RESUMO
Mantle cell lymphoma (MCL) is an incurable B-cell malignancy with an overall poor prognosis, particularly for patients that progress on targeted therapies. Novel, more durable treatment options are needed for patients with MCL. Protein arginine methyltransferase 5 (PRMT5) is overexpressed in MCL and plays an important oncogenic role in this disease via epigenetic and posttranslational modification of cell cycle regulators, DNA repair genes, components of prosurvival pathways, and RNA splicing regulators. The mechanism of targeting PRMT5 in MCL remains incompletely characterized. Here, we report on the antitumor activity of PRMT5 inhibition in MCL using integrated transcriptomics of in vitro and in vivo models of MCL. Treatment with a selective small-molecule inhibitor of PRMT5, PRT-382, led to growth arrest and cell death and provided a therapeutic benefit in xenografts derived from patients with MCL. Transcriptional reprograming upon PRMT5 inhibition led to restored regulatory activity of the cell cycle (p-RB/E2F), apoptotic cell death (p53-dependent/p53-independent), and activation of negative regulators of B-cell receptor-PI3K/AKT signaling (PHLDA3, PTPROt, and PIK3IP1). We propose pharmacologic inhibition of PRMT5 for patients with relapsed/refractory MCL and identify MTAP/CDKN2A deletion and wild-type TP53 as biomarkers that predict a favorable response. Selective targeting of PRMT5 has significant activity in preclinical models of MCL and warrants further investigation in clinical trials.
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Linfoma de Célula do Manto , Fosfatidilinositol 3-Quinases , Adulto , Humanos , Linhagem Celular Tumoral , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
Diffuse large B-cell lymphoma (DLBCL) is the most common Non-Hodgkin's lymphoma and is characterized by a remarkable heterogeneity with diverse variants that can be identified histologically and molecularly. Large-scale gene expression profiling studies have identified the germinal center B-cell (GCB-) and activated B-cell (ABC-) subtypes. Standard chemo-immunotherapy remains standard front line therapy, curing approximately two thirds of patients. Patients with refractory disease or those who relapse after salvage treatment have an overall poor prognosis highlighting the need for novel therapeutic strategies. Transducin ß-like protein 1 (TBL1) is an exchange adaptor protein encoded by the TBL1X gene and known to function as a master regulator of the Wnt signalling pathway by binding to ß-CATENIN and promoting its downstream transcriptional program. Here, we show that, unlike normal B-cells, DLBCL cells express abundant levels of TBL1 and its overexpression correlates with poor clinical outcome regardless of DLBCL molecular subtype. Genetic deletion of TBL1 and pharmacological approach using tegavivint, a first-in-class small molecule targeting TBL1 (Iterion Therapeutics), promotes DLBCL cell death in vitro and in vivo. Through an integrated genomic, biochemical, and pharmacologic analyses, we characterized a novel, ß-CATENIN independent, post-transcriptional oncogenic function of TBL1 in DLBCL where TBL1 modulates the stability of key oncogenic proteins such as PLK1, MYC, and the autophagy regulatory protein BECLIN-1 through its interaction with a SKP1-CUL1-F-box (SCF) protein supercomplex. Collectively, our data provide the rationale for targeting TBL1 as a novel therapeutic strategy in DLBCL.
