RESUMO
The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas , Células-Tronco Neurais , Neurogênese , Neurônios , Adulto , Animais , Humanos , Camundongos , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Fatores de Tempo , Transcrição GênicaRESUMO
Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.
Assuntos
Instabilidade Cromossômica , Segregação de Cromossomos , Cromossomos , Epigênese Genética , Micronúcleos com Defeito Cromossômico , Neoplasias , Animais , Humanos , Camundongos , Cromatina/genética , Instabilidade Cromossômica/genética , Cromossomos/genética , Cromossomos/metabolismo , Histonas/química , Histonas/metabolismo , Neoplasias/genética , Neoplasias/patologia , Mitose , Variações do Número de Cópias de DNA , Processamento de Proteína Pós-TraducionalRESUMO
Circular extrachromosomal DNA (ecDNA) is a form of oncogene amplification found across cancer types and associated with poor outcome in patients. ecDNA can be structurally complex and contain rearranged DNA sequences derived from multiple chromosome locations. As the structure of ecDNA can impact oncogene regulation and may indicate mechanisms of its formation, disentangling it at high resolution from sequencing data is essential. Even though methods have been developed to identify and reconstruct ecDNA in cancer genome sequencing, it remains challenging to resolve complex ecDNA structures, in particular amplicons with shared genomic footprints. We here introduce Decoil, a computational method which combines a breakpoint-graph approach with regression to reconstruct complex ecDNA and deconvolve co-occurring ecDNA elements with overlapping genomic footprints from long-read nanopore sequencing. Decoil outperforms de novo assembly and alignment-based methods in simulated long-read sequencing data for both simple and complex ecDNAs. Applying Decoil on whole genome sequencing data uncovered different ecDNA topologies and explored ecDNA structure heterogeneity in neuroblastoma tumors and cell lines, indicating that this method may improve ecDNA structural analyzes in cancer.
RESUMO
Enhancer activation is a critical step for gene activation. Here we report an epigenetic crosstalk at enhancers between the UTX (H3K27 demethylase)-MLL4 (H3K4 methyltransferase) complex and the histone acetyltransferase p300. We demonstrate that UTX, in a demethylase activity-independent manner, facilitates conversion of inactive enhancers in embryonic stem cells to an active (H3K4me1+/H3K27ac+) state by recruiting and coupling the enzymatic functions of MLL4 and p300. Loss of UTX leads to attenuated enhancer activity, characterized by reduced levels of H3K4me1 and H3K27ac as well as impaired transcription. The UTX-MLL4 complex enhances p300-dependent H3K27 acetylation through UTX-dependent stimulation of p300 recruitment, while MLL4-mediated H3K4 monomethylation, reciprocally, requires p300 function. Importantly, MLL4-generated H3K4me1 further enhances p300-dependent transcription. This work reveals a previously unrecognized cooperativity among enhancer-associated chromatin modulators, including a unique function for UTX, in establishing an "active enhancer landscape" and defines a detailed mechanism for the joint deposition of H3K4me1 and H3K27ac.
Assuntos
Cromatina/metabolismo , Proteína p300 Associada a E1A/metabolismo , Células-Tronco Embrionárias/enzimologia , Elementos Facilitadores Genéticos , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Transcrição Gênica , Ativação Transcricional , Animais , Cromatina/genética , Montagem e Desmontagem da Cromatina , Proteína p300 Associada a E1A/genética , Retroalimentação Fisiológica , Redes Reguladoras de Genes , Células HEK293 , Histona Desmetilases/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Masculino , Metilação , Camundongos , Interferência de RNA , TransfecçãoRESUMO
Leukemic transformation (LT) of myeloproliferative neoplasm (MPN) has a dismal prognosis and is largely fatal. Mutational inactivation of TP53 is the most common somatic event in LT; however, the mechanisms by which TP53 mutations promote LT remain unresolved. Using an allelic series of mouse models of Jak2/Trp53 mutant MPN, we identify that only biallelic inactivation of Trp53 results in LT (to a pure erythroleukemia [PEL]). This PEL arises from the megakaryocyte-erythroid progenitor population. Importantly, the bone morphogenetic protein 2/SMAD pathway is aberrantly activated during LT and results in abnormal self-renewal of megakaryocyte-erythroid progenitors. Finally, we identify that Jak2/Trp53 mutant PEL is characterized by recurrent copy number alterations and DNA damage. Using a synthetic lethality strategy, by targeting active DNA repair pathways, we show that this PEL is highly sensitive to combination WEE1 and poly(ADP-ribose) polymerase inhibition. These observations yield new mechanistic insights into the process of p53 mutant LT and offer new, clinically translatable therapeutic approaches.
