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The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused over 5 million deaths worldwide. Pneumonia and systemic inflammation contribute to its high mortality. Many viruses use heparan sulfate proteoglycans as coreceptors for viral entry, and heparanase (HPSE) is a known regulator of both viral entry and inflammatory cytokines. We evaluated the heparanase inhibitor Roneparstat, a modified heparin with minimum anticoagulant activity, in pathophysiology and therapy for COVID-19. We found that Roneparstat significantly decreased the infectivity of SARS-CoV-2, SARS-CoV-1, and retroviruses (human T-lymphotropic virus 1 [HTLV-1] and HIV-1) in vitro. Single-cell RNA sequencing (scRNA-seq) analysis of cells from the bronchoalveolar lavage fluid of COVID-19 patients revealed a marked increase in HPSE gene expression in CD68+ macrophages compared to healthy controls. Elevated levels of HPSE expression in macrophages correlated with the severity of COVID-19 and the expression of inflammatory cytokine genes, including IL6, TNF, IL1B, and CCL2. In line with this finding, we found a marked induction of HPSE and numerous inflammatory cytokines in human macrophages challenged with SARS-CoV-2 S1 protein. Treatment with Roneparstat significantly attenuated SARS-CoV-2 S1 protein-mediated inflammatory cytokine release from human macrophages, through disruption of NF-κB signaling. HPSE knockdown in a macrophage cell line also showed diminished inflammatory cytokine production during S1 protein challenge. Taken together, this study provides a proof of concept that heparanase is a target for SARS-CoV-2-mediated pathogenesis and that Roneparstat may serve as a dual-targeted therapy to reduce viral infection and inflammation in COVID-19. IMPORTANCE The complex pathogenesis of COVID-19 consists of two major pathological phases: an initial infection phase elicited by SARS-CoV-2 entry and replication and an inflammation phase that could lead to tissue damage, which can evolve into acute respiratory failure or even death. While the development and deployment of vaccines are ongoing, effective therapy for COVID-19 is still urgently needed. In this study, we explored HPSE blockade with Roneparstat, a phase I clinically tested HPSE inhibitor, in the context of COVID-19 pathogenesis. Treatment with Roneparstat showed wide-spectrum anti-infection activities against SARS-CoV-2, HTLV-1, and HIV-1 in vitro. In addition, HPSE blockade with Roneparstat significantly attenuated SARS-CoV-2 S1 protein-induced inflammatory cytokine release from human macrophages through disruption of NF-κB signaling. Together, this study provides a proof of principle for the use of Roneparstat as a dual-targeting therapy for COVID-19 to decrease viral infection and dampen the proinflammatory immune response mediated by macrophages.
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Tratamento Farmacológico da COVID-19 , Heparina/análogos & derivados , Linhagem Celular , Citocinas/metabolismo , Fenofibrato , Técnicas de Silenciamento de Genes , Glucuronidase/genética , Glucuronidase/metabolismo , Heparina/uso terapêutico , Humanos , Imunidade/efeitos dos fármacos , Inflamação , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , NF-kappa B , SARS-CoV-2RESUMO
Fumagillin, an unstable anti-angiogenesis mycotoxin, was synthesized into a stable lipase-labile prodrug and incorporated into integrin-targeted lipid-encapsulated nanoparticles (αvß3-Fum-PD NP). Dual anti-angiogenic therapy combining αvß3-Fum-PD NP with zoledronic acid (ZA), a long-acting osteoclast inhibitor with proposed anti-angiogenic effects, was evaluated. In vitro, αvß3-Fum-PD NP reduced (P<0.05) endothelial cell viability without impacting macrophage viability. ZA suppressed (P<0.05) macrophage viability at high dosages but not endothelial cell proliferation. 3D MR neovascular imaging of rabbit Vx2 tumors showed no effect with ZA, whereas αvß3-Fum-PD NP alone and with ZA decreased angiogenesis (P<0.05). Immunohistochemistry revealed decreased (P<0.05) microvascularity with αvß3-Fum-PD NP and ZA and further microvascular reduction (P<0.05) with dual-therapy. In vivo, ZA did not decrease tumor macrophage numbers nor cancer cell proliferation, whereas αvß3-Fum-PD-NPs reduced both measures. Dual-therapy with ZA and αvß3-Fum-PD-NP may provide enhanced neo-adjuvant utility if macrophage ZA uptake is increased. From the Clinical Editor: Although anti-angiogenesis is one of the treatment modalities in the fight against cancer, many cancers become resistant to VEGF pathway inhibitors. In this article, the authors investigated the use of dual therapy using fumagillin, integrin-targeted lipid-encapsulated nanoparticles (αvß3- Fum-PD NP) and zoledronic acid (ZA), in both in-vitro and in-vivo experiments. This combination approach may provide an insight to the design of future drugs against cancers.
