RESUMEN
PURPOSE: Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation, and chemotherapy, is often responsible for cognitive, neurologic, and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T cells directed toward the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. EXPERIMENTAL DESIGN: GD2 expression was evaluated on primary tumor biopsies of MB children by flow cytometry. GD2 expression in MB cells was also evaluated in response to an EZH2 inhibitor (tazemetostat). In in vitro and in vivo models, GD2+ MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible caspase-9. RESULTS: GD2 was expressed in 82.68% of MB tumors. The SHH and G3-G4 subtypes expressed the highest levels of GD2, whereas the WNT subtype expressed the lowest. In in vitro coculture assays, CAR.GD2 T cells were able to kill GD2+ MB cells. Pretreatment with tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T cells significantly controlled tumor growth, prolonging the overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood-brain barrier and to eliminate both blood-circulating and tumor-infiltrating CAR.GD2 T cells. CONCLUSIONS: Our experimental data indicate the potential efficacy of CAR.GD2 T-cell therapy. A phase I/II clinical trial is ongoing in our center (NCT05298995) to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.
Asunto(s)
Gangliósidos , Inmunoterapia Adoptiva , Meduloblastoma , Receptores Quiméricos de Antígenos , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Meduloblastoma/terapia , Meduloblastoma/inmunología , Meduloblastoma/patología , Meduloblastoma/genética , Meduloblastoma/metabolismo , Animales , Ratones , Gangliósidos/metabolismo , Gangliósidos/inmunología , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Inmunoterapia Adoptiva/métodos , Inmunoterapia Adoptiva/efectos adversos , Línea Celular Tumoral , Niño , Femenino , Linfocitos T/inmunología , Linfocitos T/metabolismo , Neoplasias Cerebelosas/terapia , Neoplasias Cerebelosas/inmunología , Neoplasias Cerebelosas/patología , Neoplasias Cerebelosas/metabolismo , Morfolinas/farmacología , Masculino , Preescolar , Benzamidas , Compuestos de Bifenilo , PiridonasRESUMEN
Chimeric antigen receptor T (CAR-T) cell therapy may achieve long-lasting remission in patients with B-cell malignancies not responding to conventional therapies. However, potentially severe and hard-to-manage side effects, including cytokine release syndrome (CRS), neurotoxicity and macrophage activation syndrome, and the lack of pathophysiological experimental models limit the applicability and development of this form of therapy. Here we present a comprehensive humanized mouse model, by which we show that IFNγ neutralization by the clinically approved monoclonal antibody, emapalumab, mitigates severe toxicity related to CAR-T cell therapy. We demonstrate that emapalumab reduces the pro-inflammatory environment in the model, thus allowing control of severe CRS and preventing brain damage, characterized by multifocal hemorrhages. Importantly, our in vitro and in vivo experiments show that IFNγ inhibition does not affect the ability of CD19-targeting CAR-T (CAR.CD19-T) cells to eradicate CD19+ lymphoma cells. Thus, our study provides evidence that anti-IFNγ treatment might reduce immune related adverse effect without compromising therapeutic success and provides rationale for an emapalumab-CAR.CD19-T cell combination therapy in humans.