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Linfoma Difuso de Grandes Células B , Transducina , Carcinogênese , Perfilação da Expressão Gênica , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Recidiva Local de Neoplasia , Prognóstico , Transducina/genéticaRESUMO
The surface receptor FcγRIIIA (CD16a) is encoded by the FCGR3A gene and is acquired by human NK cells during maturation. NK cells bind the Fc portion of IgG via CD16a and execute Ab-dependent cell-mediated cytotoxicity, which is critical for the effectiveness of several antitumor mAb therapies. The role of epigenetic regulatory mechanisms controlling transcriptional and posttranscriptional CD16 expression in NK cells is unknown. In this study, we compared specific patterns of DNA methylation and expression of FCGR3A with FCGR3B, which differ in cell type-specific expression despite displaying nearly identical genomic sequences. We identified a sequence within the FCGR3A promoter that selectively exhibits reduced methylation in CD16a+ NK cells versus CD16a- NK cells and neutrophils. This region contained the transcriptional start site of the most highly expressed CD16a isoform in NK cells. Luciferase assays revealed remarkable cell-type specificity and methylation-dependent activity of FCGR3A- versus FCGR3B-derived sequences. Genomic differences between FCGR3A and FCGR3B are enriched at CpG dinucleotides, and mutation of variant CpGs reversed cell-type specificity. We further identified miR-218 as a posttranscriptional negative regulator of CD16a in NK cells. Forced overexpression of miR-218 in NK cells knocked down CD16a mRNA and protein expression. Moreover, miR-218 was highly expressed in CD16a- NK cells compared with CD16a+ NK cells. Taken together, we propose a system of FCGR3A regulation in human NK cells in which CpG dinucleotide sequences and concurrent DNA methylation confer developmental and cell type-specific transcriptional regulation, whereas miR-218 provides an additional layer of posttranscriptional regulation during the maturation process.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Processamento Pós-Transcricional do RNA , Receptores de IgG/genética , Diferenciação Celular , Linhagem Celular , Ilhas de CpG , Metilação de DNA , Citometria de Fluxo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Inativação Gênica , Genes Reporter , Humanos , MicroRNAs/genética , Regiões Promotoras Genéticas , Interferência de RNA , Receptores de IgG/metabolismoRESUMO
Group 3 innate lymphoid cells (ILC3s) are important regulators of the immune system, maintaining homeostasis in the presence of commensal bacteria, but activating immune defenses in response to microbial pathogens. ILC3s are a robust source of IL-22, a cytokine critical for stimulating the antimicrobial response. We sought to identify cytokines that can promote proliferation and induce or maintain IL-22 production by ILC3s and determine a molecular mechanism for this process. We identified IL-18 as a cytokine that cooperates with an ILC3 survival factor, IL-15, to induce proliferation of human ILC3s, as well as induce and maintain IL-22 production. To determine a mechanism of action, we examined the NF-κB pathway, which is activated by IL-18 signaling. We found that the NF-κB complex signaling component, p65, binds to the proximal region of the IL22 promoter and promotes transcriptional activity. Finally, we observed that CD11c+ dendritic cells expressing IL-18 are found in close proximity to ILC3s in human tonsils in situ. Therefore, we identify a new mechanism by which human ILC3s proliferate and produce IL-22, and identify NF-κB as a potential therapeutic target to be considered in pathologic states characterized by overproduction of IL-18 and/or IL-22.
Assuntos
Proliferação de Células , Interleucina-18/metabolismo , Interleucinas/biossíntese , Linfócitos/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais , Células Dendríticas/fisiologia , Humanos , Imunidade Inata , Interleucina-15/imunologia , Interleucinas/genética , Interleucinas/imunologia , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Regiões Promotoras Genéticas , Transdução de Sinais/imunologia , Fator de Transcrição RelA/metabolismo , Interleucina 22RESUMO
Killer cell Ig-like receptors (KIRs) on NK cells have been linked to a wide spectrum of health conditions such as chronic infections, autoimmune diseases, pregnancy complications, cancers, and transplant failures. A small subset of effector memory T cells also expresses KIRs. In this study, we use modern analytic tools including genome-wide and multiplex molecular, phenotypic, and functional assays to characterize the KIR(+) T cells in human blood. We find that KIR(+) T cells primarily reside in the CD56(+) T population that is distinctively DNAM-1(high) with a genome-wide quiescent transcriptome, short telomere, and limited TCR excision circles. During CMV reactivation in bone marrow transplant recipients, KIR(+)CD56(+) T cells rapidly expanded in real-time but not KIR(+)CD56(-) T cells or KIR(+) NK cells. In CMV(+) asymptomatic donors, as much as 50% of CD56(+) T cells are KIR(+), and most are distinguishably KIR2DL2/3(+)NKG2C(+)CD57(+). Functionally, the KIR(+)CD56(+) T cell subset lyses cancer cells and CMVpp65-pulsed target cells in a dual KIR-dependent and TCR-dependent manner. Analysis of metabolic transcriptome confirms the immunological memory status of KIR(+)CD56(+) T cells in contrast to KIR(-)CD56(+) T cells that are more active in energy metabolism and effector differentiation. KIR(-)CD56(+) T cells have >25-fold higher level of expression of RORC than the KIR(+) counterpart and are a previously unknown producer of IL-13 rather than IL-17 in multiplex cytokine arrays. Our data provide fundamental insights into KIR(+) T cells biologically and clinically.
