RESUMEN
Beyond its clinical diversity and severity, acute myeloid leukemia (AML) is known for its complex molecular background and for rewiring biological processes to aid disease onset and maintenance. FLT3 mutations are among the most recurring molecular entities that cooperatively drive AML, and their inhibition is a critical molecularly oriented therapeutic strategy. Despite being a promising avenue, it still faces challenges such as intrinsic and acquired drug resistance, which led us to investigate whether and how autophagy and inflammasome interact and whether this interaction could be leveraged to enhance FLT3 inhibition as a therapeutic strategy. We observed a strong and positive correlation between the expression of key genes associated with autophagy and the inflammasome. Gene set enrichment analysis of the FLT3-ITD samples and their ex vivo response to five different FLT3 inhibitors revealed a common molecular signature compatible with autophagy and inflammasome activation across all poor responders. Inflammasome activation was also shown to strongly increase the likelihood of a poor ex vivo response to the FLT3 inhibitors quizartinib and sorafenib. These findings reveal a distinct molecular pattern within FLT3-ITD AML samples that underscores the necessity for further exploration into how approaching these supportive parallel yet altered pathways could improve therapeutic strategies.
Asunto(s)
Autofagia , Inflamasomas , Leucemia Mieloide Aguda , Inhibidores de Proteínas Quinasas , Tirosina Quinasa 3 Similar a fms , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Autofagia/efectos de los fármacos , Inflamasomas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Mutación , Femenino , Masculino , Benzotiazoles/farmacología , Persona de Mediana Edad , Sorafenib/farmacología , Resistencia a Antineoplásicos/genética , Anciano , Compuestos de FenilureaRESUMEN
Acute myeloid leukemia (AML) is the most common hematological cancer in the adult population worldwide. Approximately 35% of patients with AML present internal tandem duplication (ITD) mutations in the FMSlike tyrosine kinase 3 (FLT3) receptor associated with poor prognosis, and thus, this receptor is a relevant target for potential therapeutics. Tyrosine kinase inhibitors (TKIs) are used to treat AML; however, their molecular interactions and effects on leukemic cells are poorly understood. The present study aimed to gain insights into the molecular interactions and affinity forces of four TKI drugs (sorafenib, midostaurin, gilteritinib and quizartinib) with the wildtype (WT)FLT3 and ITDmutated (ITDFLT3) structural models of FLT3, in its inactive aspartic acidphenylalanineglycine motif (DFGout) and active aspartic acidphenylalanineglycine motif (DFGin) conformations. Furthermore, the present study evaluated the effects of the secondgeneration TKIs gilteritinib and quizartinib on cancer cell viability, apoptosis and proliferation in the MV411 (ITDFLT3) and HL60 (WTFLT3) AML cell lines. Peripheral blood mononuclear cells (PBMCs) from a healthy volunteer were included as an FLT3negative group. Molecular docking analysis indicated higher affinities of secondgeneration TKIs for WTFLT3/DFGout and WTFLT3/DFGin compared with those of the firstgeneration TKIs. However, the ITD mutation changed the affinity of all TKIs. The in vitro data supported the in silico predictions: MV411 cells presented high selective sensibility to gilteritinib and quizartinib compared with the HL60 cells, whereas the drugs had no effect on PBMCs. Thus, the current study presented novel information about molecular interactions between the FLT3 receptors (WT or ITDmutated) and some of their inhibitors. It also paves the way for the search for novel inhibitory molecules with potential use against AML.
