RESUMEN
Targeting FLT3-ITD in AML using TKI against FLT3 cannot prevent relapse even in the presence of complete remission, suggesting the resistance and/or the persistence of leukemic-initiating cells in the hematopoietic niche. By mimicking the hematopoietic niche condition with cultures at low oxygen concentrations, we demonstrate in vitro that FLT3-ITD AML cells decrease their repopulating capacity when Vps34 is inhibited. Ex vivo, AML FLT3-ITD blasts treated with Vps34 inhibitors recovered proliferation more slowly due to an increase an apoptosis. In vivo, mice engrafted with FLT3-ITD AML MV4-11 cells have the invasion of the bone marrow and blood in 2 weeks. After 4 weeks of FLT3 TKI treatment with gilteritinib, the leukemic burden had strongly decreased and deep remission was observed. When treatment was discontinued, mice relapsed rapidly. In contrast, Vps34 inhibition strongly decreased the relapse rate, and even more so in association with mobilization by G-CSF and AMD3100. These results demonstrate that remission offers the therapeutic window for a regimen using Vps34 inhibition combined with mobilization to target persistent leukemic stem cells and thus decrease the relapse rate.
RESUMEN
PURPOSE: AXL has been shown to play a pivotal role in the selective response of FLT3-ITD acute myeloid leukemia (AML) cells to FLT3 tyrosine kinase inhibitors (TKI), particularly within the bone marrow microenvironment. EXPERIMENTAL DESIGN: Herein, we compared the effect of dual FLT3/AXL-TKI gilteritinib with quizartinib through in vitro models mimicking hematopoietic niche conditions, ex vivo in primary AML blasts, and in vivo with dosing regimens allowing plasma concentration close to those used in clinical trials. RESULTS: We observed that gilteritinib maintained a stronger proapoptotic effect in hypoxia and coculture with bone marrow stromal cells compared with quizartinib, linked to a dose-dependent inhibition of AXL phosphorylation. In vivo, use of the MV4-11 cell line with hematopoietic engraftment demonstrated that gilteritinib was more effective than quizartinib at targeting leukemic cells in bone marrow. Finally, FLT3-ITD AML patient-derived xenografts revealed that this effect was particularly reproducible in FLT3-ITD AML with high allelic ratio in primary and secondary xenograft. Moreover, gilteritinib and quizartinib displayed close toxicity profile on normal murine hematopoiesis, particularly at steady state. CONCLUSIONS: Overall, these findings suggest that gilteritinib as a single agent, compared with quizartinib, is more likely to reach leukemic cells in their protective microenvironment, particularly AML clones highly dependent on FLT3-ITD signaling.
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Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Benzotiazoles/farmacología , Benzotiazoles/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pirazinas/farmacología , Pirazinas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/fisiología , Línea Celular Tumoral , Hematopoyesis , Humanos , Tirosina Quinasa del Receptor AxlRESUMEN
Protein kinases (PK) make up around 2% of the human genome and their expression profile varies depending on the organ and tissue [...].
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Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect-the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.
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Antioxidantes/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Fosforilación Oxidativa , Fase de Descanso del Ciclo Celular , Vitaminas/farmacología , alfa-Tocoferol/farmacología , Animales , Antígenos CD34/genética , Antígenos CD34/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Autorrenovación de las Células , Células Cultivadas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We present here the data showing, in standard cultures exposed to atmospheric O2 concentration, that alpha-tocopherol acetate (α-TOA) has a positive impact on primitive cells inside mesenchymal stromal cell (MstroC) population, by maintaining their proliferative capacity. α-TOA decreases the O2 consumption rate of MStroC probably by impacting respiratory chain complex II activity. This action, however, is not associated with a compensatory increase in glycolysis activity, in spite of the fact that the degradation of HIF-1α was decreased in presence of α-TOA. This is in line with a moderate enhancement of mtROS upon α-TOA treatment. However, the absence of glycolysis stimulation implies the inactivity of HIF-1α which might - if it were active - be related to the maintenance of stemness. It should be stressed that α-TOA might act directly on the gene expression as well as the mtROS themselves, which remains to be elucidated. Alpha-tocopherol acetate (α-TOA), a synthetic vitamin E ester, attenuates electron flow through electron transport chain (ETC) which is probably associated with a moderate increase in mtROS in Mesenchymal Stromal Cells. α-TOA action results in enhancement of the proliferative capacity and maintenance of the differentiation potential of the mesenchymal stem and progenitor cells.