Assuntos
Células Matadoras Naturais/imunologia , Células T Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/análise , Doenças Assintomáticas , Transplante de Medula Óssea , Antígeno CD56/análise , Antígenos CD57/análise , Linhagem Celular Tumoral , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/imunologia , Citotoxicidade Imunológica , Estudo de Associação Genômica Ampla , Humanos , Imunofenotipagem , Metaboloma , Reação em Cadeia da Polimerase Multiplex , Subfamília C de Receptores Semelhantes a Lectina de Células NK/análise , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores KIR/análise , Receptores KIR2DL2/análise , Receptores KIR2DL3/análise , Telômero/ultraestrutura , Células Th17/imunologia , Doadores de Tecidos , Transcriptoma , Ativação ViralRESUMO
Group 1 innate lymphoid cells (ILCs) comprise conventional natural killer (cNK) cells and type 1 innate lymphoid cells (ILC1s). The main functions of liver cNK cells and ILC1s not only include directly killing target cells but also regulating local immune microenvironment of the liver through the secretion of cytokines. Uncovering the intricate mechanisms by which transcriptional factors regulate and influence the functions of liver cNK cells and ILC1s, particularly within the context of liver tumors, presents a significant opportunity to amplify the effectiveness of immunotherapies against liver malignancies. Using Ncr1-drived conditional knockout mouse model, our study reveals the regulatory role of Prdm1 in shaping the composition and maturation of cNK cells. Although Prdm1 did not affect the killing function of cNK cells in an in vivo cytotoxicity model, a significant increase in cancer metastasis was observed in Prdm1 knockout mice. Interferon-gamma (IFN-γ), granzyme B, and perforin secretion decreased significantly in Prdm1-deficient cNK cells and liver ILC1s. Single-cell RNA sequencing (scRNA-seq) data also provided evidences that Prdm1 maintains functional subsets of cNK cells and liver ILC1s and facilitates communications between cNK cells, liver ILC1s, and macrophages. The present study unveiled a novel regulatory mechanism of Prdm1 in cNK cells and liver ILC1s, showing promising potential for developing innovative immune therapy strategies against liver cancer.
Assuntos
Neoplasias Hepáticas , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo , Animais , Camundongos , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/genética , Células Matadoras Naturais/imunologia , Interferon gama/metabolismo , Imunidade Inata , Linfócitos/imunologia , Vigilância Imunológica , Granzimas/metabolismo , Granzimas/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Perforina/metabolismo , Perforina/genética , Fígado/imunologia , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Microambiente Tumoral/imunologia , Antígenos LyRESUMO
ABSTRACT: Chimeric antigen receptor (CAR) T-cell therapy has revolutionized treatment for relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). Robust biomarkers and a complete understanding of CAR T-cell function in the postinfusion phase remain limited. Here, we used a 37-color spectral flow cytometry panel to perform high dimensional single-cell analysis of postinfusion samples in 26 patients treated with CD28 costimulatory domain containing commercial CAR T cells for NHL and focused on computationally gated CD8+ CAR T cells. We found that the presence of postinfusion Programmed cell death protein 1 (PD-1)+ CD8+ CAR T cells at the day 14 time point highly correlated with the ability to achieve complete response (CR) by 6 months. Further analysis identified multiple subtypes of CD8+ PD-1+ CAR T cells, including PD-1+ T cell factor 1 (TCF1)+ stem-like CAR T cells and PD-1+ T-cell immunoglobulin and mucin-domain containing-3 (TIM3)+ effector-like CAR T cells that correlated with improved clinical outcomes such as response and progression-free survival. Additionally, we identified a subset of PD-1+ CD8+ CAR+ T cells with effector-like function that was increased in patients who achieved a CR and was associated with grade 3 or higher immune effector cell-associated neurotoxicity syndrome. Here, we identified robust biomarkers of response to CD28 CAR T cells and highlight the importance of PD-1 positivity in CD8+ CAR T cells after infusion in achieving CR.
