RESUMEN
Tissue-specific differences in the expression of paralog genes, which are not essential in most cell types due to the buffering effect of the partner pair, can make for highly selective gene dependencies. To identify selective paralogous targets for acute myeloid leukemia (AML), we integrated the Cancer Dependency Map with numerous datasets characterizing protein-protein interactions, paralog relationships, and gene expression in cancer models. Here, we identified ATP1B3 as a context-specific, paralog-related dependency in AML. ATP1B3, the beta subunit of the sodium-potassium pump (Na/K-ATP pump), interacts with the alpha subunit ATP1A1 to form an essential complex for maintaining cellular homeostasis and membrane potential in all eukaryotic cells. When ATP1B3's paralog ATP1B1 is poorly expressed, elimination of ATP1B3 leads to the destabilization of the Na/K-ATP pump. ATP1B1 expression is regulated through epigenetic silencing in hematopoietic lineage cells both through histone and DNA methylation in the promoter region. Loss of ATP1B3 in AML cells induced cell death in vitro and reduced leukemia burden in vivo, which could be rescued by stabilizing ATP1A1 through overexpression of ATP1B1. ATP1B3 is thus a potential therapeutic target for AML and other hematologic malignancies with low expression of ATP1B1.
RESUMEN
Pediatric cancers are frequently driven by genomic alterations that result in aberrant transcription factor activity. Here, we used functional genomic screens to identify multiple genes within the transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex as selective dependencies for MYCN-amplified neuroblastoma, a disease of dysregulated development driven by an aberrant oncogenic transcriptional program. We characterized the DNA recruitment sites of the SAGA complex in neuroblastoma and the consequences of loss of SAGA complex lysine acetyltransferase (KAT) activity on histone acetylation and gene expression. We demonstrate that loss of SAGA complex KAT activity is associated with reduced MYCN binding on chromatin, suppression of MYC/MYCN gene expression programs, and impaired cell cycle progression. Further, we showed that the SAGA complex is pharmacologically targetable in vitro and in vivo with a KAT2A/KAT2B proteolysis targeting chimeric. Our findings expand our understanding of the histone-modifying complexes that maintain the oncogenic transcriptional state in this disease and suggest therapeutic potential for inhibitors of SAGA KAT activity in MYCN-amplified neuroblastoma.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteína Proto-Oncogénica N-Myc , Neuroblastoma , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Línea Celular Tumoral , Histona Acetiltransferasas/metabolismo , Histona Acetiltransferasas/genética , Acetilación , Histonas/metabolismo , Animales , Amplificación de Genes , Cromatina/metabolismo , Cromatina/genética , RatonesRESUMEN
Patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) have a poor prognosis with few therapeutic options. With the goal of identifying novel therapeutic targets, we used data from the Dependency Map project to identify dihydroorotate dehydrogenase (DHODH) as one of the top metabolic dependencies in T-ALL. DHODH catalyzes the fourth step of de novo pyrimidine nucleotide synthesis. Small molecule inhibition of DHODH rapidly leads to the depletion of intracellular pyrimidine pools and forces cells to rely on extracellular salvage. In the absence of sufficient salvage, this intracellular nucleotide starvation results in the inhibition of DNA and RNA synthesis, cell cycle arrest, and, ultimately, death. T lymphoblasts appear to be specifically and exquisitely sensitive to nucleotide starvation after DHODH inhibition. We have confirmed this sensitivity in vitro and in vivo in 3 murine models of T-ALL. We identified that certain subsets of T-ALL seem to have an increased reliance on oxidative phosphorylation when treated with DHODH inhibitors. Through a series of metabolic