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Células Madre Mesenquimatosas , Mitocondrias , Oxígeno/metabolismo , alfa-Tocoferol , Diferenciación Celular , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Mitocondrias/metabolismo , alfa-Tocoferol/farmacologíaRESUMEN
The cellular receptor Notch1 is a central regulator of T-cell development, and as a consequence, Notch1 pathway appears upregulated in > 65% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T-ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti-Notch therapies in T-ALL models. In this work, we report that Notch1 upregulation in T-ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1-driven T-ALL both in vitro and in vivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1-induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1-driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1-driven leukemia.
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Glutamato-Amoníaco Ligasa/genética , Glutamina/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Animales , Línea Celular Tumoral , Regulación hacia Abajo/genética , Regulación Enzimológica de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Glutamato-Amoníaco Ligasa/metabolismo , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transducción de Señal/genéticaRESUMEN
In the context of hematopoietic cell transplantation, hematopoietic stem cells and progenitor cells (HSC and HPC) are usually collected by apheresis following their mobilization by G-CSF alone or in combination with Plerixafor® when patients fail to respond to G-CSF alone. In medical practice, the quality of the hematopoietic graft is based on CD34+ cell content that is used to define "Good Mobilizer (GM)" or "Poor Mobilizer (PM)" patients but does not report the real HSC content of grafts. In this study, we assessed the HSC content within the CD34+ fraction of graft samples from 3 groups of patients: 1-GM patients receiving G-CSF only (GMG-CSF), 2-PM patients receiving G-CSF only (PMG-CSF), 3-PM patients receiving G-CSF + Plerixafor (PMG-CSF+P). Although HSC from the 3 groups of patients displayed very similar phenotypic profiles, expression of "stemness" genes and metabolic characteristics, their capacity to engraft NSG mice differed revealing differences in terms of HSC between groups. Indeed according to mobilization regimen, we observed differences in migration capacity of HSC, as well as differences in engraftment intensity depending on the initial pathology (myeloma versus lymphoma) of patients. This suggests that mobilization regimen could strongly influence the long term engraftment efficiency of hematopoietic grafts.
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Antígenos CD34/metabolismo , Factor Estimulante de Colonias de Granulocitos/metabolismo , Compuestos Heterocíclicos/uso terapéutico , Animales , Bencilaminas , Niño , Ciclamas , Femenino , Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
BACKGROUND: Mutations in CALR observed in myeloproliferative neoplasms (MPN) were recently shown to be pathogenic via their interaction with MPL and the subsequent activation of the Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) pathway. However, little is known on the impact of those variant CALR proteins on endoplasmic reticulum (ER) homeostasis. METHODS: The impact of the expression of Wild Type (WT) or mutant CALR on ER homeostasis was assessed by quantifying the expression level of Unfolded Protein Response (UPR) target genes, splicing of X-box Binding Protein 1 (XBP1), and the expression level of endogenous lectins. Pharmacological and molecular (siRNA) screens were used to identify mechanisms involved in CALR mutant proteins degradation. Coimmunoprecipitations were performed to define more precisely actors involved in CALR proteins disposal. RESULTS: We showed that the expression of CALR mutants alters neither ER homeostasis nor the sensitivity of hematopoietic cells towards ER stress-induced apoptosis. In contrast, the expression of CALR variants is generally low because of a combination of secretion and protein degradation mechanisms mostly mediated through the ER-Associated Degradation (ERAD)-proteasome pathway. Moreover, we identified a specific ERAD network involved in the degradation of CALR variants. CONCLUSIONS: We propose that this ERAD network could be considered as a potential therapeutic target for selectively inhibiting CALR mutant-dependent proliferation associated with MPN, and therefore attenuate the associated pathogenic outcomes.
