mechanism of disease will facilitate clinicians' ability to predict/monitor response and identify patients at risk of relapse.</p

Abstract 191: Azacitidine induces differentiation of acute myeloid leukemia cell lines along the granulocytic/monocytic lineage

Abstract Acute myeloid leukemia (AML) is characterized by blast cells that are unable to mature into functional, terminally-differentiated hematopoietic cells. Inducing leukemic cells to differentiate restores a natural cell death program and inhibits proliferation. Azacitidine (AZA; 5-azacytidine; Vidaza) is a cytidine nucleoside analog used clinically for the treatment of myelodysplastic syndromes (MDS) and AML. AZA therapy was recently shown to significantly increase median overall survival in higher-risk MDS and World Health Organization AML (20-30% bone marrow blasts) patients compared with conventional care regimens, and a phase III clinical trial of AZA in patients with more advanced AML has been activated. We have shown previously that AZA induces dose-dependent cytotoxicity to AML cell lines; however, at sub-micromolar AZA concentrations, complete cell kill is not achieved. To explore an additional anti-leukemic mechanism of AZA in AML, we assessed the effect of AZA on induction of AML cell differentiation in vitro. AML cell lines, encompassing several FAB classifications, were evaluated, using all-trans retinoic acid (ATRA) and 1,25-dihydroxyvitamin D3 (VD3), two potent inducers of AML cell differentiation, as control compounds. Differentiation along the granulocytic/monocytic lineage was assayed by CD11b expression (antigen detection by flow cytometry and mRNA by Luminex) and nitroblue tetrazolium (NBT) reduction. ATRA, VD3, and AZA induced CD11b RNA and protein expression and NBT reduction in HL-60 and AML-193 cell lines. Gene expression profiles (GEPs) in HL-60 and AML-193 cells revealed significant overlap in the genes regulated by AZA-treatment vs. VD3- or ATRA-treatment. GEPs of HL-60 and AML-193 cells treated with AZA strongly correlated with publicly-available gene sets representing differentiated eosinophils, neutrophils, and monocytes, but negatively correlated with those of differentiated erythrocytes and megakaryocytes. Similar studies in primary AML cells are ongoing. Our results demonstrate that AZA can induce cellular differentiation of AML cell lines along the granulocytic/monocytic lineage in vitro, and suggest that cellular differentiation may contribute as one of multiple mechanisms of AZA's anti-leukemic activity in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 191.</jats:p

Abstract 1050: Extended treatment with azacitidine maintains low DNMT1 levels and DNA methylation in MDA-MB-231 cells in vitro and in vivo

Abstract Background: Azacitidine (AZA) is approved for treatment of patients with myelodysplastic syndromes or WHO-defined acute myeloid leukemia with multi-lineage dysplasia (&amp;lt;30% blasts). The drug is administered subcutaneously or intravenously once a day during the first 7 days of a 28-day cycle. Clinical trials investigating the use of AZA in solid tumors have been reported, although response rates were poor, possibly due to suboptimal dose and schedule. An oral formulation of AZA is currently being evaluated in a Phase 1 clinical trial in solid tumors, using continuous dosing of single agent oral AZA or intermittent dosing (14 days on, 7 days off) in combination with chemotherapeutics. It is postulated that extended AZA dosing schedules may be optimal for maintaining DNA hypomethylation and inducing cell death, and thus may cause responses in patients with solid tumors. Purpose: To better understand the effects of short-term vs. extended treatment with AZA on pharmacodynamic markers. Methods: The effects of AZA dose and schedule (short-term vs. extended) on pharmacodynamic markers such as DNMT1 depletion, DNA methylation, and apoptosis were evaluated in MDA-MB-231 breast cancer cells in vitro and in vivo. For in vitro experiments, MDA-MB-231 cells were treated daily with 0.1 or 0.3μM AZA for up to 12 days, and harvested at various times during treatment, as well as up to 12 days following treatment. For in vivo studies, MDA-MB-231 tumor-bearing mice were dosed (ip) with 1 or 3mg/kg AZA daily for 3, 7, 14, 21, or 28 days and tumors were harvested during and at several time points after the dosing period. DNA and cell lysates were prepared (from cell pellets or xenograft tumors) for DNA methylation analysis (LINE-1 or EpiTech Methyl qPCR assay) and DNMT1/cleaved-PARP western blotting, respectively. Results: In both in vitro and in vivo studies, AZA caused a rapid (by 8 hours post in vivo dose), dose-dependent depletion of DNMT1 protein; when AZA treatment was halted, DNMT1 protein levels returned to basal levels within 3-4 days. Consistent with these results, AZA in vitro and in vivo caused a dose-dependent decrease in DNA methylation (LINE-1 and gene-specific) and further reduction in DNA methylation with additional days of AZA dosing. In vitro, DNA methylation returned to basal levels upon AZA removal (within 8 days); the kinetics of DNA re-methylation was slower in more hypomethylated DNA. Lastly, apoptosis (PARP cleavage) was not observed in tumors from mice until 14 or 21 days of dosing with 3mg/kg or 1mg/kg AZA, respectively. Studies are underway in other xenograft models to support these findings. Conclusions: Extended AZA treatment maintains low DNMT1 levels and DNA methylation, and induces cell death. These results provide a strong rationale for the use of extended AZA dosing schedules in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1050. doi:1538-7445.AM2012-1050</jats:p