MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatmentHaschka, Manuel D.; Karbon, Gerlinde; Soratroi, Claudia; O’Neill, Katelyn L.; Luo, Xu; Villunger, Andreas
doi: 10.1038/s41418-020-0503-6pmid: 32015503
Cells experiencing delays in mitotic progression are prone to undergo apoptosis unless they can exit mitosis before proapoptotic factors reach a critical threshold. Microtubule targeting agents (MTAs) arrest cells in mitosis and induce apoptotic cell death engaging the BCL2 network. Degradation of the antiapoptotic BCL2 family member MCL-1 is considered to set the time until onset of apoptosis upon MTA treatment. MCL1 degradation involves its interaction with one of its key binding partners, the proapoptotic BH3-only protein NOXA. Here, we report that the mitochondria-associated E3-ligase MARCH5, best known for its role in mitochondrial quality control and regulation of components of the mitochondrial fission machinery, controls the levels of MCL1/NOXA protein complexes in steady state as well as during mitotic arrest. Inhibition of MARCH5 function sensitizes cancer cells to the proapoptotic effects of MTAs by the accumulation of NOXA and primes cancer cells that may undergo slippage to escape death in mitosis to cell death in the next G1 phase. We propose that inhibition of MARCH5 may be a suitable strategy to sensitize cancer cells to antimitotic drug treatment.
PRDM16 orchestrates angiogenesis via neural differentiation in the developing brainSu, Libo; Lei, Xuepei; Ma, Hongyan; Feng, Chao; Jiang, Jing; Jiao, Jianwei
doi: 10.1038/s41418-020-0504-5pmid: 32015502
Angiogenesis plays crucial roles in maintaining the complex operation of central nervous system (CNS) development. The architecture of communication between neurogenesis and angiogenesis is essential to maintain normal brain development and function. Hence, any disruption of neuron–vascular communications may lead to the pathophysiology of cerebrovascular diseases and blood–brain barrier (BBB) dysfunction. Here we demonstrate that neural differentiation and communication are required for vascular development. Regarding the cellular and molecular mechanism, our results show that PRDM16 activity determines the production of mature neurons and their specific positions in the neocortex. In the cortical plate (CP), aberrant neurons fail to secrete modular calcium-binding protein 1 (SMOC1), an important neuronal signal that participates in neurovascular communication to regulate CNS angiogenesis. Neuronal SMOC1 interacts with TGFBR1 by activating the transcription factors phospho-Smad2/3 to convey intercellular signals to endothelial cells (ECs) in the TGF-β-Smad signaling pathway. Together, our results highlight a crucial coordinated neurovascular development process orchestrated by PRDM16 and reveal the importance of intimate communication for building the neurovascular network during brain development.
Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axisMancarella, Serena; Serino, Grazia; Dituri, Francesco; Cigliano, Antonio; Ribback, Silvia; Wang, Jingxiao; Chen, Xin; Calvisi, Diego F.; Giannelli, Gianluigi
doi: 10.1038/s41418-020-0505-4pmid: 32042099
Intrahepatic cholangiocarcinoma (iCCA) is a deadly disease with rising incidence and few treatment options. An altered expression and/or activation of NOTCH1–3 receptors has been shown to play a role in iCCA development and progression. In this study, we established a new CCA patient-derived xenograft model, which was validated by immunohistochemistry and transcriptomic analysis. The effects of Notch pathway suppression by the Crenigacestat (LY3039478)-specific inhibitor were evaluated in human iCCA cell lines and the PDX model. In vitro, LY3039478 significantly reduced Notch pathway components, including NICD1 and HES1, but not the other Notch receptors, in a panel of five different iCCA cell lines. In the PDX model, LY3039478 significantly inhibited the Notch pathway and tumor growth to the same extent as gemcitabine. Furthermore, gene expression analysis of iCCA mouse tissues treated with LY3039478 revealed a downregulation of VEGFA, HES1, and MMP13 genes. In the same tissues, DLL4 and CD31 co-localized, and their expression was significantly inhibited in the treated mice, as it happened in the case of MMP13. In an in vitro angiogenesis model, LY3039478 inhibited vessel formation, which was restored by the addition of MMP13. Finally, RNA-sequencing expression data of iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database demonstrated that NOTCH1, HES1, MMP13, DLL4, and VEGFA genes were significantly upregulated in tumors compared with adjacent nontumorous tissues. These data were confirmed by our group, using an independent cohort of iCCA specimens. Conclusion: We have developed and validated a new iCCA PDX model to test in vivo the activity of LY3039478, demonstrating its inhibitory role in Notch-dependent angiogenesis. Thus, the present data provide new knowledge on Notch signaling in iCCA, and support the inhibition of the Notch cascade as a promising strategy for the treatment of this disease.
