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1.
Clin Immunol ; 264: 110217, 2024 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-38621471

RESUMO

Common variable immunodeficiency (CVID) is an immune defect characterized by hypogammaglobulinemia and impaired development of B cells into plasma cells. As follicular helper T cells (TFH) play a central role in humoral immunity, we examined TFH cells in CVID, and investigated whether an inducible T cell co-stimulator (ICOS) agonist, vopratelimab, could modulate TFH, B cell interactions and enhance immunoglobulin production. CVID subjects had decreased TFH17 and increased TFH1 subsets; this was associated with increased transitional B cells and decreased IgG+ B and IgD-IgM-CD27+ memory B cells. ICOS expression on CVID CD4+ T cells was also decreased. However, ICOS activation of CD4+ T cells by vopratelimab significantly increased total CVID TFH, TFH2, cell numbers, as well as IL-4, IL-10 and IL-21 secretion in vitro. Vopratelimab treatment also increased plasma cells, IgG+ B cells, reduced naïve & transitional B cells and significantly increased IgG1 secretion by CVID B cells. Interestingly, vopratelimab treatment also restored IgA secretion in PBMCs from several CVID patients who had a complete lack of endogenous serum IgA. Our data demonstrate the potential of TFH modulation in restoring TFH and enhancing B cell maturation in CVID. The effects of an ICOS agonist in antibody defects warrants further investigation. This biologic may also be of therapeutic interest in other clinical settings of antibody deficiency.

2.
Mol Cancer Ther ; 22(4): 471-484, 2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-36780212

RESUMO

Tumor-associated macrophages (TAM) play an important role in maintaining the immunosuppressive state of the tumor microenvironment (TME). High levels of CD163+ TAMs specifically are associated with poor prognosis in many solid tumor types. Targeting TAMs may represent a key approach in development of the next generation of cancer immune therapeutics. Members of the leukocyte immunoglobulin-like receptor B (LILRB) family, including LILRB2 (ILT4), are known to transmit inhibitory signals in macrophages and other myeloid cells. Leveraging bulk and single cell RNA-sequencing datasets, as well as extensive immunophenotyping of human tumors, we found that LILRB2 is highly expressed on CD163+ CD11b+ cells in the TME and that LILRB2 expression correlates with CD163 expression across many tumor types. To target LILRB2, we have developed JTX-8064, a highly potent and selective antagonistic mAb. JTX-8064 blocks LILRB2 binding to its cognate ligands, including classical and nonclassical MHC molecules. In vitro, JTX-8064 drives the polarization of human macrophages and dendritic cells toward an immunostimulatory phenotype. As a result, human macrophages treated with a LILRB2 blocker are reprogrammed to increase the activation of autologous T cells in co-culture systems. Furthermore, JTX-8064 significantly potentiates the activity of anti-PD-1 in allogeneic mixed lymphocyte reaction. In a human tumor explant culture, pharmacodynamic activity of JTX-8064 was observed in monotherapy and in combination with anti-PD-1. Collectively, our work provides strong translational and preclinical rationale to target LILRB2 in cancer.


Assuntos
Neoplasias , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Macrófagos/metabolismo , Ativação Linfocitária , Técnicas de Cocultura , Linfócitos T , Microambiente Tumoral , Glicoproteínas de Membrana/genética , Receptores Imunológicos
3.
Oncoimmunology ; 11(1): 2141007, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36352891

RESUMO

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.


Assuntos
Neoplasias , Linfócitos T Reguladores , Camundongos , Animais , Humanos , Linfócitos T Reguladores/metabolismo , Receptor de Morte Celular Programada 1 , Imunoterapia/métodos , Neoplasias/terapia , Microambiente Tumoral , Fragmentos Fc das Imunoglobulinas/metabolismo , Receptores CCR8/metabolismo
4.
J Immunother Cancer ; 10(9)2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36096532

