RESUMO
ABSTRACT: The overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.
Assuntos
Proteínas de Ciclo Celular , Ferroptose , Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Animais , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Camundongos , Humanos , Ferroptose/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Camundongos Knockout , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas GADD45RESUMO
Patient-derived xenografts (PDXs) are the most valuable tool for preclinical drug testing because they retain the genetic diversity and phenotypic heterogeneity of the original tumor. Acute myeloid leukemia (AML) remains difficult to engraft in immunodeficient mice. This is particularly true for long-term frozen patient specimens. This protocol is designed to establish PDXs of human AML with improved engraftment rates. The optimized approach increases the viability of patient cells before implantation, efficiently monitors in vivo engraftment, and maximizes bone marrow collection. For complete details on the use and execution of this protocol, please refer to Salik et al. (2020) and Lynch et al. (2019).
Assuntos
Leucemia Mieloide Aguda/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Medula Óssea/patologia , Sobrevivência Celular , Dissecação , Fêmur/patologia , Congelamento , Humanos , Leucemia Mieloide Aguda/sangue , Camundongos , Baço/patologia , Tíbia/patologiaRESUMO
Signals driving aberrant self-renewal in the heterogeneous leukemia stem cell (LSC) pool determine aggressiveness of acute myeloid leukemia (AML). We report that a positive modulator of canonical WNT signaling pathway, RSPO-LGR4, upregulates key self-renewal genes and is essential for LSC self-renewal in a subset of AML. RSPO2/3 serve as stem cell growth factors to block differentiation and promote proliferation of primary AML patient blasts. RSPO receptor, LGR4, is epigenetically upregulated and works through cooperation with HOXA9, a poor prognostic predictor. Blocking the RSPO3-LGR4 interaction by clinical-grade anti-RSPO3 antibody (OMP-131R10/rosmantuzumab) impairs self-renewal and induces differentiation in AML patient-derived xenografts but does not affect normal hematopoietic stem cells, providing a therapeutic opportunity for HOXA9-dependent leukemia.
Assuntos
Leucemia Mieloide/genética , Células-Tronco Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Trombospondinas/genética , Doença Aguda , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Células K562 , Estimativa de Kaplan-Meier , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores Acoplados a Proteínas G/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células THP-1 , Trombospondinas/imunologia , Trombospondinas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Adrenocortical carcinoma (ACC) has high recurrence rates and poor prognosis with limited response to conventional cancer therapy. Recent contributions of high-throughput transcriptomic profiling identified microRNA-497 (miR-497) as significantly underexpressed, while lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) as overexpressed in ACC. miR-497 is located in the chromosomal region 17p13.1, in which there is a high frequency of loss of heterozygosity in ACC. We aim to investigate the interaction of miR-497 and MALAT1 in ACC and its functional roles in the process of tumourigenesis. In this study, we demonstrated miR-497 post-transcriptionally repressed MALAT1 while MALAT1 also competes for miR-497 binding to its molecular target, EIF4E (eukaryotic translation initiation factor 4E). We showed that overexpression of miR-497 and silencing of MALAT1 suppressed cellular proliferation and induced cell cycle arrest through downregulation of EIF4E expression. Furthermore, MALAT1 directly binds to SFPQ (splicing factor proline and glutamine rich) protein, indicating its multifaceted roles in ACC pathophysiology. This is the first study to identify the feedback axis of miR-497-MALAT1/EIF4E in ACC tumourigenesis, providing novel insights into the molecular functions of noncoding RNAs in ACC.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/patologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Sítios de Ligação , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Fator de Iniciação 4E em Eucariotos/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , HumanosRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression in a sequence-specific manner. Due to its association with an assortment of diseases, miRNAs have been extensively studied in the last decade. In this review, the current understanding of the role of miRNAs in the pathophysiology of adrenal tumors is discussed. The recent contributions of high-throughput miRNA profiling studies have identified miRNAs that have functional and molecular roles in adrenal tumorigenesis. With respect to the biological heterogeneity of adrenal tumors and the limitations of the current treatments, an improved understanding of miRNAs may hold potential diagnostic and therapeutic value to facilitate better clinical management.