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BACKGROUND: Neoadjuvant immunotherapies hold promise in muscle-invasive bladder cancer (MIBC). OBJECTIVE: To report on 2-yr disease-free (DFS) and overall (OS) survival including novel tissue-based biomarkers and circulating tumor DNA (ctDNA) in the ABACUS trial. DESIGN, SETTING, AND PARTICIPANTS: ABACUS was a multicenter, single-arm, neoadjuvant, phase 2 trial, including patients with MIBC (T2-4aN0M0) who were ineligible for or refused neoadjuvant cisplatin-based chemotherapy. INTERVENTION: Two cycles of atezolizumab were given prior to radical cystectomy. Serial tissue and blood samples were collected. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoints of pathological complete response (pCR) rate and dynamic changes to T-cell biomarkers were published previously. Secondary outcomes were 2-yr DFS and OS. A biomarker analysis correlated with relapse-free survival (RFS) was performed, which includes FOXP3, major histocompatibility complex class I, CD8/CD39, and sequential ctDNA measurements. RESULTS AND LIMITATIONS: The median follow-up time was 25 mo (95% confidence interval [CI] 25-26). Ninety-five patients received at least one cycle of atezolizumab. Eight patients did not undergo cystectomy (only one due to disease progression). The pCR rate was 31% (27/88; 95% CI 21-41). Two-year DFS and OS were 68% (95% CI 58-76) and 77% (95% CI 68-85), respectively. Two-year DFS in patients achieving a pCR was 85% (95% CI 65-94). Baseline PD-L1 and tumor mutational burden did not correlate with RFS (hazard ratio [HR] 0.60 [95% CI 0.24-1.5], p = 0.26, and 0.72 [95% CI 0.31-1.7], p = 0.46, respectively). RFS correlated with high baseline stromal CD8+ (HR 0.25 [95% CI 0.09-0.68], p = 0.007) and high post-treatment fibroblast activation protein (HR 4.1 [95% CI 1.3-13], p = 0.01). Circulating tumor DNA positivity values at baseline, after neoadjuvant therapy, and after surgery were 63% (25/40), 47% (14/30), and 14% (five/36), respectively. The ctDNA status was highly prognostic at all time points. No relapses were observed in ctDNA-negative patients at baseline and after neoadjuvant therapy. The lack of randomization and exploratory nature of the biomarker analysis are limitations of this work. CONCLUSIONS: Neoadjuvant atezolizumab in MIBC is associated with clinical responses and high DFS. CD8+ expression and serial ctDNA levels correlated with outcomes, and may contribute to personalized therapy in the future. PATIENT SUMMARY: We showed that bladder cancer patients receiving immunotherapy followed by cystectomy have good long-term outcomes. Furthermore, we found that certain biological features can predict patients who might have particular benefit from this therapy.
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Anticuerpos Monoclonales Humanizados , ADN Tumoral Circulante , Terapia Neoadyuvante , Neoplasias de la Vejiga Urinaria , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/uso terapéutico , ADN Tumoral Circulante/análisis , Cisplatino/uso terapéutico , Cistectomía/métodos , Humanos , Neoplasias de los Músculos/tratamiento farmacológico , Músculos/patología , Terapia Neoadyuvante/métodos , Invasividad Neoplásica , Recurrencia Local de Neoplasia , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/cirugíaRESUMEN
The formation of a functional organ such as the eye requires specification of the correct cell types and their terminal differentiation into cells with the appropriate morphologies and functions. Here, we show that the zinc-finger transcription factor Blimp-1 acts in secondary and tertiary pigment cells in the Drosophila retina to promote the formation of a bi-convex corneal lens with normal refractive power, and in cone cells to enable complete extension of the photoreceptor rhabdomeres. Blimp-1 expression depends on the hormone ecdysone, and loss of ecdysone signaling causes similar differentiation defects. Timely termination of Blimp-1 expression is also important, as its overexpression in the eye has deleterious effects. Our transcriptomic analysis revealed that Blimp-1 regulates the expression of many structural and secreted proteins in the retina. Blimp-1 may function in part by repressing another transcription factor; Slow border cells is highly upregulated in the absence of Blimp-1, and its overexpression reproduces many of the effects of removing Blimp-1. This work provides insight into the transcriptional networks and cellular interactions that produce the structures necessary for visual function.