RESUMO

BACKGROUND: Cancer immunotherapy has revolutionized cancer treatment. However, considering the limited success of immunotherapy to only some cancer types and patient cohorts, there is an unmet need for developing new treatments that will result in higher response rates in patients with cancer. Immunoglobulin-like transcript 2 (ILT2), a LILRB family member, is an inhibitory receptor expressed on a variety of immune cells including T cells, natural killer (NK) cells and different myeloid cells. In the tumor microenvironment, binding of class I MHC (in particular HLA-G) to ILT2 on immune cells mediates a strong inhibitory effect, which manifests in inhibition of antitumor cytotoxicity of T and NK cells, and prevention of phagocytosis of the tumor cells by macrophages. METHODS: We describe here the development and characteristics of BND-22, a novel, humanized monoclonal antibody that selectively binds to ILT2 and blocks its interaction with classical MHC I and HLA-G. BND-22 was evaluated for its binding and blocking characteristics as well as its ability to increase the antitumor activity of macrophages, T cells and NK cells in various in vitro, ex vivo and in vivo systems. RESULTS: Collectively, our data suggest that BND-22 enhances activity of both innate and adaptive immune cells, thus generating robust and comprehensive antitumor immunity. In humanized mice models, blocking ILT2 with BND-22 decreased the growth of human tumors, hindered metastatic spread to the lungs, and prolonged survival of the tumor-bearing mice. In addition, BND-22 improved the antitumor immune response of approved therapies such as anti-PD-1 or anti-EGFR antibodies. CONCLUSIONS: BND-22 is a first-in-human ILT2 blocking antibody which has demonstrated efficient antitumor activity in various preclinical models as well as a favorable safety profile. Clinical evaluation of BND-22 as a monotherapy or in combination with other therapeutics is under way in patients with cancer. TRIAL REGISTRATION NUMBER: NCT04717375.


Assuntos
Antígenos HLA-G , Neoplasias , Animais , Antígenos HLA-G/metabolismo , Humanos , Imunoterapia , Células Matadoras Naturais , Camundongos , Neoplasias/tratamento farmacológico , Linfócitos T , Microambiente Tumoral
6.
Nature ; 603(7900): 328-334, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35197632

RESUMO

Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies1. Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies2-4. Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2+ breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells.


Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Feminino , Humanos , Camundongos , Receptor ErbB-2/genética
7.
Blood ; 139(8): 1160-1176, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35201323

RESUMO

Anti-CD38 monoclonal antibodies (mAbs) represent a breakthrough in the treatment of multiple myeloma (MM), yet some patients fail to respond or progress quickly with this therapy, highlighting the need for novel approaches. In this study we compared the preclinical efficacy of SAR442085, a next-generation anti-CD38 mAb with enhanced affinity for activating Fcγ receptors (FcγR), with first-generation anti-CD38 mAb daratumumab and isatuximab. In surface plasmon resonance and cellular binding assays, we found that SAR442085 had higher binding affinity than daratumumab and isatuximab for FcγRIIa (CD32a) and FcγRIIIa (CD16a). SAR442085 also exhibited better in vitro antibody-dependent cellular cytotoxicity (ADCC) against a panel of MM cells expressing variable CD38 receptor densities including MM patients' primary plasma cells. The enhanced ADCC of SAR442085 was confirmed using NK-92 cells bearing low and high affinity FcγRIIIa (CD16a)-158F/V variants. Using MM patients' primary bone marrow cells, we confirmed that SAR442085 had an increased ability to engage FcγRIIIa, resulting in higher natural killer (NK) cell activation and degranulation against primary plasma cells than preexisting Fc wild-type anti-CD38 mAbs. Finally, using huFcgR transgenic mice that express human Fcγ receptors under the control of their human regulatory elements, we demonstrated that SAR442085 had higher NK cell-dependent in vivo antitumor efficacy and better survival than daratumumab and isatuximab against EL4 thymoma or VK*MYC myeloma cells overexpressing human CD38. These results highlight the preclinical efficacy of SAR442085 and support the current evaluation of this next-generation anti-CD38 antibody in phase I clinical development in patients with relapsed/refractory MM.


Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Antineoplásicos Imunológicos/farmacologia , Células da Medula Óssea , Glicoproteínas de Membrana/antagonistas & inibidores , Mieloma Múltiplo , Proteínas de Neoplasias/antagonistas & inibidores , ADP-Ribosil Ciclase 1/metabolismo , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos Transgênicos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Oncoimmunology ; 10(1): 1945803, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484869

RESUMO

Novel therapies are needed for effective treatment of AML. In the relapsed setting, prognosis is very poor despite salvage treatment with chemotherapy. Evidence suggests that leukemic stem cells (LSCs) cause relapse. The cell surface receptor CD123 is highly expressed in blast cells and LSCs from AML patients and is a potential therapeutic target. CD123 cross-over dual-variable domain T-cell engager (CD123-CODV-TCE) is a bispecific antibody with an innovative format. One arm targets the CD3εδ subunit of T-cell co-receptors on the surface of T cells, while the other targets CD123 on malignant cells, leading to cell-specific cytotoxic activity. Here, we describe the preclinical activity of CD123-CODV-TCE. CD123-CODV-TCE effectively binds to human and cynomolgus monkey CD3 and CD123 and is a highly potent T-cell engager. It mediates T-cell activation and T-cell-directed killing of AML cells in vitro. In vivo, CD123-CODV-TCE suppresses AML tumor growth in leukemia xenograft mouse models, where it achieves an effective half-life of 3.2 days, which is a significantly longer half-life compared to other bispecific antibodies with no associated Fc fragment. The in vitro safety profile is as expected for compounds with similar modes of action. These results suggest that CD123-CODV-TCE may be a promising therapy for patients with relapsed/refractory AML.