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Sistemas de Transporte de Aminoácidos Neutros/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Difosfonatos/administração & dosagem , Imidazóis/administração & dosagem , Integrina alfaVbeta3/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Pró-Fármacos/administração & dosagem , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/química , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/química , Linhagem Celular Tumoral , Difusão , Difosfonatos/química , Imidazóis/química , Masculino , Terapia de Alvo Molecular/métodos , Nanocápsulas/administração & dosagem , Nanocápsulas/química , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Pró-Fármacos/química , Coelhos , Resultado do Tratamento , Ácido ZoledrônicoRESUMO
T-cell malignancies are associated with frequent relapse and high morbidity, which is partly due to the lack of effective or targeted treatment options. To broaden the use of CAR-T cells in pan T-cell malignancies, we developed an allogeneic "universal" CD2-targeting CAR-T cell (UCART2), in which the CD2 antigen is deleted to prevent fratricide, and the T-cell receptor is removed to prevent GvHD. UCART2 demonstrated efficacy against T-ALL and CTCL and prolonged the survival of tumor-engrafted NSG mice in vivo. To evaluate the impact of CD2 on CAR-T function, we generated CD19 CAR-T cells (UCART19) with or without CD2 deletion, single-cell secretome analysis revealed that CD2 deletion in UCART19 reduced frequencies of the effector cytokines (Granzyme-B and IFN-γ). We also observed that UCART19ΔCD2 had reduced anti-tumor efficacy compared to UCART19 in a CD19+NALM6 xenograft model. Of note is that the reduced efficacy resulting from CD2 deletion was reversed when combined with rhIL-7-hyFc, a long-acting recombinant human interleukin-7. Treatment with rhIL-7-hyFc prolonged UCART2 persistence and increased survival in both the tumor re-challenge model and primary patient T-ALL model in vivo. Together, these data suggest that allogeneic fratricide-resistant UCART2, in combination with rhIL-7-hyFc, could be a suitable approach for treating T-cell malignancies.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Receptores de Antígenos Quiméricos , Humanos , Camundongos , Animais , Linfócitos T , Receptores de Antígenos Quiméricos/genética , Recidiva Local de Neoplasia , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T , Antígenos CD19RESUMO
BACKGROUND: Conjugation of transferrin (Tf) to imaging or nanotherapeutic agents is a promising strategy to target breast cancer. Since the efficacy of these biomaterials often depends on the overexpression of the targeted receptor, we set out to survey expression of transferrin receptor (TfR) in primary and metastatic breast cancer samples, including metastases and relapse, and investigate its modulation in experimental models. METHODS: Gene expression was investigated by datamining in twelve publicly-available datasets. Dedicated Tissue microarrays (TMAs) were generated to evaluate matched primary and bone metastases as well as and pre and post chemotherapy tumors from the same patient. TMA were stained with the FDA-approved MRQ-48 antibody against TfR and graded by staining intensity (H-score). Patient-derived xenografts (PDX) and isogenic metastatic mouse models were used to study in vivo TfR expression and uptake of transferrin. RESULTS: TFRC gene and protein expression were high in breast cancer of all subtypes and stages, and in 60-85% of bone metastases. TfR was detectable after neoadjuvant chemotherapy, albeit with some variability. Fluorophore-conjugated transferrin iron chelator deferoxamine (DFO) enhanced TfR uptake in human breast cancer cells in vitro and proved transferrin localization at metastatic sites and correlation of tumor burden relative to untreated tumor mice. CONCLUSIONS: TfR is expressed in breast cancer, primary, metastatic, and after neoadjuvant chemotherapy. Variability in expression of TfR suggests that evaluation of the expression of TfR in individual patients could identify the best candidates for targeting. Further, systemic iron chelation with DFO may upregulate receptor expression and improve uptake of therapeutics or tracers that use transferrin as a homing ligand.
