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Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma having a poor overall survival that is in need for the development of new therapeutics. In this study, we report the identification and expression of a new isoform splice variant of the tyrosine kinase receptor AXL in MCL cells. This new AXL isoform, called AXL3, lacks the ligand-binding domain of the commonly described AXL splice variants and is constitutively activated in MCL cells. Interestingly, functional characterization of AXL3, using CRISPR inhibition, revealed that only the knock down of this isoform leads to apoptosis of MCL cells. Importantly, pharmacological inhibition of AXL activity resulted in a significant decrease in the activation of well-known proproliferative and survival pathways activated in MCL cells (ie, ß-catenin, Ak strain transforming, and NF-κB). Therapeutically, preclinical studies using a xenograft mouse model of MCL indicated that bemcentinib is more effective than ibrutinib in reducing the tumor burden and to increase the overall survival. Our study highlights the importance of a previously unidentified AXL splice variant in cancer and the potential of bemcentinib as a targeted therapy for MCL.
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Linfoma de Célula do Manto , Proteínas Tirosina Quinases , Humanos , Adulto , Animais , Camundongos , Tirosina Quinase da Agamaglobulinemia , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , ApoptoseRESUMO
BACKGROUND AND AIMS: Acute myeloid leukemia (AML) is a heterogeneous malignant condition characterized by massive infiltration of poorly differentiated white blood cells in the blood stream, bone marrow, and extramedullary sites. During leukemic development, hepatosplenomegaly is expected to occur because large blood volumes are continuously filtered through these organs. We asked whether infiltration of leukemic blasts initiated a response that could be detected in the interstitial fluid phase of the spleen and liver. MATERIAL AND METHODS: We used a rat model known to mimic human AML in growth characteristics and behavior. By cannulating efferent lymphatic vessels from the spleen and liver, we were able to monitor the response of the microenvironment during AML development. RESULTS AND DISCUSSION: Flow cytometric analysis of lymphocytes showed increased STAT3 and CREB signaling in spleen and depressed signaling in liver, and proteins related to these pathways were identified with a different profile in lymph and plasma in AML compared with control. Additionally, several proteins were differently regulated in the microenvironment of spleen and liver in AML when compared with control. CONCLUSION: Interstitial fluid, and its surrogate efferent lymph, can be used to provide unique information about responses in AML-infiltered organs and substances released to the general circulation during leukemia development.
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Leucemia Mieloide Aguda , Vasos Linfáticos , Animais , Humanos , Ratos , Medula Óssea/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Fígado/metabolismo , Vasos Linfáticos/metabolismo , Baço/metabolismo , Baço/patologia , Fator de Transcrição STAT3/metabolismo , Microambiente TumoralRESUMO
BACKGROUND: Personalised medicine is a medical model that aims to provide tailor-made prevention and treatment strategies for defined groups of individuals. The concept brings new challenges to the translational step, both in clinical relevance and validity of models. We have developed a set of recommendations aimed at improving the robustness of preclinical methods in translational research for personalised medicine. METHODS: These recommendations have been developed following four main steps: (1) a scoping review of the literature with a gap analysis, (2) working sessions with a wide range of experts in the field, (3) a consensus workshop, and (4) preparation of the final set of recommendations. RESULTS: Despite the progress in developing innovative and complex preclinical model systems, to date there are fundamental deficits in translational methods that prevent the further development of personalised medicine. The literature review highlighted five main gaps, relating to the relevance of experimental models, quality assessment practices, reporting, regulation, and a gap between preclinical and clinical research. We identified five points of focus for the recommendations, based on the consensus reached during the consultation meetings: (1) clinically relevant translational research, (2) robust model development, (3) transparency and education, (4) revised regulation, and (5) interaction with clinical research and patient engagement. Here, we present a set of 15 recommendations aimed at improving the robustness of preclinical methods in translational research for personalised medicine. CONCLUSIONS: Appropriate preclinical models should be an integral contributor to interventional clinical trial success rates, and predictive translational models are a fundamental requirement to realise the dream of personalised medicine. The implementation of these guidelines is ambitious, and it is only through the active involvement of all relevant stakeholders in this field that we will be able to make an impact and effectuate a change which will facilitate improved translation of personalised medicine in the future.
