RESUMEN
BACKGROUND: Cellular immunotherapies for cancer represent a means by which a patient's immune system can be augmented with high numbers of tumor-specific T cells. Chimeric antigen receptor (CAR) therapy involves genetic engineering to 'redirect' peripheral T cells to tumor targets, showing remarkable potency in blood cancers. However, due to several resistance mechanisms, CAR-T cell therapies remain ineffective in solid tumors. We and others have shown the tumor microenvironment harbors a distinct metabolic landscape that produces a barrier to immune cell function. Further, altered differentiation of T cells within tumors induces defects in mitochondrial biogenesis, resulting in severe cell-intrinsic metabolic deficiencies. While we and others have shown murine T cell receptor (TCR)-transgenic cells can be improved through enhanced mitochondrial biogenesis, we sought to determine whether human CAR-T cells could be enabled through a metabolic reprogramming approach. MATERIALS AND METHODS: Anti-EGFR CAR-T cells were infused in NSG mice which bore A549 tumors. The tumor infiltrating lymphocytes were analyzed for exhaustion and metabolic deficiencies. Lentiviruses carrying PPAR-gamma coactivator 1α (PGC-1α), PGC-1αS571A and NT-PGC-1α constructs were used to co-transduce T cells with anti-EGFR CAR lentiviruses. We performed metabolic analysis via flow cytometry and Seahorse analysis in vitro as well as RNA sequencing. Finally, we treated therapeutically A549-carrying NSG mice with either PGC-1α or NT-PGC-1α anti-EGFR CAR-T cells. We also analyzed the differences in the tumor-infiltrating CAR-T cells when PGC-1α is co-expressed. RESULTS: Here, in this study, we show that an inhibition resistant, engineered version of PGC-1α, can metabolically reprogram human CAR-T cells. Transcriptomic profiling of PGC-1α-transduced CAR-T cells showed this approach effectively induced mitochondrial biogenesis, but also upregulated programs associated with effector functions. Treatment of immunodeficient animals bearing human solid tumors with these cells resulted in substantially improved in vivo efficacy. In contrast, a truncated version of PGC-1α, NT-PGC-1α, did not improve the in vivo outcomes. CONCLUSIONS: Our data further support a role for metabolic reprogramming in immunomodulatory treatments and highlight the utility of genes like PGC-1α as attractive candidates to include in cargo along with chimeric receptors or TCRs for cell therapy of solid tumors.
Asunto(s)
Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Animales , Ratones , Inmunoterapia Adoptiva/métodos , Receptores de Antígenos de Linfocitos T , Linfocitos T , Microambiente TumoralRESUMEN
Gene transfer technology and its application to human gene therapy greatly expanded in the last decade. One area of investigation that appears particularly promising is the transfer of new genetic material into T cells for the potential treatment of cancer. Herein, we describe several core technologies that now yield high-efficiency gene transfer into primary human T cells. These gene transfer techniques include viral-based gene transfer methods based on modified Retroviridae and non-viral methods such as DNA-based transposons and direct transfer of mRNA by electroporation. Where specific examples are cited, we emphasize the transfer of chimeric antigen receptors (CARs) to T cells, which permits engineered T cells to recognize potential tumor antigens.
RESUMEN
Several anti-PD-1/PD-L1 monoclonal antibodies (mAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these mAbs is to inhibit PD-1 on immune cells interacting with PD-L1 on tumor cells. These mAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective mAb-mediated cancer therapies. A fully human anti-PD-L1 mAb would potentially be able to block PD-1/PD-L1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 mAb. The studies reported here demonstrate (i) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (ii) IFNγ can enhance tumor cell PD-L1 expression and, in some cases, enhance ADCC tumor cell lysis; (iii) purified NK cells are potent effectors for avelumab; (iv) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (v) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (vi) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 mAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity.
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Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Anticuerpos Monoclonales Humanizados , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Biomarcadores de Tumor , Línea Celular Tumoral , Membrana Celular/metabolismo , Expresión Génica , Genotipo , Humanos , Interferón gamma/metabolismo , Interferón gamma/farmacología , Interleucina-12/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Receptores de IgG/genética , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
The transcription factor brachyury is a major driver of epithelial to mesenchymal transition in human carcinoma cells. It is overexpressed in several human tumor types versus normal adult tissues, except for testes and thyroid. Overexpression is associated with drug resistance and poor prognosis. Previous studies identified a brachyury HLA-A2 cytotoxic T-lymphocyte epitope. The studies reported here describe an enhancer epitope of brachyury. Compared to the native epitope, the agonist epitope: (a) has enhanced binding to MHC class I, (b) increased the IFN-γ production from brachyury-specific T cells, (c) generated brachyury-specific T cells with greater levels of perforin and increased proliferation, (d) generated T cells more proficient at lysing human carcinoma cells endogenously expressing the native epitope, and (e) achieved greater brachyury-specific T-cell responses in vivo in HLA-A2 transgenic mice. These studies also report the generation of a heat-killed recombinant Saccharomyces cerevisiae (yeast) vector expressing the full-length brachyury gene encoding the agonist epitope. Compared to yeast-brachyury (native) devoid of the agonist epitope, the yeast-brachyury (agonist) enhanced the activation of brachyury-specific T cells, which efficiently lysed human carcinoma cells. In addition to providing the rationale for the recombinant yeast-brachyury (agonist) as a potential vaccine in cancer therapy, these studies also provide the rationale for the use of the agonist in (a) dendritic cell (DC) vaccines, (b) adjuvant or liposomal vaccines, (c) recombinant viral and/or bacterial vaccines, (d) protein/polypeptide vaccines, (e) activation of T cells ex vivo in adoptive therapy protocols, and (f) generation of genetically engineered targeted T cells.