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Neoplasias da Mama , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Quelantes , Expressão Gênica , Terapia de Alvo Molecular , Receptores da Transferrina/metabolismo , Transferrina/metabolismoRESUMO
Mobilized peripheral blood has become the primary source of hematopoietic stem cells for both autologous and allogeneic stem cell transplantation. Granulocyte colony-stimulating factor (G-CSF) is currently the standard agent used in the allogeneic setting. Despite the high mobilization efficacy in most donors, G-CSF requires 4-5 days of daily administration, and a small percentage of the donors fail to mobilize an optimal number of stem cells necessary for a safe allogeneic stem cell transplant. In this study, we retrospectively reviewed 1361 related allogeneic donors who underwent stem cell mobilization at Washington University. We compared the standard mobilization agent G-CSF with five alternative mobilization regimens, including GM-CSF, G-CSF+GM-CSF, GM-CSF + Plerixafor, Plerixafor and BL-8040. Cytokine-based mobilization strategies (G-CSF or in combination with GM-CSF) induce higher CD34 cell yield after 4-5 consecutive days of treatment, while CXCR4 antagonists (plerixafor and BL-8040) induce significantly less but rapid mobilization on the same day. Next, using a large dataset containing the demographic and baseline laboratory data from G-CSF-mobilized donors, we established machine learning (ML)-based scoring models that can be used to predict patients who may have less than optimal stem cell yields after a single leukapheresis session. To our knowledge, this is the first prediction model at the early donor screening stage, which may help identify allogeneic stem cell donors who may benefit from alternative approaches to enhance stem cell yields, thus ensuring safe and effective stem cell transplantation.
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Transplante de Células-Tronco Hematopoéticas , Compostos Heterocíclicos , Antígenos CD34/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Mobilização de Células-Tronco Hematopoéticas , Compostos Heterocíclicos/farmacologia , Humanos , Aprendizado de Máquina , Estudos RetrospectivosRESUMO
Tumor growth and metastases are dependent on interactions between cancer cells and the local environment. Expression of the cell-cell adhesion molecule N-cadherin (Ncad) is associated with highly aggressive cancers, and its expression by osteogenic cells has been proposed to provide a molecular "dock" for disseminated tumor cells to establish in pre-metastatic niches within the bone. To test this biologic model, we conditionally deleted the Ncad gene (Cdh2) in osteolineage cells using Osx-cre (cKO). Contrary to expectations, the metastatic breast cancer cell line PyMT-BO1 was able to form tumors in bone and to induce osteolysis in cKO as well as in control mice. Despite absence of Ncad, bone marrow stromal cells isolated from cKO mice were able to engage in direct cell-cell interactions with tumor cells expressing either N- or E-cadherin. However, subcutaneous PyMT-BO1 and B16F10 tumors grew larger in cKO relative to control littermates. Cell tracking experiments using the Ai9 reporter revealed the presence of Osx+ and Ncad+ cells in the stroma of extra-skeletal tumors and in a small population of lung cells. Gene expression analysis by RNAseq of Osx+ cells isolated from extra-skeletal tumors revealed alterations of pro-tumorigenic signaling pathways in cKO cells relative to control Osx+ cells. Thus, Ncad in Osx+ cells is not necessary for the establishment of bone metastases, but in extra-skeletal tumors it regulates pro-tumorigenic support by the microenvironment.