assays, we show that leukemia cells, in the setting of nucleotide starvation, undergo changes in their mitochondrial membrane potential and may be more highly dependent on alternative fuel sources. The effect on normal T-cell development in young mice was also examined to show that DHODH inhibition does not permanently damage the developing thymus. These changes suggest a new metabolic vulnerability that may distinguish these cells from normal T cells and other normal hematopoietic cells and offer an exploitable therapeutic opportunity. The availability of clinical-grade DHODH inhibitors currently in human clinical trials suggests a potential for rapidly advancing this work into the clinic.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Animales , Ratones , Dihidroorotato Deshidrogenasa , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Linfocitos T/metabolismo , Nucleótidos/uso terapéuticoRESUMEN
Aberrant RAS/MAPK signaling is a common driver of oncogenesis that can be therapeutically targeted with clinically approved MEK inhibitors. Disease progression on single-agent MEK inhibitors is common, however, and combination therapies are typically required to achieve significant clinical benefit in advanced cancers. Here we focused on identifying MEK inhibitor-based combination therapies in neuroblastoma with mutations that activate the RAS/MAPK signaling pathway, which are rare at diagnosis but frequent in relapsed neuroblastoma. A genome-scale CRISPR-Cas9 functional genomic screen was deployed to identify genes that when knocked out sensitize RAS-mutant neuroblastoma to MEK inhibition. Loss of either CCNC or CDK8, two members of the mediator kinase module, sensitized neuroblastoma to MEK inhibition. Furthermore, small-molecule kinase inhibitors of CDK8 improved response to MEK inhibitors in vitro and in vivo in RAS-mutant neuroblastoma and other adult solid tumors. Transcriptional profiling revealed that loss of CDK8 or CCNC antagonized the transcriptional signature induced by MEK inhibition. When combined, loss of CDK8 or CCNC prevented the compensatory upregulation of progrowth gene expression induced by MEK inhibition. These findings propose a new therapeutic combination for RAS-mutant neuroblastoma and may have clinical relevance for other RAS-driven malignancies. SIGNIFICANCE: Transcriptional adaptation to MEK inhibition is mediated by CDK8 and can be blocked by the addition of CDK8 inhibitors to improve response to MEK inhibitors in RAS-mutant neuroblastoma, a clinically challenging disease.
Asunto(s)
Recurrencia Local de Neoplasia , Neuroblastoma , Adulto , Humanos , Línea Celular Tumoral , Recurrencia Local de Neoplasia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/patología , Mutación , Quinasas de Proteína Quinasa Activadas por Mitógenos , Quinasa 8 Dependiente de Ciclina/genéticaRESUMEN
Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.
Asunto(s)
Gangliósidos , Neuroblastoma , Anticuerpos Monoclonales , Niño , Humanos , Inmunoterapia , Recurrencia Local de Neoplasia/inducido químicamente , Neuroblastoma/tratamiento farmacológicoRESUMEN
Genomic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in acute lymphoblastic leukemia (ALL), suggesting new opportunities for therapeutic interventions. In this study, we identified G9a/EHMT2 as a potential target in T-ALL through the intersection of epigenome-centered shRNA and chemical screens. We subsequently validated G9a with low-throughput CRISPR-Cas9-based studies targeting the catalytic G9a SET-domain and the testing of G9a chemical inhibitors in vitro, 3D, and in vivo T-ALL models. Mechanistically we determined that G9a repression promotes lysosomal biogenesis and autophagic degradation associated with the suppression of sestrin2 (SESN2) and inhibition of glycogen synthase kinase-3 (GSK-3), suggesting that in T-ALL glycolytic dependent pathways are at least in part under epigenetic control. Thus, targeting G9a represents a strategy to exhaust the metabolic requirement of T-ALL cells.
