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Combination approaches are needed to strengthen and extend the clinical response to KRASG12C inhibitors (KRASG12Ci). Here, we assessed the antitumor responses of KRASG12C mutant lung and colorectal cancer models to combination treatment with a SOS1 inhibitor (SOS1i), BI-3406, plus the KRASG12C inhibitor, adagrasib. We found that responses to BI-3406 plus adagrasib were stronger than to adagrasib alone, comparable to adagrasib with SHP2 (SHP2i) or EGFR inhibitors and correlated with stronger suppression of RAS-MAPK signaling. BI-3406 plus adagrasib treatment also delayed the emergence of acquired resistance and elicited antitumor responses from adagrasib-resistant models. Resistance to KRASG12Ci seemed to be driven by upregulation of MRAS activity, which both SOS1i and SHP2i were found to potently inhibit. Knockdown of SHOC2, a MRAS complex partner, partially restored response to KRASG12Ci treatment. These results suggest KRASG12C plus SOS1i to be a promising strategy for treating both KRASG12Ci naive and relapsed KRASG12C-mutant tumors.
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Clinical effectiveness of KRAS G12C inhibitors (G12Cis) is limited both by intrinsic and acquired resistance, necessitating the development of combination approaches. We found that targeting proximal receptor tyrosine kinase (RTK) signaling using the SOS1 inhibitor (SOS1i) BI-3406 both enhanced the potency of and delayed resistance to G12Ci treatment, but the extent of SOS1i effectiveness was modulated by both SOS2 expression and the specific mutational landscape. SOS1i enhanced the efficacy of G12Ci and limited rebound RTK/ERK signaling to overcome intrinsic/adaptive resistance, but this effect was modulated by SOS2 protein levels. Survival of drug-tolerant persister (DTP) cells within the heterogeneous tumor population and/or acquired mutations that reactivate RTK/RAS signaling can lead to outgrowth of tumor initiating cells (TICs) that drive therapeutic resistance. G12Ci drug tolerant persister cells showed a 2-3-fold enrichment of TICs, suggesting that these could be a sanctuary population of G12Ci resistant cells. SOS1i re-sensitized DTPs to G12Ci and inhibited G12C-induced TIC enrichment. Co-mutation of the tumor suppressor KEAP1 limits the clinical effectiveness of G12Cis, and KEAP1 and STK11 deletion increased TIC frequency and accelerated the development of acquired resistance to G12Ci in situ. SOS1i both delayed acquired G12Ci resistance and limited the total number of resistant colonies regardless of KEAP1 and STK11 mutational status. These data suggest that SOS1i could be an effective strategy to both enhance G12Ci efficacy and prevent G12Ci resistance regardless of co-mutations.
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GDF15 has recently emerged as a key driver of the development of various disease conditions including cancer cachexia. Not only the tumor itself but also adverse effects of chemotherapy have been reported to contribute to increased GDF15. Although regulation of GDF15 transcription by BET domain has recently been reported, the molecular mechanisms of GDF15 gene regulation by drugs are still unknown, leaving uncertainty about the safe and effective therapeutic strategies targeting GDF15. We screened various cardiotoxic drugs and BET inhibitors for their effects on GDF15 regulation in human cardiomyocytes and cancer cell lines and analyzed in-house and public gene signature databases. We found that DNA damaging drugs induce GDF15 in cardiomyocytes more strongly than drugs with other modes of action. In cancer cells, GDF15 induction varied depending on drug- and cell type-specific gene signatures including mutations in PI3KCA, TP53, BRAF and MUC16. GDF15 suppression by BET inhibition is particularly effective in cancer cells with low activity of the PI3K/Akt axis and high extracellular concentrations of pantothenate. Our findings provide insights that the risk for GDF15 overexpression and concomitant cachexia can be reduced by a personalized selection of anticancer drugs and patients for precision medicine.