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Breast cancer bone metastases are common and incurable. Tumoral integrin ß3 (ß3) expression is induced through interaction with the bone microenvironment. Although ß3 is known to promote bone colonization, its functional role during therapy of established bone metastases is not known. We found increased numbers of ß3+ tumor cells in murine bone metastases after docetaxel chemotherapy. ß3+ tumor cells were present in 97% of post-neoadjuvant chemotherapy triple-negative breast cancer patient samples (n = 38). High tumoral ß3 expression was associated with worse outcomes in both pre- and postchemotherapy triple-negative breast cancer groups. Genetic deletion of tumoral ß3 had minimal effect in vitro, but significantly enhanced in vivo docetaxel activity, particularly in the bone. Rescue experiments confirmed that this effect required intact ß3 signaling. Ultrastructural, transcriptomic, and functional analyses revealed an alternative metabolic response to chemotherapy in ß3-expressing cells characterized by enhanced oxygen consumption, reactive oxygen species generation, and protein production. We identified mTORC1 as a candidate for therapeutic targeting of this ß3-mediated, chemotherapy-induced metabolic response. mTORC1 inhibition in combination with docetaxel synergistically attenuated murine bone metastases. Furthermore, micelle nanoparticle delivery of mTORC1 inhibitor to cells expressing activated αvß3 integrins enhanced docetaxel efficacy in bone metastases. Taken together, we show that ß3 integrin induction by the bone microenvironment promotes resistance to chemotherapy through an altered metabolic response that can be defused by combination with αvß3-targeted mTORC1 inhibitor nanotherapy. Our work demonstrates the importance of the metastatic microenvironment when designing treatments and presents new, bone-specific strategies for enhancing chemotherapeutic efficacy.
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Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Neoplasias da Mama/tratamento farmacológico , Integrina beta3/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias Ósseas/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Docetaxel/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Análise de SobrevidaRESUMO
Tumor-infiltrating myeloid cells contribute to the development of the immunosuppressive tumor microenvironment. Myeloid cell expression of arginase 1 (ARG1) promotes a protumor phenotype by inhibiting T cell function and depleting extracellular l-arginine, but the mechanism underlying this expression, especially in breast cancer, is poorly understood. In breast cancer clinical samples and in our mouse models, we identified tumor-derived GM-CSF as the primary regulator of myeloid cell ARG1 expression and local immune suppression through a gene-KO screen of breast tumor cell-produced factors. The induction of myeloid cell ARG1 required GM-CSF and a low pH environment. GM-CSF signaling through STAT3 and p38 MAPK and acid signaling through cAMP were required to activate myeloid cell ARG1 expression in a STAT6-independent manner. Importantly, breast tumor cell-derived GM-CSF promoted tumor progression by inhibiting host antitumor immunity, driving a significant accumulation of ARG1-expressing myeloid cells compared with lung and melanoma tumors with minimal GM-CSF expression. Blockade of tumoral GM-CSF enhanced the efficacy of tumor-specific adoptive T cell therapy and immune checkpoint blockade. Taken together, we show that breast tumor cell-derived GM-CSF contributes to the development of the immunosuppressive breast cancer microenvironment by regulating myeloid cell ARG1 expression and can be targeted to enhance breast cancer immunotherapy.
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Arginase/fisiologia , Neoplasias da Mama/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Tolerância Imunológica , Células Mieloides/enzimologia , Microambiente Tumoral , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , AMP Cíclico/fisiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1α (MIP-1α) are important factors that increase bone resorption and hypercalcemia in adult T-cell leukemia (ATL). We investigated the role of PTHrP and MIP-1α in the development of local osteolytic lesions in T-cell leukemia through overexpression in Jurkat T-cells. Injections of Jurkat-PTHrP and Jurkat-MIP-1α into the tibia and the left ventricle of NSG mice were performed to evaluate tumor growth and metastasis in vivo. Jurkat-pcDNA tibial neoplasms grew at a significantly greater rate and total tibial tumor burden was significantly greater than Jurkat-PTHrP neoplasms. Despite the lower tibial tumor burden, Jurkat-PTHrP bone neoplasms had significantly greater osteolysis than Jurkat-pcDNA and Jurkat-MIP-1α neoplasms. Jurkat-PTHrP and Jurkat-pcDNA cells preferentially metastasized to bone following intracardiac injection, though the overall metastatic burden was lower in Jurkat-PTHrP mice. These findings demonstrate that PTHrP induced pathologic osteolysis in T-cell leukemia but did not increase the incidence of skeletal metastasis.
