577 Pharmacological Reactivation of Mutant p53 by PRIMA-1 Induces Apoptosis and Enhances Chemotherapeutic Cytotoxicity in Pancreatic Cancer Cells

Poster Session – Phase 0, I and II 577 POSTER Pharmacological Reactivation of Mutant p53 by PRIMA-1 Induces Apoptosis and Enhances Chemotherapeutic Cytotoxicity in Pancreatic Cancer Cells P. Izetti1 , A. Hautefeuille2 , A.L. Abujamra1 , C. Brunetto de Farias1 , R. Roesler1 , G. Macedo1 , P. Hainaut3 , G. Lenz4 , A.B. Osvaldt1 , P. Ashton-Prolla1 . 1 Hospital de Cl´ınicas de Porto Alegre, Porto Alegre-− RS, Brazil; 2 International Agency for Research on Cancer, Lyon, France; 3 International Prevention Research Institute, Lyon, France; 4 Universidade Federal do Rio Grande do Sul, Porto Alegre-− RS, Brazil Background: Inactivating TP53 mutations are common events in different types of cancers, including pancreatic adenocarcinoma, where it occurs in as much as 70% of cases. The small molecule PRIMA-1 (p53 reactivation and induction of massive apoptosis) has been shown to selectively induce apoptosis in tumor cells by reactivating some p53 mutants, but its effect in pancreatic cancer is still unclear. Methods: PANC-1 and BxPC-3 (mutant TP53) and CAPAN-2 (wild-type TP53) pancreatic cell lines were used as in vitro models in this study. We tested the effects of PRIMA-1 on cell viability (MTT assay), apoptosis (nucleus staining and AnnexinV/FITC-FACS), cell cycle (BrdU incorporation in DNA synthesizing cells and PI staining) and expression of several proteins by Western blotting. PANC-1 cell lines were transfected with siRNA to TP53 (sip53) to verify if responses were p53-dependent. Effects on cell viability were also evaluated in combination with different chemotherapeutic agents. Results: PRIMA-1 selectively induced apoptosis in mutant p53 cells compared to wild-type cells and this effect was concomitant with an increase in the levels of MDM2, Bax, cleaved caspase-3 and, interestingly, a decrease of p21. Treatment with PRIMA-1 for 12h induced a G2/M arrest and a 50% reduction in DNA synthesis was detected after 24h of treatment. p53 silencing in PANC-1 cells decreased the cytotoxicity of PRIMA-1 and N-acetylcysteine completely blocked PRIMA-1-induced growth suppression and apoptosis, suggesting that PRIMA-1 exerts its effect at least in part via restoring redox-dependent effects to mutant p53. In combination therapy, PRIMA-1 enhanced the anti-tumor activity of cisplatin, gemcitabine, doxorrubicin and bortezomib against pancreatic cancer cells. Conclusions: Our data indicate that PRIMA-1 induces apoptosis in TP53 mutant pancreatic cancer cells by promoting the re-activation of p53 and inducing proapoptotic signaling pathways. The p53-reactivating agent PRIMA-1 may be clinically useful against pancreatic cancer, in combination with chemotherapeutic drugs. 578 POSTER High Content Analysis of the Epithelial-Mesenchymal Transition to Identify Novel Targets in Cancer Progression S. Lamouille1 , R. Judson1 , R. Blelloch1 , R. Derynck1 . 1 University of California, Cell and Tissue Biology, San Francisco, USA Precise spatial and temporal orchestration of the epithelial-mesencymal transition (EMT) is critical for proper development. Further, uncontrolled reactivation of EMT in the adult significantly contributes to the dissemination of cancer cells and the formation of metastases. EMT is characterized by a complex transcriptional reprogramming that leads to loss of epithelial markers such as the junctional protein E-cadherin, and gain of mesenchymal markers such as the extracellular matrix component fibronectin. In addition, EMT is accompanied by dramatic cytoskeletal and morphological changes, and the acquisition of migratory and invasive behavior. The transforming growth factor-beta (TGF-beta) is considered one of the major inducer of EMT during development as well as during cancer progression. The purpose of this study is to develop high content analyses to quantify subtle changes in cellular morphology and EMT marker expression for use in high-throughput screens for molecules that regulate this critical process. We used the murine mammary gland epithelial cells NMuMG as a model for TGF-beta-induced EMT. Using high content full well image analysis, we were able to measure EMT in 96-well plates by immunostaining for E-cadherin and fibronectin, and membrane staining to assess the cell morphology. Using this assay, we found two microRNAs that regulate TGF-beta-induced EMT (one was found to enhance EMT, and one was found to inhibit EMT). We then screened a library of siRNAs against the predicted targets of these two micro-RNAs and identified novel mRNA networks that regulate EMT. Furthermore, we find evidence to suggest that cell populations can stably exhibit various degrees and combinations of epithelial and mesenchymal characteristics, consistent with the recently published model that cell identity is not binary, but rather represents a spectrum of cell states. In conclusion, we found a fast and reliable assay to measure EMT and identify novel targets in cancer progression. This assay will provide a potent tool to screen for new drugs that could block the metastatic potential of cancer cells.

