RESUMEN
Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA sequencing revealed that >80% of cells in thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.
RESUMEN
Despite significant advances over recent years, the treatment of T cell acute lymphoblastic leukemia (T-ALL) remains challenging. We have recently shown that a subset of T-ALL cases exhibited constitutive activation of the lymphocyte-specific protein tyrosine kinase (LCK) and were consequently responsive to treatments with LCK inhibitors and degraders such as dasatinib and dasatinib-based PROTACs. Here we report the design, synthesis and in vitro/vivo evaluation of SJ45566, a potent and orally bioavailable LCK PROTAC.
Asunto(s)
Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Humanos , Administración Oral , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Animales , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral , Relación Estructura-ActividadRESUMEN
Sonic Hedgehog (SHH) is a driver of embryonic patterning that, when corrupted, triggers developmental disorders and cancers. SHH effector responses are organized through primary cilia (PC) that grow and retract with the cell cycle and in response to extracellular cues. Disruption of PC homeostasis corrupts SHH regulation, placing significant pressure on the pathway to maintain ciliary fitness. Mechanisms by which ciliary robustness is ensured in SHH-stimulated cells are not yet known. Herein, we reveal a crosstalk circuit induced by SHH activation of Phospholipase A2α that drives ciliary E-type prostanoid receptor 4 (EP4) signaling to ensure PC function and stabilize ciliary length. We demonstrate that blockade of SHH-EP4 crosstalk destabilizes PC cyclic AMP (cAMP) equilibrium, slows ciliary transport, reduces ciliary length, and attenuates SHH pathway induction. Accordingly, Ep4-/- mice display shortened neuroepithelial PC and altered SHH-dependent neuronal cell fate specification. Thus, SHH initiates coordination between distinct ciliary receptors to maintain PC function and length homeostasis for robust downstream signaling.
Asunto(s)
Cilios , Proteínas Hedgehog , Prostaglandinas , Transducción de Señal , Animales , Ratones , Cilios/metabolismo , AMP Cíclico/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Ratones Noqueados , Prostaglandinas/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/genéticaRESUMEN
3D cellular-specific epigenetic and transcriptomic reprogramming is critical to organogenesis and tumorigenesis. Here we dissect the distinct cell fitness in 2D (normoxia vs. chronic hypoxia) vs 3D (normoxia) culture conditions. We identify over 600 shared essential genes and additional context-specific fitness genes and pathways. Knockout of the VHL-HIF1 pathway results in incompatible fitness defects under normoxia vs. 1% oxygen or 3D culture conditions. Moreover, deletion of each of the mitochondrial respiratory electron transport chain complex has distinct fitness outcomes. Notably, multicellular organogenesis signaling pathways including TGFß-SMAD specifically constrict the uncontrolled cell proliferation in 3D while inactivation of epigenetic modifiers (Bcor, Kmt2d, Mettl3 and Mettl14) has opposite outcomes in 2D vs. 3D. We further identify a 3D-dependent synthetic lethality with partial loss of Prmt5 due to a reduction of Mtap expression resulting from 3D-specific epigenetic reprogramming. Our study highlights unique epigenetic, metabolic and organogenesis signaling dependencies under different cellular settings.
RESUMEN
NLRs constitute a large, highly conserved family of cytosolic pattern recognition receptors that are central to health and disease, making them key therapeutic targets. NLRC5 is an enigmatic NLR with mutations associated with inflammatory and infectious diseases, but little is known about its function as an innate immune sensor and cell death regulator. Therefore, we screened for NLRC5's role in response to infections, PAMPs, DAMPs, and cytokines. We identified that NLRC5 acts as an innate immune sensor to drive inflammatory cell death, PANoptosis, in response to specific ligands, including PAMP/heme and heme/cytokine combinations. NLRC5 interacted with NLRP12 and PANoptosome components to form a cell death complex, suggesting an NLR network forms similar to those in plants. Mechanistically, TLR signaling and NAD+ levels regulated NLRC5 expression and ROS production to control cell death. Furthermore, NLRC5-deficient mice were protected in hemolytic and inflammatory models, suggesting that NLRC5 could be a potential therapeutic target.
