RESUMO
Asciminib is a myristoyl site BCR::ABL1 inhibitor approved for chronic phase chronic myeloid leukaemia (CP-CML) patients failing ≥2 prior lines of therapy. The Australasian Leukaemia & Lymphoma Group (ALLG) conducted the ASCEND study to assess efficacy of asciminib for newly-diagnosed CP-CML. Patients commenced asciminib 40 mg twice daily (BID) and thereafter were managed according to molecular milestones. Patients with treatment failure, defined as BCR::ABL1 >10% (IS) at 3 or 6 months, or >1% at 12 or 18 months, received either imatinib, nilotinib or dasatinib in addition to asciminib. In patients with suboptimal response, defined as levels of 1-10% at 6 months, >0.1-1% at 12 months, or >0.01%-1% at 18 months, the asciminib dose was increased to 80 mg BID. With a median follow-up of 21 months (range 0-36), 82/101 patients continue asciminib. The most frequent reasons for treatment discontinuation were adverse events (6%), loss of response (4%) and withdrawn consent (5%). There were no deaths; one patient developed lymphoid blast crisis at 6 months. The co-primary endpoints were early molecular response (BCR::ABL1 ≤10% at 3 months), achieved in 93% (96% CI 86-97%), and major molecular response by 12 months achieved in 79%; (95% CI 69.7-86.8%), respectively. The cumulative incidence of MR4.5 was 53% by 24 months. One patient had 2 cerebrovascular events; no other arterial occlusive events were reported. Asciminib as frontline therapy in CP-CML produces high rates of molecular response with excellent tolerance and a low rate of discontinuation for toxicity. (ANZ Clinical Trials Registry ACTRN12620000851965).
RESUMO
Multiple signaling pathways ultimately modulate the epigenetic information embedded in the chromatin of gene promoters by recruiting epigenetic enzymes. We found that, in estrogen-regulated gene programming, the acetyltransferase CREB-binding protein (CBP) is specifically and exclusively methylated by the coactivator-associated arginine methyltransferase (CARM1) in vivo. CARM1-dependent CBP methylation and p160 coactivators were required for estrogen-induced recruitment to chromatin targets. Notably, methylation increased the histone acetyltransferase (HAT) activity of CBP and stimulated its autoacetylation. Comparative genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) studies revealed a variety of patterns by which p160, CBP, and methyl-CBP (meCBP) are recruited (or not) by estrogen to chromatin targets. Moreover, significant target gene-specific variation in the recruitment of (1) the p160 RAC3 protein, (2) the fraction of a given meCBP species within the total CBP, and (3) the relative recruitment of different meCBP species suggests the existence of a target gene-specific "fingerprint" for coregulator recruitment. Crossing ChIP-seq and transcriptomics profiles revealed the existence of meCBP "hubs" within the network of estrogen-regulated genes. Together, our data provide evidence for an unprecedented mechanism by which CARM1-dependent CBP methylation results in gene-selective association of estrogen-recruited meCBP species with different HAT activities and specifies distinct target gene hubs, thus diversifying estrogen receptor programming.
Assuntos
Proteína de Ligação a CREB/metabolismo , Cromatina/metabolismo , Estrogênios/metabolismo , Regulação da Expressão Gênica , Acetilação , Sítios de Ligação , Linhagem Celular Tumoral , Coenzimas/metabolismo , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma/genética , Histona Acetiltransferases/metabolismo , Humanos , Metilação , Ligação Proteica/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Osteosarcoma (OS) is the most common cancer of bone in children and young adults. Despite extensive research efforts, there has been no significant improvement in patient outcome for many years. An improved understanding of the biology of this cancer and how genes frequently mutated contribute to OS may help improve outcomes for patients. While our knowledge of the mutational burden of OS is approaching saturation, our understanding of how these mutations contribute to OS initiation and maintenance is less clear. Murine models of OS have now been demonstrated to be highly valid recapitulations of human OS. These models were originally based on the frequent disruption of p53 and Rb in familial OS syndromes, which are also common mutations in sporadic OS. They have been applied to significantly improve our understanding about the functions of recurrently mutated genes in disease. The murine models can be used as a platform for preclinical testing and identifying new therapeutic targets, in addition to testing the role of additional mutations in vivo. Most recently these models have begun to be used for discovery based approaches and screens, which hold significant promise in furthering our understanding of the genetic and therapeutic sensitivities of OS. In this review, we discuss the mouse models of OS that have been reported in the last 3-5 years and newly identified pathways from these studies. Finally, we discuss the preclinical utilization of the mouse models of OS for identifying and validating actionable targets to improve patient outcome.
