Got PIDD1? Natural killer cells clear polyploid cells to ensure a balanced genome.

Removal of polyploid cells is essential to preventing cancer and restricting tumor growth. A new study published in The EMBO Journal shows assembly of the NEMO-PIDDosome on extra centrioles. Activation of this protein complex leads to NF-κB activation that, in turn, induces NK cell-mediated cell clearance.

Caspase-2 is essential for proliferation and self-renewal of nucleophosmin-mutated acute myeloid leukemia.

Mutation in nucleophosmin (NPM1) causes relocalization of this normally nucleolar protein to the cytoplasm ( NPM1c+ ). Despite NPM1 mutation being the most common driver mutation in cytogenetically normal adult acute myeloid leukemia (AML), the mechanisms of NPM1c+-induced leukemogenesis remain unclear. Caspase-2 is a pro-apoptotic protein activated by NPM1 in the nucleolus. Here, we show that caspase-2 is also activated by NPM1c+ in the cytoplasm, and DNA damage-induced apoptosis is caspase-2-dependent in NPM1c+ AML but not in NPM1wt cells. Strikingly, in NPM1c+ cells, loss of caspase-2 results in profound cell cycle arrest, differentiation, and down-regulation of stem cell pathways that regulate pluripotency including impairment in the AKT/mTORC1 and Wnt signaling pathways. In contrast, there were minimal differences in proliferation, differentiation, or the transcriptional profile of NPM1wt cells with and without caspase-2. Together, these results show that caspase-2 is essential for proliferation and self-renewal of AML cells that have mutated NPM1. This study demonstrates that caspase-2 is a major effector of NPM1c+ function and may even be a druggable target to treat NPM1c+ AML and prevent relapse.

The role of the nucleolus in regulating the cell cycle and the DNA damage response.

The nucleolus has long been perceived as the site for ribosome biogenesis, but numerous studies suggest that the nucleolus carefully sequesters crucial proteins involved in multiple cellular functions. Among these, the role of nucleolus in cell cycle regulation is the most evident. The nucleolus is the first responder of growth-related signals to mediate normal cell cycle progression. The nucleolus also senses different cellular stress insults by activating diverse pathways that arrest the cell cycle, promote DNA repair, or initiate apoptosis. Here, we review the emerging concepts on how the ribosomal and nonribosomal nucleolar proteins mediate such cellular effects.

The NLRP3 inflammasome fires up heme-induced inflammation in hemolytic conditions.

Overactive inflammatory responses are central to the pathophysiology of many hemolytic conditions including sickle cell disease. Excessive hemolysis leads to elevated serum levels of heme due to saturation of heme scavenging mechanisms. Extracellular heme has been shown to activate the NLRP3 inflammasome, leading to activation of caspase-1 and release of pro-inflammatory cytokines IL-1ß and IL-18. Heme also activates the non-canonical inflammasome pathway, which may contribute to NLRP3 inflammasome formation and leads to pyroptosis, a type of inflammatory cell death. Some clinical studies indicate there is a benefit to blocking the NLRP3 inflammasome pathway in patients with sickle cell disease and other hemolytic conditions. However, a thorough understanding of the mechanisms of heme-induced inflammasome activation is needed to fully leverage this pathway for clinical benefit. This review will explore the mechanisms of heme-induced NLRP3 inflammasome activation and the role of this pathway in hemolytic conditions including sickle cell disease.

Lethal and Non-Lethal Functions of Caspases in the DNA Damage Response.

Members of the caspase family are well known for their roles in the initiation and execution of cell death. Due to their function in the removal of damaged cells that could otherwise become malignant, caspases are important players in the DNA damage response (DDR), a network of pathways that prevent genomic instability. However, emerging evidence of caspases positively or negatively impacting the accumulation of DNA damage in the absence of cell death demonstrates that caspases play a role in the DDR that is independent of their role in apoptosis. This review highlights the apoptotic and non-apoptotic roles of caspases in the DDR and how they can impact genomic stability and cancer treatment.

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1ß release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

Cellular autophagy, an unbidden effect of caspase inhibition by zVAD-fmk.

zVAD-fmk is a widely used pan-caspase inhibitor that blocks apoptosis but has undesirable side effects, including autophagy. In this issue, Needs et al. propose that zVAD-fmk induces autophagy by inhibiting the N-glycanase NGLY1 rather than caspases. NGLY1 is essential for the ERAD response and patients with inactivating mutations in NGLY1 present with neurodevelopmental defects and organ dysfunction. The ability of NGLY1 to inhibit basal levels of autophagy may contribute to this pathology. This study demonstrates possible crosstalk between protein turnover and autophagy while also underscoring the importance of specificity when using chemical tools to interrogate these pathways. Comment on https://doi.org/10.1111/febs.16345.

Caspase-2 regulates S-phase cell cycle events to protect from DNA damage accumulation independent of apoptosis.