Asunto(s)
N-Metiltransferasa de Histona-Lisina , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Metilación de ADN/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Linfocitos T/metabolismoRESUMEN
By querying metabolic pathways associated with leukemic stemness and survival in multiple AML datasets, we nominated SLC7A11 encoding the xCT cystine importer as a putative AML dependency. Genetic and chemical inhibition of SLC7A11 impaired the viability and clonogenic capacity of AML cell lines in a cysteine-dependent manner. Sulfasalazine, a broadly available drug with xCT inhibitory activity, had anti-leukemic activity against primary AML samples in ex vivo cultures. Multiple metabolic pathways were impacted upon xCT inhibition, resulting in depletion of glutathione pools in leukemic cells and oxidative stress-dependent cell death, only in part through ferroptosis. Higher expression of cysteine metabolism genes and greater cystine dependency was noted in NPM1-mutated AMLs. Among eight anti-leukemic drugs, the anthracycline daunorubicin was identified as the top synergistic agent in combination with sulfasalazine in vitro. Addition of sulfasalazine at a clinically relevant concentration significantly augmented the anti-leukemic activity of a daunorubicin-cytarabine combination in a panel of 45 primary samples enriched in NPM1-mutated AML. These results were confirmed in vivo in a patient-derived xenograft model. Collectively, our results nominate cystine import as a druggable target in AML and raise the possibility to repurpose sulfasalazine for the treatment of AML, notably in combination with chemotherapy.
Asunto(s)
Cistina , Leucemia Mieloide Aguda , Línea Celular Tumoral , Cisteína , Cistina/metabolismo , Cistina/uso terapéutico , Daunorrubicina/farmacología , Daunorrubicina/uso terapéutico , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas Nucleares , Sulfasalazina/farmacología , Sulfasalazina/uso terapéuticoRESUMEN
Leukemic blasts are immune cells gone awry. We hypothesized that dysregulation of inflammatory pathways contributes to the maintenance of their leukemic state and can be exploited as cell-intrinsic, self-directed immunotherapy. To this end, we applied genome-wide screens to discover genetic vulnerabilities in acute myeloid leukemia (AML) cells implicated in inflammatory pathways. We identified the immune modulator IRF2BP2 as a selective AML dependency. We validated AML cell dependency on IRF2BP2 with genetic and protein degradation approaches in vitro and genetically in vivo. Chromatin and global gene-expression studies demonstrated that IRF2BP2 represses IL1ß/TNFα signaling via NFκB, and IRF2BP2 perturbation results in an acute inflammatory state leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for regulated cell death, and establish IRF2BP2-mediated transcriptional repression as a mechanism for blast survival. SIGNIFICANCE: This study exploits inflammatory programs inherent to AML blasts to identify genetic vulnerabilities in this disease. In doing so, we determined that AML cells are dependent on the transcriptional repressive activity of IRF2BP2 for their survival, revealing cell-intrinsic inflammation as a mechanism priming leukemic blasts for regulated cell death. See related commentary by Puissant and Medyouf, p. 1617. This article is highlighted in the In This Issue feature, p. 1599.
Asunto(s)
Leucemia Mieloide Aguda , Humanos , Inflamación/genética , Leucemia Mieloide Aguda/genética , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
Expression of the fusion oncoprotein EWS/FLI causes Ewing sarcoma, an aggressive pediatric tumor characterized by widespread epigenetic deregulation. These epigenetic changes are targeted by novel lysine-specific demethylase-1 (LSD1) inhibitors, which are currently in early-phase clinical trials. Single-agent-targeted therapy often induces resistance, and successful clinical development requires knowledge of resistance mechanisms, enabling the design of effective combination strategies. Here, we used a genome-scale CRISPR-Cas9 loss-of-function screen to identify genes whose knockout (KO) conferred resistance to the LSD1 inhibitor SP-2509 in Ewing sarcoma cell lines. Multiple genes required for mitochondrial electron transport chain (ETC) complexes III and IV function were hits in our screen. We validated this finding using genetic and chemical approaches, including CRISPR KO, ETC inhibitors, and mitochondrial depletion. Further global transcriptional profiling revealed that altered complex III/IV function disrupted the oncogenic program mediated by EWS/FLI and LSD1 and blunted the transcriptomic response to SP-2509. IMPLICATIONS: These findings demonstrate that mitochondrial dysfunction modulates SP-2509 efficacy and suggest that new therapeutic strategies combining LSD1 with agents that prevent mitochondrial dysfunction may benefit patients with this aggressive malignancy.