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Caquexia , Neoplasias , Humanos , Miocitos Cardíacos/metabolismo , Medicina de Precisión , Fosfatidilinositol 3-Quinasas/metabolismo , Factor 15 de Diferenciación de Crecimiento/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genéticaRESUMEN
Efforts to improve the anti-tumor response to KRASG12C targeted therapy have benefited from leveraging combination approaches. Here, we compare the anti-tumor response induced by the SOS1-KRAS interaction inhibitor, BI-3406, combined with a KRASG12C inhibitor (KRASG12Ci) to those induced by KRASG12Ci alone or combined with SHP2 or EGFR inhibitors. In lung cancer and colorectal cancer (CRC) models, BI-3406 plus KRASG12Ci induces an anti-tumor response stronger than that observed with KRASG12Ci alone and comparable to those by the other combinations. This enhanced anti-tumor response is associated with a stronger and extended suppression of RAS-MAPK signaling. Importantly, BI-3406 plus KRASG12Ci treatment delays the emergence of acquired adagrasib resistance in both CRC and lung cancer models and is associated with re-establishment of anti-proliferative activity in KRASG12Ci-resistant CRC models. Our findings position KRASG12C plus SOS1 inhibition therapy as a promising strategy for treating both KRASG12C-mutated tumors as well as for addressing acquired resistance to KRASG12Ci.
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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.
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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
Importance: Characterizing rates of SARS-CoV-2 infection among vaccinated and unvaccinated persons with the same exposure is critical to understanding the association of vaccination with the risk of infection with the Delta variant. Additionally, evidence of Delta variant transmission by children to vaccinated adults has important public health implications. Objective: To characterize transmission and infection of SARS-CoV-2 among vaccinated and unvaccinated attendees of an indoor wedding reception. Design, Setting, and Participants: This cohort study included attendees at an indoor wedding reception in Minnesota in July 2021. Data were collected from REDCap surveys and routine surveillance interviews. The full list of attendees and a partial list of emails were obtained. Fifty-seven attendees completed the emailed survey. Eighteen additional attendees were identified from the state health department COVID-19 surveillance database. Exposures: Attendance at an indoor event. Main Outcomes and Measures: Risk of SARS-CoV-2 infection among vaccinated and unvaccinated attendees, identification of an index case, whole genome sequencing (WGS) to identify the COVID-19 variant, understanding of transmission patterns, and assessment of secondary transmission. The primary case definition was an individual with a positive SARS-CoV-2 test who attended the wedding in the 14 days prior to their illness. Results: Data were gathered for 75 attendees (mean [SE] age, 37.5 [13.7] years; 57 [76%] female individuals), of whom 56 (75%) were fully vaccinated, 4 (5%) were partially vaccinated, and 15 (20%) were unvaccinated. Of 62 attendees who were tested, 29 (47%) tested positive, including 16 of 46 fully vaccinated attendees (35%), 2 of 4 partially vaccinated attendees (50%), and 11 of 12 unvaccinated attendees (92%). Being unvaccinated was associated with a higher risk of infection compared with being vaccinated (risk ratio, 2.64; 95% CI, 1.71-4.06; P = .001). One unvaccinated adult required hospitalization. An unvaccinated child who was symptomatic on the event date was identified as the index case. Eleven specimens were available for WGS. All sequenced specimens were closely related and were identified as the Delta variant. WGS supported secondary transmission from a vaccinated individual with SARS-CoV-2. Conclusions and Relevance: This cohort study identified a COVID-19 Delta variant outbreak at an indoor event despite a high proportion of vaccinated attendees. It found that vaccination was associated with a reduced risk of infection.
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COVID-19/transmisión , Cobertura de Vacunación/estadística & datos numéricos , Adulto , COVID-19/epidemiología , Vacunas contra la COVID-19/inmunología , Niño , Estudios de Cohortes , Brotes de Enfermedades , Humanos , Persona de Mediana Edad , Minnesota/epidemiología , SARS-CoV-2/patogenicidad , Encuestas y CuestionariosRESUMEN
KRAS is the most frequently mutated oncogene, harboring mutations in approximately one in seven cancers. Allele-specific KRASG12C inhibitors are currently changing the treatment paradigm for patients with KRASG12C-mutated non-small cell lung cancer and colorectal cancer. The success of addressing a previously elusive KRAS allele has fueled drug discovery efforts for all KRAS mutants. Pan-KRAS drugs have the potential to address broad patient populations, including KRASG12D-, KRASG12V-, KRASG13D-, KRASG12R-, and KRASG12A-mutant or KRAS wild-type-amplified cancers, as well as cancers with acquired resistance to KRASG12C inhibitors. Here, we review actively pursued allele-specific and pan-KRAS inhibition strategies and their potential utility. SIGNIFICANCE: Mutant-selective KRASG12C inhibitors target a fraction (approximately 13.6%) of all KRAS-driven cancers. A broad arsenal of KRAS drugs is needed to comprehensively conquer KRAS-driven cancers. Conceptually, we foresee two future classes of KRAS medicines: mutant-selective KRAS drugs targeting individual variant alleles and pan-KRAS therapeutics targeting a broad range of KRAS alterations.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Mutación , Oncogenes , Medicina de Precisión , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
The Minnesota Department of Health investigated a coronavirus disease 2019 (COVID-19) outbreak at a fitness center in Olmsted County, Minnesota. Twenty-three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections (5 employees and 18 members) were identified. An epidemiological investigation supported by whole genome sequencing demonstrated that transmission of SARS-CoV-2 occurred at the fitness center despite following recommended prevention strategies.