Asunto(s)
Leucemia Mieloide Aguda , Inhibidores de Proteínas Quinasas , Estaurosporina , Tirosina Quinasa 3 Similar a fms , Humanos , Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Benzotiazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Simulación por Computador , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo , Tirosina Quinasa 3 Similar a fms/química , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Simulación del Acoplamiento Molecular , Mutación , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , Sorafenib/farmacología , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Triazinas/farmacología , Triazinas/químicaRESUMEN
Acute myeloid leukaemia (AML) is a severe haematological neoplasm that originates from the transformation of haematopoietic stem cells (HSCs) into leukaemic stem cells (LSCs). The bone marrow (BM) microenvironment, particularly that of mesenchymal stromal cells (hMSCs), plays a crucial role in the maintenance of HSCs. In this context, we explored whether alterations in the secretome of hMSCs derived from AML patients (hMSC-AML) could impact HSC gene expression. Proteomic analysis revealed that the secretome of coculture assays with hMSC-AMLs and HSC from healthy donor is altered, with increased levels of secretory leukocyte protease inhibitor (SLPI), a protein associated with important processes for maintenance of the haematopoietic niche that has already been described to be altered in several tumours. Increased SLPI expression was also observed in the BM plasma of AML patients. Transcriptome analysis of HSCs cocultured with hMSC-AML in comparison with HSCs cocultured with hMSC-HD revealed altered expression of SLPI target genes associated with the cell cycle, proliferation, and apoptosis. Important changes were identified, such as increased expression levels of CCNA2, CCNE2, CCND2, CD133 and CDK1 and decreased levels of CDKN2A and IGFBP3, among others. Overall, these findings suggest that the altered secretome of coculture assays with hMSC-AMLs and HSC from healthy donor, particularly increased SLPI expression, can contribute to gene expression changes in HSCs, potentially influencing important molecular mechanisms related to AML development and progression.
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Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Células Madre Mesenquimatosas , Inhibidor Secretorio de Peptidasas Leucocitarias , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Células Madre Mesenquimatosas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Técnicas de Cocultivo , Transcriptoma , Femenino , Masculino , Perfilación de la Expresión Génica , Persona de Mediana Edad , Proteómica/métodos , Regulación Leucémica de la Expresión Génica , Anciano , Adulto , Proliferación Celular/genéticaRESUMEN
Acute leukemias (ALs) are the most common cancers in pediatric population. There are two types of ALs: acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Some studies suggest that the Renin Angiotensin System (RAS) has a role in ALs. RAS signaling modulates, directly and indirectly, cellular activity in different cancers, affecting tumor cells and angiogenesis. Our review aimed to summarize the role of RAS in ALs and to explore future perspectives for the treatment of these hematological malignancies by modulating RAS molecules. The database including Pubmed, Scopus, Cochrane Library, and Scielo were searched to find articles about RAS molecules in ALL and in pediatric patients. The search terms were "RAS", "Acute Leukemia", "ALL", "Angiotensin-(1-7)", "Pediatric", "Cancer", "Angiotensin II", "AML". In the bone marrow, RAS has been found to play a key role in blood cell formation, affecting several processes including apoptosis, cell proliferation, mobilization, intracellular signaling, angiogenesis, fibrosis, and inflammation. Local tissue RAS modulates tumor growth and metastasis through autocrine and paracrine actions. RAS mainly acts via two molecules, Angiotensin II (Ang II) and Angiotensin (1-7) [Ang-(1-7)]. While Ang II promotes tumor cell growth and stimulates angiogenesis, Ang-(1-7) inhibits the proliferation of neoplastic cells and the angiogenesis, suggesting a potential therapeutic role of this molecule in ALL. The interaction between ALs and RAS reveals a complex network of molecules that can affect the hematopoiesis and the development of hematological cancers. Understanding these interactions could pave the way for innovative therapeutic approaches targeting RAS components.
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Angiotensina II , Leucemia-Linfoma Linfoblástico de Células Precursoras , Sistema Renina-Angiotensina , Humanos , Sistema Renina-Angiotensina/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Angiotensina II/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Transducción de Señal , Angiotensina I/metabolismo , Neovascularización Patológica/metabolismo , Animales , Fragmentos de Péptidos/metabolismoAsunto(s)
Leucemia Mieloide Aguda , Proteína Tumoral p73 , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Pronóstico , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Femenino , Masculino , Persona de Mediana Edad , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Anciano , Regulación Leucémica de la Expresión Génica , AdultoRESUMEN
Leukemias are among the most prevalent types of cancer worldwide. Bone marrow mesenchymal stem cells (MSCs) participate in the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases such as leukemias, to a yet unknown extent. Here we described the effect of secretome of bone marrow MSCs obtained from healthy donors and from patients with acute myeloid leukemia (AML) on leukemic cell lineages, sensitive (K562) or resistant (K562-Lucena) to chemotherapy drugs. Cell proliferation, viability and death were evaluated, together with cell cycle, cytokine production and gene expression of ABC transporters and cyclins. The secretome of healthy MSCs decreased proliferation and viability of both K562 and K562-Lucena cells; moreover, an increase in apoptosis and necrosis rates was observed, together with the activation of caspase 3/7, cell cycle arrest in G0/G1 phase and changes in expression of several ABC proteins and cyclins D1 and D2. These effects were not observed using the secretome of MSCs derived from AML patients. In conclusion, the secretome of healthy MSCs have the capacity to inhibit the development of leukemia cells, at least in the studied conditions. However, MSCs from AML patients seem to have lost this capacity, and could therefore contribute to the development of leukemia.