Asunto(s)
Neoplasias Óseas , Sarcoma de Ewing , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Línea Celular Tumoral , Niño , Resistencia a Medicamentos , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Mitocondrias/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologíaRESUMEN
Acute myeloid leukemia (AML) is an aggressive blood cancer with a poor prognosis. We report a comprehensive proteogenomic analysis of bone marrow biopsies from 252 uniformly treated AML patients to elucidate the molecular pathophysiology of AML in order to inform future diagnostic and therapeutic approaches. In addition to in-depth quantitative proteomics, our analysis includes cytogenetic profiling and DNA/RNA sequencing. We identify five proteomic AML subtypes, each reflecting specific biological features spanning genomic boundaries. Two of these proteomic subtypes correlate with patient outcome, but none is exclusively associated with specific genomic aberrations. Remarkably, one subtype (Mito-AML), which is captured only in the proteome, is characterized by high expression of mitochondrial proteins and confers poor outcome, with reduced remission rate and shorter overall survival on treatment with intensive induction chemotherapy. Functional analyses reveal that Mito-AML is metabolically wired toward stronger complex I-dependent respiration and is more responsive to treatment with the BCL2 inhibitor venetoclax.
Asunto(s)
Leucemia Mieloide Aguda , Proteogenómica , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , ProteómicaRESUMEN
Despite progress in the treatment of acute lymphoblastic leukemia (ALL), T-cell ALL (T-ALL) has limited treatment options, particularly in the setting of relapsed/refractory disease. Using an unbiased genome-scale CRISPR-Cas9 screen we sought to identify pathway dependencies for T-ALL which could be harnessed for therapy development. Disruption of the one-carbon folate, purine and pyrimidine pathways scored as the top metabolic pathways required for T-ALL proliferation. We used a recently developed inhibitor of SHMT1 and SHMT2, RZ-2994, to characterize the effect of inhibiting these enzymes of the one-carbon folate pathway in T-ALL and found that T-ALL cell lines were differentially sensitive to RZ-2994, with the drug inducing a S/G2 cell cycle arrest. The effects of SHMT1/2 inhibition were rescued by formate supplementation. Loss of both SHMT1 and SHMT2 was necessary for impaired growth and cell cycle arrest, with suppression of both SHMT1 and SHMT2 inhibiting leukemia progression in vivo. RZ-2994 also decreased leukemia burden in vivo and remained effective in the setting of methotrexate resistance in vitro. This study highlights the significance of the one-carbon folate pathway in T-ALL and supports further development of SHMT inhibitors for treatment of T-ALL and other cancers.
Asunto(s)
Sistemas CRISPR-Cas , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ácido Fólico/metabolismo , Glicina Hidroximetiltransferasa/antagonistas & inhibidores , Metotrexato/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Animales , Antimetabolitos Antineoplásicos/farmacología , Apoptosis , Ciclo Celular , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Pronóstico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF-driven cancers.
Asunto(s)
Neoplasias Óseas/mortalidad , Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Fusión Oncogénica/química , Proteína Proto-Oncogénica c-fli-1/química , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/química , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de Ewing/mortalidad , Neoplasias Óseas/metabolismo , Proteínas Portadoras/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas de Fusión Oncogénica/metabolismo , Estabilidad Proteica , Proteolisis , Sarcoma de Ewing/metabolismo , Transactivadores/metabolismoRESUMEN
The core cohesin subunit STAG2 is recurrently mutated in Ewing sarcoma but its biological role is less clear. Here, we demonstrate that cohesin complexes containing STAG2 occupy enhancer and polycomb repressive complex (PRC2)-marked regulatory regions. Genetic suppression of STAG2 leads to a compensatory increase in cohesin-STAG1 complexes, but not in enhancer-rich regions, and results in reprogramming of cis-chromatin interactions. Strikingly, in STAG2 knockout cells the oncogenic genetic program driven by the fusion transcription factor EWS/FLI1 was highly perturbed, in part due to altered enhancer-promoter contacts. Moreover, loss of STAG2 also disrupted PRC2-mediated regulation of gene expression. Combined, these transcriptional changes converged to modulate EWS/FLI1, migratory, and neurodevelopmental programs. Finally, consistent with clinical observations, functional studies revealed that loss of STAG2 enhances the metastatic potential of Ewing sarcoma xenografts. Our findings demonstrate that STAG2 mutations can alter chromatin architecture and transcriptional programs to promote an aggressive cancer phenotype.
