Genome-Wide CRISPR Screening for the Identification of Therapy Resistance-Associated Genes in Urothelial Carcinoma.

The application of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 technology with pooled guide RNA libraries enables genome-wide screening, which has some advantages over other screening methods using chemical DNA mutagens for inducing genetic changes, RNA interference, or arrayed screens. Here we describe the use of genome-wide knockout and transcriptional activation screening enabling the CRISPR-Cas9 system to discover resistance mechanisms to CDK4/6 inhibition in bladder cancer along with next-generation sequencing (NGS) analysis. We will describe the approach for transcriptional activation in the bladder cancer cell line T24 and provide guidance on critical points during the experimental workflow.

Targeting Cell Cycle Facilitates E1A-Independent Adenoviral Replication.

DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus.

The Oncolytic Adenovirus XVir-N-31 Joins Forces with CDK4/6 Inhibition Augmenting Innate and Adaptive Antitumor Immunity in Ewing Sarcoma.

Purpose: Ewing sarcoma (EwS) is a highly malignant pediatric tumor characterized by a non-T-cell-inflamed immune-evasive phenotype. When relapsed or metastasized, survival is poor, emphasizing the need for novel treatment strategies. Here, we analyze the novel combination approach using the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition to augment EwS immunogenicity.

Identification of the KIF and MCM protein families as novel targets for combination therapy with CDK4/6 inhibitors in bladder cancer.

CDK4/6 inhibitors have proven their potency for the treatment of cancer but only in combination with hormone or targeted therapies. The aim of this study was the identification of molecules that are involved in response mechanisms to CDK4/6 inhibitors and the development of novel combination therapies with corresponding inhibitors in bladder cancer. Genes of response to therapy and genes that confer resistance to the CDK4/6 inhibitor palbociclib were identified by performing an analysis of published literature and own published data using a CRISPR-dCas9 genome wide gain of function screen.

The Oncolytic Adenovirus XVir-N-31, in Combination with the Blockade of the PD-1/PD-L1 Axis, Conveys Abscopal Effects in a Humanized Glioblastoma Mouse Model.

Glioblastoma (GBM) is an obligatory lethal brain tumor with a median survival, even with the best standard of care therapy, of less than 20 months. In light of this fact, the evaluation of new GBM treatment approaches such as oncolytic virotherapy (OVT) is urgently needed. Based on our preliminary preclinical data, the YB-1 dependent oncolytic adenovirus (OAV) XVir-N-31 represents a promising therapeutic agent to treat, in particular, therapy resistant GBM.

Targeting the Retinoblastoma/E2F repressive complex by CDK4/6 inhibitors amplifies oncolytic potency of an oncolytic adenovirus.

CDK4/6 inhibitors (CDK4/6i) and oncolytic viruses are promising therapeutic agents for the treatment of various cancers. As single agents, CDK4/6 inhibitors that are approved for the treatment of breast cancer in combination with endocrine therapy cause G1 cell cycle arrest, whereas adenoviruses induce progression into S-phase in infected cells as an integral part of the their life cycle. Both CDK4/6 inhibitors and adenovirus replication target the Retinoblastoma protein albeit for different purposes.

Concepts in Oncolytic Adenovirus Therapy.

Oncolytic adenovirus therapy is gaining importance as a novel treatment option for the management of various cancers. Different concepts of modification within the adenovirus vector have been identified that define the mode of action against and the interaction with the tumour. Adenoviral vectors allow for genetic manipulations that restrict tumour specificity and also the expression of specific transgenes in order to support the anti-tumour effect.

The Oncolytic Adenovirus XVir-N-31 as a Novel Therapy in Muscle-Invasive Bladder Cancer.

Muscle-invasive bladder cancer represents approximately 25% of diagnosed bladder cancer cases and carries a significant risk of death. Oncolytic viruses are novel antitumor agents with the ability to selectively replicate and lyse tumor cells while sparing healthy tissue. We explored the efficiency of the oncolytic YB-1-selective adenovirus XVir-N-31 in vitro and in an orthotopic mouse model for bladder cancer by intramural injection under ultrasound guidance.

Adenovirus Particle Quantification in Cell Lysates Using Light Scattering.

