Adenovirus-Mediated Sensitization to the Cytotoxic Drugs Docetaxel and Mitoxantrone Is Dependent on Regulatory Domains in the E1ACR1 Gene-Region

Abstract

Oncolytic adenoviruses have shown promising efficacy in clinical trials targeting prostate cancers that frequently develop resistance to all current therapies. The replication-selective mutants AdΔΔ and dl922–947, defective in pRb-binding, have been demonstrated to synergise with the current standard of care, mitoxantrone and docetaxel, in prostate cancer models. While expression of the early viral E1A gene is essential for the enhanced cell killing, the specific E1A-regions required for the effects are unknown. Here, we demonstrate that replicating mutants deleted in small E1A-domains, binding pRb (dl1108), p300/CBP (dl1104) and p400/TRRAP or p21 (dl1102) sensitize human prostate cancer cells (PC-3, DU145, 22Rv1) to mitoxantrone and docetaxel. Through generation of non-replicating mutants, we demonstrate that the small E1A12S protein is sufficient to potently sensitize all prostate cancer cells to the drugs even in the absence of viral replication and the E1A transactivating domain, conserved region (CR) 3. Furthermore, the p300/CBP-binding domain in E1ACR1 is essential for drug-sensitisation in the absence (AdE1A1104) but not in the presence of the E1ACR3 (dl1104) domain. AdE1A1104 also failed to increase apoptosis and accumulation of cells in G2/M. All E1AΔCR2 mutants (AdE1A1108, dl922–947) and AdE1A1102 or dl1102 enhance cell killing to the same degree as wild type virus. In PC-3 xenografts in vivo the dl1102 mutant significantly prolongs time to tumor progression that is further enhanced in combination with docetaxel. Neither dl1102 nor dl1104 replicates in normal human epithelial cells (NHBE). These findings suggest that additional E1A-deletions might be included when developing more potent replication-selective oncolytic viruses, such as the AdΔCR2-mutants, to further enhance potency through synergistic cell killing in combination with current chemotherapeutics.

Sensitisation to mitoxantrone-induced apoptosis by the oncolytic adenovirus Ad∆∆ through Bcl-2-dependent attenuation of autophagy

“…Because of the central role for Bcl-2 in preventing both apoptosis and autophagy, we investigated its role in virus-mediated sensitisation to mitoxantrone. We used the androgen-independent PC3 and PC3M, and the androgen-sensitive 22Rv1 human prostate cancer cells 4 , 15 . PC3 and PC3M cells are metastatic prostate cancer models, which are highly insensitive to drugs.…”

The E1B19K-deleted oncolytic adenovirus mutant AdΔ19K sensitizes pancreatic cancer cells to drug-induced DNA-damage by down-regulating Claspin and Mre11

“…However, the cellular mechanisms for the increased tumour cell killing in combination with drugs are mostly unknown, both in preclinical models and in patients. Virus-induced cell lysis occurs through non-apoptotic, necrotic-like cell death mechanisms while in combination with cytotoxic drugs, drug-induced apoptosis is often enhanced [ 11 , 14 - 16 ]. It has been established that viral E1A expression is necessary for synergistic enhancement of cytotoxic drug-induced cell death and that E1A-binding to p300 and p400 is required, but not to pRb [ 14 , 17 - 19 ].…”

“…Virus-induced cell lysis occurs through non-apoptotic, necrotic-like cell death mechanisms while in combination with cytotoxic drugs, drug-induced apoptosis is often enhanced [ 11 , 14 - 16 ]. It has been established that viral E1A expression is necessary for synergistic enhancement of cytotoxic drug-induced cell death and that E1A-binding to p300 and p400 is required, but not to pRb [ 14 , 17 - 19 ]. Furthermore, the E4orf3 and E4orf6 genes are rapidly expressed after infection to prevent the cellular DNA-damage response (DDR) mainly by inactivating the Mre11-Rad50-Nbs1 (MRN) complex [ 20 , 21 ].…”

Designer Oncolytic Adenovirus: Coming of Age

“…However, increased druginduced apoptosis in response to E1A expression has been indicated in combination with several cytotoxic drugs including inhibitors of metabolism, microtubules and topoisomerases. In prostate and pancreatic models, E1A-mediated caspase 8 and 3 activation through E1A-binding to the caspase 8 inhibitor cellular (Fas-Associated protein with Death Domain (FADD))-like IL(InterLeukin)-1β-Converting enzyme (FLICE) Inhibitory Protein (cFLIP) followed by mitochondrial depolarization significantly enhanced apoptosis in response to the cytotoxic drugs (e.g., Miranda et al, 2012) [77,78].…”