Spatio-temporal characterization of the antiviral activity of the XRN1-DCP1/2 aggregation against cytoplasmic RNA viruses to prevent cell deathNg, Chen Seng; Kasumba, Dacquin M.; Fujita, Takashi; Luo, Honglin
doi: 10.1038/s41418-020-0509-0pmid: 32034313
Host nucleases are implicated in antiviral response through the processing of pathogen-derived nucleic acids. Among many host RNases, decapping enzymes DCP1 and 2, and 5′→3′ exonuclease XRN1, which are components of the RNA decay machinery, have been extensively studied in prokaryotes, plants, and invertebrates but less so in mammalian systems. As a result, the implication of XRN1 and DCPs in viral replication, in particular, the spatio-temporal dynamics during RNA viral infections remains elusive. Here, we highlight that XRN1 and DCPs play a critical role in limiting several groups of RNA viral infections. This antiviral activity was not obvious in wild-type cells but clearly observed in type I interferon (IFN-I)-deficient cells. Mechanistically, infection with RNA viruses induced the enrichment of XRN1 and DCPs in viral replication complexes (vRCs), hence forming distinct cytoplasmic aggregates. These aggregates served as sites for direct interaction between XRN1, DCP1/2, and viral ribonucleoprotein that contains viral RNA (vRNA). Although these XRN1-DCP1/2-vRC-containing foci resemble antiviral stress granules (SGs) or P-body (PB), they did not colocalize with known SG markers and did not correlate with critical PB functions. Furthermore, the presence of 5′ mono- and 5′ triphosphate structures on vRNA was not required for the formation of XRN1-DCP1/2-vRC-containing foci. On the other hand, single-, double-stranded, and higher-ordered vRNA species play a role but are not deterministic for efficient formation of XRN1-DCP1/2 foci and consequent antiviral activity in a manner proportional to RNA length. These results highlight the mechanism behind the antiviral function of XRN1-DCP1/2 in RNA viral infections independent of IFN-I response, protein kinase R and PB function.
BRUCE preserves genomic stability in the male germline of miceChe, Lixiao; Alavattam, Kris G.; Stambrook, Peter J.; Namekawa, Satoshi H.; Du, Chunying
doi: 10.1038/s41418-020-0513-4pmid: 32139899
BRUCE is a DNA damage response protein that promotes the activation of ATM and ATR for homologous recombination (HR) repair in somatic cells, making BRUCE a key protector of genomic stability. Preservation of genomic stability in the germline is essential for the maintenance of species. Here, we show that BRUCE is required for the preservation of genomic stability in the male germline of mice, specifically in spermatogonia and spermatocytes. Conditional knockout of Bruce in the male germline leads to profound defects in spermatogenesis, including impaired maintenance of spermatogonia and increased chromosomal anomalies during meiosis. Bruce-deficient pachytene spermatocytes frequently displayed persistent DNA breaks. Homologous synapsis was impaired, and nonhomologous associations and rearrangements were apparent in up to 10% of Bruce-deficient spermatocytes. Genomic instability was apparent in the form of chromosomal fragmentation, translocations, and synapsed quadrivalents and hexavalents. In addition, unsynapsed regions of rearranged autosomes were devoid of ATM and ATR signaling, suggesting an impairment in the ATM- and ATR-dependent DNA damage response of meiotic HR. Taken together, our study unveils crucial functions for BRUCE in the maintenance of spermatogonia and in the regulation of meiotic HR—functions that preserve the genomic stability of the male germline.