Assuntos
Neoplasias Ósseas , Neoplasias Experimentais , Osteossarcoma , Proteína do Retinoblastoma , Proteína Supressora de Tumor p53 , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
RECQL4 mutations are associated with Rothmund Thomson Syndrome (RTS), RAPADILINO Syndrome and Baller-Gerold Syndrome. These patients display a range of benign skeletal abnormalities such as low bone mass. In addition, RTS patients have a highly increased incidence of osteosarcoma (OS). The role of RECQL4 in normal adult bone development and homeostasis is largely uncharacterized and how mutation of RECQL4 contributes to OS susceptibility is not known. We hypothesised that Recql4 was required for normal skeletal development and both benign and malignant osteoblast function, which we have tested in the mouse. Recql4 deletion in vivo at the osteoblastic progenitor stage of differentiation resulted in mice with shorter bones and reduced bone volume, assessed at 9 weeks of age. This was associated with an osteoblast intrinsic decrease in mineral apposition rate and bone formation rate in the Recql4-deficient cohorts. Deletion of Recql4 in mature osteoblasts/osteocytes in vivo, however, did not cause a detectable phenotype. Acute deletion of Recql4 in primary osteoblasts or shRNA knockdown in an osteoblastic cell line caused failed proliferation, accompanied by cell cycle arrest, induction of apoptosis and impaired differentiation. When cohorts of animals were aged long term, the loss of Recql4 alone was not sufficient to initiate OS. We then crossed the Recql4fl/fl allele to a fully penetrant OS model (Osx-Cre p53fl/fl). Unexpectedly, the Osx-Cre p53fl/flRecql4fl/fl (dKO) animals had a significantly increased OS-free survival compared to Osx-Cre p53fl/fl or Osx-Cre p53fl/flRecql4fl/+ (het) animals. The extended survival was explained when the Recql4 status in the tumors that arose was assessed, and in no case was there complete deletion of Recql4 in the dKO OS. These data provide a mechanism for the benign skeletal phenotypes of RECQL4 mutation syndromes. We propose that tumor suppression and osteosarcoma susceptibility are most likely a function of mutant, not null, alleles of RECQL4.
Assuntos
Neoplasias Ósseas/genética , Osteoblastos/metabolismo , Osteogênese , Osteossarcoma/genética , RecQ Helicases/metabolismo , Animais , Neoplasias Ósseas/metabolismo , Proliferação de Células , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Osteossarcoma/metabolismo , RecQ Helicases/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Genome-wide profiling of transcription factors based on massive parallel sequencing of immunoprecipitated chromatin (ChIP-seq) requires nanogram amounts of DNA. Here we describe a high-fidelity, single-tube linear DNA amplification method (LinDA) for ChIP-seq and reChIP-seq with picogram DNA amounts obtained from a few thousand cells. This amplification technology will facilitate global analyses of transcription-factor binding and chromatin with very small cell populations, such as stem or cancer-initiating cells.