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Acute myeloid leukemia (AML) is a myeloid malignancy carrying a heterogeneous molecular panel of mutations participating in the blockade of differentiation and the increased proliferation of myeloid hematopoietic stem and progenitor cells. The historical "3 + 7" treatment (cytarabine and daunorubicin) is currently challenged by new therapeutic strategies, including drugs depending on the molecular landscape of AML. This panel of mutations makes it possible to combine some of these new treatments with conventional chemotherapy. For example, the FLT3 receptor is overexpressed or mutated in 80% or 30% of AML, respectively. Such anomalies have led to the development of targeted therapies using tyrosine kinase inhibitors (TKIs). In this review, we document the history of TKI targeting, FLT3 and several other tyrosine kinases involved in dysregulated signaling pathways.
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Antineoplásicos/uso terapéutico , Biomarcadores de Tumor , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Ensayos Clínicos como Asunto , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/etiología , Leucemia Mieloide Aguda/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteoma , Transducción de Señal , Transcriptoma , Resultado del TratamientoRESUMEN
BACKGROUND: Natural Killer (NK) cells are innate lymphoid cells that can be cytotoxic toward a large panel of solid tumors and hematological malignancies including chronic myeloid leukemia (CML). Such a cytotoxicity depends on various receptors. Killer immunoglobulin-like receptors (KIR) belong to these receptors and are involved in maturation process, then in the activation abilities of NK cells. METHODS: We investigated the prognostic impact of the KIR2DL5B genotype in 240 CML patients included in two clinical trials investigating tyrosine kinase inhibitors (TKI) discontinuation: STIM and STIM2. RESULTS: After adjustment for standard risk factors in CML, we found that the inhibitory receptor KIR2DL5B-positive genotype was independently related to a delayed second deep molecular remission (HR 0.54, 95% CI [0.32-0.91], P = 0.02) after TKI rechallenge but not to time to first deep molecular remission or treatment-free remission rates. CONCLUSION: These results suggest that KIR2DL5B could carry a role in lymphocyte-mediated control of leukemic residual disease control in patient with CML relapse.
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Variación Genética , Genotipo , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Receptores KIR/genética , Anciano , Antineoplásicos/uso terapéutico , Biomarcadores , Femenino , Haplotipos , Humanos , Mesilato de Imatinib/uso terapéutico , Inmunofenotipificación , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores KIR/metabolismo , Receptores KIR2DL5/genética , Inducción de Remisión , Resultado del Tratamiento , Privación de TratamientoRESUMEN
Internal tandem duplication in Fms-like tyrosine kinase 3 (FLT3-ITD) is the most frequent mutation observed in acute myeloid leukemia (AML) and correlates with poor prognosis. FLT3 tyrosine kinase inhibitors are promising for targeted therapy. Here, we investigated mechanisms dampening the response to the FLT3 inhibitor quizartinib, which is specific to the hematopoietic niche. Using AML primary samples and cell lines, we demonstrate that convergent signals from the hematopoietic microenvironment drive FLT3-ITD cell resistance to quizartinib through the expression and activation of the tyrosine kinase receptor AXL. Indeed, cytokines sustained phosphorylation of the transcription factor STAT5 in quizartinib-treated cells, which enhanced AXL expression by direct binding of a conserved motif in its genomic sequence. Likewise, hypoxia, another well-known hematopoietic niche hallmark, also enhanced AXL expression. Finally, in a xenograft mouse model, inhibition of AXL significantly increased the response of FLT3-ITD cells to quizartinib exclusively within a bone marrow environment. These data highlight a new bypass mechanism specific to the hematopoietic niche that hampers the response to quizartinib through combined upregulation of AXL activity. Targeting this signaling offers the prospect of a new therapy to eradicate resistant FLT3-ITD leukemic cells hidden within their specific microenvironment, thereby preventing relapses from FLT3-ITD clones.