Asunto(s)
Neoplasias , Receptores Quiméricos de Antígenos , Ratones , Animales , Humanos , Inmunoterapia Adoptiva/efectos adversos , Linfocitos B , Interferón gamma , Neoplasias/etiología , Síndrome de Liberación de Citoquinas , Antígenos CD19 , Tratamiento Basado en Trasplante de Células y TejidosRESUMEN
BACKGROUND: Paediatric acute myeloid leukaemia (AML) is characterized by poor outcomes in patients with relapsed/refractory disease, despite the improvements in intensive standard therapy. The leukaemic cells of paediatric AML patients show high expression of the CD123 antigen, and this finding provides the biological basis to target CD123 with the chimeric antigen receptor (CAR). However, CAR.CD123 therapy in AML is hampered by on-target off-tumour toxicity and a long "vein-to-vein" time. METHODS: We developed an off-the-shelf product based on allogeneic natural killer (NK) cells derived from the peripheral blood of healthy donors and engineered them to express a second-generation CAR targeting CD123 (CAR.CD123). RESULTS: CAR.CD123-NK cells showed significant anti-leukaemia activity not only in vitro against CD123+ AML cell lines and CD123+ primary blasts but also in two animal models of human AML-bearing immune-deficient mice. Data on anti-leukaemia activity were also corroborated by the quantification of inflammatory cytokines, namely granzyme B (Granz B), interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α), both in vitro and in the plasma of mice treated with CAR.CD123-NK cells. To evaluate and compare the on-target off-tumour effects of CAR.CD123-T and NK cells, we engrafted human haematopoietic cells (hHCs) in an immune-deficient mouse model. All mice infused with CAR.CD123-T cells died by Day 5, developing toxicity against primary human bone marrow (BM) cells with a decreased number of total hCD45+ cells and, in particular, of hCD34+CD38- stem cells. In contrast, treatment with CAR.CD123-NK cells was not associated with toxicity, and all mice were alive at the end of the experiments. Finally, in a mouse model engrafted with human endothelial tissues, we demonstrated that CAR.CD123-NK cells were characterized by negligible endothelial toxicity when compared to CAR.CD123-T cells. CONCLUSIONS: Our data indicate the feasibility of an innovative off-the-shelf therapeutic strategy based on CAR.CD123-NK cells, characterized by remarkable efficacy and an improved safety profile compared to CAR.CD123-T cells. These findings open a novel intriguing scenario not only for the treatment of refractory/resistant AML patients but also to further investigate the use of CAR-NK cells in other cancers characterized by highly difficult targeting with the most conventional T effector cells.
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Leucemia Mieloide Aguda , Receptores Quiméricos de Antígenos , Niño , Humanos , Ratones , Animales , Subunidad alfa del Receptor de Interleucina-3 , Receptores Quiméricos de Antígenos/uso terapéutico , Receptores Quiméricos de Antígenos/metabolismo , Leucemia Mieloide Aguda/patología , Inmunoterapia Adoptiva/efectos adversos , Células Asesinas Naturales , Línea Celular TumoralRESUMEN
Programmed cell death-1 (PD-1) signaling downregulates the T-cell response, promoting an exhausted state in tumor-infiltrating T cells, through mostly unveiled molecular mechanisms. Dynamin-related protein-1 (Drp1)-dependent mitochondrial fission plays a crucial role in sustaining T-cell motility, proliferation, survival, and glycolytic engagement. Interestingly, such processes are exactly those inhibited by PD-1 in tumor-infiltrating T cells. Here, we show that PD-1pos CD8+ T cells infiltrating an MC38 (murine adenocarcinoma)-derived murine tumor mass have a downregulated Drp1 activity and more elongated mitochondria compared with PD-1neg counterparts. Also, PD-1pos lymphocytic elements infiltrating a human colon cancer rarely express active Drp1. Mechanistically, PD-1 signaling directly prevents mitochondrial fragmentation following T-cell stimulation by downregulating Drp1 phosphorylation on Ser616, via regulation of the ERK1/2 and mTOR pathways. In addition, downregulation of Drp1 activity in tumor-infiltrating PD-1pos CD8+ T cells seems to be a mechanism exploited by PD-1 signaling to reduce motility and proliferation of these cells. Overall, our data indicate that the modulation of Drp1 activity in tumor-infiltrating T cells may become a valuable target to ameliorate the anticancer immune response in future immunotherapy approaches.
Asunto(s)
Linfocitos T CD8-positivos , Dinaminas/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Dinaminas/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
T cells engineered with chimeric antigen receptor (CAR-T cells) are an effective treatment in patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia or B-cell non-Hodgkin lymphoma. Despite the reported exciting clinical results, the CAR-T cell approach needs efforts to improve the safety profile, limiting the occurrence of adverse events in patients given this treatment. Besides the most common side effects, such as cytokine release syndrome and CAR-T cell-related encephalopathy syndrome, another potential issue involves the inadvertent transduction of leukemia B cells with the CAR construct during the manufacturing process, thus leading to the possibility of a peculiar mechanism of antigen masking and treatment resistance. In this study, we investigated whether the inclusion of the inducible caspase 9 (iC9) suicide gene in the CAR construct design could be an effective safety switch to control malignant CAR+ B cells, ultimately counteracting this serious adverse event. iC9 is a suicide gene able to be activated through binding with an otherwise inert small biomolecule, known as AP1903. The exposure of iC9.CAR.CD19-DAUDI lymphoma and iC9.CAR.CD19-NALM-6 leukemia cells in vitro to 20 nM of AP1903 resulted into the prompt elimination of CAR+ B-leukemia/lymphoma cell lines. The results obtained in the animal model corroborate in vitro data, since iC9.CAR.CD19+ tumor cells were controlled in vivo by the activation of the suicide gene through administration of AP1903. Altogether, our data indicate that the inclusion of the iC9 suicide gene may result in a safe CAR-T cell product, even when manufacturing starts from biological materials characterized by heavy leukemia blast contamination.