Convergence of pathway analysis and pattern recognition predicts sensitization to latest generation TRAIL therapeutics by IAP antagonismVetma, Vesna; Guttà, Cristiano; Peters, Nathalie; Praetorius, Christian; Hutt, Meike; Seifert, Oliver; Meier, Friedegund; Kontermann, Roland; Kulms, Dagmar; Rehm, Markus
doi: 10.1038/s41418-020-0512-5pmid: 32081986
Second generation TRAIL-based therapeutics, combined with sensitising co-treatments, have recently entered clinical trials. However, reliable response predictors for optimal patient selection are not yet available. Here, we demonstrate that a novel and translationally relevant hexavalent TRAIL receptor agonist, IZI1551, in combination with Birinapant, a clinically tested IAP antagonist, efficiently induces cell death in various melanoma models, and that responsiveness can be predicted by combining pathway analysis, data-driven modelling and pattern recognition. Across a panel of 16 melanoma cell lines, responsiveness to IZI1551/Birinapant was heterogeneous, with complete resistance and pronounced synergies observed. Expression patterns of TRAIL pathway regulators allowed us to develop a combinatorial marker that predicts potent cell killing with high accuracy. IZI1551/Birinapant responsiveness could be predicted not only for cell lines, but also for 3D tumour cell spheroids and for cells directly isolated from patient melanoma metastases (80–100% prediction accuracies). Mathematical parameter reduction identified 11 proteins crucial to ensure prediction accuracy, with x-linked inhibitor of apoptosis protein (XIAP) and procaspase-3 scoring highest, and Bcl-2 family members strongly represented. Applied to expression data of a cohort of n = 365 metastatic melanoma patients in a proof of concept in silico trial, the predictor suggested that IZI1551/Birinapant responsiveness could be expected for up to 30% of patient tumours. Overall, response frequencies in melanoma models were very encouraging, and the capability to predict melanoma sensitivity to combinations of latest generation TRAIL-based therapeutics and IAP antagonists can address the need for patient selection strategies in clinical trials based on these novel drugs.
Squamous differentiation requires G2/mitosis slippage to avoid apoptosisSanz-Gómez, Natalia; de Pedro, Isabel; Ortigosa, Beatriz; Santamaría, David; Malumbres, Marcos; de Cárcer, Guillermo; Gandarillas, Alberto
doi: 10.1038/s41418-020-0515-2pmid: 32080348
The cellular mechanisms controlling cell fate in self-renewal tissues remain unclear. Cell cycle failure often leads to an apoptosis anti-oncogenic response. We have inactivated Cdk1 or Polo-like-1 kinases, essential targets of the mitotic checkpoints, in the epithelia of skin and oral mucosa. Here, we show that inactivation of the mitotic kinases leading to polyploidy in vivo, produces a fully differentiated epithelium. Cells within the basal layer aberrantly differentiate and contain large or various nuclei. Freshly isolated KO cells were also differentiated and polyploid. However, sustained metaphase arrest downstream of the spindle anaphase checkpoint (SAC) due to abrogation of CDC20 (essential cofactor of anaphase-promoting complex), impaired squamous differentiation and resulted in apoptosis. Therefore, upon prolonged arrest keratinocytes need to slip beyond G2 or mitosis in order to initiate differentiation. The results altogether demonstrate that mitotic checkpoints drive squamous cell fate towards differentiation or apoptosis in response to genetic damage.