Inhibitors of the ATPase p97/VCP: From basic research to clinical applications.

Protein homeostasis deficiencies underlie various cancers and neurodegenerative diseases. The ubiquitin-proteasome system (UPS) and autophagy are responsible for most of the protein degradation in mammalian cells and, therefore, represent attractive targets for cancer therapy and that of neurodegenerative diseases. The ATPase p97, also known as VCP, is a central component of the UPS that extracts and disassembles its substrates from various cellular locations and also regulates different steps in autophagy. Several UPS- and autophagy-targeting drugs are in clinical trials. In this review, we focus on the development of various p97 inhibitors, including the ATPase inhibitors CB-5083 and CB-5339, which reached clinical trials by demonstrating effective anti-tumor activity across various tumor models, providing an effective alternative to targeting protein degradation for cancer therapy. Here, we provide an overview of how different p97 inhibitors have evolved over time both as basic research tools and effective UPS-targeting cancer therapies in the clinic.

Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells.

DNA end resection converts broken ends of double-stranded DNA (dsDNA) to 3'-single-stranded DNA (3'-ssDNA). The extent of resection regulates DNA double-strand break (DSB) repair pathway choice and thereby genomic stability. Here, we characterize an optimized immunofluorescence (IF) microscopy-based protocol for measuring ssDNA in mammalian cells by labeling genomic DNA with 5-bromo-2'-deoxyuridine (BrdU). BrdU foci can be detected under non-denaturing conditions by anti-BrdU antibody, providing an accurate and reliable readout of DNA end resection in most mammalian cell lines. For complete details on the use and execution of this protocol, please refer to Kilgas et al. (2021).

The protease SPRTN and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability.

DNA-protein crosslinks (DPCs) are a specific type of DNA lesion in which proteins are covalently attached to DNA. Unrepaired DPCs lead to genomic instability, cancer, neurodegeneration, and accelerated aging. DPC proteolysis was recently identified as a specialized pathway for DPC repair. The DNA-dependent protease SPRTN and the 26S proteasome emerged as two independent proteolytic systems. DPCs are also repaired by homologous recombination (HR), a canonical DNA repair pathway. While studying the cellular response to DPC formation, we identify ubiquitylation and SUMOylation as two major signaling events in DNA replication-coupled DPC repair. DPC ubiquitylation recruits SPRTN to repair sites, promoting DPC removal. DPC SUMOylation prevents DNA double-strand break formation, HR activation, and potentially deleterious genomic rearrangements. In this way, SUMOylation channels DPC repair toward SPRTN proteolysis, which is a safer pathway choice for DPC repair and prevention of genomic instability.

p97/VCP inhibition causes excessive MRE11-dependent DNA end resection promoting cell killing after ionizing radiation.

The ATPase p97 is a central component of the ubiquitin-proteasome degradation system. p97 uses its ATPase activity and co-factors to extract ubiquitinated substrates from different cellular locations, including DNA lesions, thereby regulating DNA repair pathway choice. Here, we find that p97 physically and functionally interacts with the MRE11-RAD50-NBS1 (MRN) complex on chromatin and that inactivation of p97 blocks the disassembly of the MRN complex from the sites of DNA damage upon ionizing radiation (IR). The inhibition of p97 function results in excessive 5'-DNA end resection mediated by MRE11 that leads to defective DNA repair and radiosensitivity. In addition, p97 inhibition by the specific small-molecule inhibitor CB-5083 increases tumor cell killing following IR both in vitro and in vivo. Mechanistically, this is mediated via increased MRE11 nuclease accumulation. This suggests that p97 inhibitors might be exploited to improve outcomes for radiotherapy patients.

The Histone Deacetylase Inhibitor Romidepsin Spares Normal Tissues While Acting as an Effective Radiosensitizer in Bladder Tumors in Vivo.

PURPOSE: Muscle-invasive bladder cancer has a 40% to 60% 5-year survival rate with radical treatment by surgical removal of the bladder or radiation therapy-based bladder preservation techniques, including concurrent chemoradiation. Elderly patients cannot tolerate current chemoradiation therapy regimens and often receive only radiation therapy, which is less effective. We urgently need effective chemotherapy agents for use with radiation therapy combinations that are nontoxic to normal tissues and tolerated by elderly patients. METHODS AND MATERIALS: We have identified histone deacetylase (HDAC) inhibitors as promising agents to study. Pan-HDAC inhibition, using panobinostat, is a good strategy for radiosensitization, but more selective agents may be more useful radiosensitizers in a clinical setting, resulting in fewer systemic side effects. Herein, we study the HDAC class I-selective agent romidepsin, which we predict to have fewer off-target effects than panobinostat while maintaining an effective level of tumor radiosensitization. RESULTS: In vitro effects of romidepsin were assessed by clonogenic assay and showed that romidepsin was effective in the nanomolar range in different bladder cancer cells and radiosensitized these cells. The radiosensitizing effect of romidepsin was confirmed in vivo using superficial xenografts. The drug/irradiation combination treatment resulted in significant tumor growth delay but did not increase the severity of acute (3.75 days) intestinal normal tissue toxicity or late toxicity at 29 weeks. Moreover, we showed that romidepsin treatment impaired both homologous recombination and nonhomologous end joining DNA repair pathways, suggesting that the disruption of DNA repair pathways caused by romidepsin is a key mechanism for its radiosensitizing effect in bladder cancer cells. CONCLUSIONS: This study demonstrates that romidepsin is an effective radiosensitizer in vitro and in vivo and does not increase the acute and late toxicity after ionizing radiation. Romidepsin is already in clinical use for the cutaneous T-cell lymphoma, but a phase 1 clinical trial of romidepsin as a radiosensitizer could be considered in muscle-invasive bladder cancer.

The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8.

The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97-ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97-ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97-ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.

The Role of Extracellular Adenosine Triphosphate in Ischemic Organ Injury.

OBJECTIVES: Ischemic tissue injury contributes to significant morbidity and mortality and is implicated in a range of pathologic conditions, including but not limited to myocardial infarction, ischemic stroke, and acute kidney injury. The associated reperfusion phase is responsible for the activation of the innate and adaptive immune system, further accentuating inflammation. Adenosine triphosphate molecule has been implicated in various ischemic conditions, including stroke and myocardial infarction. STUDY SELECTION: Adenosine triphosphate is a well-defined intracellular energy transfer and is commonly referred to as the body's "energy currency." However, Laboratory studies have demonstrated that extracellular adenosine triphosphate has the ability to initiate inflammation and is therefore referred to as a damage-associated molecular pattern. Purinergic receptors-dependent signaling, proinflammatory cytokine release, increased Ca influx into cells, and subsequent apoptosis have been shown to form a common underlying extracellular adenosine triphosphate molecular mechanism in ischemic organ injury. CONCLUSIONS: In this review, we aim to discuss the molecular mechanisms behind adenosine triphosphate-mediated ischemic tissue injury and evaluate the role of extracellular adenosine triphosphate in ischemic injury in specific organs, in order to provide a greater understanding of the pathophysiology of this complex process. We also appraise potential future therapeutic strategies to limit damage in various organs, including the heart, brain, kidneys, and lungs.