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Proteínas de Drosophila , Drosophila , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Ecdisona , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Proteínas Represoras/genética , Factores de Transcripción/genéticaRESUMEN
RIP1 kinase is proposed to play a critical role in driving necroptosis and inflammation in neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS). Preclinical studies indicated that while pharmacological inhibition of RIP1 kinase can ameliorate axonal pathology and delay disease onset in the mutant SOD1 transgenic (SOD1-Tg) mice, genetic blockade of necroptosis does not provide benefit in this mouse model. To clarify the role of RIP1 kinase activity in driving pathology in SOD1-Tg mice, we crossed SOD1-Tgs to RIP1 kinase-dead knock-in mice, and measured disease progression using functional and histopathological endpoints. Genetic inactivation of the RIP1 kinase activity in the SOD1-Tgs did not benefit the declining muscle strength or nerve function, motor neuron degeneration or neuroinflammation. In addition, we did not find evidence of phosphorylated RIP1 accumulation in the spinal cords of ALS patients. On the other hand, genetic inactivation of RIP1 kinase activity ameliorated the depletion of the neurotransmitter dopamine in a toxin model of dopaminergic neurodegeneration. These findings indicate that RIP1 kinase activity is dispensable for disease pathogenesis in the SOD1-Tg mice while inhibition of kinase activity may provide benefit in acute injury models.
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Esclerosis Amiotrófica Lateral/enzimología , Proteínas Activadoras de GTPasa/genética , Neuronas Motoras/patología , Superóxido Dismutasa-1/genética , Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/patología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Células HT29 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , NecroptosisRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Mitochondria import nearly their entire proteome from the cytoplasm by translocating precursor proteins through the translocase of the outer membrane (TOM) complex. Here, we show dynamic regulation of mitochondrial import by the ubiquitin system. Acute pharmacological inhibition or genetic ablation of the mitochondrial deubiquitinase (DUB) USP30 triggers accumulation of Ub-substrates that are normally localized inside the mitochondria. Mitochondrial import of USP30 substrates is impaired in USP30 knockout (KO) cells, suggesting that deubiquitination promotes efficient import. Upstream of USP30, the E3 ligase March5 ubiquitinates mitochondrial proteins whose eventual import depends on USP30. In USP30 KOs, exogenous March5 expression induces accumulation of unimported translocation intermediates that are degraded by the proteasomes. In USP30 KO mice, TOM subunits have reduced abundance across multiple tissues. Together these data highlight how protein import into a subcellular compartment can be regulated by ubiquitination and deubiquitination by E3 ligase and DUB machinery positioned at the gate.
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Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Tioléster Hidrolasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Animales , Transporte Biológico , Proteínas Portadoras/genética , Femenino , Células HEK293 , Células HeLa , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Tioléster Hidrolasas/genética , Factores de Tiempo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Antibodies targeting PD-1 or its ligand 1 PD-L1 such as atezolizumab, have great efficacy in a proportion of metastatic urothelial cancers1,2. Biomarkers may facilitate identification of these responding tumors3. Neoadjuvant use of these agents is associated with pathological complete response in a spectrum of tumors, including urothelial cancer4-7. Sequential tissue sampling from these studies allowed for detailed on-treatment biomarker analysis. Here, we present a single-arm phase 2 study, investigating two cycles of atezolizumab before cystectomy in 95 patients with muscle-invasive urothelial cancer (ClinicalTrials.gov identifier: NCT02662309). Pathological complete response was the primary endpoint. Secondary endpoints focused on safety, relapse-free survival and biomarker analysis. The pathological complete response rate was 31% (95% confidence interval: 21-41%), achieving the primary efficacy endpoint. Baseline biomarkers showed that the presence of preexisting activated T cells was more prominent than expected and correlated with outcome. Other established biomarkers, such as tumor mutational burden, did not predict outcome, differentiating this from the metastatic setting. Dynamic changes to gene expression signatures and protein biomarkers occurred with therapy, whereas changes in DNA alterations with treatment were uncommon. Responding tumors showed predominant expression of genes related to tissue repair after treatment, making tumor biomarker interpretation challenging in this group. Stromal factors such as transforming growth factor-ß and fibroblast activation protein were linked to resistance, as was high expression of cell cycle gene signatures after treatment.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Biomarcadores de Tumor/genética , Carcinoma de Células Transicionales/tratamiento farmacológico , Neoplasias Urológicas/tratamiento farmacológico , Anciano , Anticuerpos Monoclonales Humanizados/efectos adversos , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/patología , Reparación del ADN/efectos de los fármacos , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante/efectos adversos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Transcriptoma/genética , Factor de Crecimiento Transformador beta/genética , Neoplasias Urológicas/genética , Neoplasias Urológicas/inmunología , Neoplasias Urológicas/patología , Urotelio/efectos de los fármacos , Urotelio/patologíaRESUMEN
