RESUMO
Acute myeloid leukemia (AML) is a highly heterogenous and aggressive disease with a poor prognosis, necessitating further improvements in treatment therapies. Recently, several targeted therapies have become available for specific AML populations. To identify potential new therapeutic targets for AML, we analyzed published genome wide CRISPR-based screens to generate a gene essentiality dataset across a panel of 14 human AML cell lines while eliminating common essential genes through integration analysis with core fitness genes among 324 human cancer cell lines and DepMap databases. The key glutathione metabolic enzyme, glutamate-cysteine ligase catalytic subunit (GCLC), met the selection threshold. Using CRISPR knockout, GCLC was confirmed to be essential for the cell growth, survival, clonogenicity, and leukemogenesis in AML cells but was comparatively dispensable for normal hematopoietic stem and progenitor cells (HSPCs), indicating that GCLC is a potential therapeutic target for AML. In addition, we evaluated the essentiality of GCLC in solid tumors and demonstrated that GCLC represents a synthetic lethal target for ARID1A-deficient ovarian and gastric cancers.
RESUMO
The PBX1 homeodomain transcription factor is converted by t(1;19) chromosomal translocations in acute leukemia into the chimeric E2A-PBX1 oncoprotein. Fusion with E2A confers potent transcriptional activation and constitutive nuclear localization, bypassing the need for dimerization with protein partners that normally stabilize and regulate import of PBX1 into the nucleus, but the mechanisms underlying its oncogenic activation are incompletely defined. We demonstrate here that E2A-PBX1 self-associates through the PBX1 PBC-B domain of the chimeric protein to form higher-order oligomers in t(1;19) human leukemia cells, and that this property is required for oncogenic activity. Structural and functional studies indicate that self-association facilitates the binding of E2A-PBX1 to DNA. Mutants unable to self-associate are transformation defective, however their oncogenic activity is rescued by the synthetic oligomerization domain of FKBP, which confers conditional transformation properties on E2A-PBX1. In contrast to self-association, PBX1 protein domains that mediate interactions with HOX DNA-binding partners are dispensable. These studies suggest that oligomeric self-association may compensate for the inability of monomeric E2A-PBX1 to stably bind DNA and circumvents protein interactions that otherwise modulate PBX1 stability, nuclear localization, DNA binding, and transcriptional activity. The unique dependence on self-association for E2A-PBX1 oncogenic activity suggests potential approaches for mechanism-based targeted therapies.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinogênese/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos Par 1/química , Cromossomos Humanos Par 19/química , DNA de Neoplasias/metabolismo , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Transcrição Gênica , Translocação GenéticaRESUMO
Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional coactivator CBP increased dasatinib sensitivity by downregulating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due, in part, to upregulation of alternative pathways including WNT through a mechanism, suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or ß-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small-molecule inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL.Significance: These findings reveal mechanisms that modulate sensitivity to dasatinib and suggest therapeutic strategies to improve the outcome of patients with acute lymphoblastic leukemia.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6497/F1.large.jpg Cancer Res; 78(22); 6497-508. ©2018 AACR.
Assuntos
Antineoplásicos/farmacologia , Proteína de Ligação a CREB/metabolismo , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Ligação Proteica , Domínios Proteicos , Pirimidinas/farmacologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transcrição Gênica , beta Catenina/genéticaRESUMO
Acute lymphoblastic leukemia (ALL) is associated with significant morbidity and mortality, necessitating further improvements in diagnosis and therapy. Targeted therapies directed against chromatin regulators are emerging as promising approaches in preclinical studies and early clinical trials. Here, we demonstrate an oncogenic role for the protein lysine methyltransferase SETDB2 in leukemia pathogenesis. It is overexpressed in pre-BCR+ ALL and required for their maintenance in vitro and in vivo. SETDB2 expression is maintained as a direct target gene of the chimeric transcription factor E2A-PBX1 in a subset of ALL and suppresses expression of the cell-cycle inhibitor CDKN2C through histone H3K9 tri-methylation, thus establishing an oncogenic pathway subordinate to E2A-PBX1 that silences a major tumor suppressor in ALL. In contrast, SETDB2 was relatively dispensable for normal hematopoietic stem and progenitor cell proliferation. SETDB2 knockdown enhances sensitivity to kinase and chromatin inhibitors, providing a mechanistic rationale for targeting SETDB2 therapeutically in ALL.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p18/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Células-Tronco Neoplásicas/patologia , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
There is limited understanding of how signaling pathways are altered by oncogenic fusion transcription factors that drive leukemogenesis. To address this, we interrogated activated signaling pathways in a comparative analysis of mouse and human leukemias expressing the fusion protein E2A-PBX1, which is present in 5%-7% of pediatric and 50% of pre-B-cell receptor (preBCR+) acute lymphocytic leukemia (ALL). In this study, we describe remodeling of signaling networks by E2A-PBX1 in pre-B-ALL, which results in hyperactivation of the key oncogenic effector enzyme PLCγ2. Depletion of PLCγ2 reduced proliferation of mouse and human ALLs, including E2A-PBX1 leukemias, and increased disease-free survival after secondary transplantation. Mechanistically, E2A-PBX1 bound promoter regulatory regions and activated the transcription of its key target genes ZAP70, SYK, and LCK, which encode kinases upstream of PLCγ2. Depletion of the respective upstream kinases decreased cell proliferation and phosphorylated levels of PLCγ2 (pPLCγ2). Pairwise silencing of ZAP70, SYK, or LCK showed additive effects on cell growth inhibition, providing a rationale for combination therapy with inhibitors of these kinases. Accordingly, inhibitors such as the SRC family kinase (SFK) inhibitor dasatinib reduced pPLCγ2 and inhibited proliferation of human and mouse preBCR+/E2A-PBX1+ leukemias in vitro and in vivo Furthermore, combining small-molecule inhibition of SYK, LCK, and SFK showed synergistic interactions and preclinical efficacy in the same setting. Our results show how the oncogenic fusion protein E2A-PBX1 perturbs signaling pathways upstream of PLCγ2 and renders leukemias amenable to targeted therapeutic inhibition. Cancer Res; 76(23); 6937-49. ©2016 AACR.