Assuntos
Transtornos Mieloproliferativos , Proteína Supressora de Tumor p53 , Animais , Proteína Morfogenética Óssea 2/genética , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/metabolismo , Camundongos , Mutação , Transtornos Mieloproliferativos/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Myeloproliferative neoplasms (MPNs) transform to myelofibrosis (MF) and highly lethal acute myeloid leukemia (AML), although the actionable mechanisms driving progression remain elusive. Here, we elucidate the role of the high mobility group A1 (HMGA1) chromatin regulator as a novel driver of MPN progression. HMGA1 is upregulated in MPN, with highest levels after transformation to MF or AML. To define HMGA1 function, we disrupted gene expression via CRISPR/Cas9, short hairpin RNA, or genetic deletion in MPN models. HMGA1 depletion in JAK2V617F AML cell lines disrupts proliferation, clonogenicity, and leukemic engraftment. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2V617F mice, decreasing erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, and expansion of stem and progenitors, while preventing splenomegaly and fibrosis within the spleen and BM. RNA-sequencing and chromatin immunoprecipitation sequencing revealed HMGA1 transcriptional networks and chromatin occupancy at genes that govern proliferation (E2F, G2M, mitotic spindle) and cell fate, including the GATA2 master regulatory gene. Silencing GATA2 recapitulates most phenotypes observed with HMGA1 depletion, whereas GATA2 re-expression partially rescues leukemogenesis. HMGA1 transactivates GATA2 through sequences near the developmental enhancer (+9.5), increasing chromatin accessibility and recruiting active histone marks. Further, HMGA1 transcriptional networks, including proliferation pathways and GATA2, are activated in human MF and MPN leukemic transformation. Importantly, HMGA1 depletion enhances responses to the JAK2 inhibitor, ruxolitinib, preventing MF and prolonging survival in murine models of JAK2V617F AML. These findings illuminate HMGA1 as a key epigenetic switch involved in MPN transformation and a promising therapeutic target to treat or prevent disease progression.
Assuntos
Fator de Transcrição GATA2 , Proteína HMGA1a , Leucemia Mieloide Aguda , Transtornos Mieloproliferativos , Mielofibrose Primária , Animais , Proliferação de Células , Cromatina/genética , Fator de Transcrição GATA2/genética , Redes Reguladoras de Genes , Proteína HMGA1a/genética , Proteína HMGA1a/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Leucemia Mieloide Aguda/genética , Camundongos , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Mielofibrose Primária/genéticaRESUMO
UDP-glucose pyrophosphorylase 2 (UGP2), the enzyme that synthesizes uridine diphosphate (UDP)-glucose, rests at the convergence of multiple metabolic pathways, however, the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an important role for UGP2 in the maintenance of pancreatic ductal adenocarcinoma (PDAC) growth in both in vitro and in vivo tumor models. We found that transcription of UGP2 is directly regulated by the Yes-associated protein 1 (YAP)-TEA domain transcription factor (TEAD) complex, identifying UGP2 as a bona fide YAP target gene. Loss of UGP2 leads to decreased intracellular glycogen levels and defects in N-glycosylation targets that are important for the survival of PDACs, including the epidermal growth factor receptor (EGFR). These critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer insights into therapeutic options for otherwise intractable PDACs.