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Neoplasias Ósseas , Hipercalcemia , Leucemia-Linfoma de Células T do Adulto , Osteólise , Adulto , Animais , Humanos , Hipercalcemia/etiologia , Camundongos , Osteólise/etiologia , Proteína Relacionada ao Hormônio Paratireóideo/genéticaRESUMO
Tumor-associated macrophages (TAMs) enhance tumor growth in mice and are correlated with a worse prognosis for breast cancer patients. While early therapies sought to deplete all macrophages, current therapeutics aim to reprogram pro-tumor macrophages (M2) and preserve those necessary for anti-tumor immune responses (M1). Recent studies have shown that c-MYC (MYC) is induced in M2 macrophages in vitro and in vivo where it regulates the expression of tumor-promoting genes. In a myeloid lineage MYC KO mouse model, MYC had important roles in macrophage maturation and function leading to reduced tumor growth. We therefore hypothesized that targeted delivery of a MYC inhibitor to established M2 TAMs could reduce polarization toward an M2 phenotype in breast cancer models. Methods: In this study, we developed a MYC inhibitor prodrug (MI3-PD) for encapsulation within perfluorocarbon nanoparticles, which can deliver drugs directly to the cytosol of the target cell through a phagocytosis independent mechanism. We have previously shown that M2-like TAMs express significant levels of the vitronectin receptor, integrin ß3, and in vivo targeting and therapeutic potential was evaluated using αvß3 integrin targeted rhodamine-labeled nanoparticles (NP) or integrin αvß3-MI3-PD nanoparticles. Results: We observed that rhodamine, delivered by αvß3-rhodamine NP, was incorporated into M2 tumor promoting macrophages through both phagocytosis-independent and dependent mechanisms, while NP uptake in tumor suppressing M1 macrophages was almost exclusively through phagocytosis. In a mouse model of breast cancer (4T1-GFP-FL), M2-like TAMs were significantly reduced with αvß3-MI3-PD NP treatment. To validate this effect was independent of drug delivery to tumor cells and was specific to the MYC inhibitor, mice with integrin ß3 knock out tumors (PyMT-Bo1 ß3KO) were treated with αvß3-NP or αvß3-MI3-PD NP. M2 macrophages were significantly reduced with αvß3-MI3-PD nanoparticle therapy but not αvß3-NP treatment. Conclusion: These data suggest αvß3-NP-mediated drug delivery of a c-MYC inhibitor can reduce protumor M2-like macrophages while preserving antitumor M1-like macrophages in breast cancer.
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Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Nanopartículas/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Antineoplásicos/química , Neoplasias da Mama/patologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/imunologia , Linhagem Celular Tumoral/transplante , Avaliação Pré-Clínica de Medicamentos , Feminino , Fluorocarbonos/administração & dosagem , Fluorocarbonos/química , Técnicas de Inativação de Genes , Humanos , Integrina alfaVbeta3 , Integrina beta3 , Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Nanopartículas/química , Fagocitose , Cultura Primária de Células , Pró-Fármacos/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Adult T-cell leukemia/lymphoma has a unique relationship to bone including latency in the marrow, and development of bone invasion, osteolytic tumors and humoral hypercalcemia of malignancy. To study these conditions, we established and characterized a novel mouse model of ATL bone metastasis. Patient-derived ATL cell lines including three that do not express HTLV-1 oncoprotein Tax (ATL-ED, RV-ATL, TL-Om1), an in vitro transformed human T-cell line with high Tax expression (HT-1RV), and an HTLV-1 negative T-cell lymphoma (Jurkat) were injected intratibially into NSG mice, and were capable of proliferating and modifying the bone microenvironment. Radiography, µCT, histopathology, immunohistochemistry, plasma calcium concentrations, and qRT-PCR for several tumor-bone signaling mRNAs were performed. Luciferase-positive ATL-ED bone tumors allowed for in vivo imaging and visualization of bone tumor growth and metastasis over time. ATL-ED and HT-1RV cells caused mixed osteolytic/osteoblastic bone tumors, TL-Om1 cells exhibited minimal bone involvement and aggressive local invasion into the adjacent soft tissues, Jurkat cells proliferated within bone marrow and induced minimal bone cell response, and RV-ATL cells caused marked osteolysis. This mouse model revealed important mechanisms of human ATL bone neoplasms and will be useful to investigate biological interactions, potential therapeutic targets, and new bone-targeted agents for the prevention of ATL metastases to bone.
