RESUMEN
Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Transactivadores/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Médula Ósea/patología , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Epigénesis Genética/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacos , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A number of diazepines are known to inhibit bromo- and extra-terminal domain (BET) proteins. Their BET inhibitory activity derives from the fusion of an acetyl-lysine mimetic heterocycle onto the diazepine framework. Herein we describe a straightforward, modular synthesis of novel 1,2,3-triazolobenzodiazepines and show that the 1,2,3-triazole acts as an effective acetyl-lysine mimetic heterocycle. Structure-based optimization of this series of compounds led to the development of potent BET bromodomain inhibitors with excellent activity against leukemic cells, concomitant with a reduction in c-MYC expression. These novel benzodiazepines therefore represent a promising class of therapeutic BET inhibitors.
RESUMEN
The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.
Asunto(s)
Benzodiazepinas/uso terapéutico , Química Clic , Sistemas de Liberación de Medicamentos , Epigenómica , Leucemia/tratamiento farmacológico , Animales , Benzodiazepinas/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Leucemia/patología , Ratones , Medicina de Precisión , Distribución Tisular , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
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
Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks. Early clinical trials have shown promise, especially in acute myeloid leukaemia, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/ß-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.