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Benzotiazoles/farmacología , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/metabolismo , Compuestos de Fenilurea/farmacología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Factor de Transcripción STAT5/metabolismo , Microambiente Tumoral , Tirosina Quinasa 3 Similar a fms/metabolismo , Hipoxia de la Célula , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Factor de Transcripción STAT5/genética , Regulación hacia Arriba/efectos de los fármacos , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa del Receptor AxlRESUMEN
BACKGROUND: Atypical Myeloproliferative Neoplasms (aMPN) share characteristics of MPN and Myelodysplastic Syndromes. Although abnormalities in cytokine signaling are common in MPN, the pathophysiology of atypical MPN still remains elusive. Since deregulation of microRNAs is involved in the biology of various cancers, we studied the miRNome of aMPN patients. METHODS: MiRNome and mutations in epigenetic regulator genes ASXL1, TET2, DNMT3A, EZH2 and IDH1/2 were explored in aMPN patients. Epigenetic regulation of miR-10a and HOXB4 expression was investigated by treating hematopoietic cell lines with 5-aza-2'deoxycytidine, valproic acid and retinoic acid. Functional effects of miR-10a overexpression on cell proliferation, differentiation and self-renewal were studied by transducing CD34+ cells with lentiviral vectors encoding the pri-miR-10a precursor. RESULTS: MiR-10a was identified as the most significantly up-regulated microRNA in aMPN. MiR-10a expression correlated with that of HOXB4, sitting in the same genomic locus. The transcription of these two genes was increased by DNA demethylation and histone acetylation, both necessary for optimal expression induction by retinoic acid. Moreover, miR-10a and HOXB4 overexpression seemed associated with DNMT3A mutation in hematological malignancies. However, overexpression of miR-10a had no effect on proliferation, differentiation or self-renewal of normal hematopoietic progenitors. CONCLUSIONS: MiR-10a and HOXB4 are overexpressed in aMPN. This overexpression seems to be the result of abnormalities in epigenetic regulation mechanisms. Our data suggest that miR-10a could represent a simple marker of transcription at this genomic locus including HOXB4, widely recognized as involved in stem cell expansion.
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Expresión Génica , Proteínas de Homeodominio/genética , MicroARNs/genética , Trastornos Mieloproliferativos/genética , Factores de Transcripción/genética , Animales , Biomarcadores , Estudios de Casos y Controles , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Genotipo , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Reacción Leucemoide/genética , Ratones , Mutación , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Factores de Transcripción/metabolismoRESUMEN
Albeit tyrosine kinase inhibitors anti-Abl used in Chronic Myeloid Leukemia (CML) block the deregulated activity of the Bcr-Abl tyrosine kinase and induce remission in 90% of patients, they do not eradicate immature hematopoietic compartments of leukemic stem cells. To elucidate if autophagy is important for stem cell survival and/or proliferation, we used culture in low oxygen concentration (0.1% O2 for 7 days) followed back by non-restricted O2 supply (normoxic culture) to mimic stem cell proliferation and commitment. Knockdown of Atg7 expression, a key player in autophagy, in K562 cell line inhibited autophagy compared to control cells. Upon 7 days at 0.1% O2 both K562 and K562 shATG7 cells stopped to proliferate and a similar amount of viable cells remained. Back to non-restricted O2 supply K562 cells proliferate whereas K562 shATG7 cells exhibited strong apoptosis. Using immunomagnetic sorted normal and CML CD34+ cells, we inhibited the autophagic process by lentiviral infection expressing shATG7 or using a Vps34 inhibitor. Both, normal and CML CD34+ cells either competent or deficient for autophagy stopped to proliferate in hypoxia. Surprisingly, while normal CD34+ cells proliferate back to non restricted O2 supply, the CML CD34+ cells deficient for autophagy failed to proliferate. All together, these results suggest that autophagy is required for CML CD34+ commitment while it is dispensable for normal CD34 cells.