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Osteolytic bone lesions and hypercalcemia are common, serious complications in adult T cell leukemia/lymphoma (ATL), an aggressive T cell malignancy associated with human T cell leukemia virus type 1 (HTLV-1) infection. The HTLV-1 viral oncogene HBZ has been implicated in ATL tumorigenesis and bone loss. In this study, we evaluated the role of HBZ on ATL-associated bone destruction using HTLV-1 infection and disease progression mouse models. Humanized mice infected with HTLV-1 developed lymphoproliferative disease and continuous, progressive osteolytic bone lesions. HTLV-1 lacking HBZ displayed only modest delays to lymphoproliferative disease but significantly decreased disease-associated bone loss compared with HTLV-1-infected mice. Gene expression array of acute ATL patient samples demonstrated increased expression of RANKL, a critical regulator of osteoclasts. We found that HBZ regulated RANKL in a c-Fos-dependent manner. Treatment of HTLV-1-infected humanized mice with denosumab, a monoclonal antibody against human RANKL, alleviated bone loss. Using patient-derived xenografts from primary human ATL cells to induce lymphoproliferative disease, we also observed profound tumor-induced bone destruction and increased c-Fos and RANKL gene expression. Together, these data show the critical role of HBZ in driving ATL-associated bone loss through RANKL and identify denosumab as a potential treatment to prevent bone complications in ATL patients.
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Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Proteínas dos Retroviridae/metabolismo , Adulto , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Osso e Ossos/patologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Vírus Linfotrópico T Tipo 1 Humano , Humanos , Estimativa de Kaplan-Meier , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoclastos/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Proteínas dos Retroviridae/genética , TranscriptomaRESUMO
How specific genetic lesions contribute to transformation of non-malignant myeloproliferative neoplasms (MPNs) and myelodysplastic syndromes (MDSs) to secondary acute myeloid leukemia (sAML) are poorly understood. JARID2 is lost by chromosomal deletions in a proportion of MPN/MDS cases that progress to sAML. In this study, genetic mouse models and patient-derived xenografts demonstrated that JARID2 acts as a tumor suppressor in chronic myeloid disorders. Genetic deletion of Jarid2 either reduced overall survival of animals with MPNs or drove transformation to sAML, depending on the timing and context of co-operating mutations. Mechanistically, JARID2 recruits PRC2 to epigenetically repress self-renewal pathways in hematopoietic progenitor cells. These studies establish JARID2 as a bona fide hematopoietic tumor suppressor and highlight potential therapeutic targets.