Asunto(s)
Neoplasias Óseas/genética , Neoplasias Óseas/patología , Proteínas de Ciclo Celular/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Elementos de Facilitación Genéticos , Femenino , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones Endogámicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/genética , Factores del Dominio POU/genética , Factores del Dominio POU/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/genética , CohesinasRESUMEN
The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.
Asunto(s)
Antineoplásicos , Leucemia Mieloide Aguda , Adenosina Trifosfatasas/metabolismo , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Reparación del ADN , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Proteína que Contiene ValosinaRESUMEN
Deciphering the impact of metabolic intervention on response to anticancer therapy may elucidate a path toward improved clinical responses. Here, we identify amino acid-related pathways connected to the folate cycle whose activation predicts sensitivity to MYC-targeting therapies in acute myeloid leukemia (AML). We establish that folate restriction and deficiency of the rate-limiting folate cycle enzyme MTHFR, which exhibits reduced-function polymorphisms in about 10% of Caucasians, induce resistance to MYC targeting by BET and CDK7 inhibitors in cell lines, primary patient samples, and syngeneic mouse models of AML. Furthermore, this effect is abrogated by supplementation with the MTHFR enzymatic product CH3-THF. Mechanistically, folate cycle disturbance reduces H3K27/K9 histone methylation and activates a SPI1 transcriptional program counteracting the effect of BET inhibition. Our data provide a rationale for screening MTHFR polymorphisms and folate cycle status to nominate patients most likely to benefit from MYC-targeting therapies. SIGNIFICANCE: Although MYC-targeting therapies represent a promising strategy for cancer treatment, evidence of predictors of sensitivity to these agents is limited. We pinpoint that folate cycle disturbance and frequent polymorphisms associated with reduced MTHFR activity promote resistance to BET inhibitors. CH3-THF supplementation thus represents a low-risk intervention to enhance their effects.See related commentary by Marando and Huntly, p. 1791.This article is highlighted in the In This Issue feature, p. 1775.
Asunto(s)
Ácido Fólico/metabolismo , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Resistencia a Antineoplásicos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Células U937RESUMEN
Spleen tyrosine kinase (SYK) is a nonmutated therapeutic target in acute myeloid leukemia (AML). Attempts to exploit SYK therapeutically in AML have shown promising results in combination with chemotherapy, likely reflecting induced mechanisms of resistance to single-agent treatment in vivo. We conducted a genome-scale open reading frame (ORF) resistance screen and identified activation of the RAS-MAPK-ERK pathway as one major mechanism of resistance to SYK inhibitors. This finding was validated in AML cell lines with innate and acquired resistance to SYK inhibitors. Furthermore, patients with AML with select mutations activating these pathways displayed early resistance to SYK inhibition. To circumvent SYK inhibitor therapy resistance in AML, we demonstrate that a MEK and SYK inhibitor combination is synergistic in vitro and in vivo. Our data provide justification for use of ORF screening to identify resistance mechanisms to kinase inhibitor therapy in AML lacking distinct mutations and to direct novel combination-based strategies to abrogate these. SIGNIFICANCE: The integration of functional genomic screening with the study of mechanisms of intrinsic and acquired resistance in model systems and human patients identified resistance to SYK inhibitors through MAPK signaling in AML. The dual targeting of SYK and the MAPK pathway offers a combinatorial strategy to overcome this resistance.This article is highlighted in the In This Issue feature, p. 161.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasa Syk/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Benzamidas/farmacología , Benzamidas/uso terapéutico , Línea Celular Tumoral , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Difenilamina/análogos & derivados , Difenilamina/farmacología , Difenilamina/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Indazoles/farmacología , Indazoles/uso terapéutico , Leucemia Mieloide Aguda/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Sistemas de Lectura Abierta/genética , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirazinas/farmacología , Pirazinas/uso terapéutico , Quinasa Syk/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The development of novel structural materials with increasing mechanical requirements is a very resource-intense process if conventional methods are used. While there are high-throughput methods for the development of functional materials, this is not the case for structural materials. Their mechanical properties are determined by their microstructure, so that increased sample volumes are needed. Furthermore, new short-time characterization techniques are required for individual samples which do not necessarily measure the desired material properties, but descriptors which can later be mapped on material properties. While universal micro-hardness testing is being commonly used, it is limited in its capability to measure sample volumes which contain a characteristic microstructure. We propose to use alternative and fast deformation techniques for spherical micro-samples in combination with classical characterization techniques such as XRD, DSC or micro magnetic methods, which deliver descriptors for the microstructural state.
