Abstract B26: AdCDKN2aIRESp53: A bicistronic adenoviral vector for glioblastoma gene therapy

Abstract

Abstract One of the hallmarks of tumor cells is the ability to ignore cell death signals, including those that initiate the apoptotic pathway. This ability may stem from the functional loss of p53 and/or CDKN2a activities, a common feature of glioblastoma (GBM), one of the most lethal tumors. In fact, p53 is lost in 50% of cases while loss of CDKN2a is seen in 30% of GBMs. Restoration of the p53 and Rb/CDKN2a pathways should aid in the apoptotic cell response and cooperate to form an effective therapeutic strategy. For example, pharmacologic and gene transfer approaches that target p53 are potent anticancer therapeutics thanks to their strong proapoptotic action as well as their role in responsiveness to chemo- and radiotherapy, among other attributes. p53 gene therapy has been applied clinically and promising results have been achieved for the treatment of GBM. However, p53 may not be sufficient to bring about cell death, requiring additional factors in order to avoid arrest or senescence, responses that have been shown to worsen response to therapy. In order to accomplish the simultaneous transfer of the CDKN2a and p53 genes, we have constructed the bicistronic adenovirus AdCDKN2aIRESp53 that we expect will overcome the barriers to cell death. In parallel, we are also interested in noninvasive and real-time technology that permits monitoring of caspase 3 activity as a reporter of the proapoptotic efficacy of AdCDKN2aIRESp53. In this study, we have used the lentivirus LvGFP-DEVD-ssGluc, developed by the Thannous laboratory. Upon apoptosis, the DEVD peptide is cleaved in response to caspase 3 activation, freeing ssGluc that is secreted and can then be detected in the culture medium. The human GBM cell lines U87 and T98G were transduced with LvGFP-DEVD-ssGluc, followed by sorting until the population of GFP-positive cells reached a purity greater than 95%. Before initiating the gene therapy assays, we determined the transduction parameters, aiming to maximize gene transfer while minimizing toxicity. For the U87 or T98G cell lines, we found that a MOI (multiplicity of infection) of 10 or 30, respectively, resulted in nearly 100% LacZ-positive cells without signs of toxicity. The U87 and T98G cells were transduced with AdLacZ, AdCDKN2a, Adp53, or AdCDKN2aIRESp53 and incubated for 24 h, 48 h, and 72 h before MTT staining. At the latest time point, the bicistronic vector resulted in a 50% decrease in U87 cell staining, while AdCDKN2a or Adp53 reduced staining by 21% or 8%, respectively. In the case of T98G, the bicistronic vector conferred an astonishing 93.5% reduction in staining, yet the transfer of CDKN2a or p53 alone conferred only a 30% decrease in staining in either case. Currently, the use of the caspase reporter system is being explored. While additional experiments are required, we already see a striking difference between the cell lines. U87 (p53wt, CDKN2a-negative) was much less susceptible to the treatment involving the bicistronic AdCDKN2aIRESp53 vector than the T98G (p53-mutant, CDKN2a-negative) cells. We speculate that the p53 status may contribute to this finding. Even so, the use of either p53 or CDKN2a gene transfer alone was relatively ineffective in either cell line as compared to the treatment with the bicistronic vector. This result is consistent with our hypothesis that the remediation of two genes will be better than one. With this promising result, we plan to use this strategy to screen the proapoptotic potential of AdCDKN2aIRESp53 in several cell lines derived from a variety of tumor types. Citation Format: Daniel Vieira C. Oliveira, Eugenia Costanzi-Strauss, Bryan E. Strauss. AdCDKN2aIRESp53: A bicistronic adenoviral vector for glioblastoma gene therapy [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B26.