Multiple lines of evidence indicate that mitochondrial dysfunction is central to Parkinson's disease. Here we investigate the mechanism by which parkin, an E3 ubiquitin ligase, and USP30, a mitochondrion-localized deubiquitylase, regulate mitophagy. We find that mitochondrial damage stimulates parkin to assemble Lys 6, Lys 11 and Lys 63 chains on mitochondria, and that USP30 is a ubiquitin-specific deubiquitylase with a strong preference for cleaving Lys 6- and Lys 11-linked multimers. Using mass spectrometry, we show that recombinant USP30 preferentially removes these linkage types from intact ubiquitylated mitochondria and counteracts parkin-mediated ubiquitin chain formation in cells. These results, combined with a series of chimaera and localization studies, afford insights into the mechanism by which a balance of ubiquitylation and deubiquitylation regulates mitochondrial homeostasis, and suggest a general mechanism for organelle autophagy.
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Homeostasis , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Tioléster Hidrolasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Dominio Catalítico , Extractos Celulares , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Homeostasis/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Lisina/metabolismo , Espectrometría de Masas , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/química , Mitofagia/efectos de los fármacos , Modelos Biológicos , Peroxisomas/efectos de los fármacos , Peroxisomas/metabolismo , Especificidad por Sustrato/efectos de los fármacos , Tioléster Hidrolasas/química , Proteasas Ubiquitina-Específicas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.
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Disección/métodos , Drosophila melanogaster/embriología , Ojo/embriología , Procedimientos Quirúrgicos Oftalmológicos/veterinaria , Animales , Diferenciación Celular/fisiología , Morfogénesis , Procedimientos Quirúrgicos Oftalmológicos/métodos , Células Fotorreceptoras , PupaRESUMEN
Cells maintain healthy mitochondria by degrading damaged mitochondria through mitophagy; defective mitophagy is linked to Parkinson's disease. Here we report that USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy driven by the ubiquitin ligase parkin (also known as PARK2) and protein kinase PINK1, which are encoded by two genes associated with Parkinson's disease. Parkin ubiquitinates and tags damaged mitochondria for clearance. Overexpression of USP30 removes ubiquitin attached by parkin onto damaged mitochondria and blocks parkin's ability to drive mitophagy, whereas reducing USP30 activity enhances mitochondrial degradation in neurons. Global ubiquitination site profiling identified multiple mitochondrial substrates oppositely regulated by parkin and USP30. Knockdown of USP30 rescues the defective mitophagy caused by pathogenic mutations in parkin and improves mitochondrial integrity in parkin- or PINK1-deficient flies. Knockdown of USP30 in dopaminergic neurons protects flies against paraquat toxicity in vivo, ameliorating defects in dopamine levels, motor function and organismal survival. Thus USP30 inhibition is potentially beneficial for Parkinson's disease by promoting mitochondrial clearance and quality control.
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Proteínas Mitocondriales/metabolismo , Mitofagia/fisiología , Tioléster Hidrolasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Masculino , Proteínas Mitocondriales/genética , Neuronas/metabolismo , Enfermedad de Parkinson/fisiopatología , Proteínas Quinasas/metabolismo , Ratas , Tioléster Hidrolasas/genética , Ubiquitina-Proteína Ligasas/genética , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismo , UbiquitinaciónRESUMEN
The human brain comprises more than 100 billion neurons, each of which has an elaborate shape and a complex pattern of connections. To untangle this complexity, it is often useful to visualize one neuron at a time. Mosaic analysis with double markers (MADM) is a genetic method for labeling and manipulating individual neurons. This method was developed in mice and it allows simultaneous labeling and gene knockout in clones of somatic cells or isolated single cells in vivo. In MADM, labeling is achieved by using site-specific recombinases to induce the reconstitution of chimeric fluorescent proteins. Here we provide the standard procedure for utilizing MADM to examine lineage analysis, neural circuit tracing, and gene function. ROSA26-MADM is used as an example because the reagents are published and available. As MADM cassettes are introduced onto more chromosomes, genes located on these other chromosomes can be subjected to mosaic analysis in an analogous manner to that described below. We present detailed protocols with troubleshooting guides, as well as applications of the technique in tracing neural circuits, live imaging of developing neurons, and studying mechanisms of neuronal morphogenesis.