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Medicina de Precisão , HumanosRESUMO
Chronic myeloid leukemia (CML) is a stem cell disease of the bone marrow where mechanisms of inter-leukemic communication and cell-to-cell interactions are proposed to be important for optimal therapy response. Tunneling nanotubes (TNTs) are novel intercellular communication structures transporting different cargos with potential implications in therapy resistance. Here, we have investigated TNTs in CML cells and following treatment with the highly effective CML therapeutics tyrosine kinase inhibitors (TKIs) and interferon-α (IFNα). CML cells from chronic phase CML patients as well as the blast crisis phase cell lines, Kcl-22 and K562, formed few or no TNTs. Treatment with imatinib increased TNT formation in both Kcl-22 and K562 cells, while nilotinib or IFNα increased TNTs in Kcl-22 cells only where the TNT increase was associated with adherence to fibronectin-coated surfaces, altered morphology, and reduced movement involving ß1integrin. Ex vivo treated cells from chronic phase CML patients showed limited changes in TNT formation similarly to bone marrow cells from healthy individuals. Interestingly, in vivo nilotinib treatment in a Kcl-22 subcutaneous mouse model resulted in morphological changes and TNT-like structures in the tumor-derived Kcl-22 cells. Our results demonstrate that CML cells express low levels of TNTs, but CML therapeutics increase TNT formation in designated cell models indicating TNT functionality in bone marrow derived malignancies and their microenvironment.
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Adesão Celular/efeitos dos fármacos , Interferon-alfa/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Imunofluorescência , Humanos , Immunoblotting , Integrina beta1/metabolismo , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal adenocarcinoma (PDAC) cell lines has not been elucidated. We hypothesized that CDA regulates gemcitabine flux through its inactivation and activation pathways in PDAC cell lines. Three PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) were incubated with 10 or 100 µM gemcitabine for 60 minutes or 24 hours, with or without tetrahydrouridine, a CDA inhibitor. Extracellular inactive gemcitabine metabolite (dFdU) and intracellular active metabolite (dFdCTP) were quantified with liquid chromatography tandem mass spectrometry. Cellular expression of CDA was assessed with real-time PCR and Western blot. Gemcitabine conversion to dFdU was extensive in BxPC-3 and low in MIA PaCa-2 and PANC-1, in accordance with their respective CDA expression levels. CDA inhibition was associated with low or undetectable dFdU in all three cell lines. After 24 hours gemcitabine incubation, dFdCTP was highest in MIA PaCa-2 and lowest in BxPC-3. CDA inhibition resulted in a profound dFdCTP increase in BxPC-3 but not in MIA PaCa-2 or PANC-1. dFdCTP concentrations were not higher after exposure to 100 versus 10 µM gemcitabine when CDA activities were low (MIA PaCa-2 and PANC-1) or inhibited (BxPC-3). The results suggest a regulatory role of CDA for gemcitabine activation in PDAC cells but within limits related to the capacity in the activation pathway in the cell lines. SIGNIFICANCE STATEMENT: The importance of cytidine deaminase (CDA) for cellular gemcitabine toxicity, linking a lower activity to higher toxicity, is well described. An underlying assumption is that CDA, by inactivating gemcitabine, limits the amount available for the intracellular activation pathway. Our study is the first to illustrate this regulatory role of CDA in pancreatic ductal adenocarcinoma cell lines by quantifying intracellular and extracellular gemcitabine metabolite concentrations.