Asunto(s)
Transición Epitelial-Mesenquimal/inmunología , Epítopos de Linfocito T/inmunología , Proteínas Fetales/inmunología , Neoplasias/inmunología , Proteínas de Dominio T Box/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Neoplasias/patología , Neoplasias/terapiaRESUMEN
The MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated variable number of tandem repeats (VNTR) region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are also being evaluated that express the entire MUC1 transgene. While previous studies have described MUC1 as a tumor-associated tissue differentiation antigen, studies have now determined that the C-terminus of MUC1 (MUC1-C) is an oncoprotein, and its expression is an indication of poor prognosis in numerous tumor types. We report here the identification of nine potential CD8⺠cytotoxic T lymphocyte epitopes of MUC1, seven in the C-terminus and two in the VNTR region, and have identified enhancer agonist peptides for each of these epitopes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 major histocompatibility complex (MHC) class I alleles, which encompass the majority of the population. The agonist peptides, compared to the native peptides, more efficiently (a) generate T-cell lines from the peripheral blood mononuclear cells of cancer patients, (b) enhance the production of IFN-γ by peptide-activated human T cells, and (c) lyse human tumor cell targets in an MHC-restricted manner. The agonist epitopes described here can be incorporated into various vaccine platforms and for the ex vivo generation of human T cells. These studies provide the rationale for the T-cell-mediated targeting of the oncogenic MUC1-C, which has been shown to be an important factor in both drug resistance and poor prognosis for numerous tumor types.
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Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Mucina-1/inmunología , Fragmentos de Péptidos/inmunología , Línea Celular Tumoral , Antígeno HLA-A2/inmunología , Antígeno HLA-A24/inmunología , Antígeno HLA-A3/inmunología , Humanos , Interferón gamma/biosíntesis , Repeticiones de Minisatélite , Neoplasias/inmunologíaRESUMEN
Malignant cells may evade death from cytotoxic agents if they are in a dormant state. The host microenvironment plays important roles in cancer progression, but how niches might control cancer cell dormancy is little understood. Here we show that osteopontin (OPN), an extracellular matrix molecule secreted by osteoblasts, can function to anchor leukemic blasts in anatomic locations supporting tumor dormancy. We demonstrate that acute lymphoblastic leukemia (ALL) cells specifically adhere to OPN in vitro and secrete OPN when localized to the endosteal niche in vivo. Using intravital microscopy to perform imaging studies of the calvarial bone marrow (BM) of xenografted mice, we show that OPN is highly expressed adjacent to dormant tumor cells within the marrow. Inhibition of the OPN-signaling axis significantly increases the leukemic cell Ki-67 proliferative index and leads to a twofold increase in tumor burden in treated mice. Moreover, using cell-cycle-dependent Ara-C chemotherapy to produce minimal residual disease (MRD) in leukemic mice, we show that OPN neutralization synergizes with Ara-C to reduce detectable BM MRD. Taken together, these data suggest that ALL interacts with extracellular OPN within the malignant BM, and that this interaction induces cell cycle exit in leukemic blasts, protecting them from cytotoxic chemotherapy.
