138 The MDM2 Inhibitor Nutlin-3 But Not the p53 Activator RITA Induces Loss-of-function p53 Mutations

Poster Session – Drug Resistance and Modifiers supports the hypothesis that inhibited PARP1 is a component of the toxic DNA damage lesion. Notably, we did not isolate mutations in any genes encoding other PARP proteins, implicating PARP1 as the major mediator of this effect. Our results suggest that toxicity to normal cells in patients treated with PARP inhibitors is likely to be an on-target effect mediated via PARP1, and also identify PARP1 mutation as a possible route of resistance to PARP inhibitors in tumours. Haploid screening will be a powerful way of identifying genetic determinants of toxicity and resistance to other drugs. 138 POSTER The MDM2 Inhibitor Nutlin-3 But Not the p53 Activator RITA Induces Loss-of-function p53 Mutations M. Michaelis1 , F. Rothweiler2 , S. Barth3 , J. Cinatl2 , M. van Rikxoort2 , ¨ 5 , D. Speidel6 , J. Cinatl Jr.2 . 1 University A. von Deimling4 , F. Rodel of Kent, School of Biosciences, Canterbury, United Kingdom; 2 Goethe-University, Institut fur ¨ Medizinische Virologie, Frankfurt/Main, Germany; 3 blue-drugs GmbH, Bioinformatics, Frankfurt/Main, Germany; 4 Ruprecht-Karls-University, Department of Neuropathology, Heidelberg, Germany; 5 Goethe-University, Klinik fur ¨ Strahlentherapie und Onkologie, Frankfurt/Main, Germany; 6 Children’s Medical Research Institute, Cell Transformation, Westmead, Australia Background: Non-genotoxic p53 activators are a novel class of anti-cancer drugs. Here, resistance formation to two prototype agents, nutlin-3 (MDM2 inhibitor) and RITA (inhibits the p53−MDM2 interaction by binding to p53), was investigated. Materials and Methods: p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, melanoma) were continuously exposed to increasing concentrations of nutlin-3 or RITA to establish drug-resistant sub-lines. The resulting drug-adapted sub-lines were examined for p53 status, resistance profiles to anti-cancer drugs and radiation, and global gene expression. Results: The adaptation of p53 wild-type cancer cell lines to nutlin-3 resulted in the emergence p53-mutated sub-lines displaying a multi-drug and radiation resistance phenotype. 28 out of 35 nutlin-3-adapted sub-lines harboured various p53 mutations while only 2 out of 28 sub-lines adapted to various cytotoxic drugs harboured p53-mutations. Analysis of UKF-NB-3 and UKF-NB-3r Nutlin10 mm cells (a UKF-NB-3 sub-line harbouring a G245C p53 mutation) by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3r Nutlin10 mm cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3r Nutlin10 mm cells. Transcriptomics analyses indicated similarities between nutlin-3- and cytotoxic drug-adapted cells. A p53-wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in 10 independent experiments. Eight out of 10 resulting sub-lines harboured 6 different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations. In sharp contrast, all 11 UKF-NB-3 sub-lines adapted to RITA retained functional p53. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 investigated nutlin-3-adapted UKFNB-3 sub-lines retained their sensitivity to RITA. Repeated adaptation of the RITA-adapted sub-line UKF-NB-3r RITA10mM to nutlin-3 in resulted in p53 mutations in 4 out of 6 sub-lines. The RITA-adapted UKF-NB-3 sublines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines. These data correlate with the observed chemotherapy and irradiation sensitivity phenotypes. Conclusion: Nutlin-3-adapted cells were frequently characterised by p53 mutations and displayed (independently of their p53 status) a multidrug resistance phenotype. RITA-adapted cells retained functional p53, remained sensitive to nutlin-3, and displayed a less pronounced resistance phenotype than nutlin-3-adapted cells. 139 POSTER Histone Demethylase Inhibitor Overcomes Bortezomib Resistance in Myeloma Cells Y. Terui1 , R. Kuniyoshi1 , A. Tomida2 , K. Hatake1 . 1 Japanese Foundation for Cancer Research, Clinical Chemotherapy, Tokyo, Japan; 2 Japanese Foundation for Cancer Research, Genome Research, Tokyo, Japan Purpose: The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the treatment of multiple myeloma (MM). Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance are poorly understood. However, resistance to bortezomib as a single agent develops in the majority of patients, and activity in other malignancies has been less

