Abstract 3532: Selective eradication of colorectal cancer cells by adenovirus-based delivery of toxins

Abstract

Abstract Background: K-Ras gene mutation is an early event in the development of colorectal cancer (CRC) and occurs in ∼50% of CRC cases. We propose a strategy that exploits the Ras hyperactive pathway, rather than inhibiting it. We have previously shown that recombinant adenovirus, carrying a pro-apoptotic gene under the regulation of Ras-responsive elements (RREs; Ets/AP1), suppressed the growth of tumor cells displaying hyperactive K-Ras (Naumov, 2012; Lisiansky, 2012). Toxin-antitoxin (TA) systems are evolutionarily successful entities, prevalent in lower organisms that play important roles in a diverse range of cellular activities Aim: To establish an improved tightly regulated RRE-cassette based on the bacterial MazEF TA system. Methods: Efficient vectors for targeted gene delivery were constructed and cloned into a “1st generation” ΔE1/ΔE3 human type-5 adenoviral vector. Virus particles were produced their titer was calculated, by the end-point dilution assay, and tested for their potency. Cell death was measured qualitatively by fluorescent microscopy and colony formation assay, and quantified by MTT. Apoptosis was determined by FACS using annexin V and RedDot2 dyes. In vivo tumor formation was examined in xenografts. Ad-Py4-SV40-MazEF and Ad-ΔPY4-CMV-MazEF viruses (1×109pfu) or PBS were administrated i.p. twice with a 3-day interval between injections. Results: Adenovirus treatment induced massive cell death, in a dose-dependent manner; 73% in K-Ras-mutated compared to 22% in WT K-Ras tumor cells, at 10 MOI. The cytotoxic effect was confirmed qualitatively by colony formation assay. In the absence of RRE, MazE anti-toxin expression was increased, protecting normal cells from any possible system leakage and confirming the selectivity, specificity, and safety of the targeting system. FACS analysis showed massive cell death, 55% apoptosis and 82% dead cells, following infection with the full TA-encoding viruses. In control RRE-lacking viruses a modest toxicity was seen (18% and 10%, respectively). Impressive tumor shrinkage was demonstrated in vivo following treatment with Ad-Py4-SV40-MazEF-encoding adenovirus (61%) without any toxic side effects. The tumor volume in the Ad-ΔPY4-SV40-MazEF-treated mice (control group) was reduced only by 27% (p<0.05). No growth inhibition was seen following injection of PBS. Conclusions: A proof-of-concept for a novel cancer gene therapy by exploiting aberrant K-Ras hyperactive pathway was successfully demonstrated. The lack of toxicity holds promise for effective and safe therapy of human cancers carrying K-Ras mutations. Note: This abstract was not presented at the meeting. Citation Format: Shiran Shapira, Assaf Shapira, Dina Kazanov, Ilana Nabiochtchikov, Nadir Arber, Sarah R. Kraus. Selective eradication of colorectal cancer cells by adenovirus-based delivery of toxins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3532. doi:10.1158/1538-7445.AM2015-3532