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Acute basophilic leukaemia (ABL) is a rare subtype of acute myeloblastic leukaemia. We previously described a recurrent t(X;6)(p11;q23) translocation generating an MYB-GATA1 fusion gene in male infants with ABL. To better understand its role, the chimeric MYB-GATA1 transcription factor was expressed in CD34-positive haematopoietic progenitors, which were transplanted into immunodeficient mice. Cells expressing MYB-GATA1 showed increased expression of markers of immaturity (CD34), of granulocytic lineage (CD33 and CD117), and of basophilic differentiation (CD203c and FcϵRI). UT-7 cells also showed basophilic differentiation after MYB-GATA1 transfection. A transcriptomic study identified nine genes deregulated by both MYB-GATA1 and basophilic differentiation. Induction of three of these genes (CCL23, IL1RL1, and NTRK1) was confirmed in MYB-GATA1-expressing CD34-positive cells by reverse transcription quantitative polymerase chain reaction. Interleukin (IL)-33 and nerve growth factor (NGF), the ligands of IL-1 receptor-like 1 (IL1RL1) and neurotrophic receptor tyrosine kinase 1 (NTRK1), respectively, enhanced the basophilic differentiation of MYB-GATA1-expressing UT-7 cells, thus demonstrating the importance of this pathway in the basophilic differentiation of leukaemic cells and CD34-positive primary cells. Finally, gene reporter assays confirmed that MYB and MYB-GATA1 directly activated NTRK1 and IL1RL1 transcription, leading to basophilic skewing of the blasts. MYB-GATA1 is more efficient than MYB, because of better stability. Our results highlight the role of IL-33 and NGF receptors in the basophilic differentiation of normal and leukaemic cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Interleucina-33/fisiología , Leucemia Basofílica Aguda/etiología , Receptores de Factor de Crecimiento Nervioso/fisiología , Animales , Transformación Celular Neoplásica/genética , Femenino , Factor de Transcripción GATA1/genética , Fusión Génica/fisiología , Células Madre Hematopoyéticas/fisiología , Masculino , Ratones SCID , Trasplante de Neoplasias , Proteínas Oncogénicas v-myb/genética , Receptor trkA/metabolismo , Factores de Transcripción/metabolismo , Transfección , Trasplante HeterólogoRESUMEN
Purpose: AXL receptor tyrosine kinase has been described as a relevant molecular marker and a key player in invasiveness, especially in triple-negative breast cancer (TNBC).Experimental Design: We evaluate the antitumor efficacy of the anti-AXL monoclonal antibody 20G7-D9 in several TNBC cell xenografts or patient-derived xenograft (PDX) models and decipher the underlying mechanisms. In a dataset of 254 basal-like breast cancer samples, genes correlated with AXL expression are enriched in EMT, migration, and invasion signaling pathways.Results: Treatment with 20G7-D9 inhibited tumor growth and bone metastasis formation in AXL-positive TNBC cell xenografts or PDX, but not in AXL-negative PDX, highlighting AXL role in cancer growth and invasion. In vitro stimulation of AXL-positive cancer cells by its ligand GAS6 induced the expression of several EMT-associated genes (SNAIL, SLUG, and VIM) through an intracellular signaling implicating the transcription factor FRA-1, important in cell invasion and plasticity, and increased their migration/invasion capacity. 20G7-D9 induced AXL degradation and inhibited all AXL/GAS6-dependent cell signaling implicated in EMT and in cell migration/invasion.Conclusions: The anti-AXL antibody 20G7-D9 represents a promising therapeutic strategy in TNBC with mesenchymal features by inhibiting AXL-dependent EMT, tumor growth, and metastasis formation. Clin Cancer Res; 23(11); 2806-16. ©2016 AACR.
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Anticuerpos Antiidiotipos/administración & dosificación , Proliferación Celular/efectos de los fármacos , Proteínas Proto-Oncogénicas/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Neoplasias de la Mama Triple Negativas/terapia , Animales , Anticuerpos Antiidiotipos/inmunología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/inmunología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Xenoinjertos , Humanos , Ratones , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa del Receptor AxlRESUMEN
The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication.