Friday 9 November 2012 177

Phase 0, I and II 579 POSTER Phase I and Pharmacokinetic (PK) Study of Pazopanib in Combination with Two Schedules of Docetaxel (D) in Patients (pts) with Advanced Solid Tumors P. Hamberg1 , R.H. Mathijssen1 , P. de Bruijn1 , D. van der Biessen1 , W.J. Loos1 , S. Sleijfer1 , J. Verweij1 , M.J. de Jonge1 . 1 Erasmus Medical Center Rotterdam Daniel den Hoed Cancer Center, Medical Oncology, Rotterdam, The Netherlands Background: Pazopanib (P) is a TKI of PDGFR, VEGFR and KIT. This study aimed to determine the recommended phase II dose (RP2D) of P combined with D. To assess the impact of scheduling on tolerability, 3-weekly and weekly schedules of D were explored. Methods: Pts with progressive disease without standard therapy options, PS of 0−1 and good organ function were eligible. Pts received daily P with either 3-weekly D (D3w) or weekly D (days 1, 8, 15) q 4 weeks (D1w). P and D were alternately escalated in serial cohorts of 3−9 pts. If in 3−6 pts dose-limiting toxicity (DLT) of 33.3% within 1st cycle was seen, 3 more pts at that dose level (DL) were enrolled. RP2D was exceeded if in >33.3% of pts DLT occurred. At RP2D, an expansion cohort of 6−9 pts was studied to confirm safety. PK samples of P and D were collected in all pts in the first course, additionally in pts in the expansion phase samples were also collected in the second course. This allowed intra-individual comparison of D-PK with or without P and vice versa. Results: For D3w (23 pts) RP2D was P 400 mg and D 50 mg/m2 . Combinations of 600/50 and 400/60 P/D were not tolerated. At RP2D 1 DLT occurred in 1/15 pts: grade 5 cardiovascular toxicity. Adverse events are shown in table 1. Intrapatient comparison for PK showed a lower docetaxel clearance (CLD ) for D+P compared to D alone (mean (SD): 41 (7) vs 57 (8) L/h; p = 0.019). The Cmax of D during D+P is higher then during D alone; (mean Cmax (SD) 2085 (559) vs 1537 (310) ng/mL (p = 0.075). In D1w (14 pts) P 400 mg and D 20 mg/m2 was tolerable (1 DLT in 6 patients: dizziness requiring omission of D day 15) and the next cohort (400/25) is currently enrolling. P 400 mg + D compared to P 200 mg + D led to a lower CLD (mean (SD): 34 (10) vs 56 (18) L/h) as well as to a higher mean Cmax of docetaxel (SD): 933 (201) vs 684 (180) ng/mL. Table 1. Adverse events occurring in >30% of pts in D3w

Neutropenia Anemia Elevated AST Elevated ALT Nausea Vomiting Diarrhea Oral Hypertension Neurotoxicity Fatigue

All grades

Grade 3−4

80 95 65 50 65 35 65 50 30 50 100

70 5 5 5 0 5 0 10 5 0 15

Conclusions: Pazopanib combined with 3-weekly Docetaxel is well tolerated if doses are set at P 400 mg and D 50 mg/m2 . The RP2D for the combination with weekly D is to be determined yet. Preliminary analyses of the PK suggest a decreased CL of D due to exposure to P, an effect that seems P dose-dependent. However, interpatient variability is high. Reasons for these PK effects require further study. 580 POSTER First-in-human, Safety, Pharmacokinetic (PK) and Pharmacodynamic (PD) Results From a Phase I Study of a Selective BRAF Inhibitor (BRAFi) RG7256 in Patients with BRAF V600-mutated Advanced Solid Tumors R. Dienstmann1 , U. Lassen2 , J. Cebon3 , J. Desai4 , M.P. Brown5 , S. Evers6 , F. Su7 , W. Zhang8 , V. Meresse9 , J. Tabernero1 . 1 Hospital Valle De Hebron, Medical Oncology, Barcelona, Spain; 2 Rigshospital, Medical Oncology, Copenhagen, Denmark; 3 Austin Hospital, Oncology Unit, Heidelberg, Australia; 4 Royal Melbourne Hospital, Institute for Cancer Research, Parkville, Australia; 5 Royal Adelaide Hospital, Cancer Center, Adelaide, Australia; 6 Oncology Roche, Pharma Research & Early Development, Schlieren, Switzerland; 7 Hoffmann La Roche, Discovery Oncology, Nutley, USA; 8 Hoffmann La Roche, Pharma Research & Early Development, Basel, Switzerland; 9 Oncology Roche, Pharma Research & Early Development, Basel, Switzerland Background: Vemurafenib (Vem) is the first BRAFi to demonstrate significant survival benefit in BRAF V600-mutated metastatic melanoma