Assuntos
Anticorpos Biespecíficos , Leucemia Mieloide Aguda , Animais , Complexo CD3 , Humanos , Subunidade alfa de Receptor de Interleucina-3 , Leucemia Mieloide Aguda/tratamento farmacológico , Macaca fascicularis , Camundongos , Linfócitos T
9.
Sci Transl Med ; 13(610): eabc7804, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34516826

RESUMO

Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.


Assuntos
Citocinas , Neoplasias , Citocinas/genética , Humanos , Neoplasias/genética , Neoplasias/terapia , RNA Mensageiro
10.
Oncoimmunology ; 10(1): 1881268, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33796402

RESUMO

Immune checkpoint blockade elicits durable anti-cancer responses in the clinic, however a large proportion of patients do not benefit from treatment. Several mechanisms of innate and acquired resistance to checkpoint blockade have been defined and include mutations of MHC I and IFNγ signaling pathways. However, such mutations occur in a low frequency of patients and additional mechanisms have yet to be elucidated. In an effort to better understand acquired resistance to checkpoint blockade, we generated a mouse tumor model exhibiting in vivo resistance to anti-PD-1 antibody treatment. MC38 tumors acquired resistance to PD-1 blockade following serial in vivo passaging. Lack of sensitivity to PD-1 blockade was not attributed to dysregulation of PD-L1 or ß2M expression, as both were expressed at similar levels in parental and resistant cells. Similarly, IFNγ signaling and antigen processing and presentation pathways were functional in both parental and resistant cell lines. Unbiased gene expression analysis was used to further characterize potential resistance mechanisms. RNA-sequencing revealed substantial differences in global gene expression, with tumors resistant to anti-PD-1 displaying a marked reduction in expression of immune-related genes relative to parental MC38 tumors. Indeed, resistant tumors exhibited reduced immune infiltration across multiple cell types, including T and NK cells. Pathway analysis revealed activation of TGFß and Notch signaling in anti-PD-1 resistant tumors, and activation of these pathways was associated with poorer survival in human cancer patients. While pharmacological inhibition of TGFß and Notch in combination with PD-1 blockade decelerated tumor growth, a local mRNA-based immunotherapy potently induced regression of resistant tumors, resulting in complete tumor remission, and resensitized tumors to treatment with anti-PD-1. Overall, this study describes a novel anti-PD-1 resistant mouse tumor model and underscores the role of two well-defined signaling pathways in response to immune checkpoint blockade. Furthermore, our data highlights the potential of intratumoral mRNA therapy in overcoming acquired resistance to PD-1 blockade.


Assuntos
Imunoterapia , Neoplasias , Animais , Apresentação de Antígeno , Modelos Animais de Doenças , Humanos , Camundongos , RNA Mensageiro/genética
11.
Oncoimmunology ; 9(1): 1811605, 2020 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-33224628

RESUMO

TGFß is a pleiotropic cytokine that may have both tumor inhibiting and tumor promoting properties, depending on tissue and cellular context. Emerging data support a role for TGFß in suppression of antitumor immunity. Here we show that SAR439459, a pan-TGFß neutralizing antibody, inhibits all active isoforms of human and murine TGFß, blocks TGFß-mediated pSMAD signaling, and TGFß-mediated suppression of T cells and NK cells. In vitro, SAR439459 synergized with anti-PD1 to enhance T cell responsiveness. In syngeneic tumor models, SAR439459 treatment impaired tumor growth, while the combination of SAR439459 with anti-PD-1 resulted in complete tumor regression and a prolonged antitumor immunity. Mechanistically, we found that TGFß inhibition with PD-1 blockade augmented intratumoral CD8+ T cell proliferation, reduced exhaustion, evoked proinflammatory cytokines, and promoted tumor-specific CD8+ T cell responses. Together, these data support the hypothesis that TGFß neutralization using SAR439459 synergizes with PD-1 blockade to promote antitumor immunity and formed the basis for the ongoing clinical investigation of SAR439459 in patients with cancer (NCT03192345).