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Autorrenovação Celular/genética , Transformação Celular Neoplásica/genética , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Transtornos Mieloproliferativos/genética , Complexo Repressor Polycomb 2/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Autorrenovação Celular/fisiologia , Transformação Celular Neoplásica/patologia , Feminino , Deleção de Genes , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndromes Mielodisplásicas/patologia , Transtornos Mieloproliferativos/patologia , Proteína Proto-Oncogênica N-Myc/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Transplante HeterólogoRESUMO
PURPOSE: We hypothesized that in multiple myeloma cells (MMC), high membrane biosynthesis will induce acetate uptake in vitro and in vivo Here, we studied acetate metabolism and targeting in MMC in vitro and tested the efficacy of 11C-acetate-positron emission tomography (PET) to detect and quantitatively image myeloma treatment response in vivo EXPERIMENTAL DESIGN: Acetate fate tracking using 13C-edited-1H NMR (nuclear magnetic resonance) was performed to study in vitro acetate uptake and metabolism in MMC. Effects of pharmacological modulation of acetate transport or acetate incorporation into lipids on MMC cell survival and viability were assessed. Preclinical mouse MM models of subcutaneous and bone tumors were evaluated using 11C-acetate-PET/CT imaging and tissue biodistribution. RESULTS: In vitro, NMR showed significant uptake of acetate by MMC and acetate incorporation into intracellular metabolites and membrane lipids. Inhibition of lipid synthesis and acetate transport was toxic to MMC, while sparing resident bone cells or normal B cells. In vivo, 11C-acetate uptake by PET imaging was significantly enhanced in subcutaneous and bone MMC tumors compared with unaffected bone or muscle tissue. Likewise, 11C-acetate uptake was significantly reduced in MM tumors after treatment. CONCLUSIONS: Uptake of acetate from the extracellular environment was enhanced in MMC and was critical to cellular viability. 11C-Acetate-PET detected the presence of myeloma cells in vivo, including uptake in intramedullary bone disease. 11C-Acetate-PET also detected response to therapy in vivo Our data suggested that acetate metabolism and incorporation into lipids was crucial to MM cell biology and that 11C-acetate-PET is a promising imaging modality for MM. Clin Cancer Res; 23(2); 416-29. ©2016 AACR.
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Acetatos/metabolismo , Neoplasias Ósseas/metabolismo , Metabolismo dos Lipídeos , Mieloma Múltiplo/diagnóstico por imagem , Acetatos/isolamento & purificação , Animais , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Fluordesoxiglucose F18/uso terapêutico , Humanos , Imageamento por Ressonância Magnética , Camundongos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos/uso terapêutico , Distribuição TecidualRESUMO
Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell malignancy that occurs in HTLV-1 infected patients. Most ATL patients develop osteolytic lesions and hypercalcemia of malignancy, causing severe skeletal related complications and reduced overall survival. The HTLV-1 virus encodes 2 viral oncogenes, Tax and HBZ. Tax, a transcriptional activator, is critical to ATL development, and has been implicated in pathologic osteolysis. HBZ, HTLV-1 basic leucine zipper transcription factor, promotes tumor cell proliferation and disrupts Wnt pathway modulators; however, its role in ATL induced osteolytic bone loss is unknown. To determine if HBZ is sufficient for the development of bone loss, we established a transgenic Granzyme B HBZ (Gzmb-HBZ) mouse model. Lymphoproliferative disease including tumors, enlarged spleens and/or abnormal white cell counts developed in two-thirds of Gzmb-HBZ mice at 18 months. HBZ positive cells were detected in tumors, spleen and bone marrow. Importantly, pathologic bone loss and hypercalcemia were present at 18 months. Bone-acting factors were present in serum and RANKL, PTHrP and DKK1, key mediators of hypercalcemia and bone loss, were upregulated in Gzmb-HBZ T cells. These data demonstrate that Gzmb-HBZ mice model ATL bone disease and express factors that are current therapeutic targets for metastatic and bone resident tumors.
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Integrin ß3 is critical for tumor invasion, neoangiogenesis, and inflammation, making it a promising cancer target. However, preclinical and clinical data of integrin ß3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin ß3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin ß3 in macrophage lineage cells (ß3KOM). ß3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8(+) T cells. Integrin ß3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells, wherein integrin ß3 signaling favored STAT1 activation, an M1-polarizing signal, and suppressed M2-polarizing STAT6 activation. Finally, disruption of CD8(+) T cells, macrophages, or macrophage integrin ß3 signaling blocked the tumor-promoting effects of integrin ß3 antagonism. These results suggest that effects of integrin ß3 therapies on immune cells should be considered to improve outcomes. Cancer Res; 76(12); 3484-95. ©2016 AACR.