Assuntos
Carcinoma Ductal Pancreático/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Glicogênio/biossíntese , Neoplasias Pancreáticas/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Glicosilação , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais , Neoplasias Pancreáticas/patologia , Fatores de Transcrição de Domínio TEA/genética , Fatores de Transcrição de Domínio TEA/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/genética , Proteínas de Sinalização YAP/genética , Proteínas de Sinalização YAP/metabolismoRESUMO
Plasmacytoid dendritic cells (pDCs) are the principal natural type I interferon-producing dendritic cells. Neoplastic expansion of pDCs and pDC precursors leads to blastic plasmacytoid dendritic cell neoplasm (BPDCN), and clonal expansion of mature pDCs has been described in chronic myelomonocytic leukemia. The role of pDC expansion in acute myeloid leukemia (AML) is poorly studied. Here, we characterize patients with AML with pDC expansion (pDC-AML), which we observe in â¼5% of AML cases. pDC-AMLs often possess cross-lineage antigen expression and have adverse risk stratification with poor outcome. RUNX1 mutations are the most common somatic alterations in pDC-AML (>70%) and are much more common than in AML without pDC expansion and BPDCN. We demonstrate that pDCs are clonally related to, as well as originate from, leukemic blasts in pDC-AML. We further demonstrate that leukemic blasts from RUNX1-mutated AML upregulate a pDC transcriptional program, poising the cells toward pDC differentiation and expansion. Finally, tagraxofusp, a targeted therapy directed to CD123, reduces leukemic burden and eliminates pDCs in a patient-derived xenograft model. In conclusion, pDC-AML is characterized by a high frequency of RUNX1 mutations and increased expression of a pDC transcriptional program. CD123 targeting represents a potential treatment approach for pDC-AML.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Células Dendríticas/patologia , Leucemia Mieloide Aguda/genética , Adulto , Idoso , Crise Blástica/genética , Crise Blástica/patologia , Células Dendríticas/metabolismo , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
The interaction of menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and provides a potential opportunity for treatment of NPM1-mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. In this study, transcriptional profiling after pharmacological inhibition of the menin-MLL complex revealed specific changes in gene expression, with downregulation of the MEIS1 transcription factor and its transcriptional target gene FLT3 being the most pronounced. Combining menin-MLL inhibition with specific small-molecule kinase inhibitors of FLT3 phosphorylation resulted in a significantly superior reduction of phosphorylated FLT3 and transcriptional suppression of genes downstream of FLT3 signaling. The drug combination induced synergistic inhibition of proliferation, as well as enhanced apoptosis, compared with single-drug treatment in models of human and murine NPM1mut and MLL-r leukemias harboring an FLT3 mutation. Primary acute myeloid leukemia (AML) cells harvested from patients with NPM1mutFLT3mut AML showed significantly better responses to combined menin and FLT3 inhibition than to single-drug or vehicle control treatment, whereas AML cells with wild-type NPM1, MLL, and FLT3 were not affected by either of the 2 drugs. In vivo treatment of leukemic animals with MLL-r FLT3mut leukemia reduced leukemia burden significantly and prolonged survival compared with results in the single-drug and vehicle control groups. Our data suggest that combined menin-MLL and FLT3 inhibition represents a novel and promising therapeutic strategy for patients with NPM1mut or MLL-r leukemia and concurrent FLT3 mutation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Cocultura , Sinergismo Farmacológico , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos NOD , Proteína Meis1/biossíntese , Proteína Meis1/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Nucleofosmina , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Distribuição Aleatória , Transcrição Gênica/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/biossíntese , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Oncogenic IDH1/2 mutations produce 2-hydroxyglutarate (2HG), resulting in competitive inhibition of DNA and protein demethylation. IDH-mutant cancer cells show an inability to differentiate but whether 2HG accumulation is sufficient to perturb differentiation directed by lineage-specifying transcription factors is unknown. A MyoD-driven model was used to study the role of IDH mutations in the differentiation of mesenchymal cells. The presence of 2HG produced by oncogenic IDH2 blocks the ability of MyoD to drive differentiation into myotubes. DNA 5mC hypermethylation is dispensable while H3K9 hypermethylation is required for this differentiation block. IDH2-R172K mutation results in H3K9 hypermethylation and impaired accessibility at myogenic chromatin regions but does not result in genome-wide decrease in accessibility. The results demonstrate the ability of the oncometabolite 2HG to block transcription factor-mediated differentiation in a molecularly defined system.
Assuntos
Diferenciação Celular , Glutaratos/metabolismo , Histonas/metabolismo , Proteína MyoD/metabolismo , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina , Metilação de DNA , Glutaratos/farmacologia , Isocitrato Desidrogenase/genética , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , MutaçãoRESUMO
Genetic studies have identified recurrent somatic mutations in acute myeloid leukemia (AML) patients, including in the Wilms' tumor 1 (WT1) gene. The molecular mechanisms by which WT1 mutations contribute to leukemogenesis have not yet been fully elucidated. We investigated the role of Wt1 gene dosage in steady-state and pathologic hematopoiesis. Wt1 heterozygous loss enhanced stem cell self-renewal in an age-dependent manner, which increased stem cell function over time and resulted in age-dependent leukemic transformation. Wt1-haploinsufficient leukemias were characterized by progressive genetic and epigenetic alterations, including those in known leukemia-associated alleles, demonstrating a requirement for additional events to promote hematopoietic transformation. Consistent with this observation, we found that Wt1 depletion cooperates with Flt3-ITD mutation to induce fully penetrant AML. Our studies provide insight into mechanisms of Wt1-loss leukemogenesis and into the evolutionary events required to induce transformation of Wt1-haploinsufficient stem/progenitor cells.