Assuntos
Terapia de Imunossupressão , Receptor de Morte Celular Programada 1 , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Humanos , Tolerância Imunológica , Camundongos
12.
Front Immunol ; 11: 1771, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922390

RESUMO

Isatuximab is a monoclonal antibody targeting the transmembrane receptor and ectoenzyme CD38, a protein highly expressed on hematological malignant cells, including those in multiple myeloma (MM). Upon binding to CD38-expressing MM cells, isatuximab is thought to induce tumor cell killing via fragment crystallizable (Fc)-dependent mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), as well as via direct Fc-independent mechanisms. Here, these mechanisms of action were investigated in MM and diffuse large B-cell lymphoma (DLBCL) cell lines, as well as in peripheral blood mononuclear cells derived from healthy donors, and in MM patient-derived samples. Our findings show that isatuximab-mediated cytotoxicity occurred primarily via ADCC and ADCP in MM cell lines and via ADCC and apoptosis in DLBCL cell lines expressing high levels of CD38. We identified the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway and MM cell-secreted transforming growth factor-beta (TGF-ß) as tumor cell-related features that could suppress CD38-mediated ADCC. Furthermore, we established that isatuximab can directly activate natural killer (NK) cells and promote NK cell-mediated cytotoxicity via crosslinking of CD38 and CD16. Finally, isatuximab-induced CDC was observed in cell lines with high CD38 receptor density (>250,000 molecules/cell) and limited expression of inhibitory complement regulatory proteins (CD46, CD55, and CD59; <50,000 molecules/cell). Taken together, our findings highlight mechanistic insights for isatuximab and provide support for a range of combination therapy approaches that could be tested for isatuximab in the future.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Mieloma Múltiplo/imunologia , Apoptose/efeitos dos fármacos , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Ativação Linfocitária/efeitos dos fármacos
13.
PLoS One ; 15(8): e0235551, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833964

RESUMO

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ferro/metabolismo , Neoplasias/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Colesterol/biossíntese , Colesterol/genética , Classe III de Fosfatidilinositol 3-Quinases/genética , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Receptores de LDL/metabolismo , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
14.
Cells ; 8(12)2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31779273

RESUMO

CD38 is a transmembrane glycoprotein with ectoenzymatic activity involved in regulation of migration, signal transduction, and receptor-mediated adhesion. CD38 is highly expressed on various malignant cells, including multiple myeloma (MM), and at relatively low levels in other tissues, making it a suitable target for therapeutic antibodies. Several anti-CD38 therapies have been, or are being, developed for the treatment of MM, including daratumumab and isatuximab (SAR650984), respectively. Studies have shown that anti-CD38 therapies are effective in the treatment of relapsed/refractory MM and are well tolerated, with infusion reactions being the most common side effects. They can be used as monotherapy or in combination with immunomodulatory agents, such as pomalidomide, or proteasome inhibitors to potentiate their activity. Here we examine isatuximab and several anti-CD38 agents in development that were generated using new antibody engineering techniques and that may lead to more effective CD38 targeting. We also summarize trials assessing these antibodies in MM, other malignancies, and solid organ transplantation. Finally, we propose that further research on the mechanisms of resistance to anti-CD38 therapy and the development of biomarkers and new backbone regimens with CD38 antibodies will be important steps in building more personalized treatment for patients with MM.


Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , ADP-Ribosil Ciclase 1/química , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/uso terapêutico , Humanos , Glicoproteínas de Membrana/química , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
15.
Mol Cancer Ther ; 18(12): 2343-2356, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515294

RESUMO

The pattern recognition receptor RIG-I plays an important role in the recognition of nonself RNA and antiviral immunity. RIG-I's natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. In this study, we present comprehensive data validating the concept and utility of treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Using mRNA expression data of human tumors, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Furthermore, we confirm susceptibility of cancer cells to ppp-RNA treatment in different cellular models of human melanoma, revealing unexpected heterogeneity between cell lines in their susceptibility to RNA agonist features, including sequence, secondary structures, and presence of triphosphate. Cellular responses to RNA treatment (induction of type I IFN, FasR, MHC-I, and cytotoxicity) were demonstrated to be RIG-I dependent using KO cells. Following ppp-RNA treatment of a mouse melanoma model, we observed significant local and systemic antitumor effects and survival benefits. These were associated with type I IFN response, tumor cell apoptosis, and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen-specific CTLs following treatment with RIG-I agonists. Despite potential challenges in the generation and formulation of potent RIG-I agonists, ppp-RNA or analogues thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy.


Assuntos
Proteína DEAD-box 58/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/genética , Polifosfatos/uso terapêutico , RNA/metabolismo , Animais , Linhagem Celular Tumoral , Proteína DEAD-box 58/farmacologia , Humanos , Melanoma/patologia , Camundongos , Polifosfatos/farmacologia , Receptores Imunológicos , Transdução de Sinais , Transfecção
16.
PLoS One ; 12(9): e0185092, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28950000

RESUMO

Tumor cells display fundamental changes in metabolism and nutrient uptake in order to utilize additional nutrient sources to meet their enhanced bioenergetic requirements. Glutamine (Gln) is one such nutrient that is rapidly taken up by tumor cells to fulfill this increased metabolic demand. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS). This study has identified GLS a potential therapeutic target in breast cancer, specifically in the basal subtype that exhibits a deregulated glutaminolysis pathway. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines with a deregulated glutaminolysis pathway led to profound tumor growth inhibition in vitro and in vivo. GLS knockdown had no effect on growth and metabolite levels in non-TNBC cell lines. We rescued the anti-tumor effect of GLS knockdown using shRNA resistant cDNAs encoding both GLS isoforms and by addition of an α-ketoglutarate (αKG) analog thus confirming the critical role of GLS in TNBC. Pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) activity and an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). Finally, we found that GLS inhibition synergizes with mTOR inhibition, which introduces the possibility of a novel therapeutic strategy for TNBC. Our study revealed that GLS is essential for the survival of TNBC with a deregulated glutaminolysis pathway. The synergistic activity of GLS and mTOR inhibitors in TNBC cell lines suggests therapeutic potential of this combination for the treatment of vulnerable subpopulations of TNBC.


Assuntos
Glutaminase/metabolismo , Glutamina/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/enzimologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
17.
Oncotarget ; 8(70): 114526-114539, 2017 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-29383099

RESUMO

Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/ß-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (ß-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of ß-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on ß-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on ß-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) ß-catenin were significantly more sensitive to ß-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active ß-catenin. Finally, significant therapeutic benefit of ß-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. ß-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of ß-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant ß-catenin signaling in the maintenance of oncogenic phenotype in HCC.

18.
Cancer Cell ; 28(6): 773-784, 2015 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-26678339

RESUMO

Heterozygous mutation of IDH1 in cancers modifies IDH1 enzymatic activity, reprogramming metabolite flux and markedly elevating 2-hydroxyglutarate (2-HG). Here, we found that 2-HG depletion did not inhibit growth of several IDH1 mutant solid cancer types. To identify other metabolic therapeutic targets, we systematically profiled metabolites in endogenous IDH1 mutant cancer cells after mutant IDH1 inhibition and discovered a profound vulnerability to depletion of the coenzyme NAD+. Mutant IDH1 lowered NAD+ levels by downregulating the NAD+ salvage pathway enzyme nicotinate phosphoribosyltransferase (Naprt1), sensitizing to NAD+ depletion via concomitant nicotinamide phosphoribosyltransferase (NAMPT) inhibition. NAD+ depletion activated the intracellular energy sensor AMPK, triggered autophagy, and resulted in cytotoxicity. Thus, we identify NAD+ depletion as a metabolic susceptibility of IDH1 mutant cancers.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glioblastoma/tratamento farmacológico , Isocitrato Desidrogenase/genética , Mutação , NAD/deficiência , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática , Feminino , Glioblastoma/enzimologia , Glioblastoma/genética , Glioblastoma/patologia , Glutaratos/metabolismo , Células HEK293 , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/metabolismo , Metabolômica/métodos , Camundongos SCID , Terapia de Alvo Molecular , Nicotinamida Fosforribosiltransferase/metabolismo , Pentosiltransferases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Mol Cancer Res ; 13(6): 1009-21, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25758165

RESUMO

UNLABELLED: Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti-miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti-miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti-miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. IMPLICATIONS: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention.


Assuntos
Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Oligorribonucleotídeos Antissenso/farmacologia , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Xenoenxertos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/química , Invasividade Neoplásica , Oligorribonucleotídeos Antissenso/uso terapêutico
20.
Nucleic Acids Res ; 43(2): 1204-15, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25550434

RESUMO

Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , MicroRNAs/antagonistas & inibidores , Neoplasias/metabolismo , Oligonucleotídeos/metabolismo , Animais , Transporte Biológico , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Feminino , Humanos , Camundongos SCID , MicroRNAs/metabolismo , Neoplasias/genética , Fatores de Transcrição/fisiologia
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