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BACKGROUND: Development of precision medicine requires the identification of easily detectable and druggable biomarkers. Despite recent targeted drug approvals, prognosis of acute myeloid leukemia (AML) patients needs to be greatly improved, as relapse and refractory disease are still difficult to manage. Thus, new therapeutic approaches are needed. Based on in silico-generated preliminary data and the literature, the role of the prolactin (PRL)-mediated signaling was interrogated in AML. METHODS: Protein expression and cell viability were determined by flow cytometry. Repopulation capacity was studied in murine xenotransplantation assays. Gene expression was measured by qPCR and luciferase-reporters. SA-ß-Gal staining was used as a senescence marker. RESULTS: The prolactin receptor (PRLR) was upregulated in AML cells, as compared to their healthy counterpart. The genetic and molecular inhibition of this receptor reduced the colony-forming potential. Disruption of the PRLR signaling, either using a mutant PRL or a dominant-negative isoform of PRLR, reduced the leukemia burden in vivo, in xenotransplantation assays. The expression levels of PRLR directly correlated with resistance to cytarabine. Indeed, acquired cytarabine resistance was accompanied with the induction of PRLR surface expression. The signaling associated to PRLR in AML was mainly mediated by Stat5, in contrast to the residual function of Stat3. In concordance, Stat5 mRNA was significantly overexpressed at mRNA levels in relapse AML samples. A senescence-like phenotype, measured by SA-ß-gal staining, was induced upon enforced expression of PRLR in AML cells, partially dependent on ATR. Similar to the previously described chemoresistance-induced senescence in AML, no cell cycle arrest was observed. Additionally, the therapeutic potential of PRLR in AML was genetically validated. CONCLUSIONS: These results support the role of PRLR as a therapeutic target for AML and the further development of drug discovery programs searching for specific PRLR inhibitors.
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Despite the success of immunotherapy in several haematological neoplasms, the effectiveness in acute myeloid leukaemia (AML) is still controversial, partially due to the lack of knowledge regarding immune-related processes in this disease and similar neoplasias. In this study, we analysed the role and expression of histamine receptor 1 (HRH1) in haematological malignancies. Although the histamine receptor type 1 was widely expressed in healthy and malignant haematopoiesis, especially along the myeloid lineage, HRH1 lacked a relevant role in survival/proliferation and chemoresistance of AML cells, as analysed by HRH1 knockdown (KD) and pharmacological modulation. However, HRH1-mediated signalling was critical for the activation of the differentiation process induced by several agents including all-trans retinoic acid, establishing a role for HRH1 in myeloid differentiation. Pharmacological activation of Erk was able to partially restore differentiation capacity in HRH1 KD AML cells, suggesting that HRH1 signalling acts upstream MAPK-Erk pathway. As an indirect consequence of our results, treatment-related histamine release is not expected to confer a proliferative advantage in leukaemic cells.
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Diferenciación Celular/genética , Regulación Leucémica de la Expresión Génica , Receptores Histamínicos H1/genética , Biomarcadores , Línea Celular Tumoral , Hematopoyesis/genética , Humanos , Inmunohistoquímica , Inmunofenotipificación , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Receptores Histamínicos H1/metabolismoRESUMEN
Macrophages serve essential functions as regulators of immunity and homeostasis, and their proliferation contributes to pathogenesis of certain disorders. In this report, we show that induction of macrophage proliferation by the growth factor M-CSF is negatively modulated by agonists that activate the nuclear receptor liver X receptor (LXR), both in vitro and in vivo. Both isoforms LXR α and ß are involved in the antiproliferative actions of LXR ligands in macrophages. In contrast, M-CSF does not exert negative effects on LXR-mediated gene expression. Treatment with LXR agonists results in the accumulation of macrophages in the G(0)/G(1) phase of the cell cycle without affecting ERK-1/2 phosphorylation. The use of small interfering RNA or genetically modified mice revealed that, in contrast to other cellular models, functional expression of either the cyclin-dependent kinase inhibitor p27KIP1 or the cholesterol transporters ATP-binding cassette A1 or ATP-binding cassette G1 was not required for the antiproliferative effects of LXR agonists in macrophages. Western blot analysis revealed that protein expression of key molecules that regulate progression through the cell cycle, such as cyclins D1 and B1 and cyclin-dependent kinases 2 and 4, was downregulated upon LXR activation. These observations suggest a role for LXR agonists in limiting macrophage proliferative responses associated to pathogenic disorders.
