Targeting the Epigenome for the Treatment and Prevention

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The emerging relationships between epigenetics and malignant transformation provide impetus for the use of chromatin remodeling agents for lung cancer therapy. During the past year my research has continued to focus on:1.Evaluation of gene expression in lung and esophageal cancer and malignant pleural mesothelioma (MPM) cells mediated by DNA demethylating agents and histone deacetylase (HDAC) inhibitors.2.Examination of gene expression profiles in cultured cancer cells and primary cancer specimens following exposure to chromatin remodeling agents.3.Investigation of the mechanisms by which inhibitors of DNA methyltransferase (DNMT) and HDAC activity mediate growth arrest and apoptosis in thoracic malignancies. Previously, we reported that the DNA demethylating agent, 5 aza-2'deoxycytidine (DAC) and the HDAC inhibitor, Depsipeptide FK228 (DP) synergistically induce apoptosis, and markedly enhance expression of the NY-ESO-1 cancer-testis antigen (CTA) preferentially in cancer cells, facilitating their recognition by cytolytic T lymphocytes (CTL) specific for this CTA. Furthermore, we have observed that adoptive immunotherapy targeting a CTA induced by DAC in vivo diminishes pulmonary metastases in a syngeneic murine tumor model. These preclinical data provided the rationale for several protocols have been conducted in the Thoracic Oncology Section, Surgery Branch, NCI in an attempt to recapitulate in clinical settings drug exposure conditions that mediate apoptosis and CTA induction in cultured lung cancer cells.In a phase I dose-escalation study, 34 patients received 72h DAC infusions administered on days 1-4 of a 35 day cycle; in a phase II trial, 19 patients received 4h DP infusions administered at the maximum tolerated dose (MTD) on days 1 and 7 of a 21 day cycle. Most recently, 24 patients have been enrolled on a phase I dose-escalation study evaluating sequential DAC/DP therapy, in which DAC is administered on days 1-4, and DP is administered on days 4 and 10 of a 35 day cycle. Clinical toxicities, and response to therapy have been assessed using standard CTCAE and RECIST criteria, respectively. Plasma DAC and DP levels have been evaluated by LC-MS and HPLC techniques. Quantitative RT-PCR, methylation-specific-PCR, immunohistochemistry, and ELISA techniques have been used to assess a variety of molecular endpoints in pre-and post-treatment tumor biopsies and sera. Micro-array techniques have been used to comprehensively examine gene expression profiles in laser-captured tumor cells from FNAs of 20 patients, including 4 individuals receiving DAC infusions, 4 receiving DP infusions, and 12 receiving sequential DAC/DP therapy. Results of these arrays have been compared to array data from laser-captured tumor cells and adjacent histologically normal bronchial epithelia from 20 patients undergoing lung cancer resections on the Thoracic Surgery Service.No objective clinical responses have been observed; however, several patients have exhibited prolonged stabilization of disease following DAC, DP, or sequential DAC/DP therapy. Plasma DAC and DP concentrations typically exceeded threshold levels for gene induction and apoptosis in cultured lung cancer cells. Approximately 40- 50% of patients receiving DAC or DP infusions exhibited induction of NY-ESO-1, p16, or p21 expression in tumor biopsies; several individuals developed antibodies against NY-ESO-1 following drug treatment. Long-oligo array analysis revealed complex, heterogeneous responses to DAC, DP, and DAC/DP in lung cancer specimens. Interestingly, DAC as well as DP appeared to shift gene expression from a malignant profile to one more closely related to normal bronchial epithelial cells. Despite the apparent synergy of DAC and DP in vitro, the response to sequential DAC/DP in vivo was highly complex, possibly due to intra-tumoral drug concentrations, or influence of stromal elements on cancer cell gene expression.