Noncanonical open reading frames encode functional proteins essential for cancer cell survival.

Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442-renamed glycine-rich extracellular protein-1 (GREP1)-encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.

CREB5 Promotes Resistance to Androgen-Receptor Antagonists and Androgen Deprivation in Prostate Cancer.

Androgen-receptor (AR) inhibitors, including enzalutamide, are used for treatment of all metastatic castration-resistant prostate cancers (mCRPCs). However, some patients develop resistance or never respond. We find that the transcription factor CREB5 confers enzalutamide resistance in an open reading frame (ORF) expression screen and in tumor xenografts. CREB5 overexpression is essential for an enzalutamide-resistant patient-derived organoid. In AR-expressing prostate cancer cells, CREB5 interactions enhance AR activity at a subset of promoters and enhancers upon enzalutamide treatment, including MYC and genes involved in the cell cycle. In mCRPC, we found recurrent amplification and overexpression of CREB5. Our observations identify CREB5 as one mechanism that drives resistance to AR antagonists in prostate cancers.

PLZF, a tumor suppressor genetically lost in metastatic castration-resistant prostate cancer, is a mediator of resistance to androgen deprivation therapy.

Whole-exome sequencing of metastatic castration-resistant prostate cancer (mCRPC) reveals that 5% to 7% of tumors harbor promyelocytic leukemia zinc finger (PLZF) protein homozygous deletions. PLZF is a canonical androgen-regulated putative tumor suppressor gene whose expression is inhibited by androgen deprivation therapy (ADT). Here, we demonstrate that knockdown of PLZF expression promotes a CRPC and enzalutamide-resistant phenotype in prostate cancer cells. Reintroduction of PLZF expression is sufficient to reverse androgen-independent growth mediated by PLZF depletion. PLZF loss enhances CRPC tumor growth in a xenograft model. Bioinformatic analysis of the PLZF cistrome shows that PLZF negatively regulates multiple pathways, including the MAPK pathway. Accordingly, our data support an oncogenic program activated by ADT. This acquired mechanism together with the finding of genetic loss in CRPC implicates PLZF inactivation as a mechanism promoting ADT resistance and the CRPC phenotype.

PARP inhibitor olaparib increases the oncolytic activity of dl922-947 in in vitro and in vivo model of anaplastic thyroid carcinoma.

PARP inhibitors are mostly effective as anticancer drugs in association with DNA damaging agents. We have previously shown that the oncolytic adenovirus dl922-947 induces extensive DNA damage, therefore we hypothesized a synergistic antitumoral effect of the PARP inhibitor olaparib in association with dl922-947. Anaplastic thyroid carcinoma was chosen as model since it is a particularly aggressive tumor and, because of its localized growth, it is suitable for intratumoral treatment with oncolytic viruses. Here, we show that dl922-947 infection induces PARP activation, and we confirm in vitro and in vivo that PARP inhibition increases dl922-947 replication and oncolytic activity. In vitro, the combination with olaparib exacerbates the appearance of cell death markers, such as Annexin V positivity, caspase 3 cleavage, cytochrome C release and propidium iodide permeability. In vivo, we also observed a better viral distribution upon PARP inhibition. Changes in CD31 levels suggest a direct effect of olaparib on tumor vascularization and on the viral distribution within the tumor mass. The observation that PARP inhibition enhances the effects of dl922-947 is highly promising not only for the treatment of anaplastic thyroid carcinoma but, in general, for the treatment of other tumors that could benefit from the use of oncolytic viruses.

Ionizing radiation enhances dl922-947-mediated cell death of anaplastic thyroid carcinoma cells.

dl922-947 is an oncolytic adenovirus potentially suitable for the treatment of aggressive localized tumors, such as anaplastic thyroid carcinoma (ATC). In this study, we have analyzed the effects of dl922-947 in combination with ionizing radiations, testing different schedules of administration and observing synergistic effects only when ATC cells were irradiated 24 h prior to viral infection. Cells undergoing combined treatment exhibited a marked increase in cell death and viral replication, suggesting that irradiation blocks cells in a more permissive state for viral life cycle. We also show that dl922-947 triggers a DNA damage response, characterized by mobilization of the MRN complex (composed by Mre11-Rad50-Nbs1), accumulation of γH2AX, and activation of the checkpoint kinases ataxia telangiectasia mutated (ATM) and Chk1. Based on these observations, we speculate that the DNA damage response acts as a cellular protective mechanism to hinder viral infection and replication. To confirm this hypothesis, we demonstrate that the ATM inhibitor KU55933 increased the oncolytic activity of dl922-947 and its replication. Finally, we validate the potential therapeutic use of this approach by showing in vivo that the combined treatment slows tumor xenograft growth more potently than either irradiation or infection alone.

Inhibition of autophagy enhances the effects of E1A-defective oncolytic adenovirus dl922-947 against glioma cells in vitro and in vivo.

Oncolytic viruses represent a novel therapeutic approach for aggressive tumors, such as glioblastoma multiforme, which are resistant to available treatments. Autophagy has been observed in cells infected with oncolytic viruses; however, its role in cell death/survival is unclear. To elucidate the potential therapeutic use of autophagy modulators in association with viral therapy, we analyzed autophagy induction in human glioma cell lines U373MG and U87MG infected with the oncolytic adenovirus dl922-947. dl922-947 infection triggered an autophagic cellular response, as shown by the development of acidic vesicular organelles, LC3-I→LC3-II conversion, and reduction of p62 levels. However, on infection, the Akt/mTOR/p70s6k pathway, which negatively regulates autophagy, was activated, whereas the ERK1/2 pathway, a positive regulator of autophagy, was inhibited. Accordingly, MEK inhibition by PD98059 sensitized glioma cells to dl922-947 effects, whereas autophagy induction by rapamycin protected cells from dl922-947-induced death. Treatment with two inhibitors of autophagy, chloroquine and 3-methyladenine, increased the cytotoxic effects of dl922-947 in vitro. In vivo, the growth of U87MG-induced xenografts was further reduced by adding chloroquine to the dl922-947 treatment. In conclusion, autophagy acts as a survival response in glioma cells infected with dl922-947, thus suggesting autophagy inhibitors as adjuvant/neoadjuvant drugs in oncolytic virus-based treatments.