Asunto(s)
Proliferación Celular , Leucemia Mieloide Aguda , Células Madre Mesenquimatosas , Humanos , Células Madre Mesenquimatosas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/genética , Células K562 , Apoptosis , Secretoma/metabolismo , Persona de Mediana Edad , Femenino , Masculino , Células de la Médula Ósea/metabolismo , Linaje de la Célula/genética , Supervivencia Celular , AdultoRESUMEN
Acute myelogenous leukaemia (AML) originates and is maintained by leukaemic stem cells (LSCs) that are inherently resistant to antiproliferative therapies, indicating that a critical strategy for overcoming chemoresistance in AML therapy is to eradicate LSCs. In this work, we investigated the anti-AML activity of bortezomib (BTZ), emphasizing its anti-LSC potential, using KG-1a cells, an AML cell line with stem-like properties. BTZ presented potent cytotoxicity to both solid and haematological malignancy cells and reduced the stem-like features of KG-1a cells, as observed by the reduction in CD34- and CD123-positive cells. A reduction in NF-κB p65 nuclear staining was observed in BTZ-treated KG-1a cells, in addition to upregulation of the NF-κB inhibitor gene NFΚBIB. BTZ-induced DNA fragmentation, nuclear condensation, cell shrinkage and loss of transmembrane mitochondrial potential along with an increase in active caspase-3 and cleaved PARP-(Asp 214) level in KG-1a cells. Furthermore, BTZ-induced cell death was partially prevented by pretreatment with the pancaspase inhibitor Z-VAD-(OMe)-FMK, indicating that BTZ induces caspase-mediated apoptosis. BTZ also increased mitochondrial superoxide levels in KG-1a cells, and BTZ-induced apoptosis was partially prevented by pretreatment with the antioxidant N-acetylcysteine, indicating that BTZ induces oxidative stress-mediated apoptosis in KG-1a cells. At a dosage of 0.1 mg/kg every other day for 2 weeks, BTZ significantly reduced the percentage of hCD45-positive cells in the bone marrow and peripheral blood of NSG mice engrafted with KG-1a cells with tolerable toxicity. Taken together, these data indicate that the anti-LSC potential of BTZ appears to be an important strategy for AML treatment.
Asunto(s)
Bortezomib , Leucemia Mieloide Aguda , FN-kappa B , Células Madre Neoplásicas , Estrés Oxidativo , Bortezomib/farmacología , Estrés Oxidativo/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/metabolismo , Animales , FN-kappa B/metabolismo , Línea Celular Tumoral , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Apoptosis/efectos de los fármacos , Antineoplásicos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones SCIDRESUMEN
Phosphatidylinositol-5-phosphate 4-kinase type 2 (PIP4K2) protein family members (PIP4K2A, PIP4K2B, and PIP4K2C) participate in the generation of PIP4,5P2, which acts as a secondary messenger in signal transduction, a substrate for metabolic processes, and has structural functions. In patients with acute myeloid leukemia (AML), high PIP4K2A and PIP4K2C levels are independent markers of a worse prognosis. Recently, our research group reported that THZ-P1-2 (PIP4K2 pan-inhibitor) exhibits anti-leukemic activity by disrupting mitochondrial homeostasis and autophagy in AML models. In the present study, we characterized the expression of PIP4K2 in the myeloid compartment of hematopoietic cells, as well as in AML cell lines and clinical samples with different genetic abnormalities. In ex vivo assays, PIP4K2 expression levels were related to sensitivity and resistance to several antileukemia drugs and highlighted the association between high PIP4K2A levels and resistance to venetoclax. The combination of THZ-P1-2 and venetoclax showed potentiating effects in reducing viability and inducing apoptosis in AML cells. A combined treatment differentially modulated multiple genes, including TAp73, BCL2, MCL1, and BCL2A1. In summary, our study identified the correlation between the expression of PIP4K2 and the response to antineoplastic agents in ex vivo assays in AML and exposed vulnerabilities that may be exploited in combined therapies, which could result in better therapeutic responses.