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Técnicas Genéticas , Mosaicismo , Animales , Cruzamientos Genéticos , Disección , Femenino , Fluorescencia , Marcadores Genéticos , Humanos , Integrasas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mutación/genética , Recombinación Genética/genéticaRESUMEN
BACKGROUND: The Drosophila olfactory system exhibits very precise and stereotyped wiring that is specified predominantly by genetic programming. Dendrites of olfactory projection neurons (PNs) pattern the developing antennal lobe before olfactory receptor neuron axon arrival, indicating an intrinsic wiring mechanism for PN dendrites. These wiring decisions are likely determined through a transcriptional program. RESULTS: We find that loss of Brahma associated protein 55 kD (Bap55) results in a highly specific PN mistargeting phenotype. In Bap55 mutants, PNs that normally target to the DL1 glomerulus mistarget to the DA4l glomerulus with 100% penetrance. Loss of Bap55 also causes derepression of a GAL4 whose expression is normally restricted to a small subset of PNs. Bap55 is a member of both the Brahma (BRM) and the Tat interactive protein 60 kD (TIP60) ATP-dependent chromatin remodeling complexes. The Bap55 mutant phenotype is partially recapitulated by Domino and Enhancer of Polycomb mutants, members of the TIP60 complex. However, distinct phenotypes are seen in Brahma and Snf5-related 1 mutants, members of the BRM complex. The Bap55 mutant phenotype can be rescued by postmitotic expression of Bap55, or its human homologs BAF53a and BAF53b. CONCLUSIONS: Our results suggest that Bap55 functions through the TIP60 chromatin remodeling complex to regulate dendrite wiring specificity in PNs. The specificity of the mutant phenotypes suggests a position for the TIP60 complex at the top of a regulatory hierarchy that orchestrates dendrite targeting decisions.
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Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Dendritas/fisiología , Proteínas de Drosophila/metabolismo , Histona Acetiltransferasas/metabolismo , Neuronas/citología , Vías Olfatorias/citología , Factores de Transcripción/metabolismo , Actinas/genética , Actinas/fisiología , Animales , Animales Modificados Genéticamente , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Drosophila , Proteínas de Drosophila/genética , Regulación de la Expresión Génica/genética , Histona Acetiltransferasas/genética , Humanos , Cuerpos Pedunculados/crecimiento & desarrollo , Cuerpos Pedunculados/metabolismo , Mutación/genética , Neuronas/fisiología , Fenotipo , Proteína SMARCB1 , Factores de Transcripción/genéticaRESUMEN
Compared to the mechanisms of axon guidance, relatively little is known about the transcriptional control of dendrite guidance. The Drosophila olfactory system with its stereotyped organization provides an excellent model to study the transcriptional control of dendrite wiring specificity. Each projection neuron (PN) targets its dendrites to a specific glomerulus in the antennal lobe and its axon stereotypically to higher brain centers. Using a forward genetic screen, we identified a mutation in Rpd3 that disrupts PN targeting specificity. Rpd3 encodes a class I histone deacetylase (HDAC) homologous to mammalian HDAC1 and HDAC2. Rpd3(-/-) PN dendrites that normally target to a dorsolateral glomerulus mistarget to medial glomeruli in the antennal lobe, and axons exhibit a severe overbranching phenotype. These phenotypes can be rescued by postmitotic expression of Rpd3 but not HDAC3, the only other class I HDAC in Drosophila. Furthermore, disruption of the atypical homeodomain transcription factor Prospero (Pros) yields similar phenotypes, which can be rescued by Pros expression in postmitotic neurons. Strikingly, overexpression of Pros can suppress Rpd3(-/-) phenotypes. Our study suggests a specific function for the general chromatin remodeling factor Rpd3 in regulating dendrite targeting in neurons, largely through the postmitotic action of the Pros transcription factor.