Asunto(s)
Inflamación , Péptidos y Proteínas de Señalización Intracelular , NAD , Animales , Ratones , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , NAD/metabolismo , Humanos , Inmunidad Innata , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Ratones Noqueados , Transducción de Señal , Células HEK293 , Inflamasomas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Receptores Toll-Like/metabolismo , Masculino , Citocinas/metabolismo , Proteínas de Unión al CalcioRESUMEN
Background: Medulloblastoma (MB) is the most malignant childhood brain cancer. Group 3 MB subtype accounts for about 25% of MB diagnoses and is associated with the most unfavorable outcomes. Herein, we report that more than half of group 3 MB tumors express melanoma antigens (MAGEs), which are potential prognostic and therapeutic markers. MAGEs are tumor antigens, expressed in several types of adult cancers and associated with poorer prognosis and therapy resistance; however, their expression in pediatric cancers is mostly unknown. The aim of this study was to determine whether MAGEs are activated in pediatric MB. Methods: To determine MAGE frequency in pediatric MB, we obtained formalin-fixed paraffin-embedded tissue (FFPE) samples of 34 patients, collected between 2008 - 2015, from the Children's Medical Center Dallas pathology archives and applied our validated reverse transcription quantitative PCR (RT-qPCR) assay to measure the relative expression of 23 MAGE cancer-testis antigen genes. To validate our data, we analyzed several published datasets from pediatric MB patients and patient-derived orthotopic xenografts, totaling 860 patients. We then examined how MAGE expression affects the growth and oncogenic potential of medulloblastoma cells by CRISPR-Cas9- and siRNA-mediated gene depletion. Results: Our RT-qPCR analysis suggested that MAGEs were expressed in group 3/4 medulloblastoma. Further mining of bulk and single-cell RNA-sequencing datasets confirmed that 50-75% of group 3 tumors activate a subset of MAGE genes. Depletion of MAGEAs, B2, and Cs alter MB cell survival, viability, and clonogenic growth due to decreased proliferation and increased apoptosis. Conclusions: These results indicate that targeting MAGEs in medulloblastoma may be a potential therapeutic option for group 3 medulloblastomas. Key Points: Several Type I MAGE CTAs are expressed in >60% of group 3 MBs. Type I MAGEs affect MB cell proliferation and apoptosis. MAGEs are potential biomarkers and therapeutic targets for group 3 MBs. Importance of the Study: This study is the first comprehensive analysis of all Type I MAGE CTAs ( MAGEA , -B , and -C subfamily members) in pediatric MBs. Our results show that more than 60% of group 3 MBs express MAGE genes, which are required for the viability and growth of cells in which they are expressed. Collectively, these data provide novel insights into the antigen landscape of pediatric MBs. The activation of MAGE genes in group 3 MBs presents potential stratifying and therapeutic options.
RESUMEN
Retinoic acid (RA) is a standard-of-care neuroblastoma drug thought to be effective by inducing differentiation. Curiously, RA has little effect on primary human tumors during upfront treatment but can eliminate neuroblastoma cells from the bone marrow during post-chemo consolidation therapy-a discrepancy that has never been explained. To investigate this, we treated a large cohort of neuroblastoma cell lines with RA and observed that the most RA-sensitive cells predominantly undergo apoptosis or senescence, rather than differentiation. We conducted genome-wide CRISPR knockout screens under RA treatment, which identified BMP signaling as controlling the apoptosis/senescence vs differentiation cell fate decision and determining RA's overall potency. We then discovered that BMP signaling activity is markedly higher in neuroblastoma patient samples at bone marrow metastatic sites, providing a plausible explanation for RA's ability to clear neuroblastoma cells specifically from the bone marrow, seemingly mimicking interactions between BMP and RA during normal development.
RESUMEN
Defining genetic factors impacting chemotherapy failure can help to better predict response and identify drug resistance mechanisms. However, there is limited understanding of the contribution of inherited noncoding genetic variation on inter-individual differences in chemotherapy response in childhood acute lymphoblastic leukemia (ALL). Here we map inherited noncoding variants associated with treatment outcome and/or chemotherapeutic drug resistance to ALL cis-regulatory elements and investigate their gene regulatory potential and target gene connectivity using massively parallel reporter assays and three-dimensional chromatin looping assays, respectively. We identify 54 variants with transcriptional effects and high-confidence gene connectivity. Additionally, functional interrogation of the top variant, rs1247117, reveals changes in chromatin accessibility, PU.1 binding affinity and gene expression, and deletion of the genomic interval containing rs1247117 sensitizes cells to vincristine. Together, these data demonstrate that noncoding regulatory variants associated with diverse pharmacological traits harbor significant effects on allele-specific transcriptional activity and impact sensitivity to antileukemic agents.
