RESUMEN
OCA-B, OCA-T1, and OCA-T2 belong to a family of coactivators that bind to POU transcription factors (TFs) to regulate gene expression in immune cells. Here, we identify IκBζ (encoded by the NFKBIZ gene) as an additional coactivator of POU TFs. Although originally discovered as an inducible regulator of NF-κB, we show here that IκBζ shares a microhomology with OCA proteins and uses this segment to bind to POU TFs and octamer-motif-containing DNA. Our functional experiments suggest that IκBζ requires its interaction with POU TFs to coactivate immune-related genes. This finding is reinforced by epigenomic analysis of MYD88L265P-mutant lymphoma cells, which revealed colocalization of IκBζ with the POU TF OCT2 and NF-κB:p50 at hundreds of DNA elements harboring octamer and κB motifs. These results suggest that IκBζ is a transcriptional coactivator that can amplify and integrate the output of NF-κB and POU TFs at inducible genes in immune cells.
Asunto(s)
ADN , FN-kappa B , FN-kappa B/genética , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , ADN/genética , ADN/metabolismoRESUMEN
Inhibition of the tumour suppressive function of p53 (encoded by TP53) is paramount for cancer development in humans. However, p53 remains unmutated in the majority of cases of glioblastoma (GBM)-the most common and deadly adult brain malignancy1,2. Thus, how p53-mediated tumour suppression is countered in TP53 wild-type (TP53WT) GBM is unknown. Here we describe a GBM-specific epigenetic mechanism in which the chromatin regulator bromodomain-containing protein 8 (BRD8) maintains H2AZ occupancy at p53 target loci through the EP400 histone acetyltransferase complex. This mechanism causes a repressive chromatin state that prevents transactivation by p53 and sustains proliferation. Notably, targeting the bromodomain of BRD8 displaces H2AZ, enhances chromatin accessibility and engages p53 transactivation. This in turn enforces cell cycle arrest and tumour suppression in TP53WT GBM. In line with these findings, BRD8 is highly expressed with H2AZ in proliferating single cells of patient-derived GBM, and is inversely correlated with CDKN1A, a canonical p53 target that encodes p21 (refs. 3,4). This work identifies BRD8 as a selective epigenetic vulnerability for a malignancy for which treatment has not improved for decades. Moreover, targeting the bromodomain of BRD8 may be a promising therapeutic strategy for patients with TP53WT GBM.
Asunto(s)
Epigénesis Genética , Glioblastoma , Factores de Transcripción , Proteína p53 Supresora de Tumor , Adulto , Humanos , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Histonas/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proliferación CelularRESUMEN
Tuft cells are a rare chemosensory lineage that coordinates immune and neural responses to foreign pathogens in mucosal tissues1. Recent studies have also revealed tuft-cell-like human tumours2,3, particularly as a variant of small-cell lung cancer. Both normal and neoplastic tuft cells share a genetic requirement for the transcription factor POU2F3 (refs. 2,4), although the transcriptional mechanisms that generate this cell type are poorly understood. Here we show that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 (renamed here OCA-T1/POU2AF2 and OCA-T2/POU2AF3, respectively) is critical in the tuft cell lineage. OCA-T1 and OCA-T2 are paralogues of the B-cell-specific coactivator OCA-B; all three proteins are encoded in a gene cluster and contain a conserved peptide that binds to class II POU transcription factors and a DNA octamer motif in a bivalent manner. We demonstrate that binding between POU2F3 and OCA-T1 or OCA-T2 is essential in tuft-cell-like small-cell lung cancer. Moreover, we generated OCA-T1-deficient mice, which are viable but lack tuft cells in several mucosal tissues. These findings reveal that the POU2F3-OCA-T complex is the master regulator of tuft cell identity and a molecular vulnerability of tuft-cell-like small-cell lung cancer.
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Linaje de la Célula , Neoplasias Pulmonares , Proteínas de Neoplasias , Factores de Transcripción de Octámeros , Carcinoma Pulmonar de Células Pequeñas , Animales , Humanos , Ratones , Neoplasias Pulmonares/patología , Membrana Mucosa/patología , Familia de Multigenes/genética , Proteínas de Neoplasias/metabolismo , Motivos de Nucleótidos , Factores de Transcripción de Octámeros/metabolismo , Factores del Dominio POU/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , TransactivadoresRESUMEN
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.
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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
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
Lineage plasticity is a prominent feature of pancreatic ductal adenocarcinoma (PDA) cells, which can occur via deregulation of lineage-specifying transcription factors. Here, we show that the zinc finger protein ZBED2 is aberrantly expressed in PDA and alters tumor cell identity in this disease. Unexpectedly, our epigenomic experiments reveal that ZBED2 is a sequence-specific transcriptional repressor of IFN-stimulated genes, which occurs through antagonism of IFN regulatory factor 1 (IRF1)-mediated transcriptional activation at cooccupied promoter elements. Consequently, ZBED2 attenuates the transcriptional output and growth arrest phenotypes downstream of IFN signaling in multiple PDA cell line models. We also found that ZBED2 is preferentially expressed in the squamous molecular subtype of human PDA, in association with inferior patient survival outcomes. Consistent with this observation, we show that ZBED2 can repress the pancreatic progenitor transcriptional program, enhance motility, and promote invasion in PDA cells. Collectively, our findings suggest that high ZBED2 expression is acquired during PDA progression to suppress the IFN response pathway and to promote lineage plasticity in this disease.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Proteínas de Unión al ADN/metabolismo , Factor 1 Regulador del Interferón/metabolismo , Neoplasias Pancreáticas/patología , Factores de Transcripción/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Factor 1 Regulador del Interferón/genética , Interferón gamma/farmacología , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Regiones Promotoras Genéticas , Análisis de Supervivencia , Factores de Transcripción/genéticaRESUMEN
Small cell lung cancers (SCLC) are comprised of heterogeneous subtypes marked by lineage-specific transcription factors, including ASCL1, NEUROD1, and POU2F3. POU2F3-positive SCLC, â¼12% of all cases, are uniquely dependent on POU2F3 itself; as such, approaches to attenuate POU2F3 expression may represent new therapeutic opportunities. Here using genome-scale screens for regulators of POU2F3 expression and SCLC proliferation, we define mSWI/SNF complexes, including non-canonical BAF (ncBAF) complexes, as top dependencies specific to POU2F3-positive SCLC. Notably, clinical-grade pharmacologic mSWI/SNF inhibition attenuates proliferation of all POU2F3-positive SCLCs, while disruption of ncBAF via BRD9 degradation is uniquely effective in pure non-neuroendocrine POU2F3-SCLCs. mSWI/SNF maintains accessibility over gene loci central to POU2F3-mediated gene regulatory networks. Finally, chemical targeting of SMARCA4/2 mSWI/SNF ATPases and BRD9 decrease POU2F3-SCLC tumor growth and increase survival in vivo . Taken together, these results characterize mSWI/SNF-mediated global governance of the POU2F3 oncogenic program and suggest mSWI/SNF inhibition as a therapeutic strategy for SCLC.
RESUMEN
The POU2F3-POU2AF2/3 (OCA-T1/2) transcription factor complex is the master regulator of the tuft cell lineage and tuft cell-like small cell lung cancer (SCLC). Here, we found that the POU2F3 molecular subtype of SCLC (SCLC-P) exhibits an exquisite dependence on the activity of the mammalian switch/sucrose non-fermentable (mSWI/SNF) chromatin remodeling complex. SCLC-P cell lines were sensitive to nanomolar levels of a mSWI/SNF ATPase proteolysis targeting chimera (PROTAC) degrader when compared to other molecular subtypes of SCLC. POU2F3 and its cofactors were found to interact with components of the mSWI/SNF complex. The POU2F3 transcription factor complex was evicted from chromatin upon mSWI/SNF ATPase degradation, leading to attenuation of downstream oncogenic signaling in SCLC-P cells. A novel, orally bioavailable mSWI/SNF ATPase PROTAC degrader, AU-24118, demonstrated preferential efficacy in the SCLC-P relative to the SCLC-A subtype and significantly decreased tumor growth in preclinical models. AU-24118 did not alter normal tuft cell numbers in lung or colon, nor did it exhibit toxicity in mice. B cell malignancies which displayed a dependency on the POU2F1/2 cofactor, POU2AF1 (OCA-B), were also remarkably sensitive to mSWI/SNF ATPase degradation. Mechanistically, mSWI/SNF ATPase degrader treatment in multiple myeloma cells compacted chromatin, dislodged POU2AF1 and IRF4, and decreased IRF4 signaling. In a POU2AF1-dependent, disseminated murine model of multiple myeloma, AU-24118 enhanced survival compared to pomalidomide, an approved treatment for multiple myeloma. Taken together, our studies suggest that POU2F-POU2AF-driven malignancies have an intrinsic dependence on the mSWI/SNF complex, representing a therapeutic vulnerability.
RESUMEN
Small cell lung cancers (SCLCs) are composed of heterogeneous subtypes marked by lineage-specific transcription factors, including ASCL1, NEUROD1, and POU2F3. POU2F3-positive SCLCs, â¼12% of all cases, are uniquely dependent on POU2F3 itself; as such, approaches to attenuate POU2F3 expression may represent new therapeutic opportunities. Here using genome-scale screens for regulators of POU2F3 expression and SCLC proliferation, we define mSWI/SNF complexes as top dependencies specific to POU2F3-positive SCLC. Notably, chemical disruption of mSWI/SNF ATPase activity attenuates proliferation of all POU2F3-positive SCLCs, while disruption of non-canonical BAF (ncBAF) via BRD9 degradation is effective in pure non-neuroendocrine POU2F3-SCLCs. mSWI/SNF targets to and maintains accessibility over gene loci central to POU2F3-mediated gene regulatory networks. Finally, clinical-grade pharmacologic disruption of SMARCA4/2 ATPases and BRD9 decreases POU2F3-SCLC tumor growth and increases survival in vivo. These results demonstrate mSWI/SNF-mediated governance of the POU2F3 oncogenic program and suggest mSWI/SNF inhibition as a therapeutic strategy for POU2F3-positive SCLCs.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Factores de Transcripción , Humanos , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ratones , Línea Celular Tumoral , Proliferación Celular , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genéticaRESUMEN
The POU2F3-POU2AF2/3 transcription factor complex is the master regulator of the tuft cell lineage and tuft cell-like small cell lung cancer (SCLC). Here, we identify a specific dependence of the POU2F3 molecular subtype of SCLC (SCLC-P) on the activity of the mammalian switch/sucrose non-fermentable (mSWI/SNF) chromatin remodeling complex. Treatment of SCLC-P cells with a proteolysis targeting chimera (PROTAC) degrader of mSWI/SNF ATPases evicts POU2F3 and its coactivators from chromatin and attenuates downstream signaling. B cell malignancies which are dependent on the POU2F1/2 cofactor, POU2AF1, are also sensitive to mSWI/SNF ATPase degraders, with treatment leading to chromatin eviction of POU2AF1 and IRF4 and decreased IRF4 signaling in multiple myeloma cells. An orally bioavailable mSWI/SNF ATPase degrader significantly inhibits tumor growth in preclinical models of SCLC-P and multiple myeloma without signs of toxicity. This study suggests that POU2F-POU2AF-driven malignancies have an intrinsic dependence on the mSWI/SNF complex, representing a therapeutic vulnerability.
Asunto(s)
Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Factores de Transcripción , Humanos , Animales , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Línea Celular Tumoral , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Transducción de Señal , Regulación Neoplásica de la Expresión Génica , Factor 2 de Transcripción de Unión a OctámerosRESUMEN
A genetic mechanism accounts for the variation in tuft cell abundance seen among inbred mouse strains: alternative isoforms of OCA-T1 (Oct coactivator from tuft cells 1), a recently discovered transcriptional coactivator that specifies the tuft cell lineage (see related Research Article by Nadjsombati et al.).
Asunto(s)
Linfocitos B , Transactivadores , Ratones , Animales , Transactivadores/genética , Factores de Transcripción , Isoformas de Proteínas/genética , Linaje de la CélulaRESUMEN
During the progression of pancreatic ductal adenocarcinoma (PDAC), tumor cells are known to acquire transcriptional and morphological properties of the basal (also known as squamous) epithelial lineage, which leads to more aggressive disease characteristics. Here, we show that a subset of basal-like PDAC tumors aberrantly express p73 (TA isoform), which is a known transcriptional activator of basal lineage identity, ciliogenesis, and tumor suppression in normal tissue development. Using gain- and loss- of function experiments, we show that p73 is necessary and sufficient to activate genes related to basal identity (e.g. KRT5), ciliogenesis (e.g. FOXJ1), and p53-like tumor suppression (e.g. CDKN1A) in human PDAC models. Owing to the paradoxical combination of oncogenic and tumor suppressive outputs of this transcription factor, we propose that PDAC cells express a low level of p73 that is optimal for promoting lineage plasticity without severe impairment of cell proliferation. Collectively, our study reinforces how PDAC cells exploit master regulators of the basal epithelial lineage during disease progression.
RESUMEN
The EWS-FLI1 fusion oncoprotein deregulates transcription to initiate the paediatric cancer Ewing sarcoma. Here we used a domain-focused CRISPR screen to implicate the transcriptional repressor ETV6 as a unique dependency in this tumour. Using biochemical assays and epigenomics, we show that ETV6 competes with EWS-FLI1 for binding to select DNA elements enriched for short GGAA repeat sequences. Upon inactivating ETV6, EWS-FLI1 overtakes and hyper-activates these cis-elements to promote mesenchymal differentiation, with SOX11 being a key downstream target. We show that squelching of ETV6 with a dominant-interfering peptide phenocopies these effects and suppresses Ewing sarcoma growth in vivo. These findings reveal targeting of ETV6 as a strategy for neutralizing the EWS-FLI1 oncoprotein by reprogramming of genomic occupancy.
Asunto(s)
Sarcoma de Ewing , Niño , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismoRESUMEN
Dynamic regulation of intestinal epithelial cell (IEC) differentiation is crucial for both homeostasis and the response to helminth infection. SIRT6 belongs to the NAD+-dependent deacetylases and has established diverse roles in aging, metabolism and disease. Here, we report that IEC Sirt6 deletion leads to impaired tuft cell development and type 2 immunity in response to helminth infection, thereby resulting in compromised worm expulsion. Conversely, after helminth infection, IEC SIRT6 transgenic mice exhibit enhanced epithelial remodeling process and more efficient worm clearance. Mechanistically, Sirt6 ablation causes elevated Socs3 expression, and subsequently attenuated tyrosine 641 phosphorylation of STAT6 in IECs. Notably, intestinal epithelial overexpression of constitutively activated STAT6 (STAT6vt) in mice is sufficient to induce the expansion of tuft and goblet cell linage. Furthermore, epithelial STAT6vt overexpression remarkedly reverses the defects in intestinal epithelial remodeling caused by Sirt6 ablation. Our results reveal a novel function of SIRT6 in regulating intestinal epithelial remodeling and mucosal type 2 immunity in response to helminth infection.
Asunto(s)
Helmintiasis/inmunología , Mucosa Intestinal , Factor de Transcripción STAT6/metabolismo , Sirtuinas/metabolismo , Animales , Células Epiteliales/metabolismo , Células Caliciformes/metabolismo , Helmintiasis/metabolismo , Inmunidad Mucosa , Mucosa Intestinal/metabolismo , Intestinos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Transcripción STAT6/genética , Sirtuinas/genéticaRESUMEN
Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Desarrollo de Músculos/fisiología , Rabdomiosarcoma/genética , Rabdomiosarcoma/metabolismo , Línea Celular Tumoral , Niño , Cromatina , ADN Helicasas/metabolismo , Epigenómica , Regulación Neoplásica de la Expresión Génica , Humanos , Músculo Esquelético , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Factor de Transcripción PAX7 , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Proteómica , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
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.
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Aldehído Deshidrogenasa Mitocondrial/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Leucemia Mieloide Aguda/genética , Línea Celular Tumoral , Humanos , UbiquitinaciónRESUMEN
Reactivation of fetal hemoglobin (HbF) production benefits patients with sickle cell disease and ß-thalassemia. To identify new HbF regulators that might be amenable to pharmacologic control, we screened a protein domain-focused CRISPR-Cas9 library targeting chromatin regulators, including BTB domain-containing proteins. Speckle-type POZ protein (SPOP), a substrate adaptor of the CUL3 ubiquitin ligase complex, emerged as a novel HbF repressor. Depletion of SPOP or overexpression of a dominant negative version significantly raised fetal globin messenger RNA and protein levels with minimal detrimental effects on normal erythroid maturation, as determined by transcriptome and proteome analyses. SPOP controls HbF expression independently of the major transcriptional HbF repressors BCL11A and LRF. Finally, pharmacologic HbF inducers cooperate with SPOP depletion during HbF upregulation. Our study implicates SPOP and the CUL3 ubiquitin ligase system in controlling HbF production in human erythroid cells and may offer new therapeutic strategies for the treatment of ß-hemoglobinopathies.
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Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adulto , Femenino , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas Represoras/genética , Adulto JovenRESUMEN
Core regulatory transcription factors (CR TFs) orchestrate the placement of super-enhancers (SEs) to activate transcription of cell-identity specifying gene networks, and are critical in promoting cancer. Here, we define the core regulatory circuitry of rhabdomyosarcoma and identify critical CR TF dependencies. These CR TFs build SEs that have the highest levels of histone acetylation, yet paradoxically the same SEs also harbor the greatest amounts of histone deacetylases. We find that hyperacetylation selectively halts CR TF transcription. To investigate the architectural determinants of this phenotype, we used absolute quantification of architecture (AQuA) HiChIP, which revealed erosion of native SE contacts, and aberrant spreading of contacts that involved histone acetylation. Hyperacetylation removes RNA polymerase II (RNA Pol II) from core regulatory genetic elements, and eliminates RNA Pol II but not BRD4 phase condensates. This study identifies an SE-specific requirement for balancing histone modification states to maintain SE architecture and CR TF transcription.
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Histonas/metabolismo , Rabdomiosarcoma/genética , Factores de Transcripción/genética , Acetilación , Benzamidas/farmacología , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Elementos de Facilitación Genéticos , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/genética , Humanos , Piridinas/farmacología , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Estabilidad del ARN , Factores de Transcripción SOXE/genética , Análisis de la Célula IndividualRESUMEN
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
RING-between-RING (RBR) E3s contain RING1 domains that are structurally similar yet mechanistically distinct from canonical RING domains. Both types of E3 bind E2â¼ubiquitin (E2â¼Ub) via their RINGs but canonical RING E3s promote closed E2â¼Ub conformations required for direct Ub transfer from the E2 to substrate, while RBR RING1s promote open E2â¼Ub to favor Ub transfer to the E3 active site. This different RING/E2â¼Ub conformation determines its direct target, which for canonical RING E3s is typically a substrate or substrate-linked Ub, but is the E3 active-site cysteine in the case of RBR-type E3s. Here we show that a short extension of HHARI RING1, namely Zn2+-loop II, not present in any RING E3s, acts as a steric wedge to disrupt closed E2â¼Ub, providing a structural explanation for the distinctive RING1-dependent conformational restriction mechanism utilized by RBR E3s.