RESUMO
Acute myeloid leukemia (AML) is a devastating disease initiated and maintained by a rare subset of cells called leukemia stem cells (LSCs). LSCs are responsible for driving disease relapse, making the development of new therapeutic strategies to target LSCs urgently needed. The use of mass spectrometry-based metabolomics profiling has enabled the discovery of unique and targetable metabolic properties in LSCs. However, we do not have a comprehensive understanding of metabolite differences between LSCs and their normal counterparts, hematopoietic stem and progenitor cells (HSPCs). In this study, we used an unbiased mass spectrometry-based metabolomics analysis to define differences in metabolites between primary human LSCs and HSPCs, which revealed that LSCs have a distinct metabolome. Spermidine was the most enriched metabolite in LSCs compared with HSPCs. Pharmacological reduction of spermidine concentrations decreased LSC function but spared normal HSPCs. Polyamine depletion also decreased leukemic burden in patient-derived xenografts. Mechanistically, spermidine depletion induced LSC myeloid differentiation by decreasing eIF5A-dependent protein synthesis, resulting in reduced expression of a select subset of proteins. KAT7, a histone acetyltransferase, was one of the top candidates identified to be down-regulated by spermidine depletion. Overexpression of KAT7 partially rescued polyamine depletion-induced decreased colony-forming ability, demonstrating that loss of KAT7 is an essential part of the mechanism by which spermidine depletion targets AML clonogenic potential. Together, we identified and mechanistically dissected a metabolic vulnerability of LSCs that has the potential to be rapidly translated into clinical trials to improve outcomes for patients with AML.
Assuntos
Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Espermidina , Animais , Humanos , Camundongos , Acetiltransferases , Diferenciação Celular , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Metaboloma , Metabolômica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Espermidina/metabolismoRESUMO
PURPOSE: Small cell lung cancer (SCLC) is an aggressive and lethal form of lung cancer and the overall 5-year survival (OS) for patients is a dismal 7%. Radiation therapy (RT) provides some benefit for selected patients with SCLC but could be improved with radiosensitizing agents. In this study, we identified novel radiosensitizers for SCLC by a CRISPR-Cas9 screen and evaluated the efficacy of ATM inhibitor AZD1390 as a radiosensitizer of SCLC. METHODS AND MATERIALS: We transduced the SCLC cell line SBC5 with a custom CRISPR sgRNA library focused on druggable gene targets and treated cells with RT. Cells collected at multiple timepoints were subjected to next-generation sequencing. We determined radiosensitization both in vitro with cell lines assessed by short-term viability and clonogenic assays, and in vivo mouse models by tumor growth delay. Pharmacodynamic effects of AZD1390 were quantified by ATM-Ser1981 phosphorylation, and RT-induced DNA damage by comet assay. RESULTS: Using a CRISPR dropout screen, we identified multiple radiosensitizing genes for SCLC at various timepoints with ATM as a top determinant gene for radiosensitivity. Validation by ATM knockout (KO) demonstrated increased radiosensitivity by short-term viability assay (dose modification factor [DMF]50 = 3.25-3.73 in SBC5 ATM-KO) and clonogenic assays (DMF37 1.25-1.65 in SBC5 ATM-KO). ATM inhibition by AZD1390 effectively abrogated ATM Ser1981 phosphorylation in SCLC cell lines and increased RT-induced DNA damage. AZD1390 synergistically increased the radiosensitivity of SCLC cell lines (cell viability assay: SBC5 DMF37 = 2.19, SHP77 DMF37 = 1.56, H446 DMF37 = 3.27, KP1 DMF37 = 1.65 at 100nM; clonogenic assay: SBC5 DMF37 = 4.23, H1048 DMF37 = 1.91), and in vivo murine syngeneic, KP1, and patient-derived xenograft (PDX) models, JHU-LX108 and JHU-LX33. CONCLUSIONS: In this study, we demonstrated that genetically and pharmacologically (AZD1390) inhibiting ATM markedly enhanced RT against SCLC, providing a novel pharmacologically tractable radiosensitizing strategy for patients with SCLC.
Assuntos
Neoplasias Pulmonares , Piridinas , Quinolonas , Radiossensibilizantes , Carcinoma de Pequenas Células do Pulmão , Humanos , Animais , Camundongos , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/radioterapia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/tratamento farmacológico , RNA Guia de Sistemas CRISPR-Cas , Radiossensibilizantes/farmacologia , Radiossensibilizantes/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Linhagem Celular Tumoral , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
Abundant expression of the long noncoding (lnc) PAN (polyadenylated nuclear) RNA by the human oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) depends on a cis-element called the expression and nuclear retention element (ENE). The ENE upregulates PAN RNA by inhibiting its rapid nuclear decay through triple-helix formation with the poly(A) tail. Using structure-based bioinformatics, we identified six ENE-like elements in evolutionarily diverse viral genomes. Five are in double-stranded DNA viruses, including mammalian herpesviruses, insect polydnaviruses, and a protist mimivirus. One is in an insect picorna-like positive-strand RNA virus, suggesting that the ENE can counteract cytoplasmic as well as nuclear RNA decay pathways. Functionality of four of the ENEs was demonstrated by increased accumulation of an intronless polyadenylated reporter transcript in human cells. Identification of these ENEs enabled the discovery of PAN RNA homologs in two additional gammaherpesviruses, RRV and EHV2. Our findings demonstrate that searching for structural elements can lead to rapid identification of lncRNAs.