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Proliferación Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Macrófagos/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Animales , Benzoatos/farmacología , Bencilaminas/farmacología , Western Blotting , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Ciclina B1/metabolismo , Ciclina D1/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Regulación hacia Abajo , Citometría de Flujo , Células HEK293 , Humanos , Hidrocarburos Fluorados/farmacología , Células L , Receptores X del Hígado , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Nucleares Huérfanos/agonistas , Receptores Nucleares Huérfanos/genética , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Acute myeloid leukemia (AML) is a heterogeneous hematological cancer characterized by poor prognosis and frequent relapses. Aside from specific mutation-related changes, in AML, the overall function of lysosomes and mitochondria is drastically altered to fulfill the elevated biomass and bioenergetic demands. On the basis of previous results, in silico drug discovery screening was used to identify a new family of lysosome-/mitochondria-targeting compounds. These novel tetracyclic hits, with a cationic amphiphilic structure, specifically eradicate leukemic cells by inducing both mitochondrial damage and apoptosis, and simultaneous lysosomal membrane leakiness. Lysosomal leakiness does not only elicit canonical lysosome-dependent cell death, but also activates the terminal differentiation of AML cells through the Ca2+-TFEB-MYC signaling axis. In addition to being an effective monotherapy, its combination with the chemotherapeutic arsenic trioxide (ATO) used in other types of leukemia is highly synergistic in AML cells, widening the therapeutic window of the treatment. Moreover, the compounds are effective in a wide panel of cancer cell lines and possess adequate pharmacological properties rendering them promising drug candidates for the treatment of AML and other neoplasias.
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Despite the outstanding advances in understanding the biology underlying the pathophysiology of acute myeloid leukemia (AML) and the promising preclinical data published lastly, AML treatment still relies on a classic chemotherapy regimen largely unchanged for the past five decades. Recently, new drugs have been approved for AML, but the real clinical benefit is still under evaluation. Nevertheless, primary refractory and relapse AML continue to represent the main clinical challenge, as the majority of AML patients will succumb to the disease despite achieving a complete remission during the induction phase. As such, treatments for chemoresistant AML represent an unmet need in this disease. Although great efforts have been made to decipher the biological basis for leukemogenesis, the mechanism by which AML cells become resistant to chemotherapy is largely unknown. The identification of the signaling pathways involved in resistance may lead to new combinatory therapies or new therapeutic approaches suitable for this subset of patients. Several mechanisms of chemoresistance have been identified, including drug transporters, key secondary messengers, and metabolic regulators. However, no therapeutic approach targeting chemoresistance has succeeded in clinical trials, especially due to broad secondary effects in healthy cells. Recent research has highlighted the importance of lysosomes in this phenomenon. Lysosomes' key role in resistance to chemotherapy includes the potential to sequester drugs, central metabolic signaling role, and gene expression regulation. These results provide further evidence to support the development of new therapeutic approaches that target lysosomes in AML.
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Liver X receptors (LXR) are transcription factors from the nuclear receptor family that are activated by oxysterols and synthetic high-affinity agonists. In this study, we assessed the antitumor effects of synthetic LXR agonist TO901317 in a murine model of syngeneic Lewis Lung carcinoma. Treatment with TO901317 inhibited tumor growth in wild-type, but not in LXR-deficient mice, indicating that the antitumor effects of the agonist depends on functional LXR activity in host cells. Pharmacologic activation of the LXR pathway reduced the intratumoral abundance of regulatory T cells (Treg) and the expression of the Treg-attracting chemokine Ccl17 by MHCIIhigh tumor-associated macrophages (TAM). Moreover, gene expression profiling indicated a broad negative impact of the LXR agonist on other mechanisms used by TAM for the maintenance of an immunosuppressive environment. In studies exploring the macrophage response to GM-CSF or IL4, activated LXR repressed IRF4 expression, resulting in subsequent downregulation of IRF4-dependent genes including Ccl17. Taken together, this work reveals the combined actions of the LXR pathway in the control of TAM responses that contribute to the antitumoral effects of pharmacologic LXR activation. Moreover, these data provide new insights for the development of novel therapeutic options for the treatment of cancer. SIGNIFICANCE: This study reveals unrecognized roles of LXR in the transcriptional control of the tumor microenvironment and suggests use of a synthetic LXR agonist as a novel therapeutic strategy to stimulate antitumor activity.