Asunto(s)
Farmacogenética , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Proto-Oncogénicas , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Niño , Resistencia a Antineoplásicos/genética , Variación Genética , Línea Celular Tumoral , Vincristina/uso terapéutico , Vincristina/farmacología , Polimorfismo de Nucleótido Simple , Alelos , Cromatina/metabolismo , Cromatina/genética , Transactivadores/genética , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacosRESUMEN
BACKGROUND: The innate immune system serves as the first line of host defense. Transforming growth factor-ß-activated kinase 1 (TAK1) is a key regulator of innate immunity, cell survival, and cellular homeostasis. Because of its importance in immunity, several pathogens have evolved to carry TAK1 inhibitors. In response, hosts have evolved to sense TAK1 inhibition and induce robust lytic cell death, PANoptosis, mediated by the RIPK1-PANoptosome. PANoptosis is a unique innate immune inflammatory lytic cell death pathway initiated by an innate immune sensor and driven by caspases and RIPKs. While PANoptosis can be beneficial to clear pathogens, excess activation is linked to pathology. Therefore, understanding the molecular mechanisms regulating TAK1 inhibitor (TAK1i)-induced PANoptosis is central to our understanding of RIPK1 in health and disease. RESULTS: In this study, by analyzing results from a cell death-based CRISPR screen, we identified protein phosphatase 6 (PP6) holoenzyme components as regulators of TAK1i-induced PANoptosis. Loss of the PP6 enzymatic component, PPP6C, significantly reduced TAK1i-induced PANoptosis. Additionally, the PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 had redundant roles in regulating TAK1i-induced PANoptosis, and their combined depletion was required to block TAK1i-induced cell death. Mechanistically, PPP6C and its regulatory subunits promoted the pro-death S166 auto-phosphorylation of RIPK1 and led to a reduction in the pro-survival S321 phosphorylation. CONCLUSIONS: Overall, our findings demonstrate a key requirement for the phosphatase PP6 complex in the activation of TAK1i-induced, RIPK1-dependent PANoptosis, suggesting this complex could be therapeutically targeted in inflammatory conditions.
Asunto(s)
Fosfoproteínas Fosfatasas , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Humanos , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Necroptosis , Inmunidad InnataRESUMEN
The cellular plasticity of neuroblastoma is defined by a mixture of two major cell states, adrenergic (ADRN) and mesenchymal (MES), which may contribute to therapy resistance. However, how neuroblastoma cells switch cellular states during therapy remains largely unknown and how to eradicate neuroblastoma regardless of their cell states is a clinical challenge. To better understand the lineage switch of neuroblastoma in chemoresistance, we comprehensively defined the transcriptomic and epigenetic map of ADRN and MES types of neuroblastomas using human and murine models treated with indisulam, a selective RBM39 degrader. We showed that cancer cells not only undergo a bidirectional switch between ADRN and MES states, but also acquire additional cellular states, reminiscent of the developmental pliancy of neural crest cells. The lineage alterations are coupled with epigenetic reprogramming and dependency switch of lineage-specific transcription factors, epigenetic modifiers and targetable kinases. Through targeting RNA splicing, indisulam induces an inflammatory tumor microenvironment and enhances anticancer activity of natural killer cells. The combination of indisulam with anti-GD2 immunotherapy results in a durable, complete response in high-risk transgenic neuroblastoma models, providing an innovative, rational therapeutic approach to eradicate tumor cells regardless of their potential to switch cell states.
RESUMEN
The CRISPR toolbox continues to expand at a rapid pace, leaving researchers scrambling to assess the latest tools in their systems of interest. McGee et al.1 have developed a modular assembly platform with standardized and interchangeable components for rapid construction and deployment of novel CRISPR constructs.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
Dysregulated pre-mRNA splicing and metabolism are two hallmarks of MYC-driven cancers. Pharmacological inhibition of both processes has been extensively investigated as potential therapeutic avenues in preclinical and clinical studies. However, how pre-mRNA splicing and metabolism are orchestrated in response to oncogenic stress and therapies is poorly understood. Here, we demonstrate that jumonji domain containing 6, arginine demethylase, and lysine hydroxylase, JMJD6, acts as a hub connecting splicing and metabolism in MYC-driven human neuroblastoma. JMJD6 cooperates with MYC in cellular transformation of murine neural crest cells by physically interacting with RNA binding proteins involved in pre-mRNA splicing and protein homeostasis. Notably, JMJD6 controls the alternative splicing of two isoforms of glutaminase (GLS), namely kidney-type glutaminase (KGA) and glutaminase C (GAC), which are rate-limiting enzymes of glutaminolysis in the central carbon metabolism in neuroblastoma. Further, we show that JMJD6 is correlated with the anti-cancer activity of indisulam, a 'molecular glue' that degrades splicing factor RBM39, which complexes with JMJD6. The indisulam-mediated cancer cell killing is at least partly dependent on the glutamine-related metabolic pathway mediated by JMJD6. Our findings reveal a cancer-promoting metabolic program is associated with alternative pre-mRNA splicing through JMJD6, providing a rationale to target JMJD6 as a therapeutic avenue for treating MYC-driven cancers.
