Poster Session – New Molecular Targets Friday 9 November 2012 163

ADAM17 with a monoclonal antibody may be a new approach to treatTNBC. Compared to low molecular weight ADAM17 inhibitors, a mono-clonal antibody should be more specific. Furthermore, it may display addi-tional anti-tumor activities such as antibody-dependent cellular cytoxicity.Acknowledgement: The authors thank Science Foundation Ireland StrategicResearch Cluster Award (08/SRC/B1410) to MTCI for funding this work.

528 POSTERThe BRD-inhibitor OTX015 Shows Pre-clinical Activity in Diffuse

Large B-cell Lymphoma (DLBCL)

P. Bonetti1, M. Ponzoni2, M.G. Tibiletti3, A. Stathis4, P. Heirat5,E. Zucca4, F. Bertoni1. 1Institute of Oncology Research (IOR), Bellinzona,Switzerland; 2San Raffaele H Scientific Institute, Milan, Italy; 3InsubriaUniversity, Varese, Italy; 4Oncology Institute of Southern Switzerland(IOSI), Bellinzona, Switzerland; 5Onocethix SA, Lausanne, Switzerland

Background: DLBCL is the most common type of lymphoma. Despite thetherapeutic improvements, 40% of patients cannot be cured. Identificationof new treatment modalities is highly needed. Bromodomain-containingproteins exert important roles in the regulation of the chromatin structure.Inhibitors of BRD2/3/4, members of the Bromodomain and Extraterminal(BET) family, have recently shown antitumor activity in different hematologi-cal malignancies directly regulating MYC expression and activity. We reporthere preclinical data obtained with OTX015, a novel selective BRD2/3/4inhibitor, in a large panel of DLBCL cell lines.Material and Methods: Established human DLBCL cell lines derived fromdiffuse large B-cell lymphomas (DLBCL) were treated with increasing dosesof OTX015 (OncoEthix SA) and MTT assays were performed after 72 hoursexposure. For cell cycle analysis cells were stained with propidium iodideand analyzed for DNA content using a FACScan flow cytometer. RNAextracted using the Qiagen RNA easy kit and reverse-transcribed usingthe Superscript First-Strand Synthesis System for RT-PCR kit accordingto the manufacturer’s instructions. RT-PCR was performed on using FastSYBR Green Master Mix on a StepOnePlus Real-Time PCR System.Results: We assessed the anti-proliferative activity of OTX015 in a panelof 13 DLBCL cell lines and observed that the majority (9 of 13) werehighly sensitive, with IC50 between 70 to 500 nM. There was no apparentdifference based upon the cell of origin of the DLBCL cell lines. OTX105severely affected the cell cycle, inducing a G1-arrest in a dose-dependentmanner in five DLBCL cell lines without an increase of cell death. In orderto understand the mechanism of action of OTX015 we assessed MYClevels in five DLBCL cell lines before and after treatment. We observed adose-dependent suppression of MYC by the BRD-inhibitor in 4 of 5 celllines, strongly correlating with G1-arrest. Expression of MYC was reducedby OTX015 within 1 hr after treatment suggesting a direct effect on theMYC locus. To determine whether the suppression of MYC by OTX015was reversible, DLBCL cell lines were treated for 2hr with OTX015 and thenthe inhibitor was removed from the media. We observed a time-dependentrestoration of MYC expression to untreated levels after 2−3 hours. In oneof the most sensitive cell lines no MYC down-regulation was observedafter treatment, suggesting that alternative pathways are affected by BRD-inhibition.Conclusion: OTX015 is a potent BRD-inhibitor with clear anti-proliferativeactivity on a large number of DLBCL cell lines. An important andreversible abrogation of MYC gene expression appears as the possiblemain mechanism of action. These data strongly suggest the opportunity offurther preclinical and clinical evaluation of OTX015 in diffuse large B-celllymphoma.

529 POSTERTargeting SIRT1 Activity: a Novel Strategy for Acute Myeloid

Leukemia Therapy

S.T. Nawrocki1, K.R. Kelly1, C.M. Espitia1, C. Bachier2, W.G. Bornmann3,J.S. Carew1. 1The Institute for Drug Development CTRC at UTHSCSA,Medicine, San Antonio TX, USA; 2The Methodist Specialty & TransplantHospital, BMT, San Antonio TX, USA; 3M.D. Anderson Cancer Center,Experimental Therapeutics, Houston TX, USA

Background: Acute myeloid leukemia (AML) is a highly aggressivedisease and novel strategies are urgently required in order to improve thesurvivorship of patients. The sirtuin deacetylases (SIRTs) are membersof the Class III family of histone deacetylases (HDACs) with essentialroles in the regulation of genes that are critical for longevity, cellproliferation, metabolism, tumor suppression, and apoptosis. ElevatedSIRT expression has been reported in several types of cancer and maypromote pathogenesis and drug resistance by increasing the lifespan andsurvival capacity of malignant cells. However, the therapeutic potentialof disrupting SIRT function as an anticancer strategy remains to berigorously investigated. We hypothesized that SIRT1 is a regulator of AMLpathogenesis that can be targeted for therapeutic benefit.