Assuntos
Citidina Desaminase/metabolismo , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/metabolismo , Humanos , GencitabinaRESUMO
The bone marrow (BM) provides a protective microenvironment to support the survival of leukemic cells and influence their response to therapeutic agents. In acute myeloid leukemia (AML), the high rate of relapse may in part be a result of the inability of current treatment to effectively overcome the protective influence of the BM niche. To better understand the effect of the BM microenvironment on drug responses in AML, we conducted a comprehensive evaluation of 304 inhibitors, including approved and investigational agents, comparing ex vivo responses of primary AML cells in BM stroma-derived and standard culture conditions. In the stroma-based conditions, the AML patient cells exhibited significantly reduced sensitivity to 12% of the tested compounds, including topoisomerase II, B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2), and many tyrosine kinase inhibitors (TKIs). The loss of TKI sensitivity was most pronounced in patient samples harboring FLT3 or PDGFRB alterations. In contrast, the stroma-derived conditions enhanced sensitivity to Janus kinase (JAK) inhibitors. Increased cell viability and resistance to specific drug classes in the BM stroma-derived conditions was a result of activation of alternative signaling pathways mediated by factors secreted by BM stromal cells and involved a switch from BCL2 to BCLXL-dependent cell survival. Moreover, the JAK1/2 inhibitor ruxolitinib restored sensitivity to the BCL2 inhibitor venetoclax in AML patient cells ex vivo in different model systems and in vivo in an AML xenograft mouse model. These findings highlight the potential of JAK inhibitors to counteract stroma-induced resistance to BCL2 inhibitors in AML.
Assuntos
Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirazóis/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Nitrilas , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazóis/farmacologia , Pirimidinas , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologia , Sulfonamidas/farmacologia , Células Tumorais CultivadasRESUMO
Internal tandem duplications in the tyrosine kinase receptor FLT3 (FLT3-ITD) are among the most common lesions in acute myeloid leukemia and there exists a need for new forms of treatment. Using ex vivo drug sensitivity screening, we found that FLT3-ITD+ patient cells are particularly sensitive to HSP90 inhibitors. While it is well known that HSP90 is important for FLT3-ITD stability, we found that HSP90 family members play a much more complex role in FLT3-ITD signaling than previously appreciated. First, we found that FLT3-ITD activates the unfolded protein response, leading to increased expression of GRP94/HSP90B1. This results in activation of a nefarious feedback loop, in which GRP94 rewires FLT3-ITD signaling by binding and retaining FLT3-ITD in the endoplasmic reticulum, leading to aberrant activation of downstream signaling pathways and further inducing the unfolded protein response. Second, HSP90 family proteins protect FLT3-ITD+ acute myeloid leukemia cells against apoptosis by alleviating proteotoxic stress, and treatment with HSP90 inhibitors results in proteotoxic overload that triggers unfolded protein response-induced apoptosis. Importantly, leukemic stem cells are strongly dependent upon HSP90 for their survival, and the HSP90 inhibitor ganetespib causes leukemic stem cell exhaustion in patient-derived mouse xenograft models. Taken together, our study reveals a molecular basis for HSP90 addiction of FLT3-ITD+ acute myeloid leukemia cells and provides a rationale for including HSP90 inhibitors in the treatment regime for FLT3-ITD+ acute myeloid leukemia.
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BACKGROUND: Uterine serous carcinoma (USC) is a rare but aggressive subtype of endometrial carcinoma. Large-scale comprehensive efforts have resulted in an improved molecular understanding of its pathogenesis, and the p53 pathway has been proposed as a key player and is potentially targetable. Here we attempt to further portray the p53 pathway in USC by assessing p53 isoform expression. METHODS: We applied quantitative Real-Time PCRs (RT-qPCR) for expression analyses of total p53 mRNA as well as quantitative distinction of p53ß, p53γ, and the total mRNA of amino-terminal truncated Δ40p53 and Δ133p53 in a retrospective cohort of 37 patients with USC. TP53 mutation status was assessed by targeted massive parallel sequencing. Findings were correlated with clinical data. RESULTS: The p53 isoform expression landscape in USCs was heterogeneous and dominated by total Δ133p53, while the distinct p53ß and p53γ variants were found at much lower levels. The isoform expression profiles varied between samples, while their expression was independent of TP53 mutation status. We found high relative p53γ expression to be associated with reduced progression-free survival (PFS). CONCLUSIONS: This is the first indication that elevated p53γ expression is associated with reduced PFS in USC. This single-center study may offer some insight in the landscape of p53 isoform expression in USC, but further validation studies are crucial to understand the context-dependent and tissue-specific role of the p53 isoform network in gynecological cancer.
