Oncolytic Adenoviral Mutants with E1B19K Gene Deletions Enhance Gemcitabine-induced Apoptosis in Pancreatic Carcinoma Cells and Anti-Tumor Efficacy In vivo

Abstract

Purpose: Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal.We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency. Experimental Design: Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdDE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts. Results: The DE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G 1 /S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdDE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction. Conclusions: Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways.These findings imply that less toxic doses than currently practiced in the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants.

The oncolytic adenovirus AdΔΔ enhances selective cancer cell killing in combination with DNA-damaging drugs in pancreatic cancer models

“…Potent sensitization to drugs was observed with all mutants with the anti-apoptotic E1B19K gene deleted and to a lesser degree in combination with ONYX-015, deleted in the second viral anti-apoptotic gene E1B55K. We hypothesize that by attenuating the viral anti-apoptotic defense mechanisms, cell killing through drug-induced apoptosis is promoted in pancreatic cancer cells, as previously demonstrated for mutants with only the E1B19K deletion, 30 and also for the AdDD mutant in this study. Notably, the E1B19K deletion attenuates replication and spread, and prevents replication in the presence of TNF in normal cells and tissue, but not in the majority of cancer cells.…”

“…On the other hand, the singledeleted AdD19K mutant was highly efficacious, and had prolonged survival in combination with gemcitabine from 71 days for virus alone to 129 days for the combination (Po0.01) as previously demonstrated. 30 Similar results were obtained in the SUIT-2 model with significantly prolonged time to progression when suboptimal doses of AdDD (1Â10 9 v.p.) and gemcitabine (15 mg kg À1 ) were combined; from 25 days for both virus and drug alone to 445 days when combined (Po0.01; Figure 6c).…”

“…30 We also observed an increase in caspase 3 activation with AdDD in prostate cancer cells. 31 In agreement with these observations, at early time points both Ad5tg and AdDD genome amplification was attenuated in the presence of gemcitabine, but increased over time and was not significantly lower 72 h post infection (Figures 5a and b; dashed lines).…”

“…Notably, the E1B19K deletion attenuates replication and spread, and prevents replication in the presence of TNF in normal cells and tissue, but not in the majority of cancer cells. [28][29][30]35,36 We recently engineered the adenoviral mutant AdDD to replicate only in cells with disrupted cell cycle regulation by deleting the pRbbinding E1ACR2 region in wild-type Ad5. 31 This deletion restricts replication mainly to cancer cells as demonstrated for E1ACR2-deleted viruses.…”

“…28,29 In addition, when E1B19K-deleted mutants were combined with gemcitabine cell killing was greatly enhanced through drug-induced apoptosis. 30 To improve on efficacy and specificity, we generated a doubledeleted mutant (AdDD) by taking advantage of the high-anticancer efficacy of E1ACR2-deleted viruses and the apoptosis promoting function of E1B19K-deleted mutants as well as the retention of the intact E3 region for sustained replication in vivo. 31 While the E1B19K deletion alone is not sufficiently selective for cancer cells, we found that AdDD was highly selective and could significantly enhance cell killing in combination with chemotherapeutics.…”