Methods: We tested our hypothesis by genetically and pharmacologicallyinhibiting SIRT1 activity using shRNA and tenovin-6, respectively, in AMLcells and xenograft mouse models. We quantified the consequential impactof impaired SIRT1 function on cell proliferation and viability, apoptosis, geneexpression, metabolism, and animal survival.Results: We demonstrated that SIRT1 was consistently expressed atsignificantly higher levels in AML cell lines and primary AML blasts ascompared with normal controls. Cells with targeted SIRT1 knockdowndisplayed an altered gene expression and metabolic profiles as comparedwith non-targeted controls. Impaired SIRT1 expression achieved by shRNAsignificantly retarded disease progression in a xenograft mouse model.We investigated the efficacy and pharmacodynamic effects of the smallmolecule SIRT1 inhibitor tenovin-6 in AML cell lines, primary blasts frompatients with AML, and mouse models. Treatment with tenovin-6 inducedapoptosis, disrupted cell cycle progression, and dramatically diminishedAML clonogenic survival. Tenovin-6 promoted a dose-dependent increasein the acetylated levels of several SIRT1-regulated genes and triggered theinduction of their transcriptional targets. The anticancer activity of tenovin-6 was not directly correlated with the status of the SIRT1 target andtherapeutic regulator p53, but differential downstream effects on BH3-onlyapoptotic regulators were observed in cells with intact versus impaired p53function. Targeted knockdown of PUMA with shRNA significantly reducedthe pro-apoptotic effects of tenovin-6, indicating that it is a critical mediatorof its anti-leukemic activity. Administration of tenovin-6 to mice implantedwith AML cells was well-tolerated and led to a highly significant reductionin disease burden and increase in overall survival.Conclusions: Our collective findings demonstrate that SIRT1 is a noveltherapeutic target in AML. Further investigation aimed to elucidate thesafety, efficacy, and mechanism of action of tenovin-6 is warranted.

530 POSTERChk1 Inhibitor Targets Replicative Stress in Melanomas

B. Gabrielli1, K. Brooks1, V. Oakes1, B. Edwards1, J. Chen1,P. Mukhopadhyay1. 1University of Queensland, Diamantina Institute,Brisbane Qld, Australia

There are few effective treatments for metastatic melanoma. Chk1inhibitors are being trialed for their efficacy in enhancing conventionalchemotherapeutic agents, but their effectiveness as single agents isrelatively unexplored. We have examined the effectiveness of two novelChk1 selective inhibitors, AR-323 and AR-678, in a large panel (40) ofmelanoma cell lines and normal cell types in vitro, and investigated themechanism promoting their cytotoxicity in the drug-sensitive cell lines. Wedemonstrate that these drugs display single agent activity, with IC50s inthe nanomolar range. The drugs produce cytotoxic effects in cell lines thatare most sensitive to these drugs, whereas normal cells are only sensitiveto these drugs at the higher concentrations where they have cytostaticactivity. The cytotoxic effect is related to inhibition of S phase Chk1 whichdrives cells prematurely from late S phase, resulting in apoptosis eitherprior to or during an aberrant mitosis. Time lapse imaging has shownthat the timing of cell death is related to the drug sensitivity of the cellline. The sensitivity to the Chk1 inhibitors was correlated with the levelof endogenous DNA damage indicating replicative stress, and increasingendogenous replicative stress increased drug sensitivity. Chk1 inhibitorsare viable single agent therapies which target melanoma cells with highlevels of endogenous DNA damage. This sensitivity suggests that Chk1 isa critical component of an adaptation to replicative stress in these cells. Italso suggests that markers of DNA damage may be useful in identifyingthe melanomas and potentially other tumour types that are more likely tobe sensitive to Chk1 inhibitors as single agents.

531 POSTERCalcium Channel Transient Receptor Potential V6 (TRPV6) as a

Potential Therapeutic Target for Some Breast Cancers

A.A. Peters1, P.T. Simpson2, J.J. Bassett1, J.M. Lee1, S. Song2,M.O. Parat1, S.R. Lakhani3, P.A. Kenny4, S.J. Roberts-Thomson1,G.R. Monteith1. 1The University of Queensland, School of Pharmacy,Brisbane, Australia; 2The University of Queensland, UQ Centre forClinical Research, Brisbane, Australia; 3The University of Queensland,UQ Centre for Clinical Research and School of Medicine, Brisbane,Australia; 4Albert Einstein College of Medicine, Department ofDevelopmental and Molecular Biology, New York, USA

Background: TRPV6 is an important calcium channel, which is highlyselective for Ca2+ and has high basal activity. In prostate tumors TRPV6over-expression is linked to enhanced invasiveness and proliferation. Whilesome studies have demonstrated that TRPV6 is over-expressed in breasttumors the mechanism for TRPV6 over-expression is unknown and a linkbetween TRPV6 expression and breast cancer aggressiveness has notbeen demonstrated.