Assuntos
Cistadenocarcinoma Seroso/genética , Proteína Supressora de Tumor p53/genética , Neoplasias Uterinas/genética , Idoso , Idoso de 80 Anos ou mais , Cistadenocarcinoma Seroso/mortalidade , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Intervalo Livre de Progressão , Isoformas de Proteínas/genética , RNA Mensageiro/análise , Estudos Retrospectivos , Proteína Supressora de Tumor p53/fisiologia , Neoplasias Uterinas/mortalidadeRESUMO
Cancer stem cells (CSCs) are a challenge in cancer treatment due to their therapy resistance. We demonstrated that enhanced Notch signaling in breast cancer promotes self-renewal of CSCs that display high glycolytic activity and aggressive hormone-independent tumor growth in vivo. We took advantage of the glycolytic phenotype and the dependence on Notch activity of the CSCs and designed nanoparticles to target the CSCs. Mesoporous silica nanoparticles were functionalized with glucose moieties and loaded with a γ-secretase inhibitor, a potent interceptor of Notch signaling. Cancer cells and CSCs in vitro and in vivo efficiently internalized these particles, and particle uptake correlated with the glycolytic profile of the cells. Nanoparticle treatment of breast cancer transplants on chick embryo chorioallantoic membranes efficiently reduced the cancer stem cell population of the tumor. Our data reveal that specific CSC characteristics can be utilized in nanoparticle design to improve CSC-targeted drug delivery and therapy.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/administração & dosagem , Glucose/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores Notch/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Antineoplásicos/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Células MCF-7 , Nanopartículas/administração & dosagem , Nanopartículas/química , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Linhagem Celular/transplante , Síndromes Mielodisplásicas/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Anemia Refratária/diagnóstico , Anemia Refratária/etiologia , Anemia Refratária/patologia , Animais , Linhagem Celular/metabolismo , Epigenômica , Humanos , Hidrogéis , Hibridização in Situ Fluorescente/métodos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Animais , Mutação , Síndromes Mielodisplásicas/complicações , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologiaRESUMO
Antibodies play a fundamental role in diagnostic immunophenotyping of leukemias and in cell-targeting therapy. However, this versatility is not reflected in imaging diagnostics. In the present study, we labeled antihuman mAbs monochromatically against selected human myeloid markers expressed on acute myeloid leukemia (AML) cells, all with the same near-infrared fluorochrome. In a novel "multiplexing" strategy, we then combined these mAbs to overcome the limiting target-to-background ratio to image multiple xenografts of AML. Time-domain imaging was used to discriminate autofluorescence from the distinct fluorophore-conjugated antibodies. Imaging with multiplexed mAbs demonstrated superior imaging of AML to green fluorescent protein or bioluminescence and permitted evaluation of therapeutic efficacy with the standard combination of anthracycline and cytarabine in primary patient xenografts. Multiplexing mAbs against CD11b and CD11c provided surrogate imaging biomarkers of differentiation therapy in an acute promyelocytic leukemia model treated with all-trans retinoic acid combined with the histone-deacetylase inhibitor valproic acid. We present herein an optimizedapplication of multiplexed immunolabeling in vivo for optical imaging of AML cellxenografts that provides reproducible, highly accurate disease staging and monitoring of therapeutic effects.