Assuntos
Tolerância Imunológica , Integrina beta3/fisiologia , Neoplasias/imunologia , Animais , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição STAT1/fisiologia , Fator de Transcrição STAT6/fisiologia , Quinase Syk/metabolismo , Microambiente TumoralRESUMO
The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow-disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a "chemotherapy framing" dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas/farmacologia , Receptores CXCR4/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Materiais Biomiméticos/administração & dosagem , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Epitopos/metabolismo , Furanos/administração & dosagem , Furanos/farmacologia , Humanos , Cetonas/administração & dosagem , Cetonas/farmacologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Metástase Neoplásica , Ligação Proteica/efeitos dos fármacos , Proteínas/administração & dosagem , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multiple myeloma (MM) is a malignant disorder of differentiated B-cells for which standard care involves the inhibition of the proteasome. All clinically used proteasome inhibitors, including the chemotherapeutic drug bortezomib, target the catalytic active sites of the proteasome and inhibit protein proteolysis by competing with substrate binding. However, nearly all (~97%) patients become intolerant or resistant to treatments within a few years, after which the average survival time is less than 1 year. We describe herein the inhibition of the human proteasome via a noncompetitive mechanism by the imidazoline scaffold, TCH-13. Consistent with a mechanism distinct from that of competitive inhibitors, TCH-013 acts additively with and overcomes resistance to bortezomib. Importantly, TCH-013 induces apoptosis in a panel of myeloma and leukemia cell lines, but in contrast, normal lymphocytes, primary bone marrow stromal cells (hBMSC), and macrophages are resistant to its cytotoxic effects. TCH-013 was equally effective in blocking MM cell growth in co-cultures of MM cells with hBMSC isolated from CD138 negative bone marrow (BM) samples of MM patients. The cellular activity translated well in vivo where TCH-013 delayed tumor growth in an MM xenograft model to a similar extent as bortezomib.
Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Imidazóis/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirazinas/farmacologia , Animais , Bortezomib , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Proteínas I-kappa B/metabolismo , Imidazóis/química , Camundongos , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Hedgehog (Hh) signaling is implicated in bone development and cellular transformation. Here we show that inhibition of Hh pathway activity inhibits tumor growth through effects on the microenvironment. Pharmacologic inhibition of the Hh effector Smoothened (Smo) increased trabecular bone in vivo and inhibited osteoclastogenesis in vitro. In addition, enhanced Hh signaling due to heterozygosity of the Hh inhibitory receptor Patched (Ptch1(+/-)) increased bone resorption, suggesting direct regulation of osteoclast (OC) activity by the Hh pathway. Ptch1(+/-) mice had increased bone metastatic and subcutaneous tumor growth, suggesting that increased Hh activation in host cells promoted tumor growth. Subcutaneous growth of Hh-resistant tumor cells was inhibited by LDE225, a novel orally bioavailable SMO antagonist, consistent with effects on tumor microenvironment. Knockdown of the Hh ligand Sonic Hh (SHH) in these cells decreased subcutaneous tumor growth and decreased stromal cell production of interleukin-6, indicating that tumor-derived Hh ligands stimulated tumor growth in a paracrine fashion. Together our findings show that inhibition of the Hh pathway can reduce tumor burden, regardless of tumor Hh responsiveness, through effects on tumor cells, OCs, and stromal cells within the tumor microenvironment. Hh may be a promising therapeutic target for solid cancers and bone metastases.
Assuntos
Neoplasias Ósseas/prevenção & controle , Osso e Ossos/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Neoplasias Experimentais/metabolismo , Microambiente Tumoral , Alcaloides de Veratrum/farmacologia , Animais , Neoplasias Ósseas/secundário , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Proteínas Hedgehog/antagonistas & inibidores , Heterozigoto , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/patologia , Osteoclastos/efeitos dos fármacos , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais , Receptor SmoothenedRESUMO
The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12-/- OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12-/-, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent αIIbß3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin ß3 gene knockout (Itgb3-/-) OCs, but its effects were substantially blunted in P2ry12-/- OCs. In vivo, P2ry12-/- mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss.