RESUMO
Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.
Assuntos
Antígenos CD , Linfócitos T CD8-Positivos , Antígenos de Histocompatibilidade Classe I , Proteína do Gene 3 de Ativação de Linfócitos , Subfamília D de Receptores Semelhantes a Lectina de Células NK , Receptor de Morte Celular Programada 1 , Animais , Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Camundongos Endogâmicos C57BL , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Citotoxicidade Imunológica , Proliferação de Células , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/imunologiaRESUMO
Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TFs), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key mechanism restraining TEFF biology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced TEFF responses without compromising memory or exhaustion precursors. Fli1 restrained TEFF lineage differentiation by binding to cis-regulatory elements of effector-associated genes. Loss of Fli1 increased chromatin accessibility at ETS:RUNX motifs, allowing more efficient Runx3-driven TEFF biology. CD8+ T cells lacking Fli1 provided substantially better protection against multiple infections and tumors. These data indicate that Fli1 safeguards the developing CD8+ T cell transcriptional landscape from excessive ETS:RUNX-driven TEFF cell differentiation. Moreover, genetic deletion of Fli1 improves TEFF differentiation and protective immunity in infections and cancer.
Assuntos
Linfócitos T CD8-Positivos/citologia , Proteína Proto-Oncogênica c-fli-1/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular , Doença Crônica , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Epigênese Genética , Redes Reguladoras de Genes , Infecções/imunologia , Camundongos , Neoplasias/imunologiaRESUMO
During chronic infection and cancer, a self-renewing CD8+ T cell subset maintains long-term immunity and is critical to the effectiveness of immunotherapy. These stem-like CD8+ T cells diverge from other CD8+ subsets early after chronic viral infection. However, pathways guarding stem-like CD8+ T cells against terminal exhaustion remain unclear. Here, we show that the gene encoding transcriptional repressor BACH2 is transcriptionally and epigenetically active in stem-like CD8+ T cells but not terminally exhausted cells early after infection. BACH2 overexpression enforced stem-like cell fate, whereas BACH2 deficiency impaired stem-like CD8+ T cell differentiation. Single-cell transcriptomic and epigenomic approaches revealed that BACH2 established the transcriptional and epigenetic programs of stem-like CD8+ T cells. In addition, BACH2 suppressed the molecular program driving terminal exhaustion through transcriptional repression and epigenetic silencing. Thus, our study reveals a new pathway that enforces commitment to stem-like CD8+ lineage and prevents an alternative terminally exhausted cell fate.
Assuntos
Infecções por Arenaviridae/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Epigênese Genética , Células Precursoras de Linfócitos T/metabolismo , Transcrição Gênica , Animais , Infecções por Arenaviridae/genética , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/virologia , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Linhagem da Célula , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/patogenicidade , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Células Precursoras de Linfócitos T/imunologia , Células Precursoras de Linfócitos T/virologia , Transdução de SinaisRESUMO
CD8+ T cell exhaustion (Tex) limits disease control during chronic viral infections and cancer. Here, we investigated the epigenetic factors mediating major chromatin-remodeling events in Tex-cell development. A protein-domain-focused in vivo CRISPR screen identified distinct functions for two versions of the SWI/SNF chromatin-remodeling complex in Tex-cell differentiation. Depletion of the canonical SWI/SNF form, BAF, impaired initial CD8+ T cell responses in acute and chronic infection. In contrast, disruption of PBAF enhanced Tex-cell proliferation and survival. Mechanistically, PBAF regulated the epigenetic and transcriptional transition from TCF-1+ progenitor Tex cells to more differentiated TCF-1- Tex subsets. Whereas PBAF acted to preserve Tex progenitor biology, BAF was required to generate effector-like Tex cells, suggesting that the balance of these factors coordinates Tex-cell subset differentiation. Targeting PBAF improved tumor control both alone and in combination with anti-PD-L1 immunotherapy. Thus, PBAF may present a therapeutic target in cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos , Montagem e Desmontagem da Cromatina , Cromatina , Diferenciação Celular , Epigênese GenéticaRESUMO
Interferon-gamma (IFN-γ) has pleiotropic effects on cancer immune checkpoint blockade (ICB), including roles in ICB resistance. We analyzed gene expression in ICB-sensitive versus ICB-resistant tumor cells and identified a strong association between interferon-mediated resistance and expression of Ripk1, a regulator of tumor necrosis factor (TNF) superfamily receptors. Genetic interaction screening revealed that in cancer cells, RIPK1 diverted TNF signaling through NF-κB and away from its role in cell death. This promoted an immunosuppressive chemokine program by cancer cells, enhanced cancer cell survival, and decreased infiltration of T and NK cells expressing TNF superfamily ligands. Deletion of RIPK1 in cancer cells compromised chemokine secretion, decreased ARG1+ suppressive myeloid cells linked to ICB failure in mice and humans, and improved ICB response driven by CASP8-killing and dependent on T and NK cells. RIPK1-mediated resistance required its ubiquitin scaffolding but not kinase function. Thus, cancer cells co-opt RIPK1 to promote cell-intrinsic and cell-extrinsic resistance to immunotherapy.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores de Checkpoint Imunológico , Interferons , Neoplasias , Proteína Serina-Treonina Quinases de Interação com Receptores , Animais , Imunoterapia , Interferon gama/metabolismo , Interferons/metabolismo , Camundongos , NF-kappa B/metabolismo , Neoplasias/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
The clinical benefit of T cell immunotherapies remains limited by incomplete understanding of T cell differentiation and dysfunction. We generated an epigenetic and transcriptional atlas of T cell differentiation from healthy humans that included exhausted CD8 T cells and applied this resource in three ways. First, we identified modules of gene expression and chromatin accessibility, revealing molecular coordination of differentiation after activation and between central memory and effector memory. Second, we applied this healthy molecular framework to three settings-a neoadjuvant anti-PD1 melanoma trial, a basal cell carcinoma scATAC-seq dataset, and autoimmune disease-associated SNPs-yielding insights into disease-specific biology. Third, we predicted genome-wide cis-regulatory elements and validated this approach for key effector genes using CRISPR interference, providing functional annotation and demonstrating the ability to identify targets for non-coding cellular engineering. These studies define epigenetic and transcriptional regulation of human T cells and illustrate the utility of interrogating disease in the context of a healthy T cell atlas.
Assuntos
Epigenômica , Ativação Linfocitária , Linfócitos T CD8-Positivos , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Humanos , Ativação Linfocitária/genéticaRESUMO
Several rRNA-modifying enzymes install rRNA modifications while participating in ribosome assembly. Here, we show that 18S rRNA methyltransferase DIMT1 is essential for acute myeloid leukemia (AML) proliferation through a noncatalytic function. We reveal that targeting a positively charged cleft of DIMT1, remote from the catalytic site, weakens the binding of DIMT1 to rRNA and mislocalizes DIMT1 to the nucleoplasm, in contrast to the primarily nucleolar localization of wild-type DIMT1. Mechanistically, rRNA binding is required for DIMT1 to undergo liquid-liquid phase separation, which explains the distinct nucleoplasm localization of the rRNA binding-deficient DIMT1. Re-expression of wild-type or a catalytically inactive mutant E85A, but not the rRNA binding-deficient DIMT1, supports AML cell proliferation. This study provides a new strategy to target DIMT1-regulated AML proliferation via targeting this essential noncatalytic region.
Assuntos
Leucemia Mieloide Aguda , Metiltransferases , Humanos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Leucemia Mieloide Aguda/genética , Metiltransferases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S/metabolismoRESUMO
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.
Assuntos
Epigênese Genética , Glioblastoma , Fatores de Transcrição , Proteína Supressora de Tumor p53 , Adulto , Humanos , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proliferação de CélulasRESUMO
The transformed state in acute leukemia requires gene regulatory programs involving transcription factors and chromatin modulators. Here, we uncover an IRF8-MEF2D transcriptional circuit as an acute myeloid leukemia (AML)-biased dependency. We discover and characterize the mechanism by which the chromatin "reader" ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their lineage-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo and associates with MYC and IRF8 enhancer elements that we define in cell lines and in patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcription coactivator also necessary for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential therapeutic target for modulating essential transcriptional programs in AML.
Assuntos
Fatores Reguladores de Interferon/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Fatores Reguladores de Interferon/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Proto-Oncogene Mas , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplantation settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels.. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells, and its perturbation might offer new opportunities for treatment of hemoglobinopathies.
Assuntos
DNA/genética , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Fatores de Transcrição/genética , Animais , Sítios de Ligação , Células COS , Sistemas CRISPR-Cas , Chlorocebus aethiops , DNA/metabolismo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/transplante , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Hemoglobina Fetal/metabolismo , Feto , Edição de Genes , Células HEK293 , Xenoenxertos , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos , Modelos Moleculares , Células-Tronco Embrionárias Murinas/citologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Sequencing efforts led to the identification of somatic mutations that could affect the self-renewal and differentiation of cancer-initiating cells. One such recurrent mutation targets the binding pocket of the ubiquitin ligase Fbxw7. Missense FBXW7 mutations are prevalent in various tumors, including T cell acute lymphoblastic leukemia (T-ALL). To study the effects of such lesions, we generated animals carrying regulatable Fbxw7 mutant alleles. Here, we show that these mutations specifically bolster cancer-initiating cell activity in collaboration with Notch1 oncogenes but spare normal hematopoietic stem cell function. We were also able to show that FBXW7 mutations specifically affect the ubiquitylation and half-life of c-Myc protein, a key T-ALL oncogene. Using animals carrying c-Myc fusion alleles, we connected Fbxw7 function to c-Myc abundance and correlated c-Myc expression to leukemia-initiating activity. Finally, we demonstrated that small-molecule-mediated suppression of MYC activity leads to T-ALL remission, suggesting an effective therapeutic strategy.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Modelos Animais de Doenças , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Receptor Notch1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.
Assuntos
Transformação Celular Neoplásica/genética , Cromatina/genética , Linfoma de Células B/genética , Oncogenes , Receptores Notch/genética , Neoplasias de Mama Triplo Negativas/genética , Sítios de Ligação , Linhagem da Célula/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Ciclina D1/genética , Ciclina D1/metabolismo , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293 , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Mutação , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
ABSTRACT: The switch from fetal hemoglobin (γ-globin, HBG) to adult hemoglobin (ß-globin, HBB) gene transcription in erythroid cells serves as a paradigm for a complex and clinically relevant developmental gene regulatory program. We previously identified HIC2 as a regulator of the switch by inhibiting the transcription of BCL11A, a key repressor of HBG production. HIC2 is highly expressed in fetal cells, but the mechanism of its regulation is unclear. Here we report that HIC2 developmental expression is controlled by microRNAs (miRNAs), as loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG messenger RNA. We identified the adult-expressed let-7 miRNA family as a direct posttranscriptional regulator of HIC2. Ectopic expression of let-7 in fetal cells lowered HIC2 levels, whereas inhibition of let-7 in adult erythroblasts increased HIC2 production, culminating in decommissioning of a BCL11A erythroid enhancer and reduced BCL11A transcription. HIC2 depletion in let-7-inhibited cells restored BCL11A-mediated repression of HBG. Together, these data establish that fetal hemoglobin silencing in adult erythroid cells is under the control of a miRNA-mediated inhibitory pathway (let-7 ⣠HIC2 ⣠BCL11A ⣠HBG).
Assuntos
Hemoglobina Fetal , Fatores de Transcrição Kruppel-Like , MicroRNAs , Proteínas Repressoras , Humanos , Globinas beta/genética , Globinas beta/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Eritroblastos/metabolismo , Eritroblastos/citologia , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , gama-Globinas/genética , gama-Globinas/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transcrição GênicaRESUMO
MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Mieloma Múltiplo/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Antineoplásicos/química , Azepinas/química , Azepinas/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/genética , Ativação Transcricional/efeitos dos fármacos , Triazóis/química , Triazóis/farmacologiaRESUMO
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.
Assuntos
Leucemia Mieloide Aguda/enzimologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Acetilação , Animais , Antineoplásicos/farmacologia , Proliferação de Células , Elementos Facilitadores Genéticos , Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Células Hep G2 , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Células NIH 3T3 , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Células THP-1 , Células U937RESUMO
The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity. Our model involves multiple layers of previously underappreciated cooperativity in BE steps including: (i) Cas9 binding can potentially expose both DNA strands for 'capture' by the deaminase, a feature that is enhanced by guide RNA mismatches; (ii) after strand capture, the intrinsic activity of the DNA deaminase can tune window size and base editing efficiency; (iii) Cas9 defines the boundaries of editing on each strand, with deamination blocked by Cas9 binding to either the PAM or the protospacer and (iv) non-canonical edits on the guide RNA bound strand can be further elicited by changing which strand is nicked by Cas9. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C > T and G > A transitions over >65 bp with significant potential for targeted gene diversification.
Assuntos
Proteína 9 Associada à CRISPR , Citidina Desaminase , Escherichia coli , Edição de Genes , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Citidina Desaminase/metabolismo , DNA/genética , Escherichia coli/metabolismo , Mutação , RNA Guia de Sistemas CRISPR-Cas , Humanos , AnimaisRESUMO
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.
Assuntos
Rabdomiossarcoma , Fator de Ligação a CCAAT/genética , Diferenciação Celular/genética , Aberrações Cromossômicas , Rabdomiossarcoma/genética , Fatores de TranscriçãoRESUMO
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.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatologia , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/fisiopatologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Humanos , Pulmão/patologia , Camundongos , Receptor IGF Tipo 1/metabolismoRESUMO
The switch from fetal hemoglobin (HbF) to adult hemoglobin (HbA) is a paradigm for developmental gene expression control with relevance to sickle cell disease and ß-thalassemia. Polycomb repressive complex (PRC) proteins regulate this switch, and an inhibitor of PRC2 has entered a clinical trial for HbF activation. Yet, how PRC complexes function in this process, their target genes, and relevant subunit composition are unknown. Here, we identified the PRC1 subunit BMI1 as a novel HbF repressor. We uncovered the RNA binding proteins LIN28B, IGF2BP1, and IGF2BP3 genes as direct BMI1 targets, and demonstrate that they account for the entirety of BMI1's effect on HbF regulation. BMI1 functions as part of the canonical PRC1 (cPRC1) subcomplex as revealed by the physical and functional dissection of BMI1 protein partners. Lastly, we demonstrate that BMI1/cPRC1 acts in concert with PRC2 to repress HbF through the same target genes. Our study illuminates how PRC silences HbF, highlighting an epigenetic mechanism involved in hemoglobin switching.