Assuntos
Imunoprecipitação da Cromatina , Cromatina/metabolismo , DNA/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de DNA/métodos , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Cromatina/genética , DNA/genética , Humanos , CamundongosRESUMO
Chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) is increasingly used to map protein-chromatin interactions at global scale. The comparison of ChIP-seq profiles for RNA polymerase II (PolII) established in different biological contexts, such as specific developmental stages or specific time-points during cell differentiation, provides not only information about the presence/accumulation of PolII at transcription start sites (TSSs) but also about functional features of transcription, including PolII stalling, pausing and transcript elongation. However, annotation and normalization tools for comparative studies of multiple samples are currently missing. Here, we describe the R-package POLYPHEMUS, which integrates TSS annotation with PolII enrichment over TSSs and coding regions, and normalizes signal intensity profiles. Thereby POLYPHEMUS facilitates to extract information about global PolII action to reveal changes in the functional state of genes. We validated POLYPHEMUS using a kinetic study on retinoic acid-induced differentiation and a publicly available data set from a comparative PolII ChIP-seq profiling in Caenorhabditis elegans. We demonstrate that POLYPHEMUS corrects the data sets by normalizing for technical variation between samples and reveal the potential of the algorithm in comparing multiple data sets to infer features of transcription regulation from dynamic PolII binding profiles.
Assuntos
Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , RNA Polimerase II/metabolismo , Software , Algoritmos , Animais , Sítios de Ligação , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Diferenciação Celular , Cromatina/genética , Interpretação Estatística de Dados , Análise de Sequência de DNA , Sítio de Iniciação de TranscriçãoRESUMO
Retinoic acid (RA) triggers physiological processes by activating heterodimeric transcription factors (TFs) comprising retinoic acid receptor (RARα, ß, γ) and retinoid X receptor (RXRα, ß, γ). How a single signal induces highly complex temporally controlled networks that ultimately orchestrate physiological processes is unclear. Using an RA-inducible differentiation model, we defined the temporal changes in the genome-wide binding patterns of RARγ and RXRα and correlated them with transcription regulation. Unexpectedly, both receptors displayed a highly dynamic binding, with different RXRα heterodimers targeting identical loci. Comparison of RARγ and RXRα co-binding at RA-regulated genes identified putative RXRα-RARγ target genes that were validated with subtype-selective agonists. Gene-regulatory decisions during differentiation were inferred from TF-target gene information and temporal gene expression. This analysis revealed six distinct co-expression paths of which RXRα-RARγ is associated with transcription activation, while Sox2 and Egr1 were predicted to regulate repression. Finally, RXRα-RARγ regulatory networks were reconstructed through integration of functional co-citations. Our analysis provides a dynamic view of RA signalling during cell differentiation, reveals RAR heterodimer dynamics and promiscuity, and predicts decisions that diversify the RA signal into distinct gene-regulatory programs.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Genômica/métodos , Receptores do Ácido Retinoico/metabolismo , Receptor X Retinoide alfa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologia , Sequência de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Ligação Proteica/genética , Receptor Cross-Talk/efeitos dos fármacos , Receptores do Ácido Retinoico/genética , Receptor X Retinoide alfa/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Receptor gama de Ácido RetinoicoRESUMO
Co-transcriptional R loops arise from stalling of RNA polymerase, leading to the formation of stable DNA:RNA hybrids. Unresolved R loops promote genome instability but are counteracted by helicases and nucleases. Here, we show that branchpoint translocases are a third class of R-loop-displacing enzyme in vitro. In cells, deficiency in the Fanconi-anemia-associated branchpoint translocase FANCM causes R-loop accumulation, particularly after treatment with DNA:RNA-hybrid-stabilizing agents. This correlates with FANCM localization at R-loop-prone regions of the genome. Moreover, other branchpoint translocases associated with human disease, such as SMARCAL1 and ZRANB3, and those from lower organisms can also remove R loops in vitro. Branchpoint translocases are more potent than helicases in resolving R loops, indicating their evolutionary important role in R-loop suppression. In human cells, FANCM, SMARCAL1, and ZRANB3 depletion causes additive effects on R-loop accumulation and DNA damage. Our work reveals a mechanistic basis for R-loop displacement that is linked to genome stability.