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Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Línea Celular Tumoral , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Apoptosis , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Fosfotransferasas (Aceptor de Grupo Alcohol)/farmacologíaRESUMEN
BACKGROUND: Acute myeloid leukemia (AML) is a malignant disorder of hematopoietic stem and progenitor cells, characterized by accumulation of immature blasts in the bone marrow and peripheral blood of affected patients. Response to chemotherapy treatment in patients with AML is wide-ranging, and to date there are no adequate molecular biomarkers used to predict clinical outcome. OBJECTIVE: The aim of this study was to identify potential protein biomarkers which could help predict response to induction treatment in AML patients. METHODS: Peripheral blood samples were obtained from 15 AML patients both before and after treatment. A comparative proteomic analysis was performed using 2D gel electrophoresis followed by Mass Spectrometry. RESULTS: This comparative proteomic study, combined with a protein network analysis, revealed several proteins that could be considered potential biomarkers of poor prognosis in AML: GAPDH which favors increased glucose metabolism; eEF1A1 and Annexin A1 that promote proliferation and migration, cofilin 1 which plays a role in the activation of apoptosis; and GSTP1 which is involved in the processes of detoxification and chemoresistance. CONCLUSIONS: This study gives an insight into a panel of protein biomarkers with prognostic potential that should be further investigated.
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Quimioterapia de Inducción , Leucemia Mieloide Aguda , Humanos , Proteómica/métodos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Médula Ósea/patología , Pronóstico , Proteínas/metabolismo , Biomarcadores/metabolismoRESUMEN
BACKGROUND: Voltage-dependent anion selective channels (VDACs) are the most abundant mitochondrial outer membrane proteins, encoded in mammals by three genes, VDAC1, 2 and 3, mostly ubiquitously expressed. As 'mitochondrial gatekeepers', VDACs control organelle and cell metabolism and are involved in many diseases. Despite the presence of numerous VDAC pseudogenes in the human genome, their significance and possible role in VDAC protein expression has not yet been considered. RESULTS: We investigated the relevance of processed pseudogenes of human VDAC genes, both in physiological and in pathological contexts. Using high-throughput tools and querying many genomic and transcriptomic databases, we show that some VDAC pseudogenes are transcribed in specific tissues and pathological contexts. The obtained experimental data confirm an association of the VDAC1P8 pseudogene with acute myeloid leukemia (AML). CONCLUSIONS: Our in-silico comparative analysis between the VDAC1 gene and its VDAC1P8 pseudogene, together with experimental data produced in AML cellular models, indicate a specific over-expression of the VDAC1P8 pseudogene in AML, correlated with a downregulation of the parental VDAC1 gene.