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Crisis Blástica/metabolismo , Médula Ósea/metabolismo , Osteoblastos/metabolismo , Osteopontina/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Microambiente Tumoral , Adulto , Animales , Anticuerpos Neutralizantes/farmacología , Antimetabolitos Antineoplásicos/farmacología , Crisis Blástica/tratamiento farmacológico , Crisis Blástica/genética , Crisis Blástica/patología , Médula Ósea/patología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Niño , Preescolar , Citarabina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Neoplasia Residual/genética , Neoplasia Residual/metabolismo , Neoplasia Residual/patología , Osteoblastos/patología , Osteopontina/antagonistas & inhibidores , Osteopontina/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Transducción de Señal , Trasplante HeterólogoRESUMEN
Ovarian cancer patients frequently develop resistance to chemotherapy regiments using Taxol and carboplatin. One of the resistance factors that protects cancer cells from Taxol-based therapy is multidrug resistance 1 (MDR1). micro(mi)RNAs are small noncoding RNAs that negatively regulate protein expression. Members of the let-7 family of miRNAs are downregulated in many human cancers, and low let-7 expression has been correlated with resistance to microtubule targeting drugs (Taxanes), although little is known that would explain this activity. We now provide evidence that, although let-7 is not a universal sensitizer of cancer cells to Taxanes, it affects acquired resistance of cells to this class of drugs by targeting IMP-1, resulting in destabilization of the mRNA of MDR1. Introducing let-7g into ADR-RES cells expressing both IMP-1 and MDR1 reduced expression of both proteins rendering the cells more sensitive to treatment with either Taxol or vinblastine without affecting the sensitivity of the cells to carboplatin, a non-MDR1 substrate. This effect could be reversed by reintroducing IMP-1 into let-7g high/MDR1 low cells causing MDR1 to again become stabilized. Consistently, many relapsed ovarian cancer patients tested before and after chemotherapy were found to downregulate let-7 and to co-upregulate IMP-1 and MDR1, and the increase in the expression levels of both proteins after chemotherapy negatively correlated with disease-free time before recurrence. Our data point at IMP-1 and MDR1 as indicators for response to therapy, and at IMP-1 as a novel therapeutic target for overcoming multidrug resistance of ovarian cancer.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , MicroARNs/genética , Neoplasias Ováricas/genética , Proteínas de Unión al ARN/genética , Taxoides/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Supervivencia sin Enfermedad , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Inmunohistoquímica , Hibridación in Situ , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Taxoides/uso terapéuticoRESUMEN
MicroRNAs (miRNAs or miRs) are small noncoding RNAs capable of regulating gene expression at the translational level. Current evidence suggests that a significant portion of the human genome is regulated by microRNAs, and many reports have demonstrated that microRNA expression is deregulated in human cancer. The let-7 family of microRNAs, first discovered in Caenorhabditis elegans, is functionally conserved from worms to humans. The human let-7 family contains 13 members located on nine different chromosomes, and many human cancers have deregulated let-7 expression. A growing body of evidence suggests that restoration of let-7 expression may be a useful therapeutic option in cancers, where its expression has been lost. In this review, we discuss the role of let-7 in normal development and differentiation, and provide an overview of the relationship between deregulated let-7 expression and tumorigenesis. The regulation of let-7 expression, cancer-relevant let-7 targets, and the relationship between let-7 and drug sensitivity are highlighted.
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Diferenciación Celular/fisiología , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/fisiología , Neoplasias/genética , Animales , Antineoplásicos/farmacocinética , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/fisiología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Resistencia a Antineoplásicos/genética , Evolución Molecular , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Larva , Masculino , MicroARNs/genética , MicroARNs/uso terapéutico , Familia de Multigenes , Neoplasias/patología , Neoplasias/terapia , ARN Neoplásico/genética , Especificidad de la EspecieRESUMEN
MicroRNAs (miRNA) are small RNA molecules of approximately 20 to 22 nucleotides that reduce expression of proteins through mRNA degradation and/or translational silencing. Each known miRNA has a large number of predicted targets. Members of the let-7/miR-98 family of miRNAs are up-regulated at the end of embryonic development. Let-7 is often down-regulated early during cancer development, suggesting that let-7-regulated oncofetal genes (LOG) may become reexpressed in cancer cells. Using comparative bioinformatics, we have identified 12 conserved LOGs that include HMGA2 and IMP-1/CRD-BP. IMP-1 has growth-promoting activities through stabilization of c-myc mRNA. We experimentally confirmed that IMP-1 is a direct let-7 target that promotes cell growth and motility of tumor cells, and we confirmed by proteomics analysis that IMP-1 and HMGA2 are major miRNA targets. Our data suggest that a substantial part of the growth inhibitory activities of let-7 comes from suppressing the expression of IMP-1. LOGs could be novel therapeutic targets and potential biomarkers for cancer treatment.
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Regulación de la Expresión Génica , MicroARNs/genética , Adenocarcinoma , Animales , Antígenos de Neoplasias/genética , División Celular , Línea Celular Tumoral , Movimiento Celular , Cartilla de ADN , Silenciador del Gen , Humanos , Neoplasias Pulmonares , Ratones , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
FADD is essential for death receptor (DR)-induced apoptosis. However, it is also critical for cell cycle progression and proliferation, activities that are regulated by phosphorylation of its C-terminal Ser194, which has also been implicated in sensitizing cancer cells to chemotherapeutic drugs and in regulating FADD's intracellular localization. We now demonstrate that casein kinase Ialpha (CKIalpha) phosphorylates FADD at Ser194 both in vitro and in vivo. FADD-CKIalpha association regulates the subcellular localization of FADD, and phosphorylated FADD was found to colocalize with CKIalpha on the spindle poles in metaphase. Inhibition of CKIalpha diminished FADD phosphorylation, prevented the ability of Taxol to arrest cells in mitosis, and blocked mitogen-induced proliferation of mouse splenocytes. In contrast, a low level of cycling splenocytes from mice expressing FADD with a mutated phosphorylation site was insensitive to CKI inhibition. These data suggest that phosphorylation of FADD by CKI is a crucial event during mitosis.