Assuntos
Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Mutação , Proteínas Repressoras/genética , Animais , Carcinogênese/genética , Carcinogênese/patologia , Autorrenovação Celular , Deleção de Genes , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , Leucopoese , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Células Mieloides/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas WT1 , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Epigenetic regulators are recurrently mutated and aberrantly expressed in acute myeloid leukemia (AML). Targeted therapies designed to inhibit these chromatin-modifying enzymes, such as the histone demethylase lysine-specific demethylase 1 (LSD1) and the histone methyltransferase DOT1L, have been developed as novel treatment modalities for these often refractory diseases. A common feature of many of these targeted agents is their ability to induce myeloid differentiation, suggesting that multiple paths toward a myeloid gene expression program can be engaged to relieve the differentiation blockade that is uniformly seen in AML. We performed a comparative assessment of chromatin dynamics during the treatment of mixed lineage leukemia (MLL)-AF9-driven murine leukemias and MLL-rearranged patient-derived xenografts using 2 distinct but effective differentiation-inducing targeted epigenetic therapies, the LSD1 inhibitor GSK-LSD1 and the DOT1L inhibitor EPZ4777. Intriguingly, GSK-LSD1 treatment caused global gains in chromatin accessibility, whereas treatment with EPZ4777 caused global losses in accessibility. We captured PU.1 and C/EBPα motif signatures at LSD1 inhibitor-induced dynamic sites and chromatin immunoprecipitation coupled with high-throughput sequencing revealed co-occupancy of these myeloid transcription factors at these sites. Functionally, we confirmed that diminished expression of PU.1 or genetic deletion of C/EBPα in MLL-AF9 cells generates resistance of these leukemias to LSD1 inhibition. These findings reveal that pharmacologic inhibition of LSD1 represents a unique path to overcome the differentiation block in AML for therapeutic benefit.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Leucemia Aguda Bifenotípica/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Leucemia Aguda Bifenotípica/genética , Leucemia Aguda Bifenotípica/metabolismo , Leucemia Aguda Bifenotípica/patologia , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas/genética , Elementos de Resposta , Transativadores/genéticaRESUMO
Suboptimal functional persistence limits the efficacy of adoptive T-cell therapies. CD28-based chimeric antigen receptors (CAR) impart potent effector function to T cells but with a limited lifespan. We show here that the genetic disruption of SUV39H1, which encodes a histone-3, lysine-9 methyl-transferase, enhances the early expansion, long-term persistence, and overall antitumor efficacy of human CAR T cells in leukemia and prostate cancer models. Persisting SUV39H1-edited CAR T cells demonstrate improved expansion and tumor rejection upon multiple rechallenges. Transcriptional and genome accessibility profiling of repeatedly challenged CAR T cells shows improved expression and accessibility of memory transcription factors in SUV39H1-edited CAR T cells. SUV39H1 editing also reduces expression of inhibitory receptors and limits exhaustion in CAR T cells that have undergone multiple rechallenges. Our findings thus demonstrate the potential of epigenetic programming of CAR T cells to balance their function and persistence for improved adoptive cell therapies. SIGNIFICANCE: T cells engineered with CD28-based CARs possess robust effector function and antigen sensitivity but are hampered by limited persistence, which may result in tumor relapse. We report an epigenetic strategy involving disruption of the SUV39H1-mediated histone-silencing program that promotes the functional persistence of CD28-based CAR T cells. See related article by López-Cobo et al., p. 120. This article is featured in Selected Articles from This Issue, p. 5.