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Leucemia Mieloide Aguda , Seudogenes , Canales Aniónicos Dependientes del Voltaje , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mitocondrias , Seudogenes/genética , Transcriptoma , Canales Aniónicos Dependientes del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
Exogenous enzymes are added to diets to improve nutrient utilization and feed efficiency. A study was conducted to evaluate the effects of dietary exogenous enzyme products with amylolytic (Amaize, Alltech) and proteolytic (Vegpro, Alltech) activity on performance, excretion of purine derivatives, and ruminal fermentation of dairy cows. A total of 24 Holstein cows, 4 of which were ruminally cannulated (161 ± 88 d in milk, 681 ± 96 body weight, and 35.2 ± 5.2 kg/d of milk yield), were blocked by milk yield, days in milk, and body weight, and then distributed in a replicated 4 × 4 Latin square design. Experimental periods lasted 21 d, of which the first 14 d were allowed for treatment adaptation and the last 7 d were used for data collection. Treatments were as follows: (1) control (CON) with no feed additives, (2) amylolytic enzyme product added at 0.5 g/kg diet dry matter (DM; AML), (3) amylolytic enzyme product at 0.5 g/kg of diet DM and proteolytic enzyme product at 0.2 g/kg of diet DM (low level; APL), and (4) amylolytic enzyme products added at 0.5 g/kg diet DM and proteolytic enzyme product at 0.4 g/kg of diet DM (high level; APH). Data were analyzed using the mixed procedure of SAS (version 9.4; SAS Institute Inc.). Differences between treatments were analyzed by orthogonal contrasts: CON versus all enzyme groups (ENZ); AML versus APL+APH; and APL versus APH. Dry matter intake was not affected by treatments. Sorting index for feed particles with size <4 mm was lower for ENZ group than for CON. Total-tract apparent digestibility of DM and nutrients (organic matter, starch, neutral detergent fiber, crude protein, and ether extract) were similar between CON and ENZ. Starch digestibility was greater in cows fed APL and APH treatments (86.3%) compared with those in the AML group (83.6%). Neutral detergent fiber digestibility was greater in APH cows compared with those in the APL group (58.1 and 55.2%, respectively). Ruminal pH and NH3-N concentration were not affected by treatments. Molar percentage of propionate tended to be greater in cows fed ENZ treatments than in those fed CON. Molar percentage of propionate was greater in cows fed AML than those fed the blends of amylase and protease (19.2 and 18.5%, respectively). Purine derivative excretions in urine and milk were similar in cows fed ENZ and CON. Uric acid excretion tended to be greater in cows consuming APL and APH than in those in the AML group. Serum urea N concentration tended to be greater in cows fed ENZ than in those fed CON. Milk yield was greater in cows fed ENZ treatments compared with CON (32.0, 33.1, 33.1, and 33.3 kg/d for CON, AML, APL, and APH, respectively). Fat-corrected milk and lactose yields were higher when feeding ENZ. Feed efficiency tended to be greater in cows fed ENZ than in those fed CON. Feeding ENZ benefited cows' performance, whereas the effects on nutrient digestibility were more pronounced when the combination of amylase and protease was fed at the highest dose.
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Enfermedades de los Bovinos , Leucemia Mieloide Aguda , Femenino , Bovinos , Animales , Lactancia , Péptido Hidrolasas/metabolismo , Propionatos/metabolismo , Fermentación , Detergentes/metabolismo , Digestión , Leche/metabolismo , Dieta/veterinaria , Nutrientes , Almidón/metabolismo , Peso Corporal , Amilasas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/veterinaria , Rumen/metabolismo , Alimentación Animal/análisis , Zea mays/metabolismo , Enfermedades de los Bovinos/metabolismoRESUMEN
The treatment of acute leukemia is challenging because of the genetic heterogeneity between and within patients. Leukemic stem cells (LSCs) are relatively drug-resistant and frequently relapse. Their plasticity and capacity to adapt to extracellular stress, in which mitochondrial metabolism and autophagy play important roles, further complicates treatment. Genetic models of phosphatidylinositol-5-phosphate 4-kinase type 2 protein (PIP4K2s) inhibition have demonstrated the relevance of these enzymes in mitochondrial homeostasis and autophagic flux. Here, we uncovered the cellular and molecular effects of THZ-P1-2, a pan-inhibitor of PIP4K2s, in acute leukemia cells. THZ-P1-2 reduced cell viability and induced DNA damage, apoptosis, loss of mitochondrial membrane potential, and the accumulation of acidic vesicular organelles. Protein expression analysis revealed that THZ-P1-2 impaired autophagic flux. In addition, THZ-P1-2 induced cell differentiation and showed synergistic effects with venetoclax. In primary leukemia cells, LC-MS/MS-based proteome analysis revealed that sensitivity to THZ-P1-2 is associated with mitochondrial metabolism, cell cycle, cell-of-origin (hematopoietic stem cell and myeloid progenitor), and the TP53 pathway. The minimal effects of THZ-P1-2 observed in healthy CD34+ cells suggest a favorable therapeutic window. Our study provides insights into the pharmacological inhibition of PIP4K2s targeting mitochondrial homeostasis and autophagy, shedding light on a new class of drugs for acute leukemia.