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COVID-19 , Centros de Acondicionamiento , Brotes de Enfermedades , Humanos , Minnesota/epidemiología , SARS-CoV-2RESUMEN
It has taken four decades of research to see the first major breakthrough for KRAS-driven cancers. In particular, the last decade has seen a paradigm shift with the discovery of druggable pockets on KRAS and clinical efficacy with covalent KRASG12C inhibitors, culminating in the first approval of sotorasib monotherapy as second-line treatment in KRASG12C-driven non-small-cell lung cancer. Nevertheless, 85% of all KRAS-mutated cancers still lack novel agents. In this review, we will outline the structure, function, and post-translational modifications of KRAS and highlight the various approaches being adopted to drug KRAS, ranging from selective to pan concepts. The range of molecular modalities being explored, including PROTACs and glues, will also be described. Finally, an outlook toward the next wave of KRAS drugs and the challenges of resistance will be given.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Son of Sevenless (SOS) is a guanine nucleotide exchange factor that activates the important cell signaling switch KRAS. SOS acts as a pacemaker for KRAS, the beating heart of cancer, by catalyzing the "beating" from the KRAS(off) to the KRAS(on) conformation. Activating mutations in SOS1 are common in Noonan syndrome and oncogenic alterations in KRAS drive 1 in seven human cancers. Promising clinical efficacy has been observed for selective KRASG12C inhibitors, but the vast majority of oncogenic KRAS alterations remain undrugged. The discovery of a druggable pocket on SOS1 has led to potent SOS1 inhibitors such as BI-3406. SOS1 inhibition leads to antiproliferative effects against all major KRAS mutants. The first SOS1 inhibitor has entered clinical trials for KRAS-mutated cancers. In this review, we provide an overview of SOS1 function, its association with cancer and RASopathies, known SOS1 activators and inhibitors, and a future perspective is provided.
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Antineoplásicos/química , Proteínas Mutantes/química , Neoplasias/terapia , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína SOS1/antagonistas & inhibidores , Acetonitrilos/farmacología , Antineoplásicos/farmacología , Regulación de la Expresión Génica , Humanos , Mutación , Marcapaso Artificial , Piperazinas/farmacología , Conformación Proteica , Piridinas/farmacología , Pirimidinas/farmacología , Proteína SOS1/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
KRAS is the most frequently mutated driver of pancreatic, colorectal, and non-small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF-MEK-ERK cascade, has shown limited success because of activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective, and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1, thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. SIGNIFICANCE: To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS-mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins, G12D, G12V, G12C, and G13D.See related commentary by Zhao et al., p. 17.This article is highlighted in the In This Issue feature, p. 1.
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Neoplasias Pulmonares , Proteínas Proto-Oncogénicas p21(ras) , Línea Celular Tumoral , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Mutación , Nucleótidos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations. Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients. An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL. However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors. Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and in vivo. We show that BCL6 knock-out in SU-DHL-4 cells in vitro results in an anti-proliferative response 4-7 days after Cas9 induction that was characterized by cell cycle (G1) arrest. Conditional BCL6 deletion in established DLBCL tumors in vivo induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics. Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/Cas9 systems for probing oncogene addiction.