Assuntos
Linfócitos T CD8-Positivos , Imunoterapia Adotiva , Linfoma não Hodgkin , Receptor de Morte Celular Programada 1 , Humanos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfoma não Hodgkin/terapia , Linfoma não Hodgkin/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Imunoterapia Adotiva/métodos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Adulto , Antígenos CD19/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Resultado do TratamentoRESUMO
IL-22 is a type 2 receptor cytokine in IL-10 family. IL-22 is usually secreted by innate and adaptive immune cells and takes its effects on non-hematopoietic cells. Through activate STAT3 pathway, IL-22 plays an important role in infection clearance and tissue regeneration, which is critical for barrier integrate and homeostasis. Abnormal activation of IL-22 signal was observed in inflammation diseases, autoimmune diseases, and cancers. We review the recent discoveries about the mechanism and regulation of IL-22 signal pathway from the perspective of intestinal micro-environment. Diet-based IL-22 target therapeutic strategies and their potential clinical significance will also be discussed.
Assuntos
Interleucinas , Intestinos , Humanos , Interleucinas/metabolismo , Citocinas/metabolismo , Dieta , Imunidade Inata , Inflamação , Interleucina 22RESUMO
BACKGROUND: Mantle cell lymphoma (MCL) is a rare B-cell non-Hodgkin lymphoma subtype which remains incurable despite multimodal approach including chemoimmunotherapy followed by stem cell transplant, targeted approaches such as the BTK inhibitor ibrutinib, and CD19 chimeric antigen receptor (CAR) T cells. CD74 is a nonpolymorphic type II integral membrane glycoprotein identified as an MHC class II chaperone and a receptor for macrophage migration inhibitory factor. Our group previously reported on CD74's abundant expression in MCL and its ability to increase via pharmacological inhibition of autophagosomal degradation. Milatuzumab, a fully humanized anti-CD74 monoclonal antibody, demonstrated significant activity in preclinical lymphoma models but failed to provide meaningful benefits in clinical trials mainly due to its short half-life. We hypothesized that targeting CD74 using a CAR-T cell would provide potent and durable anti-MCL activity. METHODS: We engineered a second generation anti-CD74 CAR with 4-1BB and CD3ζ signaling domains (74bbz). Through in silico and rational mutagenesis on the scFV domain, the 74bbz CAR was functionally optimized for superior antigen binding affinity, proliferative signaling, and cytotoxic activity against MCL cells in vitro and in vivo. RESULTS: Functionally optimized 74bbz CAR-T cells (clone 42105) induced significant killing of MCL cell lines, and primary MCL patient samples including one relapse after commercial CD19 CAR-T cell therapy with direct correlation between antigen density and cytotoxicity. It significantly prolonged the survival of an animal model established in NOD-SCIDγc-/- (NSG) mice engrafted with a human MCL cell line Mino subcutaneously compared to controls. Finally, while CD74 is also expressed on normal immune cell subsets, treatment with 74bbz CAR-T cells resulted in minimal cytotoxicity against these cells both in vitro and in vivo. CONCLUSIONS: Targeting CD74 with 74bbz CAR-T cells represents a new cell therapy to provide a potent and durable and anti-MCL activity.
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Epstein-Barr virus (EBV) is a ubiquitous herpes virus associated with various cancers. EBV establishes latency with life-long persistence in memory B-cells and can reactivate lytic infection placing immunocompromised individuals at risk for EBV-driven lymphoproliferative disorders (EBV-LPD). Despite the ubiquity of EBV, only a small percentage of immunocompromised patients (~20%) develop EBV-LPD. Engraftment of immunodeficient mice with peripheral blood mononuclear cells (PBMCs) from healthy EBV-seropositive donors leads to spontaneous, malignant, human B-cell EBV-LPD. Only about 20% of EBV+ donors induce EBV-LPD in 100% of engrafted mice (High-Incidence, HI), while another 20% of donors never generate EBV-LPD (No-Incidence, NI). Here, we report HI donors to have significantly higher basal T follicular helper (Tfh) and regulatory T-cells (Treg), and depletion of these subsets prevents/delays EBV-LPD. Transcriptomic analysis of CD4+ T cells from ex vivo HI donor PBMC revealed amplified cytokine and inflammatory gene signatures. HI vs. NI donors showed a marked reduction in IFNγ production to EBV latent and lytic antigen stimulation. In addition, we observed abundant myeloid-derived suppressor cells in HI donor PBMC that decreased CTL proliferation in co-cultures with autologous EBV+ lymphoblasts. Our findings identify potential biomarkers that may identify individuals at risk for EBV-LPD and suggest possible strategies for prevention.