Asunto(s)
Neuroblastoma , Precursores del ARN , Sulfonamidas , Humanos , Animales , Ratones , Precursores del ARN/genética , Precursores del ARN/metabolismo , Glutaminasa/genética , Reprogramación Metabólica , Histona Demetilasas con Dominio de Jumonji/metabolismoRESUMEN
MGA (Max-gene associated) is a dual-specificity transcription factor that negatively regulates MYC-target genes to inhibit proliferation and promote differentiation. Loss-of-function mutations in MGA have been commonly identified in several hematological neoplasms, including acute myeloid leukemia (AML) with RUNX1::RUNX1T1, however, very little is known about the impact of these MGA alterations on normal hematopoiesis or disease progression. We show that representative MGA mutations identified in patient samples abolish protein-protein interactions and transcriptional activity. Using a series of human and mouse model systems, including a newly developed conditional knock-out mouse strain, we demonstrate that loss of MGA results in upregulation of MYC and E2F targets, cell cycle genes, mTOR signaling, and oxidative phosphorylation in normal hematopoietic cells, leading to enhanced proliferation. The loss of MGA induces an open chromatin state at promoters of genes involved in cell cycle and proliferation. RUNX1::RUNX1T1 expression in Mga-deficient murine hematopoietic cells leads to a more aggressive AML with a significantly shortened latency. These data show that MGA regulates multiple pro-proliferative pathways in hematopoietic cells and cooperates with the RUNX1::RUNX1T1 fusion oncoprotein to enhance leukemogenesis.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteínas de Unión al ADN , Leucemia Mieloide Aguda , Mutación , Proteínas Proto-Oncogénicas , Proteína 1 Compañera de Translocación de RUNX1 , Animales , Humanos , Ratones , Proliferación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones Noqueados , Proteínas de Fusión Oncogénica/genética , Proteína 1 Compañera de Translocación de RUNX1/genética , Factores de Transcripción/genéticaRESUMEN
Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.
Asunto(s)
Complejos Multiproteicos , Mutación , Neoplasias , Proteína SMARCB1 , Animales , Femenino , Humanos , Masculino , Ratones , Línea Celular Tumoral , Sistemas CRISPR-Cas , Edición Génica , Neoplasias/genética , Neoplasias/metabolismo , Proteína SMARCB1/deficiencia , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteolisis , Ubiquitina/metabolismoRESUMEN
UBA5 encodes for the E1 enzyme of the UFMylation cascade, which plays an essential role in ER homeostasis. The clinical phenotypes of UBA5-associated encephalopathy include developmental delays, epilepsy and intellectual disability. To date, there is no humanized neuronal model to study the cellular and molecular consequences of UBA5 pathogenic variants. We developed and characterized patient-derived cortical organoid cultures and identified defects in GABAergic interneuron development. We demonstrated aberrant neuronal firing and microcephaly phenotypes in patient-derived organoids. Mechanistically, we show that ER homeostasis is perturbed along with exacerbated unfolded protein response pathway in cells and organoids expressing UBA5 pathogenic variants. We also assessed two gene expression modalities that augmented UBA5 expression to rescue aberrant molecular and cellular phenotypes. Our study provides a novel humanized model that allows further investigations of UBA5 variants in the brain and highlights novel systemic approaches to alleviate cellular aberrations for this rare, developmental disorder.
RESUMEN
Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA-sequencing revealed that most cells in mature thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.
RESUMEN
Innate immunity provides the first line of defense through multiple mechanisms, including pyrogen production and cell death. While elevated body temperature during infection is beneficial to clear pathogens, heat stress (HS) can lead to inflammation and pathology. Links between pathogen exposure, HS, cytokine release, and inflammation have been observed, but fundamental innate immune mechanisms driving pathology during pathogen exposure and HS remain unclear. Here, we use multiple genetic approaches to elucidate innate immune pathways in infection or LPS and HS models. Our results show that bacteria and LPS robustly increase inflammatory cell death during HS that is dependent on caspase-1, caspase-11, caspase-8, and RIPK3 through the PANoptosis pathway. Caspase-7 also contributes to PANoptosis in this context. Furthermore, NINJ1 is an important executioner of this cell death to release inflammatory molecules, independent of other pore-forming executioner proteins, gasdermin D, gasdermin E, and MLKL. In an in vivo HS model, mortality is reduced by deleting NINJ1 and fully rescued by deleting key PANoptosis molecules. Our findings suggest that therapeutic strategies blocking NINJ1 or its upstream regulators to prevent PANoptosis may reduce the release of inflammatory mediators and benefit patients.