Assuntos
Estruturas R-Loop , RNA , Humanos , DNA Helicases/genéticaRESUMO
The loss of p53 function is a central event in the genesis of osteosarcoma (OS). How mutation of p53 enables OS development from osteoblastic lineage cells is poorly understood. We and others have reported a key role for elevated and persistent activation of the cAMP/PKA/Creb1 pathway in maintenance of OS. In view of the osteoblast lineage being the cell of origin of OS, we sought to determine how these pathways interact within the context of the normal osteoblast. Normal osteoblasts (p53 WT) rapidly underwent apoptosis in response to acute elevation of cAMP levels or activity, whereas p53-deficient osteoblasts tolerated this aberrant cAMP/Creb level and activity. Using the p53 activating small-molecule Nutlin-3a and cAMP/Creb1 activator forskolin, we addressed the question of how p53 responds to the activation of cAMP. We observed that p53 acts dominantly to protect cells from excessive cAMP accumulation. We identify a Creb1-Cbp complex that functions together with and interacts with p53. Finally, translating these results we find that a selective small-molecule inhibitor of the Creb1-Cbp interaction demonstrates selective toxicity to OS cells where this pathway is constitutively active. This highlights the cAMP/Creb axis as a potentially actionable therapeutic vulnerability in p53-deficient tumors such as OS. These results define a mechanism through which p53 protects normal osteoblasts from excessive or abnormal cAMP accumulation, which becomes fundamentally compromised in OS.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Osteoblastos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Proteínas de Membrana/metabolismo , Camundongos , Osteossarcoma/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Retinoic acid receptor (RAR) signaling regulates bone structure and hematopoiesis through intrinsic and extrinsic mechanisms. This study aimed to establish how early in the osteoblast lineage loss of RARγ (Rarg) disrupts the bone marrow microenvironment. Bone structure was analyzed by micro-computed tomography (µCT) in Rarg-/- mice and mice with Rarg conditional deletion in Osterix-Cre-targeted osteoblast progenitors or Prrx1-Cre-targeted mesenchymal stem cells. Rarg-/- tibias exhibited less trabecular and cortical bone and impaired longitudinal and radial growth. The trabecular bone and longitudinal, but not radial, growth defects were recapitulated in Prrx1:RargΔ/Δ mice but not Osx1:RargΔ/Δ mice. Although both male and female Prrx1:RargΔ/Δ mice had low trabecular bone mass, males exhibited increased numbers of trabecular osteoclasts and Prrx1:RargΔ/Δ females had impaired mineral deposition. Both male and female Prrx1:RargΔ/Δ growth plates were narrower than controls and their epiphyses contained hypertrophic chondrocyte islands. Flow cytometry revealed that male Prrx1:RargΔ/Δ bone marrow exhibited elevated pro-B and pre-B lymphocyte numbers, accompanied by increased Cxcl12 expression in bone marrow cells. Prrx1:RargΔ/Δ bone marrow also had elevated megakaryocyte-derived Vegfa expression accompanied by smaller sinusoidal vessels. Thus, RARγ expression by Prrx1-Cre-targeted cells directly regulates endochondral bone formation and indirectly regulates tibial vascularization. Furthermore, RARγ expression by Prrx1-Cre-targeted cells extrinsically regulates osteoclastogenesis and B lymphopoiesis in male mice. © 2018 American Society for Bone and Mineral Research.
Assuntos
Linfócitos B/metabolismo , Osso e Ossos/metabolismo , Linfopoese , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Receptores do Ácido Retinoico/metabolismo , Animais , Desenvolvimento Ósseo , Medula Óssea/irrigação sanguínea , Medula Óssea/patologia , Osso Esponjoso/metabolismo , Osso Cortical/metabolismo , Feminino , Masculino , Camundongos Transgênicos , Tamanho do Órgão , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Tíbia/patologia , Receptor gama de Ácido RetinoicoRESUMO
Mutations in the P53 pathway are a hallmark of human cancer. The identification of pathways upon which p53-deficient cells depend could reveal therapeutic targets that may spare normal cells with intact p53. In contrast to P53 point mutations in other cancer, complete loss of P53 is a frequent event in osteosarcoma (OS), the most common cancer of bone. The consequences of p53 loss for osteoblastic cells and OS development are poorly understood. Here we use murine OS models to demonstrate that elevated Pthlh (Pthrp), cAMP levels and signalling via CREB1 are characteristic of both p53-deficient osteoblasts and OS. Normal osteoblasts survive depletion of both PTHrP and CREB1. In contrast, p53-deficient osteoblasts and OS depend upon continuous activation of this pathway and undergo proliferation arrest and apoptosis in the absence of PTHrP or CREB1. Our results identify the PTHrP-cAMP-CREB1 axis as an attractive pathway for therapeutic inhibition in OS.