Assuntos
Anticorpos Monoclonais , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/imunologia , Animais , Antraciclinas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Linhagem Celular Tumoral , Citarabina/administração & dosagem , Corantes Fluorescentes , Proteínas de Fluorescência Verde , Células HL-60 , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/imunologia , Medições Luminescentes , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Imagem Óptica/métodos , Tretinoína/administração & dosagem , Ácido Valproico/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The environments that harbor hematopoietic stem and progenitor cells are critical to explore for a better understanding of hematopoiesis during health and disease. These compartments often are inaccessible for controlled and rapid experimentation, thus limiting studies to the evaluation of conventional cell culture and transgenic animal models. Here we describe the manufacture and image-guided monitoring of an engineered microenvironment with user-defined properties that recruits hematopoietic progenitors into the implant. Using intravital imaging and fluorescence molecular tomography, we show in real time that the cell homing and retention process is efficient and durable for short- and long-term engraftment studies. Our results indicate that bone marrow stromal cells, precoated on the implant, accelerate the formation of new sinusoidal blood vessels with vascular integrity at the microcapillary level that enhances the recruitment hematopoietic progenitor cells to the site. This implantable construct can serve as a tool enabling the study of hematopoiesis.
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Células-Tronco Hematopoéticas/citologia , Neoplasias/patologia , Nicho de Células-Tronco , Alicerces Teciduais , Microambiente Tumoral , Animais , Matriz Extracelular , Humanos , Hidrogéis , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Microscopia Confocal , Tomografia/métodosRESUMO
BACKGROUND: The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy. RESULTS: Nutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells. CONCLUSIONS: Altogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML.
Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Regulação Leucêmica da Expressão Gênica , Proteínas de Choque Térmico HSP27/metabolismo , Histonas/metabolismo , Imidazóis/farmacologia , Lactamas Macrocíclicas/farmacologia , Piperazinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Linhagem Celular Tumoral , Sinergismo Farmacológico , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Histonas/genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Cultura Primária de Células , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells in the bone marrow and the peripheral blood. Nearly half of the AML patients relapse after standard induction therapy, and new forms of therapy are urgently needed. Chimeric antigen receptor (CAR) T therapy has so far not been successful in AML due to lack of efficacy and safety. Indeed, the most attractive antigen targets are stem cell markers such as CD33 or CD123. We demonstrate that CD37, a mature B cell marker, is expressed in AML samples, and its presence correlates with the European LeukemiaNet (ELN) 2017 risk stratification. We repurpose the anti-lymphoma CD37CAR for the treatment of AML and show that CD37CAR T cells specifically kill AML cells, secrete proinflammatory cytokines, and control cancer progression in vivo. Importantly, CD37CAR T cells display no toxicity toward hematopoietic stem cells. Thus, CD37 is a promising and safe CAR T cell AML target.
Assuntos
Imunoterapia Adotiva , Leucemia Mieloide Aguda , Receptores de Antígenos Quiméricos , Humanos , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Animais , Imunoterapia Adotiva/métodos , Camundongos , Tetraspaninas/imunologia , Linhagem Celular Tumoral , Linfócitos T/imunologia , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos de Diferenciação Mielomonocítica/imunologia , Feminino , Masculino , Antígenos de NeoplasiasRESUMO
BACKGROUND: Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and the evasion of host immune responses. Currently, debulking surgery and platinum-based chemotherapy are the treatment cornerstones, although recurrence is common. As the clinical efficacy of immune checkpoint blockade is low, new immunotherapeutic strategies are needed. Chimeric antigen receptor (CAR) T cell therapy empowers patients' own T cells to fight and eradicate cancer, and has been tested against various targets in OC. A promising candidate is the MUC16 ectodomain. This ectodomain remains on the cell surface after cleavage of cancer antigen 125 (CA125), the domain distal from the membrane, which is currently used as a serum biomarker for OC. CA125 itself has not been tested as a possible CAR target. In this study, we examined the suitability of the CA125 as a target for CAR T cell therapy. METHODS: We tested a series of antibodies raised against the CA125 extracellular repeat domain of MUC16 and adapted them to the CAR format. Comparisons between these candidates, and against an existing CAR targeting the MUC16 ectodomain, identified K101 as having high potency and specificity. The K101CAR was subjected to further biochemical and functional tests, including examination of the effect of soluble CA125 on its activity. Finally, we used cell lines and advanced orthotopic patient-derived xenograft (PDX) models to validate, in vivo, the efficiency of our K101CAR construct. RESULTS: We observed a high efficacy of K101CAR T cells against cell lines and patient-derived tumors, in vitro and in vivo. We also demonstrated that K101CAR functionality was not impaired by the soluble antigen. Finally, in direct comparisons, K101CAR, which targets the CA125 extracellular repeat domains, was shown to have similar efficacy to the previously validated 4H11CAR, which targets the MUC16 ectodomain. CONCLUSIONS: Our in vitro and in vivo results, including PDX studies, demonstrate that the CA125 domain of MUC16 represents an excellent target for treating MUC16-positive malignancies.