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VIH-1/fisiología , Recombinasa Rad51/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/química , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacología , ADN/química , ADN/metabolismo , Reparación del ADN , Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Morfolinas/química , Morfolinas/metabolismo , Morfolinas/farmacología , Unión Proteica , Pirroles/química , Pirroles/metabolismo , Pirroles/farmacología , Recombinasa Rad51/química , Recombinasa Rad51/genética , Estilbenos/química , Estilbenos/metabolismo , Estilbenos/farmacología , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
A tyrosine kinase network composed of the TAM receptor AXL and the cytoplasmic kinases LYN and SYK is involved in nilotinib-resistance of chronic myeloid leukaemia (CML) cells. Here, we show that the E3-ubiquitin ligase CBL down-regulation occurring during prolonged drug treatment plays a critical role in this process. Depletion of CBL in K562 cells increases AXL and LYN protein levels, promoting cell resistance to nilotinib. Conversely, forced expression of CBL in nilotinib-resistant K562 cells (K562-rn) dramatically reduces AXL and LYN expression and resensitizes K562-rn cells to nilotinib. A similar mechanism was found to operate in primary CML CD34(+) cells. Mechanistically, the E3-ligase CBL counteracts AXL/SYK signalling, promoting LYN transcription by controlling AXL protein stability. Surprisingly, the role of AXL in resistance was independent of its ligand GAS6 binding and its TK activity, in accordance with a scaffold activity for this receptor being involved in this cellular process. Collectively, our results demonstrate a pivotal role for CBL in the control of a tyrosine kinase network mediating resistance to nilotinib treatment in CML cells.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos , Familia-src Quinasas/metabolismo , Estabilidad de Enzimas , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ligandos , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl/genética , Interferencia de ARN , Proteínas Tirosina Quinasas Receptoras/genética , Quinasa Syk , Factores de Tiempo , Transfección , Familia-src Quinasas/genética , Tirosina Quinasa del Receptor AxlRESUMEN
ATF6α, a membrane-anchored transcription factor from the endoplasmic reticulum (ER) that modulates the cellular response to stress as an effector of the unfolded-protein response (UPR), is a key player in the development of tumors of different origin. ATF6α activation has been linked to oncogenic transformation and tumor maintenance; however, the mechanism(s) underlying this phenomenon remains elusive. Here, using a phenotypic small interfering RNA (siRNA) screening, we identified a novel role for ATF6α in chemoresistance and defined the protein disulfide isomerase A5 (PDIA5) as necessary for ATF6α activation upon ER stress. PDIA5 contributed to disulfide bond rearrangement in ATF6α under stress conditions, thereby leading to ATF6α export from the ER and activation of its target genes. Further analysis of the mechanism demonstrated that PDIA5 promotes ATF6α packaging into coat protein complex II (COPII) vesicles and that the PDIA5/ATF6α activation loop is essential to confer chemoresistance on cancer cells. Genetic and pharmacological inhibition of the PDIA5/ATF6α axis restored sensitivity to the drug treatment. This work defines the mechanisms underlying the role of ATF6α activation in carcinogenesis and chemoresistance; furthermore, it identifies PDIA5 as a key regulator ATF6α-mediated cellular functions in cancer.
Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Antineoplásicos/farmacología , Benzamidas/farmacología , Piperazinas/farmacología , Proteína Disulfuro Isomerasas/metabolismo , Pirimidinas/farmacología , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Supervivencia Celular/efectos de los fármacos , Cistina/metabolismo , Resistencia a Antineoplásicos , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Mesilato de Imatinib , Transporte de Proteínas , Respuesta de Proteína DesplegadaRESUMEN
Chronic myeloid leukemia disease (CML) found effective therapy by treating patients with tyrosine kinase inhibitors (TKI), which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs) can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs) derived from CD34⺠blood cells isolated from CML patients (CML-iPSCs) as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents.
Asunto(s)
Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Hematopoyesis/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Humanos , Células Madre Pluripotentes Inducidas/citología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Células Tumorales CultivadasRESUMEN
In BCR-ABL-expressing cells, sphingolipid metabolism is altered. Because the first step of sphingolipid biosynthesis occurs in the endoplasmic reticulum (ER), our objective was to identify ABL targets in the ER. A phosphoproteomic analysis of canine pancreatic ER microsomes identified 49 high scoring phosphotyrosine-containing peptides. These were then categorized in silico and validated in vitro. We demonstrated that the ER-resident human protein serine palmitoyltransferase long chain-1 (SPTLC1), which is the first enzyme of sphingolipid biosynthesis, is phosphorylated at Tyr(164) by the tyrosine kinase ABL. Inhibition of BCR-ABL using either imatinib or shRNA-mediated silencing led to the activation of SPTLC1 and to increased apoptosis in both K562 and LAMA-84 cells. Finally, we demonstrated that mutation of Tyr(164) to Phe in SPTLC1 increased serine palmitoyltransferase activity. The Y164F mutation also promoted the remodeling of cellular sphingolipid content, thereby sensitizing K562 cells to apoptosis. Our observations provide a mechanistic explanation for imatinib-mediated cell death and a novel avenue for therapeutic strategies.