Normand Richard

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 µM, 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 acid of KG-1a cells was 65∶35, RNA∶DNA.

Immunomodulatory Drug CC-4047 is a Cell-type and Stimulus-Selective Transcriptional Inhibitor of Cyclooxygenase 2

COX2 (prostaglandin G/H synthase, PTGS2) is a well-validated target in the fields of both oncology and inflammation. Despite their significant toxicity profile, non-steroidal anti-inflammatory drugs (NSAIDs) have become standard of care in the treatment of many COX2-mediated inflammatory conditions. In this report, we show that one IMiDs® immunomodulatory drug, CC-4047, can reduce the levels of COX2 and the production of prostaglandins (PG) in human LPS-stimulated monocytes. The inhibition of COX2 by CC-4047 occurs at the level of gene transcription, by reducing the LPS-stimulated transcriptional activity at the COX2 gene. Because it is a transcriptional rather than an enzymatic inhibitor of COX2, CC-4047 inhibition of PG production is not susceptible to competition by exogenous arachadonic acid (AA). The distinct mechanisms of action allow CC-4047 and a COX2-selective NSAID to work additively to block PG secretion from monocytes. CC-4047 does not, however, block COX2 induction in or prostacyclin secretion from IL-1β stimulated human umbilical vein endothelial cells (HUVEC) cells, nor does it inhibit COX1 in either monocytes or HUVEC cells. CC-4047 also inhibits COX2 and PG production in monocytes derived from patients with sickle cell disease (SCD). Taken together, the data in this manuscript suggest CC-4047 will provide important anti-inflammatory benefit to patients and will improve the safety of NSAIDs in the treatment of SCD or other inflammatory conditions.

Histone deacetylase inhibitor MGCD0103 synergizes with gemcitabine in human pancreatic cells

Histone deacetylase inhibitors are a group of recently developed compounds that modulate cell growth and survival. We evaluated the effects of the histone deacetylase inhibitor MGCD0103 on growth of pancreatic carcinoma models following single agent treatment and in combination with gemcitabine. MGCD0103 inhibited tumor cell growth and acted synergistically with gemcitabine to enhance its cytotoxic effects. Gene expression analysis identified the cell cycle pathway as one of the most highly modulated gene groups. Our data suggest that MGCD0103 + gemcitabine might be an effective treatment for gemcitabine‐refractory pancreatic cancer. (Cancer Sci 2011; 102: 1201–1207)

Azacitidine and decitabine have different mechanisms of action in non-small cell lung cancer cell lines

Azacitidine (AZA) and decitabine (DAC) are cytidine azanucleoside analogs with clinical activity in myelodysplastic syndromes (MDS) and potential activity in solid tumors. To better understand the mechanism of action of these drugs, we examined the effects of AZA and DAC in a panel of non-small cell lung cancer (NSCLC) cell lines. Of 5 NSCLC lines tested in a cell viability assay, all were sensitive to AZA (EC50 of 1.8-10.5 µM), while only H1299 cells were equally sensitive to DAC (EC50 of 5.1 µM). In the relatively DAC-insensitive cell line A549, both AZA and DAC caused DNA methyltransferase I depletion and DNA hypomethylation; however, only AZA significantly induced markers of DNA damage and apoptosis, suggesting that mechanisms in addition to, or other than, DNA hypomethylation are important for AZA-induced cell death. Cell cycle analysis indicated that AZA induced an accumulation of cells in sub-G1 phase, whereas DAC mainly caused an increase of cells in G2/M. Gene expression analysis of AZA- and DAC-treated cells revealed strikingly different profiles, with many genes distinctly regulated by each drug. In summary, while both AZA and DAC caused DNA hypomethylation, distinct effects were demonstrated on regulation of gene expression, cell cycle, DNA damage, and apoptosis.

Abstract LB-59: Amrubicin, a third-generation synthetic anthracycline, is active in anthracycline-resistant tumors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Amrubicin, a third generation synthetic anthracycline and a potent topoisomerase (topo) II inhibitor, is approved in Japan for the treatment of lung cancer (both small-cell and non-small cell) and is being evaluated in a phase III trial in the North America and Europe for the second line treatment of SCLC. The aim of these studies was to evaluate the cross-resistance of amrubicin with other anthracyclines (doxorubicin, epirubicin) and the topo-II inhibitor etoposide, and to identify potential mechanisms of resistance to amrubicin. The doxorubicin-resistant cell lines H69-AR, MES-SA-DX5 & NCI-ADR/RES were confirmed to be resistant to doxorubicin (∼25-fold relative resistance), however, their resistance to amrubicin was low or absent (∼2-fold relative resistance). Additionally, a number of primary human ovarian and breast tumor explants that were resistant to doxorubicin and/or etoposide retained sensitivity to amrubicin. Unlike doxorubicin, amrubicin induced DNA damage, G2M cell cycle arrest and apoptosis in both doxorubicin-sensitive and -resistant lines at or below clinically relevant plasma concentrations (∼10 uM). Furthermore, in contrast to doxorubicin, fluorescence time-lapse imaging demonstrated relatively small differences in amrubicin uptake and accumulation kinetics in doxorubicin-sensitive and resistant cell lines. Using gene expression profiling studies, we showed that a greater number of genes were regulated by amrubicin than by doxorubicin in both OVCAR8 and NCI-ADR/RES lines. Amrubicin is a moderate P-glycoprotein (Pgp) substrate; however, we demonstrated that the inhibitory effect and accumulation of amrubicin is not solely modulated by Pgp in cell lines over-expressing efflux pumps including Pgp. We postulate that the high intracellular accumulation and retention of amrubicin is a result of rapid influx due to the high intrinsic permeability and lipophilic properties of amrubicin. This may explain why amrubicin overcomes pleiotropic drug resistance. In summary, we demonstrated that amrubicin has a distinct mode of action compared with other anthracyclines, is less susceptible to typical anthracycline resistance mechanisms and therefore, may be useful in the treatment of anthracycline-resistant tumors. 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 LB-59.

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

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 AZA9s 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.