RESUMEN
Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Sistemas CRISPR-Cas , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Regulación de la Expresión Génica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dominios Proteicos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
During neural tube (NT) development, the notochord induces an organizer, the floorplate, which secretes Sonic Hedgehog (SHH) to pattern neural progenitors. Conversely, NT organoids (NTOs) from embryonic stem cells (ESCs) spontaneously form floorplates without the notochord, demonstrating that stem cells can self-organize without embryonic inducers. Here, we investigated floorplate self-organization in clonal mouse NTOs. Expression of the floorplate marker FOXA2 was initially spatially scattered before resolving into multiple clusters, which underwent competition and sorting, resulting in a stable "winning" floorplate. We identified that BMP signaling governed long-range cluster competition. FOXA2+ clusters expressed BMP4, suppressing FOXA2 in receiving cells while simultaneously expressing the BMP-inhibitor NOGGIN, promoting cluster persistence. Noggin mutation perturbed floorplate formation in NTOs and in the NT in vivo at mid/hindbrain regions, demonstrating how the floorplate can form autonomously without the notochord. Identifying the pathways governing organizer self-organization is critical for harnessing the developmental plasticity of stem cells in tissue engineering.
Asunto(s)
Proteína Morfogenética Ósea 4 , Tubo Neural , Notocorda , Organoides , Animales , Ratones , Organoides/metabolismo , Organoides/citología , Tubo Neural/metabolismo , Tubo Neural/citología , Notocorda/metabolismo , Notocorda/citología , Proteína Morfogenética Ósea 4/metabolismo , Transducción de Señal , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Morfogenéticas Óseas/metabolismoRESUMEN
Loss-of-function genetic tools are widely applied for validating therapeutic targets, but their utility remains limited by incomplete on- and uncontrolled off-target effects. We describe artificial RNA interference (ARTi) based on synthetic, ultra-potent, off-target-free shRNAs that enable efficient and inducible suppression of any gene upon introduction of a synthetic target sequence into non-coding transcript regions. ARTi establishes a scalable loss-of-function tool with full control over on- and off-target effects.
Asunto(s)
Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Genetic networks are characterized by extensive buffering. During tumor evolution, disruption of functional redundancies can create de novo vulnerabilities that are specific to cancer cells. Here, we systematically search for cancer-relevant paralog interactions using CRISPR screens and publicly available loss-of-function datasets. Our analysis reveals >2,000 candidate dependencies, several of which we validate experimentally, including CSTF2-CSTF2T, DNAJC15-DNAJC19, FAM50A-FAM50B, and RPP25-RPP25L. We provide evidence that RPP25L can physically and functionally compensate for the absence of RPP25 as a member of the RNase P/MRP complexes in tRNA processing. Our analysis also reveals unexpected redundancies between sex chromosome genes. We show that chrX- and chrY-encoded paralogs, such as ZFX-ZFY, DDX3X-DDX3Y, and EIF1AX-EIF1AY, are functionally linked. Tumor cell lines from male patients with loss of chromosome Y become dependent on the chrX-encoded gene. We propose targeting of chrX-encoded paralogs as a general therapeutic strategy for human tumors that have lost the Y chromosome.
Asunto(s)
Neoplasias , Oncogenes , ARN Helicasas DEAD-box/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Masculino , Antígenos de Histocompatibilidad Menor/metabolismo , Neoplasias/genética , Proteínas de Unión al ARN/genética , Cromosomas Sexuales/metabolismo , Cromosoma X , Cromosoma YRESUMEN
Oncogenic alterations in human epidermal growth factor receptor 2 (HER2) occur in approximately 2% of patients with non-small cell lung cancer and predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. Most clinical-grade tyrosine kinase inhibitors are limited by either insufficient selectivity against wild-type (WT) epidermal growth factor receptor (EGFR), which is a major cause of dose-limiting toxicity or by potency against HER2 exon 20 mutant variants. Here we report the discovery of covalent tyrosine kinase inhibitors that potently inhibit HER2 exon 20 mutants while sparing WT EGFR, which reduce tumor cell survival and proliferation in vitro and result in regressions in preclinical xenograft models of HER2 exon 20 mutant non-small cell lung cancer, concomitant with inhibition of downstream HER2 signaling. Our results suggest that HER2 exon 20 insertion-driven tumors can be effectively treated by a potent and highly selective HER2 inhibitor while sparing WT EGFR, paving the way for clinical translation.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/genética , Exones/genética , Genes erbB-2 , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Receptor ErbB-2/genéticaRESUMEN
SMARCA4/BRG1 and SMARCA2/BRM, the two mutually exclusive catalytic subunits of the BAF complex, display a well-established synthetic lethal relationship in SMARCA4-deficient cancers. Using CRISPR-Cas9 screening, we identify SMARCA4 as a novel dependency in SMARCA2-deficient esophageal squamous cell carcinoma (ESCC) models, reciprocal to the known synthetic lethal interaction. Restoration of SMARCA2 expression alleviates the dependency on SMARCA4, while engineered loss of SMARCA2 renders ESCC models vulnerable to concomitant depletion of SMARCA4. Dependency on SMARCA4 is linked to its ATPase activity, but not to bromodomain function. We highlight the relevance of SMARCA4 as a drug target in esophageal cancer using an engineered ESCC cell model harboring a SMARCA4 allele amenable to targeted proteolysis and identify SMARCA4-dependent cell models with low or absent SMARCA2 expression from additional tumor types. These findings expand the concept of SMARCA2/SMARCA4 paralog dependency and suggest that pharmacological inhibition of SMARCA4 represents a novel therapeutic opportunity for SMARCA2-deficient cancers.