Assuntos
Neoplasias Ósseas/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , AMP Cíclico/metabolismo , Regulação Neoplásica da Expressão Gênica , Osteossarcoma/genética , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/deficiênciaRESUMO
How MYC promotes the development of cancer remains to be fully understood. Here, we report that the Zn(2+)-finger transcription factor ASCIZ (ATMIN, ZNF822) synergizes with MYC to activate the expression of dynein light chain (DYNLL1, LC8) in the murine Eµ-Myc model of lymphoma. Deletion of Asciz or Dynll1 prevented the abnormal expansion of pre-B cells in pre-cancerous Eµ-Myc mice and potentiated the pro-apoptotic activity of MYC in pre-leukemic immature B cells. Constitutive loss of Asciz or Dynll1 delayed lymphoma development in Eµ-Myc mice, and induced deletion of Asciz in established lymphomas extended the survival of tumor-bearing mice. We propose that ASCIZ-dependent upregulation of DYNLL1 levels is essential for the development and expansion of MYC-driven lymphomas by enabling the survival of pre-neoplastic and malignant cells.
Assuntos
Dineínas/genética , Regulação Neoplásica da Expressão Gênica , Linfoma de Células B/genética , Células Precursoras de Linfócitos B/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/genética , Animais , Apoptose , Linfócitos B/imunologia , Linfócitos B/patologia , Ciclo Celular/genética , Diferenciação Celular , Proliferação de Células , Dineínas do Citoplasma , Modelos Animais de Doenças , Dineínas/deficiência , Humanos , Linfoma de Células B/imunologia , Linfoma de Células B/mortalidade , Linfoma de Células B/patologia , Camundongos , Células Precursoras de Linfócitos B/imunologia , Proteínas Proto-Oncogênicas c-myc/imunologia , Transdução de Sinais , Análise de Sobrevida , Fatores de Transcrição/deficiênciaRESUMO
Osteosarcoma (OS) survival rates have plateaued in part due to a lack of new therapeutic options. Here we demonstrate that bromodomain inhibitors (BETi), JQ1, I-BET151, I-BET762, exert potent anti-tumour activity against primary and established OS cell lines, mediated by inhibition of BRD4. Strikingly, unlike previous observations in long-term established human OS cell lines, the antiproliferative activity of JQ1 in primary OS cells was driven by the induction of apoptosis, not cell cycle arrest. In further contrast, JQ1 activity in OS was mediated independently of MYC downregulation. We identified that JQ1 suppresses the transcription factor FOSL1 by displacement of BRD4 from its locus. Loss of FOSL1 phenocopied the antiproliferative effects of JQ1, identifying FOSL1 suppression as a potential novel therapeutic approach for OS. As a monotherapy JQ1 demonstrated significant anti-tumour activity in vivo in an OS graft model. Further, combinatorial treatment approaches showed that JQ1 increased the sensitivity of OS cells to doxorubicin and induced potent synergistic activity when rationally combined with CDK inhibitors. The greater level of activity achieved with the combination of BETi with CDK inhibitors demonstrates the efficacy of this combination therapy. Taken together, our studies show that BET inhibitors are a promising new therapeutic for OS.
Assuntos
Apoptose/efeitos dos fármacos , Osteossarcoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Azepinas/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Osteossarcoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição/metabolismo , Triazóis/farmacologiaRESUMO
In osteosarcoma, knockdown of the parathyroid hormone-related protein (PTHrP) receptor reduces activation through cyclic AMP-dependent protein kinase A (PKA) and substantially decreases tumor differentiation, invasion, and proliferation in vivo. These findings complement other evidence supporting a central role of the PKA pathway in osteosarcoma biology and pathogenesis.