Assuntos
Leucemia , Receptores de Antígenos Quiméricos , Masculino , Humanos , Linfócitos T , Receptores de Antígenos de Linfócitos T , Histonas/metabolismo , Antígenos CD28/genética , Antígenos CD28/metabolismo , Imunoterapia Adotiva , Leucemia/metabolismo , Metilação , Ensaios Antitumorais Modelo de Xenoenxerto , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
INTRODUCTION: Small Cell Lung Cancer (SCLC) can be classified into transcriptional subtypes with distinct degrees of neuroendocrine (NE) differentiation. Recent evidence supports plasticity among subtypes with a bias toward adoption of low-NE states during disease progression or upon acquired chemotherapy resistance. Here, we identify a role for SMARCA4, the catalytic subunit of the SWI/SNF complex, as a regulator of subtype shift in SCLC. METHODS: ATACseq and RNAseq experiments were performed in SCLC cells after pharmacological inhibition of SMARCA4. DNA binding of SMARCA4 was characterized by ChIPseq in high-NE SCLC patient derived xenografts (PDXs). Enrichment analyses were applied to transcriptomic data. Combination of FHD-286 and afatinib was tested in vitro and in a set of chemo-resistant SCLC PDXs in vivo. RESULTS: SMARCA4 expression positively correlates with that of NE genes in both SCLC cell lines and patient tumors. Pharmacological inhibition of SMARCA4 with FHD-286 induces the loss of NE features and downregulates neuroendocrine and neuronal signaling pathways while activating non-NE factors. SMARCA4 binds to gene loci encoding NE-lineage transcription factors ASCL1 and NEUROD1 and alters chromatin accessibility, enhancing NE programs. Enrichment analysis applied to high-confidence SMARCA4 targets confirmed neuron related pathways as the top GO Biological processes regulated by SMARCA4 in SCLC. In parallel, SMARCA4 also controls REST, a known suppressor of the NE phenotype, by regulating SRRM4-dependent REST transcript splicing. Furthermore, SMARCA4 inhibition drives ERBB pathway activation in SCLC, rendering SCLC tumors sensitive to afatinib. CONCLUSIONS: This study nominates SMARCA4 as a key regulator of the NE state plasticity and defines a novel therapeutic strategy for SCLC.
Assuntos
DNA Helicases , Neoplasias Pulmonares , Proteínas Nucleares , Carcinoma de Pequenas Células do Pulmão , Fatores de Transcrição , Humanos , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , DNA Helicases/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Linhagem Celular Tumoral , Animais , Camundongos , Regulação Neoplásica da Expressão Gênica , Proteínas RepressorasRESUMO
Chronic hepatitis B virus (HBV) infection is an incurable global health threat responsible for causing liver disease and hepatocellular carcinoma. During the genesis of infection, HBV establishes an independent minichromosome consisting of the viral covalently closed circular DNA (cccDNA) genome and host histones. The viral X gene must be expressed immediately upon infection to induce degradation of the host silencing factor, Smc5/6. However, the relationship between cccDNA chromatinization and X gene transcription remains poorly understood. Establishing a reconstituted viral minichromosome platform, we found that nucleosome occupancy in cccDNA drives X transcription. We corroborated these findings in cells and further showed that the chromatin destabilizing molecule CBL137 inhibits X transcription and HBV infection in hepatocytes. Our results shed light on a long-standing paradox and represent a potential new therapeutic avenue for the treatment of chronic HBV infection.
RESUMO
Driver gene mutations can increase the metastatic potential of the primary tumor1-3, but their role in sustaining tumor growth at metastatic sites is poorly understood. A paradigm of such mutations is inactivation of SMAD4 - a transcriptional effector of TGFß signaling - which is a hallmark of multiple gastrointestinal malignancies4,5. SMAD4 inactivation mediates TGFß's remarkable anti- to pro-tumorigenic switch during cancer progression and can thus influence both tumor initiation and metastasis6-14. To determine whether metastatic tumors remain dependent on SMAD4 inactivation, we developed a mouse model of pancreatic ductal adenocarcinoma (PDAC) that enables Smad4 depletion in the pre-malignant pancreas and subsequent Smad4 reactivation in established metastases. As expected, Smad4 inactivation facilitated the formation of primary tumors that eventually colonized the liver and lungs. By contrast, Smad4 reactivation in metastatic disease had strikingly opposite effects depending on the tumor's organ of residence: suppression of liver metastases and promotion of lung metastases. Integrative multiomic analysis revealed organ-specific differences in the tumor cells' epigenomic state, whereby the liver and lungs harbored chromatin programs respectively dominated by the KLF and RUNX developmental transcription factors, with Klf4 depletion being sufficient to reverse Smad4's tumor-suppressive activity in liver metastases. Our results show how epigenetic states favored by the organ of residence can influence the function of driver genes in metastatic tumors. This organ-specific gene-chromatin interplay invites consideration of anatomical site in the interpretation of tumor genetics, with implications for the therapeutic targeting of metastatic disease.