Asunto(s)
ADN Helicasas/antagonistas & inhibidores , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Supervivencia Celular/genética , ADN Helicasas/genética , Epigénesis Genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/patología , Edición Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Humanos , Mutación con Pérdida de Función , Terapia Molecular Dirigida/métodos , Proteínas Nucleares/genética , ARN Guía de Kinetoplastida/genética , ARN Interferente Pequeño/metabolismo , Mutaciones Letales Sintéticas , Factores de Transcripción/deficienciaRESUMEN
Genotype specific vulnerabilities of cancer cells constitute a promising strategy for the development of new therapeutics. Deletions of non-essential genes in tumors can generate unique vulnerabilities which could be exploited therapeutically. The MTAP gene is recurrently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. Recent studies have uncovered an increased dependency of MTAP-deleted cancer cells on the function of a PRMT5 containing complex, including WDR77, PRMT5 and the kinase RIOK1. As RIOK1 kinase activity constitutes a potential therapeutic target, we wanted to test if MTAP deletion confers increased sensitivity to RIOK1 inhibition. Using CRISPR/Cas9-mediated genome engineering we generated analog sensitive alleles of RIOK1 in isogenic cell lines differing only by MTAP status. While we were able to independently confirm an increased dependency of MTAP-deleted cells on PRMT5, we did not detect a differential requirement for RIOK1 kinase activity between MTAP-proficient and deficient cells. Our results reveal that the kinase activity of RIOK1 is required for the survival of cancer cell lines irrespective of their MTAP status and cast doubt on the therapeutic exploitability of RIOK1 in the context of MTAP-deleted cancers.
RESUMEN
The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P. amoebophila by in silico analysis. Two of these Protochlamydiaouter membrane proteins, PomS (pc1489) and PomT (pc1077), are highly abundant in outer membrane preparations of this organism. Here we show that all four members of this putative porin family are toxic when expressed in the heterologous host Escherichia coli. Immunofluorescence analysis using antibodies against heterologously expressed PomT and PomS purified directly from elementary bodies, respectively, demonstrated the location of both proteins in the outer membrane of P. amoebophila. The location of the most abundant protein PomS was further confirmed by immuno-transmission electron microscopy. We could show that pomS is transcribed, and the corresponding protein is present in the outer membrane throughout the complete developmental cycle, suggesting an essential role for P. amoebophila. Lipid bilayer measurements demonstrated that PomS functions as a porin with anion-selectivity and a pore size similar to the Chlamydiaceae MOMP. Taken together, our results suggest that PomS, possibly in concert with PomT and other members of this porin family, is the functional equivalent of MOMP in P. amoebophila. This work contributes to our understanding of the adaptations of symbiotic and pathogenic chlamydiae to their different eukaryotic hosts.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Chlamydiaceae/citología , Chlamydiaceae/metabolismo , Porinas/metabolismo , Simbiosis , Amoeba/microbiología , Proteínas Bacterianas/genética , Chlamydiaceae/genética , Chlamydiaceae/fisiología , Escherichia coli/genética , Membrana Dobles de Lípidos/metabolismo , Porinas/genética , Transporte de Proteínas , Transcripción GenéticaRESUMEN
RNA-binding proteins play crucial roles in directing RNA translation to neuronal synapses. Staufen2 (Stau2) has been implicated in both dendritic RNA localization and synaptic plasticity in mammalian neurons. Here, we report the identification of functionally relevant Stau2 target mRNAs in neurons. The majority of Stau2-copurifying mRNAs expressed in the hippocampus are present in neuronal processes, further implicating Stau2 in dendritic mRNA regulation. Stau2 targets are enriched for secondary structures similar to those identified in the 3' UTRs of Drosophila Staufen targets. Next, we show that Stau2 regulates steady-state levels of many neuronal RNAs and that its targets are predominantly downregulated in Stau2-deficient neurons. Detailed analysis confirms that Stau2 stabilizes the expression of one synaptic signaling component, the regulator of G protein signaling 4 (Rgs4) mRNA, via its 3' UTR. This study defines the global impact of Stau2 on mRNAs in neurons, revealing a role in stabilization of the levels of synaptic targets.
Asunto(s)
Neuronas/metabolismo , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3' , Animales , Células Cultivadas , Hipocampo/citología , Hipocampo/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs.