RESUMEN
Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancies, owing in part to its propensity for metastasis. Here, we used an organoid culture system to investigate how transcription and the enhancer landscape become altered during discrete stages of disease progression in a PDA mouse model. This approach revealed that the metastatic transition is accompanied by massive and recurrent alterations in enhancer activity. We implicate the pioneer factor FOXA1 as a driver of enhancer activation in this system, a mechanism that renders PDA cells more invasive and less anchorage-dependent for growth in vitro, as well as more metastatic in vivo. In this context, FOXA1-dependent enhancer reprogramming activates a transcriptional program of embryonic foregut endoderm. Collectively, our study implicates enhancer reprogramming, FOXA1 upregulation, and a retrograde developmental transition in PDA metastasis.
Asunto(s)
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Elementos de Facilitación Genéticos , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Epigenómica , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Organoides/metabolismo , Páncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologíaRESUMEN
The lineage-specific transcription factor (TF) MEF2C is often deregulated in leukemia. However, strategies to target this TF have yet to be identified. Here, we used a domain-focused CRISPR screen to reveal an essential role for LKB1 and its Salt-Inducible Kinase effectors (SIK3, in a partially redundant manner with SIK2) to maintain MEF2C function in acute myeloid leukemia (AML). A key phosphorylation substrate of SIK3 in this context is HDAC4, a repressive cofactor of MEF2C. Consequently, targeting of LKB1 or SIK3 diminishes histone acetylation at MEF2C-bound enhancers and deprives leukemia cells of the output of this essential TF. We also found that MEF2C-dependent leukemias are sensitive to on-target chemical inhibition of SIK activity. This study reveals a chemical strategy to block MEF2C function in AML, highlighting how an oncogenic TF can be disabled by targeting of upstream kinases.
Asunto(s)
Leucemia Mieloide Aguda/enzimología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Acetilación , Animales , Antineoplásicos/farmacología , Proliferación Celular , Elementos de Facilitación Genéticos , Regulación Enzimológica de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células HEK293 , Células Hep G2 , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Ratones , Células 3T3 NIH , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Células THP-1 , Células U937RESUMEN
Recurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3-FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3-FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3-FOXO1 to block myo-differentiation in RMS. Unlike most genes evaluated in our screen, we found that loss of any of the three subunits of the Nuclear Factor Y (NF-Y) complex leads to a myo-differentiation phenotype that resembles the effect of inactivating PAX3-FOXO1. While the transcriptomes of NF-Y- and PAX3-FOXO1-deficient RMS cells bear remarkable similarity to one another, we found that these two transcription factors occupy nonoverlapping sites along the genome: NF-Y preferentially occupies promoters, whereas PAX3-FOXO1 primarily binds to distal enhancers. By integrating multiple functional approaches, we map the PAX3 promoter as the point of intersection between these two regulators. We show that NF-Y occupies CCAAT motifs present upstream of PAX3 to function as a transcriptional activator of PAX3-FOXO1 expression in RMS. These findings reveal a critical upstream role of NF-Y in the oncogenic PAX3-FOXO1 pathway, highlighting how a broadly essential transcription factor can perform tumor-specific roles in governing cellular state.
Asunto(s)
Rabdomiosarcoma , Factor de Unión a CCAAT/genética , Diferenciación Celular/genética , Aberraciones Cromosómicas , Rabdomiosarcoma/genética , Factores de TranscripciónRESUMEN
Small cell lung cancer (SCLC) is widely considered to be a tumor of pulmonary neuroendocrine cells; however, a variant form of this disease has been described that lacks neuroendocrine features. Here, we applied domain-focused CRISPR screening to human cancer cell lines to identify the transcription factor (TF) POU2F3 (POU class 2 homeobox 3; also known as SKN-1a/OCT-11) as a powerful dependency in a subset of SCLC lines. An analysis of human SCLC specimens revealed that POU2F3 is expressed exclusively in variant SCLC tumors that lack expression of neuroendocrine markers and instead express markers of a chemosensory lineage known as tuft cells. Using chromatin- and RNA-profiling experiments, we provide evidence that POU2F3 is a master regulator of tuft cell identity in a variant form of SCLC. Moreover, we show that most SCLC tumors can be classified into one of three lineages based on the expression of POU2F3, ASCL1, or NEUROD1. Our CRISPR screens exposed other unique dependencies in POU2F3-expressing SCLC lines, including the lineage TFs SOX9 and ASCL2 and the receptor tyrosine kinase IGF1R (insulin-like growth factor 1 receptor). These data reveal POU2F3 as a cell identity determinant and a dependency in a tuft cell-like variant of SCLC, which may reflect a previously unrecognized cell of origin or a trans-differentiation event in this disease.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatología , Factores de Transcripción de Octámeros/genética , Factores de Transcripción de Octámeros/metabolismo , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/fisiopatología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular , Línea Celular Tumoral , Linaje de la Célula , Humanos , Pulmón/patología , Ratones , Receptor IGF Tipo 1/metabolismoRESUMEN
The bromodomain and extraterminal (BET) protein BRD4 is a therapeutic target in acute myeloid leukemia (AML). Here, we demonstrate that the AML maintenance function of BRD4 requires its interaction with NSD3, which belongs to a subfamily of H3K36 methyltransferases. Unexpectedly, AML cells were found to only require a short isoform of NSD3 that lacks the methyltransferase domain. We show that NSD3-short is an adaptor protein that sustains leukemia by linking BRD4 to the CHD8 chromatin remodeler, by using a PWWP chromatin reader module, and by employing an acidic transactivation domain. Genetic targeting of NSD3 or CHD8 mimics the phenotypic and transcriptional effects of BRD4 inhibition. Furthermore, BRD4, NSD3, and CHD8 colocalize across the AML genome, and each is released from super-enhancer regions upon chemical inhibition of BET bromodomains. These findings suggest that BET inhibitors exert therapeutic effects in leukemia by evicting BRD4-NSD3-CHD8 complexes from chromatin to suppress transcription.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Dominio Catalítico , Proteínas de Ciclo Celular , Proliferación Celular , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Células HL-60 , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Proteínas Nucleares/química , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Factores de Transcripción/genéticaRESUMEN
Lineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a salt-inducible kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF myocyte enhancer factor (MEF2C). In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. In this study, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells under in vitro and in vivo conditions. Similar phenotypes were obtained when cells were exposed to YKL-05-099, which caused cell-cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele of SIK3, we found that the antiproliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated 2 different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progression in vivo and extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-addicted AML and provide a rationale for developing druglike inhibitors of SIK3 for definitive preclinical investigation and for studies in human patients.
Asunto(s)
Compuestos de Anilina/farmacología , Leucemia Mieloide Aguda/prevención & control , Factores de Transcripción MEF2/metabolismo , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Apoptosis , Ciclo Celular , Proliferación Celular , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Factores de Transcripción MEF2/genética , Ratones , Ratones Endogámicos C57BL , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in â¼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos/fisiología , Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide Aguda/fisiopatología , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Proliferación Celular , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos/genética , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/genéticaRESUMEN
Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, it is reported that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating roles in the activation of MAPK pathways and the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. The findings nominate the targeting of EN1 and downstream pathways in aggressive PDA.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Neoplasias Pancreáticas/genética , Regulación de la Expresión Génica , Carcinoma Ductal Pancreático/genéticaRESUMEN
Hundreds of genes become aberrantly silenced in acute myeloid leukemia (AML), with most of these epigenetic changes being of unknown functional consequence. Here, we demonstrate how gene silencing can lead to an acquired dependency on the DNA repair machinery in AML. We make this observation by profiling the essentiality of the ubiquitination machinery in cancer cell lines using domain-focused CRISPR screening, which revealed Fanconi anemia (FA) proteins UBE2T and FANCL as unique dependencies in AML. We demonstrate that these dependencies are due to a synthetic lethal interaction between FA proteins and aldehyde dehydrogenase 2 (ALDH2), which function in parallel pathways to counteract the genotoxicity of endogenous aldehydes. We show DNA hypermethylation and silencing of ALDH2 occur in a recurrent manner in human AML, which is sufficient to confer FA pathway dependency. Our study suggests that targeting of the ubiquitination reaction catalyzed by FA proteins can eliminate ALDH2-deficient AML. SIGNIFICANCE: Aberrant gene silencing is an epigenetic hallmark of human cancer, but the functional consequences of this process are largely unknown. In this study, we show how an epigenetic alteration leads to an actionable dependency on a DNA repair pathway through the disabling of genetic redundancy.This article is highlighted in the In This Issue feature, p. 2113.