RESUMO
Aging is associated with a decline in the number and fitness of adult stem cells 1-4 . Aging-associated loss of stemness is posited to suppress tumorigenesis 5,6 , but this hypothesis has not been tested in vivo . Here, using physiologically aged autochthonous genetically engineered mouse models and primary cells 7,8 , we demonstrate aging suppresses lung cancer initiation and progression by degrading stemness of the alveolar cell of origin. This phenotype is underpinned by aging-associated induction of the transcription factor NUPR1 and its downstream target lipocalin-2 in the cell of origin in mice and humans, leading to a functional iron insufficiency in the aged cells. Genetic inactivation of the NUPR1-lipocalin-2 axis or iron supplementation rescue stemness and promote tumorigenic potential of aged alveolar cells. Conversely, targeting the NUPR1- lipocalin-2 axis is detrimental to young alveolar cells via induction of ferroptosis. We find that aging-associated DNA hypomethylation at specific enhancer sites associates with elevated NUPR1 expression, which is recapitulated in young alveolar cells by inhibition of DNA methylation. We uncover that aging drives a functional iron insufficiency, which leads to loss of stemness and tumorigenesis, but promotes resistance to ferroptosis. These findings have significant implications for the therapeutic modulation of cellular iron homeostasis in regenerative medicine and in cancer prevention. Furthermore, our findings are consistent with a model whereby most human cancers initiate in young individuals, revealing a critical window for such cancer prevention efforts.
RESUMO
Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs.
RESUMO
Neuroendocrine (NE) transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis. Up to date, even if patients at high risk of transformation can be identified by the occurrence of Tumor Protein P53 (TP53) and Retinoblastoma Transcriptional Corepressor 1 (RB1) mutations in their tumors, no therapeutic strategies are available to prevent or delay histological transformation. Upregulation of the cell cycle kinase Cell Division Cycle 7 (CDC7) occurred in tumors during the initial steps of NE transformation, already after TP53/RB1 co-inactivation, leading to induced sensitivity to the CDC7 inhibitor simurosertib. CDC7 inhibition suppressed NE transdifferentiation and extended response to targeted therapy in in vivo models of NE transformation by inducing the proteasome-mediated degradation of the MYC Proto-Oncogen (MYC), implicated in stemness and histological transformation. Ectopic overexpression of a degradation-resistant MYC isoform reestablished the NE transformation phenotype observed on targeted therapy, even in the presence of simurosertib. CDC7 inhibition also markedly extended response to standard cytotoxics (cisplatin, irinotecan) in lung and prostate small cell carcinoma models. These results nominate CDC7 inhibition as a therapeutic strategy to constrain lineage plasticity, as well as to effectively treat NE tumors de novo or after transformation. As simurosertib clinical efficacy trials are ongoing, this concept could be readily translated for patients at risk of transformation.
Assuntos
Proteínas de Ciclo Celular , Neoplasias Pulmonares , Neoplasias da Próstata , Proteínas Proto-Oncogênicas c-myc , Humanos , Masculino , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Linhagem Celular Tumoral , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Camundongos , Animais , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/tratamento farmacológico , Proteólise/efeitos dos fármacos , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína LigasesRESUMO
DNA amplifications in cancer do not only harbor oncogenes. We sought to determine whether passenger coamplifications could create collateral therapeutic vulnerabilities. Through an analysis of >3,000 cancer genomes followed by the interrogation of CRISPR-Cas9 loss-of-function screens across >700 cancer cell lines, we determined that passenger coamplifications are accompanied by distinct dependency profiles. In a proof-of-principle study, we demonstrate that the coamplification of the bona fide passenger gene DEAD-Box Helicase 1 (DDX1) creates an increased dependency on the mTOR pathway. Interaction proteomics identified tricarboxylic acid (TCA) cycle components as previously unrecognized DDX1 interaction partners. Live-cell metabolomics highlighted that this interaction could impair TCA activity, which in turn resulted in enhanced mTORC1 activity. Consequently, genetic and pharmacologic disruption of mTORC1 resulted in pronounced cell death in vitro and in vivo. Thus, structurally linked coamplification of a passenger gene and an oncogene can result in collateral vulnerabilities. SIGNIFICANCE: We demonstrate that coamplification of passenger genes, which were largely neglected in cancer biology in the past, can create distinct cancer dependencies. Because passenger coamplifications are frequent in cancer, this principle has the potential to expand target discovery in oncology. This article is featured in Selected Articles from This Issue, p. 384.