Assuntos
Linfócitos B/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Transdução de SinaisRESUMO
Chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease-mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia.
Assuntos
Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Células-Tronco Hematopoéticas , Histona-Lisina N-Metiltransferase/genética , Leucemia Aguda Bifenotípica/genética , Proteína de Leucina Linfoide-Mieloide/genética , Animais , Antígenos CD34/imunologia , Separação Celular , Técnicas de Introdução de Genes , Genoma Humano , Humanos , Camundongos , Microscopia Confocal , Mutagênese Sítio-Dirigida , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transdução Genética , TransfecçãoRESUMO
Acute lymphoblastic leukemia (ALL) is the most common childhood cancer; however, its genetic diversity limits investigation into the molecular pathogenesis of disease and development of therapeutic strategies. Here, we engineered mice that conditionally express the E2A-PBX1 fusion oncogene, which results from chromosomal translocation t(1;19) and is present in 5% to 7% of pediatric ALL cases. The incidence of leukemia in these mice varied from 5% to 50%, dependent on the Cre-driving promoter (Cd19, Mb1, or Mx1) used to induce E2A-PBX1 expression. Two distinct but highly similar subtypes of B cell precursor ALLs that differed by their pre-B cell receptor (pre-BCR) status were induced and displayed maturation arrest at the pro-B/large pre-B II stages of differentiation, similar to human E2A-PBX1 ALL. Somatic activation of E2A-PBX1 in B cell progenitors enhanced self-renewal and led to acquisition of multiple secondary genomic aberrations, including prominent spontaneous loss of Pax5. In preleukemic mice, conditional Pax5 deletion cooperated with E2A-PBX1 to expand progenitor B cell subpopulations, increasing penetrance and shortening leukemia latency. Recurrent secondary activating mutations were detected in key signaling pathways, most notably JAK/STAT, that leukemia cells require for proliferation. These data support conditional E2A-PBX1 mice as a model of human ALL and suggest targeting pre-BCR signaling and JAK kinases as potential therapeutic strategies.
Assuntos
Linfócitos B/metabolismo , Diferenciação Celular , Proliferação de Células , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Proteínas de Fusão Oncogênica/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Animais , Linfócitos B/patologia , Linhagem Celular Tumoral , Proteínas de Homeodomínio/genética , Humanos , Janus Quinases/genética , Janus Quinases/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismoRESUMO
MLL fusion proteins in leukemia induce aberrant transcriptional elongation and associated chromatin perturbations; however, the upstream signaling pathways and activators that recruit or retain MLL oncoproteins at initiated promoters are unknown. Through functional and comparative genomic studies, we identified an essential role for NF-κB signaling in MLL leukemia. Suppression of NF-κB led to robust antileukemia effects that phenocopied loss of functional MLL oncoprotein or associated epigenetic cofactors. The NF-κB subunit RELA occupies promoter regions of crucial MLL target genes and sustains the MLL-dependent leukemia stem cell program. IKK/NF-κB signaling is required for wild-type and fusion MLL protein retention and maintenance of associated histone modifications, providing a molecular rationale for enhanced efficacy in therapeutic targeting of this pathway in MLL leukemias.
Assuntos
Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Leucemia/metabolismo , Proteína de Leucina Linfoide-Mieloide/fisiologia , NF-kappa B/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatina/metabolismo , Relação Dose-Resposta a Droga , Genômica , Histona-Lisina N-Metiltransferase , Humanos , Quinase I-kappa B/metabolismo , Leucemia/genética , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Prognóstico , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Tempo , Fator de Transcrição RelA/metabolismo , Transcrição GênicaRESUMO
Transforming acidic coiled-coil protein 3 (TACC3) was reported to be important for regulating mitotic spindle assembly and chromosome segregation. While the protein level of TACC3 was shown to be altered during cell cycle progression, the molecular mechanism in controlling TACC3 level is unclear. Here, we show that TACC3 protein level can be regulated by Cdh1, a well known activator of anaphase-promoting complex/cyclosome. We identified Cdh1 as an interacting partner of TACC3 by a yeast array screen. Both in vitro and in vivo binding studies indicated that TACC3 can form complexes with Cdh1. Depletion of endogenous Cdh1 prolonged TACC3 protein level during mitotic exit. Alteration of Cdh1 level by ectopic overexpression or siRNA knockdown correlated well with an increase or decrease of ubiquitinated TACC3, respectively. Furthermore, the domain mapping studies of TACC3 revealed that multiple domains are involved in Cdh1-regulated degradation of TACC3. Altogether, our findings suggest that Cdh1 controls TACC3 protein stability during mitotic exit.