Wednesday 7 November 2012

43

impressive. To overcome bortezomib resistance, we compared differential gene expression profiles of bortezomib-resistant IM-9 and bortezomibsensitive IM-9 multiple myeloma cell lines in response to bortezomib. Methods: The differential gene expression profiles of bortezomib-resistant IM-9 and bortezomib-sensitive IM-9 multiple myeloma cell lines in response to bortezomib was performed using Affymetrix GeneChip. To confirm the results, real-time PCR and Western blot analysis were performed. bortezomib-resistant IM-9 cells were treated with or without bortezomib and/or histone demethylase inhibitor, trans-2-phenylcyclopropylamine hydrochloride. Results: At concentrations that effectively inhibited proteasome activity (maximum dose with 100nM), bortezomib induced cell death in bortezomibsensitive IM-9 cells, but not in bortezomib-resistant IM-9. In comparison of differential gene expression profiles between bortezomib-resistant IM-9 and bortezomib-sensitive IM-9 cells, we showed overexpression of KDM3A and KDM5B, which are associated with chromatin-mediated reversible drugtolerant state. Moreover, trans-2-phenylcyclopropylamine hydrochloride overcomes bortezomib resistance in a dose-dependent manner. Conclusion: Histone lysine-specific demethylase inhibitors as combination with bortezomib may be useful for overcoming bortezomib-resistance in myeloma cells. 140 POSTER Unraveling BRAF Inhibitor (PLX4032) Resistance Mechanisms A. Azimi1 , C. Hertzman Johansson1 , M. Pernemalm1 , R. Touminen1 , J. Lehtio¨ 1 , J. Hansson1 , S. Egyhazi1 . 1 Karolinska University Hospital, Oncology & Pathology, Stockholm, Sweden Introduction: The BRAF V600E mutation is observed in about 50% of cutaneous malignant melanomas, and leads to hyper activation of the mitogen-activated protein kinase (MAPK) pathway. Several novel therapeutic agents targeting this pathway are emerging such as the BRAF inhibitor Vemurafenib (PLX4032), which specifically inhibits V600 mutated BRAF proteins. Despite outstanding initial clinical effects on tumor shrinkage recurring tumors that are resistant to Vemurafenib (PLX4032) eventually develop. Material and Methods: To unravel the underlying mechanisms of this acquired resistance, we have established several Vemurafenib/PLX4720 (PLX4032 analog) resistant sub lines of the A375 melanoma cell line (hemozygous for the BRAF V600E mutation). Basal proteome differences between parental A375 and the resistant sub lines were studied using liquid chromatography tandem mass spectrometry based proteomics. Group and pairwise comparison of A375 and the BRAFi resistant cell lines protein profiles using DAVID Functional Annotation analysis have shown alterations in Integrin signaling, phospho proteome, nucleotide transfer, etc. Results: The proteomics results are compared with identified differences in key signaling pathways such as MAPK, Phosphatidylinositol 3-kinases (PI3K), nuclear factor kappa-light-chain-enhancer of activated B cells (NFúB) studied by qPCR pathway arrays. Interestingly, there are several differences observed, including down regulation of the PTEN tumor suppressor gene in a PLX4032/PLX4720 resistant sub line. Moreover, several of the known resistance mechanisms, activating mutations in NRAS or KRAS were not observed in our resistant sub lines. There are 14 proteins as top candidates of BRAFi resistance drivers that are overlapping between univariate and multivariate analysis that are most differentially expressed in resistant sublines compared to original A375. More in depth biological pathway analysis is ongoing with focus on pathway effectors that are overlapping between proteomics and qPCR pathway arrays. Conclusion: So far, up regulation of phospho-ERK and down regulation of PTEN at protein level suggests co-activation of MAPK and PI3K pathways that may be one of BRAF inhibition resistance mechanisms. 141 POSTER MM-121, an Anti-ErbB3 Antibody, Inhibits PI3K/AKT Signaling and Viability in Platinum-resistant Ovarian Cells and in Primary Ascites Derived From Chemo-resistant Ovarian Cancer Patients M. Curley1 , A. Kalra1 , A. Fulgham1 , D. Xiao1 , J. Allen1 , M. Wainszelbaum1 , G. Garcia1 , W. Kubasek1 , G. MacBeath1 . 1 Merrimack Pharmaceuticals, Research, Cambridge, USA Background: Despite initial response to platinum-based drugs and taxanes, most ovarian cancer patients eventually become resistant to these treatments. It has been shown that resistance to platinum-based drugs can arise through upregulation of ErbB3 signaling and activation of the PI3K/AKT pathway. Here, we investigate the role of ligand-mediated signaling as a mechanism of ErbB3 activation and the use of ovarian ascites-derived cells to assess mechanisms of resistance. We also evaluate the ability of MM-121, an anti-ErbB3 antibody, to block ligand-mediated