RESUMEN
Genetic modification of human leukemic cell lines using CRISPR-Cas9 has become a staple of gene-function studies. Single-cell cloning of modified cells is frequently used to facilitate studies of gene function. Inherent in this approach is an assumption that the genetic drift, amplified in some cell lines by mutations in DNA replication and repair machinery, as well as non-genetic factors will not introduce significant levels of experimental cellular heterogeneity in clones derived from parental populations. In this study, we characterize the variation in cell death of fifty clonal cell lines generated from human Jurkat and MOLT-4 T-cells edited by CRISPR-Cas9. We demonstrate a wide distribution of sensitivity to chemotherapeutics between non-edited clonal human leukemia T-cell lines, and also following CRISPR-Cas9 editing at the NLRP1 locus, or following transfection with non-targeting sgRNA controls. The cell death sensitivity profile of clonal cell lines was consistent across experiments and failed to revert to the non-clonal parental phenotype. Whole genome sequencing of two clonal cell lines edited by CRISPR-Cas9 revealed unique and shared genetic variants, which had minimal read support in the non-clonal parental population and were not suspected CRISPR-Cas9 off-target effects. These variants included genes related to cell death and drug metabolism. The variation in cell death phenotype of clonal populations of human T-cell lines may be a consequence of T-cell line genetic instability, and to a lesser extent clonal heterogeneity in the parental population or CRISPR-Cas9 off-target effects not predicted by current models. This work highlights the importance of genetic variation between clonal T-cell lines in the design, conduct, and analysis of experiments to investigate gene function after single-cell cloning.
Asunto(s)
Muerte Celular , Células Clonales , Linfocitos T/efectos de los fármacos , Antineoplásicos/farmacología , Línea Celular , Heterogeneidad Genética , Humanos , Quimioterapia de Inducción , Linfocitos T/patologíaRESUMEN
Monosomy 7 or deletion of 7q (del(7q)) are common clonal cytogenetic abnormalities associated with high grade myelodysplastic syndrome (MDS) arising in inherited and acquired bone marrow failure. Current non-transplant approaches to treat marrow failure may be complicated by stimulation of clonal outgrowth. To study the biological consequences of del(7q) within the context of a failing marrow, we generated induced pluripotent stem cells (iPSCs) derived from patients with Shwachman Diamond Syndrome (SDS), a bone marrow failure disorder with MDS predisposition, and genomically engineered a 7q deletion. The TGFß pathway was the top differentially regulated pathway in transcriptomic analysis of SDS versus SDSdel(7q) iPSCs. SMAD2 phosphorylation was increased in SDS relative to wild type cells consistent with hyperactivation of the TGFbeta pathway in SDS. Phospho-SMAD2 levels were reduced following 7q deletion in SDS cells and increased upon restoration of 7q diploidy. Inhibition of the TGFbeta pathway rescued hematopoiesis in SDS-iPSCs and in bone marrow hematopoietic cells from SDS patients while it had no impact on the SDSdel(7q) cells. These results identified a potential targetable vulnerability to improve hematopoiesis in an MDS-predisposition syndrome, and highlight the importance of the germline context of somatic alterations to inform precision medicine approaches to therapy.