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Mantle cell lymphoma (MCL) is an incurable B-cell malignancy that comprises up to 6% of non-Hodgkin lymphomas diagnosed annually and is associated with a poor prognosis. The average overall survival of patients with MCL is 5 years, and for most patients who progress on targeted agents, survival remains at a dismal 3 to 8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated to improve treatment outcomes and quality of life. The protein arginine methyltransferase 5 (PRMT5) enzyme is overexpressed in MCL and promotes growth and survival. Inhibition of PRMT5 drives antitumor activity in MCL cell lines and preclinical murine models. PRMT5 inhibition reduced the activity of prosurvival AKT signaling, which led to the nuclear translocation of FOXO1 and modulation of its transcriptional activity. Chromatin immunoprecipitation and sequencing identified multiple proapoptotic BCL-2 family members as FOXO1-bound genomic loci. We identified BAX as a direct transcriptional target of FOXO1 and demonstrated its critical role in the synergy observed between the selective PRMT5 inhibitor, PRT382, and the BCL-2 inhibitor, venetoclax. Single-agent and combination treatments were performed in 9 MCL lines. Loewe synergy scores showed significant levels of synergy in most MCL lines tested. Preclinical, in vivo evaluation of this strategy in multiple MCL models showed therapeutic synergy with combination venetoclax/PRT382 treatment with an increased survival advantage in 2 patient-derived xenograft models (P ≤ .0001, P ≤ .0001). Our results provide mechanistic rationale for the combination of PRMT5 inhibition and venetoclax to treat patients with MCL.
Assuntos
Antineoplásicos , Compostos Bicíclicos Heterocíclicos com Pontes , Linfoma de Célula do Manto , Sulfonamidas , Animais , Humanos , Camundongos , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Qualidade de VidaRESUMO
BACKGROUND: Mantle cell lymphoma (MCL) is a rare, highly heterogeneous type of B-cell non-Hodgkin's lymphoma. The sumoylation pathway is known to be upregulated in many cancers including lymphoid malignancies. However, little is known about its oncogenic role in MCL. METHODS: Levels of sumoylation enzymes and sumoylated proteins were quantified in MCL cell lines and primary MCL patient samples by scRNA sequencing and immunoblotting. The sumoylation enzyme SAE2 was genetically and pharmacologically targeted with shRNA and TAK-981 (subasumstat). The effects of SAE2 inhibition on MCL proliferation and cell cycle were evaluated using confocal microscopy, live-cell microscopy, and flow cytometry. Immunoprecipitation and orbitrap mass spectrometry were used to identify proteins targeted by sumoylation in MCL cells. RESULTS: MCL cells have significant upregulation of the sumoylation pathway at the level of the enzymes SAE1 and SAE2 which correlated with poor prognosis and induction of mitosis associated genes. Selective inhibition of SAE2 with TAK-981 results in significant MCL cell death in vitro and in vivo with mitotic dysregulation being an important mechanism of action. We uncovered a sumoylation program in mitotic MCL cells comprised of multiple pathways which could be directly targeted with TAK-981. Centromeric localization of topoisomerase 2A, a gene highly upregulated in SAE1 and SAE2 overexpressing MCL cells, was lost with TAK-981 treatment likely contributing to the mitotic dysregulation seen in MCL cells. CONCLUSIONS: This study not only validates SAE2 as a therapeutic target in MCL but also opens the door to further mechanistic work to uncover how to best use desumoylation therapy to treat MCL and other lymphoid malignancies.