Adenoviral vector production for therapeutic applications is a well-established routine process. However, current methods for measurement of adenovirus particle titers as a quality characteristic require highly purified virus preparations. While purified virus is typically obtained in the last step of downstream purification, rapid and reliable methods for adenovirus particle quantification in intermediate products and crude lysates to allow for optimization and validation of cell cultures and intermediate downstream processing steps are currently not at hand.

YB-1 dependent oncolytic adenovirus efficiently inhibits tumor growth of glioma cancer stem like cells.

Background: The brain cancer stem cell (CSC) model describes a small subset of glioma cells as being responsible for tumor initiation, conferring therapy resistance and tumor recurrence. In brain CSC, the PI3-K/AKT and the RAS/mitogen activated protein kinase (MAPK) pathways are found to be activated. In consequence, the human transcription factor YB-1, knowing to be responsible for the emergence of drug resistance and driving adenoviral replication, is phosphorylated and activated.

YB-1 dependent virotherapy in combination with temozolomide as a multimodal therapy approach to eradicate malignant glioma.

The human Y-box binding protein 1 (YB-1) is known to be a promising target for cancer therapy. We have demonstrated that YB-1 plays an important role in the adenoviral life cycle by regulating the adenoviral E2-gene expression. Thus, we studied the oncolytic effect of the recombinant adenovirus Ad-Delo3-RGD, in which the transactivation domain CR3 of the E1A protein is ablated to enable viral replication only in YB-1 positive cancer cells.

Impact of radiation therapy on the oncolytic adenovirus dl520: implications on the treatment of glioblastoma.

Background And Purpose: Viral oncolytic therapy is emerging as a new form of anticancer therapy and has shown promising preclinical results, especially in combination with radio- and chemotherapy. We recently reported that nuclear localization of the human transcription factor YB-1 in multidrug-resistant cells facilitates E1-independent adenoviral replication. The aim of this study was to evaluate the combined treatment of the conditionally-replicating adenovirus dl520 and radiotherapy in glioma cell lines in vitro and in human tumor xenografts.

Inhibition of the multidrug-resistant phenotype by targeting YB-1 with a conditionally oncolytic adenovirus: implications for combinatorial treatment regimen with chemotherapeutic agents.

Bearing in mind the limited success of available treatment modalities for the therapy of multidrug-resistant tumor cells, alternative and complementary strategies need to be developed. It is known that the transcriptional activation of genes, such as MDR1 and MRP1, which play a major role in the development of a multidrug-resistant phenotype in tumor cells, involves the Y-box protein YB-1. Thus, YB-1 is a promising target for new therapeutic approaches to defeat multidrug resistance.

Characterization of the recombinant adenovirus vector AdYB-1: implications for oncolytic vector development.

Conditionally replicating adenoviruses are a promising new modality for the treatment of cancer. However, early clinical trials demonstrate that the efficacy of current vectors is limited. Interestingly, DNA replication and production of viral particles do not always correlate with virus-mediated cell lysis and virus release depending on the vector utilized for infection.

Novel three-pronged strategy to enhance cancer cell killing in glioblastoma cell lines: histone deacetylase inhibitor, chemotherapy, and oncolytic adenovirus dl520.

Resistance to radiation and chemotherapy remains an obstacle to the treatment of brain tumors. We have demonstrated that the replication-deficient adenovirus d1520, which lacks the E1A 13S protein, replicates efficiently and exhibits oncolytic potential in multidrug-resistant cells with nuclear localization of the human transcription factor YB-1. However, besides others, key factors regarding oncolytic virotherapy are limited tumor transduction rate and low replication efficiency.

Multidrug-resistant cancer cells facilitate E1-independent adenoviral replication: impact for cancer gene therapy.

Resistance to chemotherapy is responsible for a failure of current treatment regimens in cancer patients. We have reported previously that the Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1, which plays a major role in the development of a multidrug resistant-tumor phenotype. YB-1 predicts drug resistance and patient outcome in breast cancer.

YB-1 relocates to the nucleus in adenovirus-infected cells and facilitates viral replication by inducing E2 gene expression through the E2 late promoter.

The adenovirus early proteins E1A and E1B-55kDa are key regulators of viral DNA replication, and it was thought that targeting of p53 by E1B-55kDa is essential for this process. Here we have identified a previously unrecognized function of E1B for adenovirus replication. We found that E1B-55kDa is involved in targeting the transcription factor YB-1 to the nuclei of adenovirus type 5-infected cells where it is associated with viral inclusion bodies believed to be sites of viral transcription and replication.