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Bromodomain and extra-terminal (BET) protein inhibitors have been reported as treatment options for acute myeloid leukemia (AML) in preclinical models and are currently being evaluated in clinical trials. This work presents a novel potent and selective BET inhibitor (BI 894999), which has recently entered clinical trials (NCT02516553). In preclinical studies, this compound is highly active in AML cell lines, primary patient samples, and xenografts. HEXIM1 is described as an excellent pharmacodynamic biomarker for target engagement in tumors as well as in blood. Mechanistic studies show that BI 894999 targets super-enhancer-regulated oncogenes and other lineage-specific factors, which are involved in the maintenance of the disease state. BI 894999 is active as monotherapy in AML xenografts, and in addition leads to strongly enhanced antitumor effects in combination with CDK9 inhibitors. This treatment combination results in a marked decrease of global p-Ser2 RNA polymerase II levels and leads to rapid induction of apoptosis in vitro and in vivo. Together, these data provide a strong rationale for the clinical evaluation of BI 894999 in AML.
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Antineoplásicos/administración & dosificación , Elementos de Facilitación Genéticos/efectos de los fármacos , Flavonoides/administración & dosificación , Perfilación de la Expresión Génica/métodos , Leucemia Mieloide Aguda/tratamiento farmacológico , Piperidinas/administración & dosificación , Proteínas/antagonistas & inhibidores , Pirazinas/administración & dosificación , Triazoles/administración & dosificación , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Regulación hacia Abajo , Sinergismo Farmacológico , Quimioterapia Combinada , Flavonoides/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Piperidinas/farmacología , Pirazinas/farmacología , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/genética , Factores de Transcripción , Triazoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Interactions between a new potent Bromodomain and extraterminal domain (BET) inhibitor BI 894999 and the polo-like kinase (PLK) inhibitor volasertib were studied in acute myeloid leukemia cell lines in vitro and in vivo. We provide data for the distinct mechanisms of action of these two compounds with a potential utility in AML based on gene expression, cell cycle profile and modulation of PD biomarkers such as MYC and HEXIM1. In contrast to BI 894999, volasertib treatment neither affects MYC nor HEXIM1 expression, but augments and prolongs the decrease of MYC expression caused by BI 894999 treatment. In vitro combination of both compounds leads to a decrease in S-Phase and to increased apoptosis. In vitro scheduling experiments guided in vivo experiments in disseminated AML mouse models. Co-administration of BI 894999 and volasertib dramatically reduces tumor burden accompanied by long-term survival of tumor-bearing mice and eradication of AML cells in mouse bone marrow. Together, these preclinical findings provide evidence for the strong synergistic effect of BI 894999 and volasertib, warranting future clinical studies in patients with AML to investigate this paradigm.
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Derivados del Benceno/farmacología , Leucemia Mieloide Aguda/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/antagonistas & inhibidores , Pteridinas/farmacología , Animales , Línea Celular , Sinergismo Farmacológico , Genes myc , Humanos , Leucemia Mieloide Aguda/genética , RatonesRESUMEN
Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, has attracted interest as a target for pharmacological intervention in malignant diseases. Here, we describe BI 853520, a novel ATP-competitive inhibitor distinguished by high potency and selectivity. In vitro, the compound inhibits FAK autophosphorylation in PC-3 prostate carcinoma cells with an IC50 of 1 nmol/L and blocks anchorage-independent proliferation of PC-3 cells with an EC50 of 3 nmol/L, whereas cells grown in conventional surface culture are 1000-fold less sensitive. In mice, the compound shows long half-life, high volume of distribution and high oral bioavailability; oral dosing of immunodeficient mice bearing subcutaneous PC-3 prostate adenocarcinoma xenografts resulted in rapid, long-lasting repression of FAK autophosphorylation in tumor tissue. Daily oral administration of BI 853520 to nude mice at doses of 50 mg/kg was well tolerated for prolonged periods of time. In a diverse panel of 16 subcutaneous adenocarcinoma xenograft models in nude mice, drug treatment resulted in a broad spectrum of outcomes, ranging from group median tumor growth inhibition values >100% and tumor regression in subsets of animals to complete lack of sensitivity. Biomarker analysis indicated that high sensitivity is linked to a mesenchymal tumor phenotype, initially defined by loss of E-cadherin expression and subsequently substantiated by gene set enrichment analysis. Further, we obtained microRNA expression profiles for 13 models and observed that hsa-miR-200c-3p expression is strongly correlated with efficacy (R2 = 0.889). BI 853520 is undergoing evaluation in early clinical trials.