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Antígeno Ca-125 , Proteínas de Membrana , Feminino , Humanos , Antígeno Ca-125/metabolismo , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
MicroRNAs play critical roles in tumorigenesis and metastasis. Here, we report the dual functions of miR-182 and miR-203 in our previously described prostate cell model. MiR-182 and miR-203 were completely repressed during epithelial to mesenchymal transition (EMT) from prostate epithelial EP156T cells to the progeny mesenchymal nontransformed EPT1 cells. Re-expression of miR-182 or miR-203 in EPT1 cells and prostate cancer PC3 cells induced mesenchymal to epithelial transition (MET) features. Simultaneously, miR-182 and miR-203 provided EPT1 cells with the ability to self-sufficiency of growth signals, a well-recognized oncogenic feature. Gene expression profiling showed high overlap of the genes affected by miR-182 and miR-203. SNAI2 was identified as a common target of miR-182 and miR-203. Knock-down of SNAI2 in EPT1 cells phenocopied re-expression of either miR-182 or miR-203 regarding both MET and self-sufficiency of growth signals. Strikingly, considerable overlaps of changed genes were found between the re-expression of miR-182/203 and knock-down of SNAI2. Finally, P-cadherin was identified as a direct target of SNAI2. We conclude that miR-182 and miR-203 induce MET features and growth factor independent growth via repressing SNAI2 in prostate cells. Our findings shed new light on the roles of miR-182/203 in cancer related processes.
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MicroRNAs/metabolismo , Próstata/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Animais , Caderinas/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , Próstata/citologia , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução GenéticaRESUMO
Glioblastoma multiforme (GBM), the most aggressive brain malignancy, is characterized by extensive cellular proliferation, angiogenesis, and single-cell infiltration into the brain. We have previously shown that a xenograft model based on serial xenotransplantation of human biopsy spheroids in immunodeficient rodents maintains the genotype and phenotype of the original patient tumor. The present work further extends this model for optical assessment of tumor engraftment and growth using bioluminescence imaging (BLI). A method for successful lentiviral transduction of the firefly luciferase gene into multicellular spheroids was developed and implemented to generate optically active patient tumor cells. Luciferase-expressing spheroids were injected into the brains of immunodeficient mice. BLI photon counts and tumor volumes from magnetic resonance imaging (MRI) were correlated. Luciferase-expressing tumors recapitulated the histopathologic hallmarks of human GBMs and showed proliferation rates and microvessel density counts similar to those of wild-type xenografts. Moreover, we detected widespread invasion of luciferase-positive tumor cells in the mouse brains. Herein we describe a novel optically active model of GBM that closely mimics human pathology with respect to invasion, angiogenesis, and proliferation indices. The model may thus be routinely used for the assessment of novel anti-GBM therapeutic approaches implementing well-established and cost-effective optical imaging strategies.
Assuntos
Biópsia/métodos , Glioblastoma/diagnóstico , Medições Luminescentes/métodos , Imageamento por Ressonância Magnética/métodos , Imagem Molecular/métodos , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos SCID , Transplante de Neoplasias , Transplante HeterólogoRESUMO
NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157-165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157-165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1157-165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Proteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias/biossíntese , Antígenos de Superfície/biossíntese , Complexo CD3/imunologia , Linhagem Celular Tumoral , Epitopos/imunologia , Feminino , Antígeno HLA-A2/imunologia , Humanos , Fragmentos de Imunoglobulinas/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Melanoma/imunologia , Melanoma/metabolismo , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Distribuição Aleatória , Proteínas Recombinantes de Fusão/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imaging-based experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.