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Killer immunoglobulin-like receptors (KIRs) play an essential role in the regulation of natural killer cell functions. KIR genes are highly polymorphic in nature, showing both haplotypic and allelic variations among people. We demonstrated in both in vitro and in vivo models a significant heterogeneity in function among different KIR2DL1 alleles, including their ability to inhibit YT-Indy cells from degranulation, interferon gamma production, and cytotoxicity against target cells expressing the HLA-Cw6 ligand. Subsequent experiments showed that the molecular determinant was an arginine residue at position 245 (R245) in its transmembrane domain that mechanistically affects both the efficiency of inhibitory signaling and durability of surface expression. Specifically, in comparison with R245-negative alleles, KIR2DL1 that included R245 recruited more Src-homology-2 domain-containing protein tyrosine phosphatase 2 and beta-arrestin 2, showed higher inhibition of lipid raft polarization at immune synapse, and had less down-regulation of cell-surface expression upon interaction with its ligand. Thus, our findings provide novel insights into the molecular determinant of KIR2DL1 and conceivably a fundamental understanding of KIR2DL1 allelic polymorphism in human disease susceptibility, transplant outcome, and donor selection.
Assuntos
Alelos , Arginina/genética , Receptores KIR2DL1/genética , Arginina/metabolismo , Arginina/fisiologia , Arrestinas/genética , Arrestinas/metabolismo , Linhagem Celular Tumoral , Testes Imunológicos de Citotoxicidade , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Antígenos HLA-C/genética , Antígenos HLA-C/metabolismo , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Microscopia de Fluorescência , Mutação , Polimorfismo Genético , Receptores KIR2DL1/metabolismo , Receptores KIR2DL1/fisiologia , Transdução de Sinais/imunologia , Transfecção , beta-Arrestina 2 , beta-ArrestinasRESUMO
PURPOSE: Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPTi) are currently in development, but may be limited as single-agent therapy due to compound-specific toxicity and cancer metabolic plasticity allowing resistance development. To potentially lower the doses of NAMPTis required for therapeutic benefit against acute myeloid leukemia (AML), we performed a genome-wide CRISPRi screen to identify rational disease-specific partners for a novel NAMPTi, KPT-9274. EXPERIMENTAL DESIGN: Cell lines and primary cells were analyzed for cell viability, self-renewal, and responses at RNA and protein levels with loss-of-function approaches and pharmacologic treatments. In vivo efficacy of combination therapy was evaluated with a xenograft model. RESULTS: We identified two histone deacetylases (HDAC), HDAC8 and SIRT6, whose knockout conferred synthetic lethality with KPT-9274 in AML. Furthermore, HDAC8-specific inhibitor, PCI-34051, or clinical class I HDAC inhibitor, AR-42, in combination with KPT-9274, synergistically decreased the survival of AML cells in a dose-dependent manner. AR-42/KPT-9274 cotreatment attenuated colony-forming potentials of patient cells while sparing healthy hematopoietic cells. Importantly, combined therapy demonstrated promising in vivo efficacy compared with KPT-9274 or AR-42 monotherapy. Mechanistically, genetic inhibition of SIRT6 potentiated the effect of KPT-9274 on PARP-1 suppression by abolishing mono-ADP ribosylation. AR-42/KPT-9274 cotreatment resulted in synergistic attenuation of homologous recombination and nonhomologous end joining pathways in cell lines and leukemia-initiating cells. CONCLUSIONS: Our findings provide evidence that HDAC8 inhibition- or shSIRT6-induced DNA repair deficiencies are potently synergistic with NAMPT targeting, with minimal toxicity toward normal cells, providing a rationale for a novel-novel combination-based treatment for AML.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Citocinas/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Sirtuínas/antagonistas & inibidores , Acrilamidas/farmacologia , Acrilamidas/uso terapêutico , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Técnicas de Inativação de Genes , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Fenilbutiratos/farmacologia , Fenilbutiratos/uso terapêutico , Reparo de DNA por Recombinação/efeitos dos fármacos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: Under the influence of interferon-gamma (IFN-gamma), mesenchymal stromal cells (MSCs) are conditional antigen-presenting cells, which have immunosuppressive potential. Apart from IFN-gamma upregulation of major histocompatibility complexes class I and II (MHC-I and MHC-II) expression, the underlying kinetics and mechanisms have not been described previously. This information is helpful to delineate how human MSCs can be modulated by IFN-gamma in different clinical scenarios. MATERIALS AND METHODS: Here, we demonstrated that IFN-gamma-treated MSCs underwent classical signal transduction pathway via phosphorylation of signal transducers and activators of transcription-1, activation of interferon regulatory factor-1, and class II transactivator comparable to that of primary human blood macrophages. RESULTS: IFN-gamma markedly induced expression of MHC-I instantly, while its effects on MHC-II were less dramatic and delayed up to 4 days. This is due to a slower intracellular transport of the MHC-II antigen to the membrane surface. More important is that MSCs showed a reduction in their proliferation by 50% without evidence of cell death after prolonged IFN-gamma treatment for 8 days. High-dose IFN-gamma-treated MSCs (500 U/mL) could initiate T-cell activation as indicated by expression of CD25 and proliferation of allogeneic T cells. CONCLUSIONS: The summative IFN-gamma effects will adversely affect the immunoprivilege status and lifespan of MSCs.
