Abstract A4: Identification of novel histone deacetylase inhibitor-regulated drug targets and a phase I/II trial of vorinostat in combination with 5-fluorouracil in metastatic colorectal cancer

Abstract

Abstract Histone deacetylase inhibitors (HDACi) represent a novel class of agents that are undergoing clinical evaluation in colorectal cancer (CRC). Previously, we identified that HDACi including vorinostat (Vor) and LBH589 induced downregulation of the 5-FU target enzyme thymidylate synthase (TS) resulting in the sensitization of CRC cells to 5-FU. There were two goals in the present study: Firstly to identify additional genes in CRC cells that are differentially regulated by two clinically advanced HDACi, Vor and LBH589, furthering our understanding of their mechanism of action and to identify potential targets for novel HDACi-based drug combinations. Secondly, to extend our preclinical observations and evaluate the safety and feasibility of combining Vor with 5-FU in a phase I/II trial in patients with metastatic CRC (mCRC) and elevated intratumoral TS who had failed 5-FU-based therapy. HCT116 and HT29 CRC cells treated with Vor and LBH589 were analyzed using the Illumina Human-6 V2 BeadChip array. Differentially expressed genes (DEGs) were subject to hierarchical clustering, venn diagram analysis and interpreted using Ingenuity® Pathway Analysis. Results from microarray analysis were validated by quantitative PCR (qPCR) and analyzed in a panel of 8 CRC cell lines in vitro and in HCT116 and HT29 xenograft models. Bioinformatic analysis of the microarray results revealed significant similarity in DEGs following treatment with the two HDACi tested within each cell line. However, analysis of in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 DEGs modulated by both Vor and LBH589 were identified in both CRC cell lines analyzed and further validated in a panel of 8 CRC cell lines and in HCT116 and HT29 xenograft models treated with LBH589. This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel HDACi-based drug combinations in CRC. We concurrently conducted a Phase I/II clinical trial to determine the safety and feasibility of combining Vor with 5-FU in patients with mCRC and elevated intratumoral TS. Patients who had failed all standard therapeutic options were eligible. Intratumoral TS mRNA expression and peripheral blood mononuclear cell (PBMC) histone acetylation were measured before and after 6 consecutive days of Vor treatment at 400 mg PO daily. 5-FU/LV were given on days 6 and 7 and repeated every 2 wks, along with continuous daily Vor. Ten patients were enrolled and three dose levels were explored in the phase I portion of the study. Two dose-limiting toxicities (DLTs) were observed at the starting dose level, resulting in de-escalation to levels −1 and −2. Given the occurrence of two DLTs at each of the dose levels, we were unable to establish a maximum tolerated dose (MTD). Two patients achieved significant disease stabilization for 4 and 6 months. Grade 3 and 4 toxicities included fatigue, thrombocytopenia and mucositis. Intratumoral TS downregulation ≥50% was observed in one patient only. Acetylation of histone 3 was observed in PBMCs following Vor treatment. The study failed to establish a MTD and was terminated. The presence of PBMC histone acetylation indicates biological activity of Vor, however, consistent reductions in intratumoral TS mRNA were not observed. Alternate Vor dose-scheduling may alleviate the toxicity, achieve optimal TS downregulation and improve efficacy. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A4