SAFB2 Enables the Processing of Suboptimal Stem-Loop Structures in Clustered Primary miRNA Transcripts.

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.

Extra centrosomes induce PIDD1-mediated inflammation and immunosurveillance.

Unscheduled increases in ploidy underlie defects in tissue function, premature aging, and malignancy. A concomitant event to polyploidization is the amplification of centrosomes, the main microtubule organization centers in animal cells. Supernumerary centrosomes are frequent in tumors, correlating with higher aggressiveness and poor prognosis. However, extra centrosomes initially also exert an onco-protective effect by activating p53-induced cell cycle arrest. If additional signaling events initiated by centrosomes help prevent pathology is unknown. Here, we report that extra centrosomes, arising during unscheduled polyploidization or aberrant centriole biogenesis, induce activation of NF-κB signaling and sterile inflammation. This signaling requires the NEMO-PIDDosome, a multi-protein complex composed of PIDD1, RIPK1, and NEMO/IKKγ. Remarkably, the presence of supernumerary centrosomes suffices to induce a paracrine chemokine and cytokine profile, able to polarize macrophages into a pro-inflammatory phenotype. Furthermore, extra centrosomes increase the immunogenicity of cancer cells and render them more susceptible to NK-cell attack. Hence, the PIDDosome acts as a dual effector, able to engage not only the p53 network for cell cycle control but also NF-κB signaling to instruct innate immunity.

RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation.

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.

BCL-2-family protein tBID can act as a BAX-like effector of apoptosis.

During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we report that tBID can also mediate mitochondrial permeabilization by itself, resulting in release of cytochrome c and mitochondrial DNA, caspase activation and apoptosis even in absence of BAX and BAK. This previously unrecognized activity of tBID depends on helix 6, homologous to the pore-forming regions of BAX and BAK, and can be blocked by pro-survival BCL-2 proteins. Importantly, tBID-mediated mitochondrial permeabilization independent of BAX and BAK is physiologically relevant for SMAC release in the immune response against Shigella infection. Furthermore, it can be exploited to kill leukaemia cells with acquired venetoclax resistance due to lack of active BAX and BAK. Our findings define tBID as an effector of mitochondrial permeabilization in apoptosis and provide a new paradigm for BCL-2 proteins, with implications for anti-bacterial immunity and cancer therapy.

Chronic spindle assembly checkpoint activation causes myelosuppression and gastrointestinal atrophy.

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.

Centriolar distal appendages activate the centrosome-PIDDosome-p53 signalling axis via ANKRD26.

Centrosome amplification results into genetic instability and predisposes cells to neoplastic transformation. Supernumerary centrosomes trigger p53 stabilization dependent on the PIDDosome (a multiprotein complex composed by PIDD1, RAIDD and Caspase-2), whose activation results in cleavage of p53's key inhibitor, MDM2. Here, we demonstrate that PIDD1 is recruited to mature centrosomes by the centriolar distal appendage protein ANKRD26. PIDDosome-dependent Caspase-2 activation requires not only PIDD1 centrosomal localization, but also its autoproteolysis. Following cytokinesis failure, supernumerary centrosomes form clusters, which appear to be necessary for PIDDosome activation. In addition, in the context of DNA damage, activation of the complex results from a p53-dependent elevation of PIDD1 levels independently of centrosome amplification. We propose that PIDDosome activation can in both cases be promoted by an ANKRD26-dependent local increase in PIDD1 concentration close to the centrosome. Collectively, these findings provide a paradigm for how centrosomes can contribute to cell fate determination by igniting a signalling cascade.

The PIDDosome activates p53 in response to supernumerary centrosomes.