Clozapine impairs insulin action by up-regulating Akt phosphorylation and Ped/Pea-15 protein abundance.

Clinical and experimental evidence indicates that atypical antipsychotics impair glucose metabolism. We investigated whether clozapine may directly affect insulin action by analyzing insulin signaling in vitro and in vivo. Clozapine reduced insulin-stimulated glucose uptake in PC12 and in L6 cells, representative models of neuron and skeletal muscle, respectively. Consistently, clozapine reduced insulin effect on insulin receptor (IR) by 40% and on IR substrate-1 (IRS1) tyrosine phosphorylation by 60%. Insulin-stimulated Akt phosphorylation was also reduced by about 40%. Moreover, insulin-dependent phosphorylation of protein kinase C-ζ (PKC-ζ) was completely blunted in clozapine-treated cells. Interestingly, clozapine treatment was accompanied by an insulin-independent increase of Akt phosphorylation, with no change of IR, IRS1, and PKC-ζ basal phosphorylation. The cellular abundance of Ped/Pea-15, an Akt substrate and inducer of insulin resistance, was also increased following clozapine exposure, both in the absence and in the presence of cyclohexymide, a protein synthesis inhibitor. Similar as in cellular models, in the caudate-putamen and in the tibialis muscle of clozapine-treated C57/BL/KsJ mice, Akt phosphorylation and Ped/Pea-15 protein levels were increased and PKC-ζ phosphorylation was decreased. Thus, in these experimental models, clozapine deranged Akt function and up-regulated Ped/Pea-15, thereby inhibiting insulin stimulation of PKC-ζ and of glucose uptake.

AZD1152 negatively affects the growth of anaplastic thyroid carcinoma cells and enhances the effects of oncolytic virus dl922-947.

Novel therapeutic approaches are required for the treatment of anaplastic thyroid carcinoma (ATC), an incurable disease resistant to current available therapies. Aurora B is an important mitotic kinase involved in chromosome segregation and cytokinesis. It is overexpressed in many cancers including ATC and represents a potential target for chemotherapy. The effects of AZD1152, a specific Aurora B kinase inhibitor, have been evaluated against ATC, showing G(2)/M accumulation, polyploidy and subsequent cell death by mitotic catastrophe upon drug treatment. Only three administrations of AZD1152 significantly reduced the growth of ATC tumour xenogratfs. Oncolytic viruses in association with other forms of treatment have proven highly promising in preclinical and clinical reports. The oncolytic adenovirus dl922-947 is active against ATC cells, and we have evaluated the effects of the association between AZD1152 and dl922-947. In cells treated with virus and drug, we report additive/synergistic killing effects. Interestingly, the phosphorylation of histone H3 (Ser10), the main Aurora B substrate, is inhibited by dl922-947 in a dose-dependent manner, and completely abolished in association with AZD1152. The combined treatment significantly inhibited the growth of ATC tumour xenografts with respect to single treatments. Our data demonstrate that the Aurora B inhibitor AZD1152, alone or in combination with oncolytic virus dl922-947, could represent a novel therapeutic option for the treatment of ATC.

Aurora A and B kinases--targets of novel anticancer drugs.

The Aurora Kinases are highly related serine-threonine kinases, essential for accurate and equal segregation of genomic material during mitosis. A large number of studies have linked the aberrant expression of Aurora kinases to cancer, leading to the development of specific Aurora kinases inhibitors. Several small molecules inhibit with a similar efficacy both Aurora A and Aurora B, however, in most cases the effects resemble Aurora B disruption by genetic methods, indicating that Aurora B represents an effective therapeutic target. These drugs are currently under preclinical or clinical evaluation and are reviewed in this article. The relevant patents are discussed.

PED/PEA-15 modulates coxsackievirus-adenovirus receptor expression and adenoviral infectivity via ERK-mediated signals in glioma cells.

Glioblastoma multiforme (GBM) is the most aggressive human brain tumor, and is highly resistant to chemo- and radiotherapy. Selectively replicating oncolytic viruses represent a novel approach for the treatment of neoplastic diseases. Coxsackievirus-adenovirus receptor (CAR) is the primary receptor for adenoviruses, and loss or reduction of CAR greatly decreases adenoviral entry. Understanding the mechanisms regulating CAR expression and localization will contribute to increase the efficacy of oncolytic adenoviruses. Two glioma cell lines (U343MG and U373MG) were infected with the oncolytic adenovirus dl922-947. U373MG cells were more susceptible to cell death after viral infection, compared with U343MG cells. The enhanced sensitivity was paralleled by increased adenoviral entry and CAR mRNA and protein levels in U373MG cells. In addition, U373MG cells displayed a decreased ERK1/2 (extracellular signal-regulated kinase-1/2) nuclear-to-cytosolic ratio, compared with U343MG cells. Intracellular content of PED/PEA-15, an ERK1/2-interacting protein, was also augmented in these cells. Both ERK2 overexpression and genetic silencing of PED/PEA-15 by antisense oligonucleotides increased ERK nuclear accumulation and reduced CAR expression and adenoviral entry. Our data indicate that dl922-947 could represent an useful tool for the treatment of GBM and that PED/PEA-15 modulates CAR expression and adenoviral entry, by sequestering ERK1/2.