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Leucemia Mieloide Aguda , Espectrometría de Masas en Tándem , Humanos , Cromatografía Liquida , Autofagia , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Apoptosis , HomeostasisRESUMEN
Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are characterized by risk of relapses, poor survival, unwanted side effects and high toxicity with the current therapies. In light of these facts, there are efforts to develop new drugs specific for deregulated molecules that participate in leukemia pathogenesis. Hematopoietic cell kinase (HCK), an Src kinase family member, is overexpressed on hematopoietic stem cells of MDS and de novo AML patients and involved in the oncogenic process. Thus, we investigated in vitro, ex vivo and in vivo effects of a novel chemical compound targeting HCK inhibition (iHCK-37), in combination with the most used drugs for the treatment of MDS and de novo AML, 5-Azacytidine and Cytarabine. Herein, the combination treatment with iHCK-37 and 5-Azacytidine or Cytarabine demonstrated additive effects against leukemia cells, compared to either drug alone. iHCK-37 plus 5-Azacytidine or Cytarabine treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to reduction of ERK and AKT phosphorylation, and increased BAX and decreased BCL-XL protein expression. Moreover, treatment with iHCK-37 reduced MDS and AML CD34-positive cell numbers inside a 3D-structure but did not affect normal CD34-positive cell numbers. In vivo analysis showed that leukemic mice treated with iHCK-37 had reduced ERK and AKT proteins phosphorylation levels and leukocyte numbers. In conclusion, the iHCK-37 inhibitor has anti-neoplastic activity in leukemia cells without altering apoptosis and survival rate of normal cells, suggesting on-target malignant cell killing activity as a single agent or in combination with 5-Azacytidine or Cytarabine.
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Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Animales , Azacitidina/farmacología , Azacitidina/uso terapéutico , Citarabina/farmacología , Citarabina/uso terapéutico , Leucemia Mieloide Aguda/metabolismo , Ratones , Síndromes Mielodisplásicos/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-hckRESUMEN
Acute myeloid leukemia (AML) is characterized by the presence of leukemia stem cells (LSCs), and failure to fully eradicate this population contributes to disease persistence/relapse. Prior studies have characterized metabolic vulnerabilities of LSCs, which demonstrate preferential reliance on oxidative phosphorylation (OXPHOS) for energy metabolism and survival. In the present study, using both genetic and pharmacologic strategies in primary human AML specimens, we show that signal transducer and activator of transcription 3 (STAT3) mediates OXPHOS in LSCs. STAT3 regulates AML-specific expression of MYC, which in turn controls transcription of the neutral amino acid transporter gene SLC1A5. We show that genetic inhibition of MYC or SLC1A5 acts to phenocopy the impairment of OXPHOS observed with STAT3 inhibition, thereby establishing this axis as a regulatory mechanism linking STAT3 to energy metabolism. Inhibition of SLC1A5 reduces intracellular levels of glutamine, glutathione, and multiple tricarboxylic acid (TCA) cycle metabolites, leading to reduced TCA cycle activity and inhibition of OXPHOS. Based on these findings, we used a novel small molecule STAT3 inhibitor, which binds STAT3 and disrupts STAT3-DNA, to evaluate the biological role of STAT3. We show that STAT3 inhibition selectively leads to cell death in AML stem and progenitor cells derived from newly diagnosed patients and patients who have experienced relapse while sparing normal hematopoietic cells. Together, these findings establish a STAT3-mediated mechanism that controls energy metabolism and survival in primitive AML cells.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/metabolismo , Leucemia Mieloide Aguda/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Supervivencia Celular , Humanos , Células Madre Neoplásicas/citología , Fosforilación Oxidativa , Células Tumorales CultivadasRESUMEN
Flavonoids are ubiquitous groups of polyphenolic compounds present in most natural products and plants. These substances have been shown to have promising chemopreventive and chemotherapeutic properties with multiple target interactions and multiple pathway regulations against various human cancers. Polyphenolic flavonoid compounds can block the initiation or reverse the promotion stage of multistep carcinogenesis. Quercetin is one of the most abundant flavonoids found in fruits and vegetables and has been shown to have multiple properties capable of reducing cell growth in cancer cells. Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) therapy remains a challenge for hematologists worldwide, and the outcomes for patients with both disorders continue to be poor. This scenario indicates the increasing demand for innovative drugs and rational combinative therapies. Herein, we discuss the multitarget effects of the flavonoid quercetin, a naturally occurring flavonol, on AML and MDS.
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Leucemia Mieloide Aguda/tratamiento farmacológico , Síndromes Mielodisplásicos/tratamiento farmacológico , Quercetina/uso terapéutico , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicos/metabolismo , Quercetina/química , Quercetina/farmacología , Especies Reactivas de OxígenoRESUMEN
Acute myeloid leukemia (AML) belongs to a group of hematological cancer whose relapse cases are often associated with chemoresistance that impairs treatment success and contributes to a poor outcome. For this reason, there is an urgent need for the development of new therapeutic strategies. Herein, we explore the combination of venetoclax, a BCL2 inhibitor, and embelin, an XIAP inhibitor, in the AML cell lines. Combinatory treatment of venetoclax and embelin potentiated cytotoxic effects of these drugs, demonstrating that both in combination present lower IC50 values than single treatment of either venetoclax or embelin alone in both cell lines analyzed. The combinatory treatment further increased the apoptosis-inducing properties of both compounds. Computer simulations suggest that embelin binds to both BIR2 and BIR3 domains of XIAP, reinforcing this inhibitory apoptosis protein as an embelin target. Although all AML cell lines presented similar basal levels of XIAP, the combinatory treatment effectively inhibited XIAP expression in OCI-AML3 cells. In conclusion, the inhibition of both apoptosis inhibitory players, BCL2 and XIAP, by venetoclax and embelin, respectively, potentiated their cytotoxic effects in AML cell lines.
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Antineoplásicos/farmacología , Benzoquinonas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Sulfonamidas/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Sinergismo Farmacológico , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Non-T cell activation linker (NTAL) membrane protein depletion from lipid rafts by alkylphospholipids or downregulation by shRNA knockdown decreases cell viability through regulation of the Akt/PI3K pathway in mantle cell lymphoma and acute promyelocytic leukemia cells. Here, we confirmed that the knockdown of NTAL in acute myeloid leukemia (AML) cell lines was associated with decreased cell proliferation and survival. Similarly, a xenograft model using AML cells transduced with NTAL-shRNA and transplanted into immunodeficient mice led to a 1.8-fold decrease in tumor burden. Using immunoprecipitation, LC-MS/MS analysis, and label-free protein quantification, we identified interactors of NTAL in two AML cell lines. By evaluating the gene expression signatures of the NTAL protein interactors using the PREdiction of Clinical Outcomes from Genomic Profiles database, we found that 12 NTAL interactors could predict overall survival in AML, in at least two independent cohorts. In addition, patients with AML exhibiting a high expression of NTAL and its interactors were associated with a leukemic granulocyte-macrophage progenitor-like state. Taken together, our data provide evidence that NTAL and its protein interactors are relevant to AML cell proliferation and survival and represent potential therapeutic targets for granulocyte-macrophage progenitor-like leukemias.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Humanos , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Masculino , Ratones Endogámicos NOD , Ratones SCID , Fosforilación , Mapas de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Análisis de Supervivencia , TranscriptomaRESUMEN
Acute myeloid leukemia (AML) is a heterogeneous clonal disease characterized by the proliferation and accumulation of myeloid blast cells in the bone marrow, which eventually lead to hematopoietic failure. Chemoresistance presents as a major burden for therapy of AML patients. p53 is the most important tumor suppressor protein that regulates cellular response to various stress. It is also important for hematopoietic stem cell development and hematopoiesis. Mutation or deletion of TP53 has been found to be linked to cancer progression, therapy-related resistance, and poor prognosis. TP53 mutation occurs in less than 10% of AML patients; however, it represents a subset of AML with therapy resistance and poor outcome. In addition, there is a subgroup of patients with low-frequency TP53 mutations. The percentage ranges from 1% to 3% of all AML patients. These patients have outcomes comparable to those of the high-frequency TP53 mutation patients. TP53-mutated clones isolated from the parental cells exhibit a survival advantage under drug treatment compared with cells with wild-type TP53, and have a higher population of leukemia stem cell (LSC) marker-positive cells, a characteristic of chemo-resistant cells. Therefore, low-frequency TP53 mutation, which is currently underappreciated, is an important prognosis factor for AML patients. Epigenetic drugs, such as hypomethylating agent and histone deacetylase inhibitors, have been found effective in targeting TP53-mutated AML. Histone deacetylase inhibitors can preferentially target the TP53-mutated subpopulation by reactivating p53-targeted genes and by eradicating LSC marker-positive cells. Therefore, combined treatment with epigenetic drugs may represent a new therapeutic strategy for treatments of TP53-mutated AML.