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Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe I/análise , Interferon gama/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Ativação Linfocitária , Células-Tronco Mesenquimais/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
BACKGROUND: Human mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells) are multipotent cells with potential therapeutic value. Owing to their osteogenic capability, MSCs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. However, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human MSCs remains unknown. This study investigated whether proliferation and osteogenic differentiation of MSCs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings. RESULTS: We selected lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Streptococcus pyogenes as our toxins of choice. Our findings showed both LPS and LTA did not affect MSC proliferation, but prolonged LPS challenge upregulated the osteogenic differentiation of MSCs, as assessed by alkaline phosphatase activity and calcium deposition. Because toll-like receptors (TLRs), in particularly TLR4 and TLR2, are important for the cellular responsiveness to LPS and LTA respectively, we evaluated their expression profiles serially from MSCs to osteoblasts by quantitative PCR. We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12. But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression. This peculiar response to LPS suggests a possible adaptive mechanism when MSCs are subjected to continuous exposure with bacteria. CONCLUSION: In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment.
Assuntos
Toxinas Bacterianas/farmacologia , Regulação para Baixo , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Receptores Toll-Like/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Ácidos Teicoicos/química , Ácidos Teicoicos/farmacologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
There are substances that kill cancer cells, but induce T cell proliferation, like thalidomide. To find more of these, a new anticancer drug screening strategy is vital. In this study we report the development of a differential cytotoxicity screening or evaluation platform using the CellTiter-Glo (Promega, Annandale, NSW, Australia) luminescent cell viability assay (ATP assay) and also the CellTiter 96 AQueous (Promega) one solution cell proliferation assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay]. The results suggested the platform consisting of the combination of the ATP assay be used for quantifying peripheral blood mononuclear cells, while the more economic MTS colorimetric assay is well suited to be used detecting cell viability of cancer cells. In addition, we found paclitaxel (Taxol, MP Biomedicals Australasia Pty Ltd., Seven Hills, NSW, Australia) to be a useful control for this routine screening methodology. Taxol exhibits the desirable in vitro feature of differential cytotoxicity that spares the immunological cells, when used at a concentration that will kill the majority of the cancer cell population.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Trifosfato de Adenosina/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Colorimetria , Corantes , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Indicadores e Reagentes , Monócitos/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Paclitaxel/farmacologia , Própole/farmacologia , Reprodutibilidade dos Testes , Sais de Tetrazólio , Tiazóis , Azul TripanoRESUMO
Multiple myeloma (MM) is an incurable hematologic malignancy of plasma cells, with an estimated 30,000 new cases diagnosed each year in the United States, signifying the need for new therapeutic approaches. We hypothesized that targeting MM using a bispecific antibody (biAb) to simultaneously engage both innate and adaptive cytolytic immune cells could present potent antitumor activity. We engineered a biAb by fusing an anti-CS1 single-chain variable fragment (scFv) and an anti-NKG2D scFv (CS1-NKG2D biAb). Although NKG2D is a potent activation receptor ubiquitously expressed on mostly cytolytic immune cells including NK cells, CD8+ T cells, γδ T cells, and NKT cells, the CS1 tumor-associated antigen on MM represents a promising target. CS1-NKG2D biAb engaged human MM cell lines and NKG2D+ immune cells, forming immune synapses. In effector cells, CS1-NKG2D biAb triggered the phosphorylation of AKT, a downstream protein kinase of the activated NKG2D-DAP10 complex. The EC50 values of CS1-NKG2D biAb for CS1high and for CS1low MM cell lines with effector PBMCs were 10-12 and 10-9 mol/L, respectively. CS1-NKG2D biAb acted through multiple types of immune cells, and this induced cytotoxicity was both CS1- and NKG2D-specific. In vivo, survival was significantly prolonged using CS1-NKG2D biAb in a xenograft NOD-SCIDIL2γc-/- (NSG) mouse model engrafted with both human PBMCs and MM cell lines. Collectively, we demonstrated that the CS1-NKG2D biAb facilitated an enhanced immune synapse between CS1+ MM cells and NKG2D+ cytolytic innate and antigen-specific effector cells, which, in turn, activated these immune cells for improved clearance of MM. Cancer Immunol Res; 6(7); 776-87. ©2018 AACR.