Centrosomes, the main microtubule-organizing centers in animal cells, are replicated exactly once during the cell division cycle to form the poles of the mitotic spindle. Supernumerary centrosomes can lead to aberrant cell division and have been causally linked to chromosomal instability and cancer. Here, we report that an increase in the number of mature centrosomes, generated by disrupting cytokinesis or forcing centrosome overduplication, triggers the activation of the PIDDosome multiprotein complex, leading to Caspase-2-mediated MDM2 cleavage, p53 stabilization, and p21-dependent cell cycle arrest. This pathway also restrains the extent of developmentally scheduled polyploidization by regulating p53 levels in hepatocytes during liver organogenesis. Taken together, the PIDDosome acts as a first barrier, engaging p53 to halt the proliferation of cells carrying more than one mature centrosome to maintain genome integrity.

DNA-PKcs-PIDDosome: a nuclear caspase-2-activating complex with role in G2/M checkpoint maintenance.

Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.

MOMP in the absence of BH3-only proteins.

The minimum requirement for mitochondrial apoptosis has been controversial ever since the discovery of BCL-2 as a cell death regulator. In this issue ofGenes&Development, O'Neill and colleagues (pp. 973-988) end a long-standing debate by creating a cellular system free of BCL-2 family proteins, thereby identifying the outer mitochondrial membrane rather than BH3-only proteins as the only requirement for BAX/BAK activation and mitochondrial outer membrane permeabilization (MOMP).

Interrogating the relevance of mitochondrial apoptosis for vertebrate development and postnatal tissue homeostasis.

"Programmed cell death or 'apoptosis' is critical for organogenesis during embryonic development and tissue homeostasis in the adult. Its deregulation can contribute to a broad range of human pathologies, including neurodegeneration, cancer, or autoimmunity…" These or similar phrases have become generic opening statements in many reviews and textbooks describing the physiological relevance of apoptotic cell death. However, while the role in disease has been documented beyond doubt, facilitating innovative drug discovery, we wonder whether the former is really true. What goes wrong in vertebrate development or in adult tissue when the main route to apoptotic cell death, controlled by the BCL2 family, is impaired? Such scenarios have been mimicked by deletion of one or more prodeath genes within the BCL2 family, and gene targeting studies in mice exploring the consequences have been manifold. Many of these studies were geared toward understanding the role of BCL2 family proteins and mitochondrial apoptosis in disease, whereas fewer focused in detail on their role during normal development or tissue homeostasis, perhaps also due to an irritating lack of phenotype. Looking at these studies, the relevance of classical programmed cell death by apoptosis for development appears rather limited. Together, these many studies suggest either highly selective and context-dependent contributions of mitochondrial apoptosis or significant redundancy with alternative cell death mechanisms, as summarized and discussed here.

HPV-induced host epigenetic reprogramming is lost upon progression to high-grade cervical intraepithelial neoplasia.

The impact of a pathogen on host disease can only be studied in samples covering the entire spectrum of pathogenesis. Persistent oncogenic human papilloma virus (HPV) infection is the most common cause for cervical cancer. Here, we investigate HPV-induced host epigenome-wide changes prior to development of cytological abnormalities. Using cervical sample methylation array data from disease-free women with or without an oncogenic HPV infection, we develop the WID (Women's cancer risk identification)-HPV, a signature reflective of changes in the healthy host epigenome related to high-risk HPV strains (AUC = 0.78, 95% CI: 0.72-0.85, in nondiseased women). Looking at HPV-associated changes across disease development, HPV-infected women with minor cytological alterations (cervical intraepithelial neoplasia grade 1/2, CIN1/2), but surprisingly not those with precancerous changes or invasive cervical cancer (CIN3+), show an increased WID-HPV index, indicating the WID-HPV may reflect a successful viral clearance response absent in progression to cancer. Further investigation revealed the WID-HPV is positively associated with apoptosis (ρ = 0.48; P < .001) and negatively associated with epigenetic replicative age (ρ = -0.43; P < .001). Taken together, our data suggest the WID-HPV captures a clearance response associated with apoptosis of HPV-infected cells. This response may be dampened or lost with increased underlying replicative age of infected cells, resulting in progression to cancer.

PIDD1 in cell cycle control, sterile inflammation and cell death.

The death fold domain-containing protein PIDD1 has recently attracted renewed attention as a regulator of the orphan cell death-related protease, Caspase-2. Caspase-2 can activate p53 to promote cell cycle arrest in response to centrosome aberrations, and its activation requires formation of the PIDDosome multi-protein complex containing multimers of PIDD1 and the adapter RAIDD/CRADD at its core. However, PIDD1 appears to be able to engage with multiple client proteins to promote an even broader range of biological responses, such as NF-κB activation, translesion DNA synthesis or cell death. PIDD1 shows features of inteins, a class of self-cleaving proteins, to create different polypeptides from a common precursor protein that allow it to serve these diverse functions. This review summarizes structural information and molecular features as well as recent experimental advances that highlight the potential pathophysiological roles of this unique death fold protein to highlight its drug-target potential.

PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. This multi-protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome-induced p53-activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro-tumorigenic effect of PIDDosome-mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts poor survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and that tumor cell density may serve as a novel prognostic marker for recurrence-free survival in HCC patients.

Polyploidy control in hepatic health and disease.

A balanced increase in DNA content (ploidy) is observed in some human cell types, including bone-resorbing osteoclasts, platelet-producing megakaryocytes, cardiomyocytes or hepatocytes. The impact of increased hepatocyte ploidy on normal physiology and diverse liver pathologies is still poorly understood. Recent findings suggest swift genetic adaptation to hepatotoxic stress and the protection from malignant transformation as beneficial effects. Herein, we discuss the molecular mechanisms regulating hepatocyte polyploidisation and its implication for different liver diseases and hepatocellular carcinoma. We report on centrosomes' role in limiting polyploidy by activating the p53 signalling network (via the PIDDosome multiprotein complex) and we discuss the role of this pathway in liver disease. Increased hepatocyte ploidy is a hallmark of hepatic inflammation and may play a protective role against liver cancer. Our evolving understanding of hepatocyte ploidy is discussed from the perspective of its potential clinical application for risk stratification, prognosis, and novel therapeutic strategies in liver disease and hepatocellular carcinoma.

Checkpoint kinase 1 is essential for fetal and adult hematopoiesis.

Checkpoint kinase 1 (CHK1) is critical for S-phase fidelity and preventing premature mitotic entry in the presence of DNA damage. Tumor cells have developed a strong dependence on CHK1 for survival, and hence, this kinase has developed into a promising drug target. Chk1 deficiency in mice results in blastocyst death due to G2/M checkpoint failure showing that it is an essential gene and may be difficult to target therapeutically. Here, we show that chemical inhibition of CHK1 kills murine and human hematopoietic stem and progenitor cells (HSPCs) by the induction of BCL2-regulated apoptosis. Cell death in HSPCs is independent of p53 but requires the BH3-only proteins BIM, PUMA, and NOXA. Moreover, Chk1 is essential for definitive hematopoiesis in the embryo. Noteworthy, cell death inhibition in HSPCs cannot restore blood cell formation as HSPCs lacking CHK1 accumulate DNA damage and stop dividing. Moreover, conditional deletion of Chk1 in hematopoietic cells of adult mice selects for blood cells retaining CHK1, suggesting an essential role in maintaining functional hematopoiesis. Our findings establish a previously unrecognized role for CHK1 in establishing and maintaining hematopoiesis.

At a Crossroads to Cancer: How p53-Induced Cell Fate Decisions Secure Genome Integrity.

P53 is known as the most critical tumor suppressor and is often referred to as the guardian of our genome. More than 40 years after its discovery, we are still struggling to understand all molecular details on how this transcription factor prevents oncogenesis or how to leverage current knowledge about its function to improve cancer treatment. Multiple cues, including DNA-damage or mitotic errors, can lead to the stabilization and nuclear translocation of p53, initiating the expression of multiple target genes. These transcriptional programs may be cell-type- and stimulus-specific, as is their outcome that ultimately imposes a barrier to cellular transformation. Cell cycle arrest and cell death are two well-studied consequences of p53 activation, but, while being considered critical, they do not fully explain the consequences of p53 loss-of-function phenotypes in cancer. Here, we discuss how mitotic errors alert the p53 network and give an overview of multiple ways that p53 can trigger cell death. We argue that a comparative analysis of different types of p53 responses, elicited by different triggers in a time-resolved manner in well-defined model systems, is critical to understand the cell-type-specific cell fate induced by p53 upon its activation in order to resolve the remaining mystery of its tumor-suppressive function.

Perturbing mitosis for anti-cancer therapy: is cell death the only answer?

Interfering with mitosis for cancer treatment is an old concept that has proven highly successful in the clinics. Microtubule poisons are used to treat patients with different types of blood or solid cancer since more than 20 years, but how these drugs achieve clinical response is still unclear. Arresting cells in mitosis can promote their demise, at least in a petri dish. Yet, at the molecular level, this type of cell death is poorly defined and cancer cells often find ways to escape. The signaling pathways activated can lead to mitotic slippage, cell death, or senescence. Therefore, any attempt to unravel the mechanistic action of microtubule poisons will have to investigate aspects of cell cycle control, cell death initiation in mitosis and after slippage, at single-cell resolution. Here, we discuss possible mechanisms and signaling pathways controlling cell death in mitosis or after escape from mitotic arrest, as well as secondary consequences of mitotic errors, particularly sterile inflammation, and finally address the question how clinical efficacy of anti-mitotic drugs may come about and could be improved.

The resurrection of the PIDDosome - emerging roles in the DNA-damage response and centrosome surveillance.

The PIDDosome is often used as the alias for a multi-protein complex that includes the p53-induced death domain protein 1 (PIDD1), the bipartite linker protein CRADD (also known as RAIDD) and the pro-form of an endopeptidase belonging to the caspase family, i.e. caspase-2. Yet, PIDD1 variants can also interact with a number of other proteins that include RIPK1 (also known as RIP1) and IKBKG (also known as NEMO), PCNA and RFC5, as well as nucleolar components such as NPM1 or NCL. This promiscuity in protein binding is facilitated mainly by autoprocessing of the full-length protein into various fragments that contain different structural domains. As a result, multiple responses can be mediated by protein complexes that contain a PIDD1 domain. This suggests that PIDD1 acts as an integrator for multiple types of stress that need instant attention. Examples are various types of DNA lesion but also the presence of extra centrosomes that can foster aneuploidy and, ultimately, promote DNA damage. Here, we review the role of PIDD1 in response to DNA damage and also highlight novel functions of PIDD1, such as in centrosome surveillance and scheduled polyploidisation as part of a cellular differentiation program during organogenesis.

Only the strong survive.

Whether apoptosis is relevant for interclonal competition of T cells after antigen encounter has remained uncertain. In this issue of Immunity, Wensveen et al. (2010) establish a critical role for the proapoptotic BH3-only protein Noxa in this selection process.

The miR-15 family reinforces the transition from proliferation to differentiation in pre-B cells.

Precursor B lymphocytes expand upon expression of a pre-B cell receptor (pre-BCR), but then transit into a resting state in which immunoglobulin light chain gene recombination is initiated. This bi-phasic sequence is orchestrated by the IL-7 receptor (IL-7R) and pre-BCR signaling, respectively, but little is known about microRNAs fine-tuning these events. Here, we show that pre-B cells lacking miR-15 family functions exhibit prolonged proliferation due to aberrant expression of the target genes cyclin E1 and D3. As a consequence, they fail to trigger the transcriptional reprogramming normally accompanying their differentiation, resulting in a developmental block at the pre-B cell stage. Intriguingly, our data indicate that the miR-15 family is suppressed by both IL-7R and pre-BCR signaling, suggesting it is actively integrated into the regulatory circuits of developing B cells. These findings identify the miR-15 family as a novel element required to promote the switch from pre-B cell proliferation to differentiation.