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Resistencia a Antineoplásicos , Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Mutación , Células Madre Neoplásicas , Proteína p53 Supresora de Tumor , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Inhibition of the H3K79 histone methyltransferase DOT1L has exhibited encouraging preclinical and early clinical activity in KMT2A (MLL)-rearranged leukemia, supporting the development of combinatorial therapies. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination with a protein synthesis inhibitor. EZH2 is the catalytic subunit in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has been reported to have preclinical activity in KMT2A-r leukemia. When combined with DOT1L inhibition, however, we observed both synergistic and antagonistic effects. Interestingly, antagonistic effects were not due to PRC2-mediated de-repression of HOXA9. HOXA cluster genes are key canonical targets of both KMT2A and the PRC2 complex. The independence of the HOXA cluster from PRC2 repression in KMT2A-r leukemia thus affords important insights into leukemia biology. Further studies revealed that EZH2 inhibition counteracted the effect of DOT1L inhibition on ribosomal gene expression. We thus identified a previously unrecognized role of DOT1L in regulating protein production. Decreased translation was one of the earliest effects measurable after DOT1L inhibition and specific to KMT2A-rearranged cell lines. H3K79me2 chromatin immunoprecipitation sequencing patterns over ribosomal genes were similar to those of the canonical KMT2A-fusion target genes in primary AML patient samples. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (omacetaxine), supporting further preclinical/clinical development of this combination. In summary, we discovered a novel epigenetic regulation of a metabolic process-protein synthesis-that plays a role in leukemogenesis and affords a combinatorial therapeutic opportunity.
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Epigénesis Genética , Regulación Leucémica de la Expresión Génica , Reordenamiento Génico , Leucemia Mieloide Aguda/metabolismo , Biosíntesis de Proteínas , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteína de la Leucemia Mieloide-Linfoide/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/genética , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismoRESUMEN
PURPOSE: The prognosis of AML patients with chemotherapy is poor, especially those who are insensitive to and resistant to chemotherapy drugs. To clarify the underlying pathogenesis of AML and provide new therapeutic targets for clinical treatment, we explore the role of circRNA in leukemia. METHODS: High-throughput circRNA sequencing analysis was performed in patients with leukemia and healthy donors. RT-qPCR and western blot analysis were used to determine expression of GSK3ß. RNA pull-down assay was used to detect miRNAs pulled down by hsa_circ_0121582. RNA immunoprecipitation assay was performed to evaluate the binding capacity between TET1 and hsa_circ_0121582. RESULTS: A new and highly stable circRNA was found, which was derived from the reverse splicing of GSK3ß exon 1 to exon 7, and hsa_circ_0121582 was down-regulated in leukemia cells. In gain-of-function experiments, the up-regulated hsa_circ_0121582 inhibited the proliferation of leukemia cells in vitro and in vivo. In the cytoplasm, hsa_circ_0121582 could act as a sponge for miR-224, attenuate the inhibiting effect of miR-224 on GSK3ß, and thus up-regulate the expression level of GSK3ß. In addition, hsa_circ_0121582 could bind to GSK3ß promoter in the nucleus, and recruit DNA demethylase TET1 to ensuring the transcription of GSK3ß. The upregulated GSK3ß inhibited the Wnt/ß-catenin signaling pathway, and reduced the aggregation of ß-catenin in the nucleus, thus inhibited the proliferation of leukemia cells. CONCLUSIONS: This study found that hsa_circ_0121582 was involved in the inhibition of tumor proliferation, and the restoration of hsa_circ_0121582 could be an effective treatment strategy for patients with leukemia.