Asunto(s)
Aldehído Deshidrogenasa Mitocondrial/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Leucemia Mieloide Aguda/genética , Línea Celular Tumoral , Humanos , UbiquitinaciónRESUMEN
The aberrant expression of squamous lineage markers in pancreatic ductal adenocarcinoma (PDA) has been correlated with poor clinical outcomes. However, the functional role of this putative transdifferentiation event in PDA pathogenesis remains unclear. Here, we show that expression of the transcription factor TP63 (ΔNp63) is sufficient to install and sustain the enhancer landscape and transcriptional signature of the squamous lineage in human PDA cells. We also demonstrate that TP63-driven enhancer reprogramming promotes aggressive tumor phenotypes, including enhanced cell motility and invasion, and an accelerated growth of primary PDA tumors and metastases in vivo. This process ultimately leads to a powerful addiction of squamous PDA cells to continuous TP63 expression. Our study demonstrates the functional significance of squamous transdifferentiation in PDA and reveals TP63-based reprogramming as an experimental tool for investigating mechanisms and vulnerabilities linked to this aberrant cell fate transition.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Carcinoma de Células Escamosas/genética , Elementos de Facilitación Genéticos , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Carcinogénesis/genética , Carcinoma Ductal Pancreático/patología , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Humanos , Ratones , Fenotipo , Transcripción GenéticaRESUMEN
Targeting of general coactivators is an emerging strategy to interfere with oncogenic transcription factors (TFs). However, coactivator perturbations often lead to pleiotropic effects by influencing numerous TFs. Here we identify TAF12, a subunit of TFIID and SAGA coactivator complexes, as a selective requirement for acute myeloid leukemia (AML) progression. We trace this dependency to a direct interaction between the TAF12/TAF4 histone-fold heterodimer and the transactivation domain of MYB, a TF with established roles in leukemogenesis. Ectopic expression of the TAF4 histone-fold fragment can efficiently squelch TAF12 in cells, suppress MYB, and regress AML in mice. Our study reveals a strategy for potent MYB inhibition in AML and highlights how an oncogenic TF can be selectively neutralized by targeting a general coactivator complex.
Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/metabolismo , Animales , Humanos , Ratones Transgénicos , OncogenesRESUMEN
The Mixed Lineage Leukemia gene (MLL) is altered in leukemia by chromosomal translocations to produce oncoproteins composed of the MLL N-terminus fused to the C-terminus of a partner protein. Here, we used domain-focused CRISPR screening to identify ZFP64 as an essential transcription factor in MLL-rearranged leukemia. We show that the critical function of ZFP64 in leukemia is to maintain MLL expression via binding to the MLL promoter, which is the most enriched location of ZFP64 occupancy in the human genome. The specificity of ZFP64 for MLL is accounted for by an exceptional density of ZFP64 motifs embedded within the MLL promoter. These findings demonstrate how a sequence anomaly of an oncogene promoter can impose a transcriptional addiction in cancer.
Asunto(s)
Proteínas de Unión al ADN/genética , Leucemia Bifenotípica Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Translocación Genética , Células A549 , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Regulación Leucémica de la Expresión Génica , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células K562 , Leucemia Bifenotípica Aguda/metabolismo , Leucemia Bifenotípica Aguda/patología , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Células THP-1 , Factores de Transcripción/metabolismo , Trasplante HeterólogoRESUMEN
CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-Cas9-induced mutations to the 5' exons of candidate genes, but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies. Here we overcome this limitation by targeting CRISPR-Cas9 mutagenesis to exons encoding functional protein domains. This generates a higher proportion of null mutations and substantially increases the potency of negative selection. We also show that the magnitude of negative selection can be used to infer the functional importance of individual protein domains of interest. A screen of 192 chromatin regulatory domains in murine acute myeloid leukemia cells identifies six known drug targets and 19 additional dependencies. A broader application of this approach may allow comprehensive identification of protein domains that sustain cancer cells and are suitable for drug targeting.