In addition to its classical role in apoptosis, accumulating evidence suggests that caspase-2 has non-apoptotic functions, including regulation of cell division. Loss of caspase-2 is known to increase proliferation rates but how caspase-2 is regulating this process is currently unclear. We show that caspase-2 is activated in dividing cells in G1-phase of the cell cycle. In the absence of caspase-2, cells exhibit numerous S-phase defects including delayed exit from S-phase, defects in repair of chromosomal aberrations during S-phase, and increased DNA damage following S-phase arrest. In addition, caspase-2-deficient cells have a higher frequency of stalled replication forks, decreased DNA fiber length, and impeded progression of DNA replication tracts. This indicates that caspase-2 protects from replication stress and promotes replication fork protection to maintain genomic stability. These functions are independent of the pro-apoptotic function of caspase-2 because blocking caspase-2-induced cell death had no effect on cell division, DNA damage-induced cell cycle arrest, or DNA damage. Thus, our data supports a model where caspase-2 regulates cell cycle and DNA repair events to protect from the accumulation of DNA damage independently of its pro-apoptotic function.

Noncanonical Roles of Caspase-4 and Caspase-5 in Heme-Driven IL-1ß Release and Cell Death.

Excessive release of heme from RBCs is a key pathophysiological feature of several disease states, including bacterial sepsis, malaria, and sickle cell disease. This hemolysis results in an increased level of free heme that has been implicated in the inflammatory activation of monocytes, macrophages, and the endothelium. In this study, we show that extracellular heme engages the human inflammatory caspases, caspase-1, caspase-4, and caspase-5, resulting in the release of IL-1ß. Heme-induced IL-1ß release was further increased in macrophages from patients with sickle cell disease. In human primary macrophages, heme activated caspase-1 in an inflammasome-dependent manner, but heme-induced activation of caspase-4 and caspase-5 was independent of canonical inflammasomes. Furthermore, we show that both caspase-4 and caspase-5 are essential for heme-induced IL-1ß release, whereas caspase-4 is the primary contributor to heme-induced cell death. Together, we have identified that extracellular heme is a damage-associated molecular pattern that can engage canonical and noncanonical inflammasome activation as a key mediator of inflammation in macrophages.

Targeting apoptotic caspases in cancer.

Members of the caspase family of proteases play essential roles in the initiation and execution of apoptosis. These caspases are divided into two groups: the initiator caspases (caspase-2, -8, -9 and -10), which are the first to be activated in response to a signal, and the executioner caspases (caspase-3, -6, and -7) that carry out the demolition phase of apoptosis. Many conventional cancer therapies induce apoptosis to remove the cancer cell by engaging these caspases indirectly. Newer therapeutic applications have been designed, including those that specifically activate individual caspases using gene therapy approaches and small molecules that repress natural inhibitors of caspases already present in the cell. For such approaches to have maximal clinical efficacy, emerging insights into non-apoptotic roles of these caspases need to be considered. This review will discuss the roles of caspases as safeguards against cancer in the context of the advantages and potential limitations of targeting apoptotic caspases for the treatment of cancer.

Caspase-2 Substrates: To Apoptosis, Cell Cycle Control, and Beyond.

Caspase-2 belongs to the caspase family of proteins responsible for essential cellular functions including apoptosis and inflammation. Uniquely, caspase-2 has been identified as a tumor suppressor, but how it regulates this function is still unknown. For many years, caspase-2 has been considered an "orphan" caspase because, although it is able to induce apoptosis, there is an abundance of conflicting evidence that questions its necessity for apoptosis. Recent evidence supports that caspase-2 has non-apoptotic functions in the cell cycle and protection from genomic instability. It is unclear how caspase-2 regulates these opposing functions, which has made the mechanism of tumor suppression by caspase-2 difficult to determine. As a protease, caspase-2 likely exerts its functions by proteolytic cleavage of cellular substrates. This review highlights the known substrates of caspase-2 with a special focus on their functional relevance to caspase-2's role as a tumor suppressor.

Inflammatory caspase regulation: maintaining balance between inflammation and cell death in health and disease.

Members of the mammalian inflammatory caspase family, including caspase-1, caspase-4, caspase-5, caspase-11, and caspase-12, are key regulators of the innate immune response. Most studies to date have focused on the role of caspase-1 in the maturation of the proinflammatory cytokine interleukin-1ß and its upstream regulation by the inflammasome signaling complexes. However, an emerging body of research has supported a role for caspase-4, caspase-5, and caspase-11 in both regulating caspase-1 activation and inducing the inflammatory form of cell death called pyroptosis. This inflammatory caspase pathway appears essential for the regulation of cytokine processing. Consequently, insight into this noncanonical pathway may reveal important and, to date, understudied targets for the treatment of autoinflammatory disorders where the inflammasome pathway is dysregulated. Here, we will discuss the mechanisms of inflammasome and inflammatory caspase activation and how these pathways intersect to promote pathogen clearance.

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation.

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

Direct pro-apoptotic role for NPM1 as a regulator of PIDDosome formation.

Despite being frequently mutated or deregulated in acute myeloid leukemia (AML) and many other cancers, the mechanisms by which nucleophosmin (NPM1) regulates oncogenesis remain elusive. We found that NPM1 plays a direct and conserved role in DNA damage-induced assembly of the PIDDosome complex, the activating platform for caspase-2. This function is carried in the nucleolus and is essential for caspase-2-mediated apoptosis in response to a variety of DNA injuries.

NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus.

The PIDDosome (PIDD-RAIDD-caspase-2 complex) is considered to be the primary signaling platform for caspase-2 activation in response to genotoxic stress. Yet studies of PIDD-deficient mice show that caspase-2 activation can proceed in the absence of PIDD. Here we show that DNA damage induces the assembly of at least two distinct activation platforms for caspase-2: a cytoplasmic platform that is RAIDD dependent but PIDD independent, and a nucleolar platform that requires both PIDD and RAIDD. Furthermore, the nucleolar phosphoprotein nucleophosmin (NPM1) acts as a scaffold for PIDD and is essential for PIDDosome assembly in the nucleolus after DNA damage. Inhibition of NPM1 impairs caspase-2 processing, apoptosis, and caspase-2-dependent inhibition of cell growth, demonstrating that the NPM1-dependent nucleolar PIDDosome is a key initiator of the caspase-2 activation cascade. Thus we have identified the nucleolus as a novel site for caspase-2 activation and function.

Detection of Initiator Caspase Induced Proximity in Single Cells by Caspase Bimolecular Fluorescence Complementation.

The caspase family of proteases includes key regulators of apoptosis and inflammation. The caspases can be divided into two groups, the initiator caspases and the executioner caspases. Initiator caspases include caspase-2, caspase-8, and caspase-9 and are activated by proximity-induced dimerization upon recruitment to large molecular weight protein complexes called activation platforms. This protocol describes an imaging-based technique called caspase Bimolecular Fluorescence Complementation (BiFC) that measures induced proximity of initiator caspases. This method uses nonfluorescent fragments of the fluorescent protein Venus fused to initiator caspase monomers. When the caspase is recruited to its activation platform, the resulting induced proximity of the caspase monomers facilitates refolding of the Venus fragments into the full molecule, reconstituting its fluorescence. Thus, the assembly of initiator caspase activation platforms can be followed in single cells in real time. Induced proximity is the most apical step in the activation of initiator caspases, and therefore, caspase BiFC is a robust and specific method to measure initiator caspase activation.

Induction of cell death by the novel proteasome inhibitor marizomib in glioblastoma in vitro and in vivo.

New therapies for glioblastoma (GBM) are needed, as five-year survival is <10%. The proteasome inhibitor marizomib (MRZ) has inhibitory and death-inducing properties unique from previous inhibitors such as bortezomib (BTZ), and has not been well examined in GBM. We evaluated the mechanism of death and in vivo properties of MRZ in GBM. The activation kinetics of initiator caspases 2, 8, and 9 were assessed using chemical and knockdown strategies to determine their contribution to cell death. Blood brain barrier permeance and proteasome inhibition by MRZ and BTZ were examined in an orthotopic GBM model. Blockade of caspase 9, relative to other caspases, was most protective against both MRZ and BTZ. Only MRZ increased the proteasome substrate p27 in orthotopic brain tumors after a single injection, while both MRZ and BTZ increased p21 levels after multiple treatments. Cleavage of caspase substrate lamin A was increased in orthotopic brain tumors from mice treated with MRZ or BTZ and the histone deacetylase inhibitor vorinostat. Our data indicate that MRZ induces caspase 9-dependent death in GBM, suggesting drug efficacy biomarkers and possible resistance mechanisms. MRZ reaches orthotopic brain tumors where it inhibits proteasome function and increases death in combination with vorinostat.

Mesenchymal Stromal Cells for Linked Delivery of Oncolytic and Apoptotic Adenoviruses to Non-small-cell Lung Cancers.

Oncolytic adenoviruses (OAdV) represent a promising strategy for cancer therapy. Despite their activity in preclinical models, to date the clinical efficacy remains confined to minor responses after intratumor injection. To overcome these limitations, we developed an alternative approach using the combination of the OAdv ICOVIR15 with a replication incompetent adenoviral vector carrying the suicide gene of inducible Caspase 9 (Ad.iC9), both of which are delivered by mesenchymal stromal cells (MSCs). We hypothesized that coinfection with ICOVIR15 and Ad.iC9 would allow MSCs to replicate both vectors and deliver two distinct types of antitumor therapy to the tumor, amplifying the cytotoxic effects of the two viruses, in a non-small-cell lung cancer (NSCLC) model. We showed that MSCs can replicate and release both vectors, enabling significant transduction of the iC9 gene in tumor cells. In the in vivo model using human NSCLC xenografts, MSCs homed to lung tumors where they released both viruses. The activation of iC9 by the chemical inducer of dimerization (CID) significantly enhanced the antitumor activity of the ICOVIR15, increasing the tumor control and translating into improved overall survival of tumor-bearing mice. These data support the use of this innovative approach for the treatment of NSCLC.