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The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.
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Proteolisis , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Concentración 50 Inhibidora , Cinética , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-6/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-6/química , Pirimidinas/farmacología , Relación Estructura-Actividad , Ubiquitinación/efectos de los fármacosRESUMEN
Localized protein translation is critical in many biological contexts, particularly in highly polarized cells, such as neurons, to regulate gene expression in a spatiotemporal manner. The cytoplasmic polyadenylation element-binding (CPEB) family of RNA-binding proteins has emerged as a key regulator of mRNA transport and local translation required for early embryonic development, synaptic plasticity, and long-term memory (LTM). Drosophila Orb and Orb2 are single members of the CPEB1 and CPEB2 subfamilies of the CPEB proteins, respectively. At present, the identity of the mRNA targets they regulate is not fully known, and the binding specificity of the CPEB2 subfamily is a matter of debate. Using transcriptome-wide UV cross-linking and immunoprecipitation, we define the mRNA-binding sites and targets of Drosophila CPEBs. Both Orb and Orb2 bind linear cytoplasmic polyadenylation element-like sequences in the 3' UTRs of largely overlapping target mRNAs, with Orb2 potentially having a broader specificity. Both proteins use their RNA-recognition motifs but not the Zinc-finger region for RNA binding. A subset of Orb2 targets is translationally regulated in cultured S2 cells and fly head extracts. Moreover, pan-neuronal RNAi knockdown of these targets suggests that a number of these targets are involved in LTM. Our results provide a comprehensive list of mRNA targets of the two CPEB proteins in Drosophila, thus providing insights into local protein synthesis involved in various biological processes, including LTM.
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The Piwi pathway is deeply conserved amongst animals because one of its essential functions is to repress transposons. However, many Piwi-interacting RNAs (piRNAs) do not base-pair to transposons and remain mysterious in their targeting function. The sheer number of piRNA cluster (piC) loci in animal genomes and infrequent piRNA sequence conservation also present challenges in determining which piC loci are most important for development. To address this question, we determined the piRNA expression patterns of piC loci across a wide phylogenetic spectrum of animals, and reveal that most genic and intergenic piC loci evolve rapidly in their capacity to generate piRNAs, regardless of known transposon silencing function. Surprisingly, we also uncovered a distinct set of piC loci with piRNA expression conserved deeply in Eutherian mammals. We name these loci Eutherian-Conserved piRNA cluster (ECpiC) loci. Supporting the hypothesis that conservation of piRNA expression across ~100 million years of Eutherian evolution implies function, we determined that one ECpiC locus generates abundant piRNAs antisense to the STOX1 transcript, a gene clinically associated with preeclampsia. Furthermore, we confirmed reduced piRNAs in existing mouse mutations at ECpiC-Asb1 and -Cbl, which also display spermatogenic defects. The Asb1 mutant testes with strongly reduced Asb1 piRNAs also exhibit up-regulated gene expression profiles. These data indicate ECpiC loci may be specially adapted to support Eutherian reproduction.
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Mamíferos/genética , Familia de Multigenes , ARN Interferente Pequeño/genética , Animales , Evolución Molecular , Mamíferos/clasificaciónRESUMEN
In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA-binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between the cytoplasm and nuclei and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific splicing of various salm-dependent sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis.
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Empalme Alternativo/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Músculo Esquelético/metabolismo , Miofibrillas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo/genética , Animales , Drosophila , Drosophila melanogasterRESUMEN
Phenotypic differences between closely related species are thought to arise primarily from changes in gene expression due to mutations in cis-regulatory sequences (enhancers). However, it has remained unclear how frequently mutations alter enhancer activity or create functional enhancers de novo. Here we use STARR-seq, a recently developed quantitative enhancer assay, to determine genome-wide enhancer activity profiles for five Drosophila species in the constant trans-regulatory environment of Drosophila melanogaster S2 cells. We find that the functions of a large fraction of D. melanogaster enhancers are conserved for their orthologous sequences owing to selection and stabilizing turnover of transcription factor motifs. Moreover, hundreds of enhancers have been gained since the D. melanogaster-Drosophila yakuba split about 11 million years ago without apparent adaptive selection and can contribute to changes in gene expression in vivo. Our finding that enhancer activity is often deeply conserved and frequently gained provides functional insights into regulatory evolution.