Assuntos
Anticorpos Biespecíficos/farmacologia , Citotoxicidade Imunológica , Mieloma Múltiplo/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Família de Moléculas de Sinalização da Ativação Linfocitária/antagonistas & inibidores , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Animais , Biomarcadores , Linhagem Celular Tumoral , Feminino , Humanos , Sinapses Imunológicas , Imunofenotipagem , Interferon gama/metabolismo , Camundongos , Mieloma Múltiplo/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Subpopulações de Linfócitos T/metabolismoRESUMO
OBJECTIVES: Ganoderma lucidum (GL) is one of the most commonly used Chinese herbs in the oriental community, with more than 30% of pediatric cancer patients taking GL. The immunomodulating and anticancer effects exerted by GL extracts have been demonstrated by in vitro and in vivo studies. There was, however, no comparison between the immunomodulating effects of GL mycelium extract (GL-M) and spore extracts on human immune cells. Dendritic cells (DCs) are professional antigen-presenting cells and their role in DC-based tumor vaccine has been well defined. The possibility of GL as natural adjuvant for human DCs remains unknown. DESIGN: This study explored the differential effect of GL-M and GL spore extract (GL-S) on proliferation and Th1/Th2 cytokine mRNA expression of human peripheral blood mononuclear cells (PBMCs) and monocytes. Their effects on the phenotypic and functional maturation of human monocyte-derived DCs were also investigated. RESULTS: GL-M induced the proliferation of PBMCs and monocytes, whereas GL-S showed a mild suppressive effect. Both extracts could stimulate Th1 and Th2 cytokine mRNA expression, but GL-M was a relatively stronger Th1 stimulator. Different from GL-S, GL-M enhanced maturation of DCs in terms of upregulation of CD40, CD80, and CD86, and also reduced fluorescein isothiocyanate-dextran endocytosis. Interestingly, GLM- treated DCs only modestly enhanced lymphocyte proliferation in allogenic mixed lymphocyte culture with mild enhancement in Th development. CONCLUSION: These findings provide evidences that GL-M has immunomodulating effects on human immune cells and therefore can be used as a natural adjuvant for cancer immunotherapy with DCs.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Reishi , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Neoplasias/prevenção & controle , RNA Mensageiro/metabolismo , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). To identify recipient risk factors, a genome-wide study was performed including 481,820 single-nucleotide polymorphisms (SNPs). Two GVHD susceptibility loci (rs17114803 and rs17114808) within the SUFU gene were identified in the discovery cohort (p = 2.85 × 10(-5)). The incidence of acute GVHD among patients homozygous for CC at SUFU rs17114808 was 69%, which was significantly higher than the 8% rate observed in CT heterozygous patients (p = 0.0002). In an independent validation cohort of 100 patients, 50% of the patients with the CC genotype developed GVHD compared to 8% of the patients with either CT or TT genotype (p = 0.01). In comparison to CC dendritic cells, those from CT expressed higher levels of SUFU mRNA and protein, had lower levels of surface HLA-DR, and induced less allogeneic mixed leukocyte response (MLR). Ectopic expression of SUFU in THP-1 derived DCs reduced HLA-DR expression and suppressed MLR, whereas silencing of SUFU enhanced HLA-DR expression and increased MLR. Thus our findings provide novel evidence that recipient SUFU germline polymorphism is associated with acute GVHD and is a novel molecular target for GVHD prevention and treatment.