Abstract 3303: Selective eradication of cancer cells by adenovirus-based delivery of cytotoxic agents: an alternative method for targeting pancreatic cancer.
Authors: Shapira Shiran , Kazanov Dina , Arber Nadir , Kraus Sarah R. 
Published: August 2014

Background: Most cases of pancreatic cancer (PC) are diagnosed at the metastatic stage and current therapies are inefficient. K-Ras mutations are present in 95% of the cases. Ras is a difficult target to inhibit thus, we propose an alternative strategy which does not inhibit the Ras pathway but exploits it. We have previously shown that recombinant adenovirus, which carries the pro-apoptotic PUMA gene under the control of Ets and AP1/Ras- responsive elements (RREs; Py2-SV40-PUMA), suppressed the growth of cancer cells expressing mutated Ras. We have constructed additional vectors; Py4-; Py5-SV40-PUMA containing several RREs repeats, which were proven effective in selectively targeting Ras-mutated tumor cells (Naumov et al., 2012; Lisiansky et al., 2012). In this study, we utilized a MazE-MazF (MazEF) unique toxin-antitoxin (TA) system encoded from the E. coli genome. Under silent conditions the antitoxin (antidote) inhibits the toxin and the toxin-antidote complex acts as a repressor for the TA operon, whereas in activation conditions, proteolytic degradation of the antidote outpaces its synthesis.

Aim: Improve the therapeutic efficacy and specificity of the adenoviral vectors by substituting the lethal gene with more toxic agents that are highly regulated.

Methods: To achieve efficient DNA delivery into mammalian cells; the ribonuclease MazF-mCherry fusion gene, truncated derivative of Pseudomonas exotoxin (PE38), the full regulated cassette encoding for the MazEF TA system, and the corresponding control vectors were cloned into a “first generation” ΔE1/ΔE3 human type-5 adenoviral-vector under the control of RRE or SV40 minimal promoter. Virus particles were propagated in HEK293 toxin-resistant packaging cells. Tumor cells harboring wild type (WT) and mutated K-Ras were used to test inhibition of cell proliferation, viability, and toxin-expression upon treatment. Apoptosis was detected by FACS using the RedDot 2 and Annexin V stains, measuring the fraction of dead cells.

Results: Two modified helper cell lines and efficient vectors for targeted gene delivery were established. Virus particles were produced and their potency was tested. Specific massive cell death (&gt;50%) at low MOIs was induced in cells exhibiting activated K-Ras upon treatment, compared to WT-K-Ras cells. Induction of cell death was measured qualitatively by fluorescent microscopy and quantified by the MTT assay. Viral infection induced a marked inhibition of cell growth and induction of apoptosis in cells expressing high levels of Ras activity whereas WT-K-Ras cells remained unaffected.

Conclusions: These novel adenoviral vectors carrying either, PE38, MazF or MazEF genes targeting the K-Ras pathway can serve to selectively and efficiently kill PC cells harboring K-Ras mutations while sparing normal cells with WT Ras, thereby improving the outcome of this devastating disease.

Citation Format: Shiran Shapira, Dina Kazanov, Nadir Arber, Sarah R. Kraus. Selective eradication of cancer cells by adenovirus-based delivery of cytotoxic agents: an alternative method for targeting pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3303. doi:10.1158/1538-7445.AM2013-3303