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The impact of aging on intestinal stem cells (ISCs) has not been fully elucidated. In this study, we identified widespread epigenetic and transcriptional alterations in old ISCs. Using a reprogramming algorithm, we identified a set of key transcription factors (Egr1, Irf1, FosB) that drives molecular and functional differences between old and young states. Overall, by dissecting the molecular signature of aged ISCs, our study identified transcription factors that enhance the regenerative capacity of ISCs.
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There is limited knowledge on the benefit of the α-subunit-specific PI3K inhibitor alpelisib in later lines of therapy for advanced estrogen receptor-positive (ER+) HER2- and triple-negative breast cancer (TNBC). We conducted a phase II multicohort study of alpelisib monotherapy in patients with advanced PI3K pathway mutant ER+HER2- and TNBC. In the intention-to-treat ER+ cohort, the overall response rate was 30% and the clinical benefit rate was 36%. A decline in PI3K pathway mutant circulating tumor DNA (ctDNA) levels from baseline to week 8 while on therapy was significantly associated with a partial response, clinical benefit, and improved progression-free-survival [HR 0.24; 95% confidence interval (CI), 0.083-0.67, P = 0.0065]. Detection of ESR1 mutations at baseline in plasma was also associated with clinical benefit and improved progression-free survival (HR 0.22; 95% CI, 0.078-0.60, P = 0.003). SIGNIFICANCE: Alpelisib monotherapy displayed efficacy in heavily pretreated ER+ breast cancer with PIK3CA mutations. PIK3CA mutation dynamics in plasma during treatment and ESR1 mutations detected in plasma at baseline were candidate biomarkers predictive of benefit from alpelisib, highlighting the utility of ctDNA assays in this setting. This article is highlighted in the In This Issue feature, p. 2007.
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Neoplasias de la Mama , Neoplasias de la Mama Triple Negativas , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Fosfatidilinositol 3-Quinasa Clase I/genética , Femenino , Humanos , Mutación , Fosfatidilinositol 3-Quinasas/genética , Receptor ErbB-2/genética , Tiazoles , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
BACKGROUND: Metastatic breast cancer (mBC) is a heterogenous disease with increasing availability of targeted therapies as well as emerging genomic markers of therapeutic resistance, necessitating timely and accurate molecular characterization of disease. As a minimally invasive test, analysis of circulating tumour DNA (ctDNA) is well positioned for real-time genomic profiling to guide treatment decisions. Here, we report the results of a prospective testing program established to assess the feasibility of ctDNA analysis to guide clinical management of mBC patients. METHODS AND FINDINGS: Two hundred thirty-four mBC patients (median age 54 years) were enrolled between June 2015 and October 2018 at the Peter MacCallum Cancer Centre, Melbourne, Australia. Median follow-up was 15 months (range 1-46). All patient samples at the time of enrolment were analysed in real time for the presence of somatic mutations. Longitudinal plasma testing during the course of patient management was also undertaken in a subset of patients (n = 67, 28.6%), according to clinician preference, for repeated molecular profiling or disease monitoring. Detection of somatic mutations from patient plasma was performed using a multiplexed droplet digital PCR (ddPCR) approach to identify hotspot mutations in PIK3CA, ESR1, ERBB2, and AKT1. In parallel, subsets of samples were also analysed via next-generation sequencing (targeted panel sequencing and low-coverage whole-genome sequencing [LC-WGS]). The sensitivity of ddPCR and targeted panel sequencing to identify actionable mutations was compared. Results were discussed at a multidisciplinary breast cancer meeting prior to treatment decisions. ddPCR and targeted panel sequencing identified at least 1 actionable mutation at baseline in 80/234 (34.2%) and 62/159 (39.0%) of patients tested, respectively. Combined, both methods detected an actionable alteration in 104/234 patients (44.4%) through baseline or serial ctDNA testing. LC-WGS was performed on 27 patients from the cohort, uncovering several recurrently amplified regions including 11q13.3 encompassing CCND1. Increasing ctDNA levels were associated with inferior overall survival, whether assessed by ddPCR, targeted sequencing, or LC-WGS. Overall, the ctDNA results changed clinical management in 40 patients including the direct recruitment of 20 patients to clinical trials. Limitations of the study were that it was conducted at a single site and that 31.3% of participants were lost to follow-up. CONCLUSION: In this study, we found prospective ctDNA testing to be a practical and feasible approach that can guide clinical trial enrolment and patient management in mBC.
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Neoplasias de la Mama/genética , ADN Tumoral Circulante/genética , Metástasis de la Neoplasia/genética , Australia , Biomarcadores de Tumor/sangre , Ácidos Nucleicos Libres de Células/genética , ADN Tumoral Circulante/sangre , Fosfatidilinositol 3-Quinasa Clase I/genética , Estudios de Cohortes , Receptor alfa de Estrógeno/genética , Femenino , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex/métodos , Mutación , Medicina de Precisión/métodos , Proteínas Proto-Oncogénicas c-akt/genética , Receptor ErbB-2/genéticaRESUMEN
BACKGROUND: Metastatic breast cancer remains incurable. Next-generation sequencing (NGS) offers the ability to identify actionable genomic alterations in tumours which may then be matched with targeted therapies, but the implementation and utility of this approach is not well defined for patients with metastatic breast cancer. METHODS: We recruited patients with advanced breast cancer of any subtype for prospective targeted NGS of their most recent tumour samples, using a panel of 108 breast cancer-specific genes. Genes were classified as actionable or non-actionable using the European Society of Medical Oncology Scale for Clinical Actionability of Molecular Targets (ESCAT) guidelines. RESULTS: Between February 2014 and May 2019, 322 patients were enrolled onto the study, with 72% (n = 234) of patients successfully sequenced (n = 357 samples). The majority (74%, n = 171) of sequenced patients were found to carry a potentially actionable alteration, the most common being a PIK3CA mutation. Forty-three percent (n = 74) of patients with actionable alterations were referred for a clinical trial or referred for confirmatory germline testing or had a change in therapy outside of clinical trials. We found alterations in AKT1, BRCA2, CHEK2, ESR1, FGFR1, KMT2C, NCOR1, PIK3CA and TSC2 to be significantly enriched in our metastatic population compared with primary breast cancers. Concordance between primary and metastatic samples for key driver genes (TP53, ERBB2 amplification) was > 75%. Additionally, we found that patients with a higher number of mutations had a significantly worse overall survival. CONCLUSION: Genomic profiling of patients with metastatic breast cancer can have clinical implications and should be considered in all suitable patients.
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Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Proteína BRCA2/genética , Neoplasias de la Mama/tratamiento farmacológico , Fosfatidilinositol 3-Quinasa Clase I/genética , Femenino , Humanos , Persona de Mediana Edad , Terapia Molecular Dirigida , Metástasis de la Neoplasia , Pronóstico , Estudios Prospectivos , Adulto JovenRESUMEN
Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.
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Histona Acetiltransferasas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Línea Celular Tumoral , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis.
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Regulación Leucémica de la Expresión Génica , Histonas/genética , Leucemia Bifenotípica Aguda/genética , Metiltransferasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Acetilación , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Proteínas de Ciclo Celular , Proliferación Celular , Cromatina/química , Cromatina/metabolismo , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Femenino , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , Leucemia Bifenotípica Aguda/metabolismo , Leucemia Bifenotípica Aguda/patología , Masculino , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Cultivo Primario de Células , Unión Proteica , Proteómica/métodos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/patología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Cellular dedifferentiation is the regression of a cell from a specialized state to a more multipotent state and is implicated in cancer. However, the transcriptional network that prevents differentiated cells from reacquiring stem cell fate is so far unclear. Neuroblasts (NBs), the Drosophila neural stem cells, are a model for the regulation of stem cell self-renewal and differentiation. Here we show that the Drosophila zinc finger transcription factor Nervous fingers 1 (Nerfin-1) locks neurons into differentiation, preventing their reversion into NBs. Following Prospero-dependent neuronal specification in the ganglion mother cell (GMC), a Nerfin-1-specific transcriptional program maintains differentiation in the post-mitotic neurons. The loss of Nerfin-1 causes reversion to multipotency and results in tumors in several neural lineages. Both the onset and rate of neuronal dedifferentiation in nerfin-1 mutant lineages are dependent on Myc- and target of rapamycin (Tor)-mediated cellular growth. In addition, Nerfin-1 is required for NB differentiation at the end of neurogenesis. RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP) analysis show that Nerfin-1 administers its function by repression of self-renewing-specific and activation of differentiation-specific genes. Our findings support the model of bidirectional interconvertibility between neural stem cells and their post-mitotic progeny and highlight the importance of the Nerfin-1-regulated transcriptional program in neuronal maintenance.