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Benzoatos/farmacología , Bencilaminas/farmacología , Hidrocarburos Fluorados/farmacología , Sulfonamidas/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Macrófagos Asociados a Tumores/efectos de los fármacos , Animales , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Receptores X del Hígado/agonistas , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Células RAW 264.7 , Linfocitos T Reguladores/patología , Transcriptoma/efectos de los fármacos , Microambiente Tumoral/inmunología , Macrófagos Asociados a Tumores/metabolismo , Macrófagos Asociados a Tumores/patologíaRESUMEN
BACKGROUND: Despite great efforts to identify druggable molecular targets for AML, there remains an unmet need for more effective therapies. METHODS: An in silico screening was performed using Connectivity Maps to identify FDA-approved drugs that may revert an early leukaemic transformation gene signature. Hit compounds were validated in AML cell lines. Cytotoxic effects were assessed both in primary AML patient samples and healthy donor blood cells. Xenotransplantation assays were undertaken to determine the effect on engraftment of hit compounds. The mechanism of action responsible for the antileukaemic effect was studied focussing on lysosomes and mitochondria. FINDINGS: We identified a group of antihistamines (termed ANHAs) with distinct physicochemical properties associated with their cationic-amphiphilic nature, that selectively killed leukaemic cells. ANHAs behaved as antileukaemic agents against primary AML samples ex vivo, sparing healthy cells. Moreover, ANHAs severely impaired the in vivo leukaemia regeneration capacity. ANHAs' cytotoxicity relied on simultaneous mitochondrial and lysosomal disruption and induction of autophagy and apoptosis. The pharmacological effect was exerted based on their physicochemical properties that permitted the passive targeting of both organelles, without the involvement of active molecular recognition. INTERPRETATION: Dual targeting of lysosomes and mitochondria constitutes a new promising therapeutic approach for leukaemia treatment, supporting the further clinical development. FUND: This work was funded by the Fundación Mutua Madrileña (RMR), CaixaImpulse (RMR), the Spanish Ministry of Economy (RMR), the Josep Carreras International Leukaemia Foundation (RMR), l'Obra Social "La Caixa" (RMR), and Generalitat de Catalunya (IJC).
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Antineoplásicos/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Lisosomas/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Animales , Antineoplásicos/química , Biomarcadores , Línea Celular Tumoral , Modelos Animales de Enfermedad , Antagonistas de los Receptores Histamínicos/química , Humanos , Leucemia Mieloide Aguda , Lisosomas/metabolismo , Ratones , Mitocondrias/metabolismo , Modelos Biológicos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SCOPE: Previous studies have proposed that phytosterols activate liver X receptors (LXR) in the intestine, thereby reducing intestinal cholesterol absorption and promoting fecal cholesterol excretion. METHODS AND RESULTS: In the present study, we examined the effects of dietary phytosterol supplementation on intestinal cholesterol absorption and fecal neutral sterol excretion in LXRαß-deficient mice, and wild-type mice treated with synthetic high-affinity LXRαß agonists. LXRαß deficiency led to an induction of intestinal cholesterol absorption and liver cholesterol accumulation. Phytosterol feeding resulted in an approximately 40% reduction of intestinal cholesterol absorption both in wild-type and LXRαß-deficient mice, reduced dietary cholesterol accumulation in liver and promoted the excretion of fecal cholesterol-derived compounds. Furthermore, phytosterols produced additive inhibitory effects on cholesterol absorption in mice treated with LXRαß agonists. CONCLUSIONS: Our data confirm the effect of LXR in regulating intestinal cholesterol absorption and demonstrate that the cholesterol-lowering effects of phytosterols occur in an LXR-independent manner.
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Colesterol/metabolismo , Absorción Intestinal/efectos de los fármacos , Receptores X del Hígado/fisiología , Fitosteroles/farmacología , Animales , Ratones , Ratones Endogámicos C57BLRESUMEN
Low levels of high-density lipoprotein cholesterol (HDLc) have been associated with breast cancer risk, but several epidemiologic studies have reported contradictory results with regard to the relationship between apolipoprotein (apo) A-I and breast cancer. We aimed to determine the effects of human apoA-I overexpression and administration of specific apoA-I mimetic peptide (D-4F) on tumour progression by using mammary tumour virus-polyoma middle T-antigen transgenic (PyMT) mice as a model of inherited breast cancer. Expression of human apoA-I in the mice did not affect tumour onset and growth in PyMT transgenic mice, despite an increase in the HDLc level. In contrast, D-4F treatment significantly increased tumour latency and inhibited the development of tumours. The effects of D-4F on tumour development were independent of 27-hydroxycholesterol. However, D-4F treatment reduced the plasma oxidized low-density lipoprotein (oxLDL) levels in mice and prevented oxLDL-mediated proliferative response in human breast adenocarcinoma MCF-7 cells. In conclusion, our study shows that D-4F, but not apoA-I-containing HDL, hinders tumour growth in mice with inherited breast cancer in association with a higher protection against LDL oxidative modification.