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Caspasa 9 , Genes Transgénicos Suicidas , Inmunoterapia Adoptiva/métodos , Leucemia de Células B , Linfoma de Células B , Receptores Quiméricos de Antígenos/uso terapéutico , Animales , Línea Celular Tumoral , Humanos , RatonesRESUMEN
Chimeric antigen receptor T-cells (CAR T-cells) for the treatment of relapsing/refractory B-cell precursor acute lymphoblastic leukemia have led to exciting clinical results. However, CAR T-cell approaches revealed a potential risk of CD19-/CAR+ leukemic relapse due to inadvertent transduction of leukemia cells. BACKGROUND: METHODS: We evaluated the impact of a high percentage of leukemia blast contamination in patient-derived starting material (SM) on CAR T-cell drug product (DP) manufacturing. In vitro as well as in vivo models were employed to identify characteristics of the construct associated with better profile of safety in case of inadvertent B-cell leukemia transduction during CAR T-cell manufacturing. RESULTS: The presence of large amounts of CD19+ cells in SM did not affect the transduction level of DPs, as well as the CAR T-cell rate of expansion at the end of standard production of 14 days. DPs were deeply characterized by flow cytometry and molecular biology for Ig-rearrangements, showing that the level of B-cell contamination in DPs did not correlate with the percentage of CD19+ cells in SM, in the studied patient cohort. Moreover, we investigated whether CAR design may affect the control of CAR+ leukemia cells. We provided evidences that CAR.CD19 short linker (SL) prevents complete epitope masking in CD19+CAR+ leukemia cells and we demonstrated in vitro and in vivo that CD19 +CAR(SL)+leukemic cells are killed by CAR.CD19 T-cells. CONCLUSIONS: Taken together, these data suggest that a VL-VH SL may result in a safe CAR-T product, even when manufacturing starts from biological materials characterized by heavy contamination of leukemia blasts.
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Epítopos/inmunología , Leucemia de Células B/inmunología , Receptores Quiméricos de Antígenos/inmunología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
The prognosis of many patients with chemotherapy-refractory or multiply relapsed CD30+ non-Hodgkin Lymphoma (NHL) or Hodgkin lymphoma (HL) still remains poor, and novel therapeutic approaches are warranted to address this unmet clinical need. In light of this consideration, we designed and pre-clinically validated a Chimeric Antigen Receptor (CAR) construct characterized by a novel anti-CD30 single-chain variable-fragment cassette, linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. We found that CAR.CD30 T-cells exhibit remarkable cytolytic activity in vitro against HL and NHL cell lines, with sustained proliferation and pro-inflammatory cytokine production, even after multiple and sequential lymphoma cell challenges. CAR.CD30 T-cells also demonstrated anti-lymphoma activity in two in vivo xenograft immune-deficient mouse models of metastatic HL and NHL. We observed that administration of CAR.CD30 T-cells, incorporating the CD28.OX40 costimulatory domains and manufactured in the presence of IL7 and IL15, were associated with the best overall survival in the treated mice, along with the establishment of a long-term immunological memory, able to protect mice from further tumor re-challenge. Our data indicate that, in the context of in vivo systemic metastatic xenograft mouse models, the costimulatory machinery of CD28.OX40 is crucial for improving persistence, in vivo expansion and proliferation of CAR.CD30 T-cells upon tumor encounter. CD28.OX40 costimulatory combination is ultimately responsible for the antitumor efficacy of the approach, paving the way to translate this therapeutic strategy in patients with CD30+ HL and NHL.
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Antígenos CD28 , Receptores Quiméricos de Antígenos , Animales , Humanos , Inmunoterapia Adoptiva , Ratones , Receptores de Antígenos de Linfocitos T , Linfocitos TRESUMEN
The antidiabetic drug phenformin displays potent anticancer activity in different tumors, but its mechanism of action remains elusive. Using Shh medulloblastoma as model, we show here that at clinically relevant concentrations, phenformin elicits a significant therapeutic effect through a redox-dependent but complex I-independent mechanism. Phenformin inhibits mitochondrial glycerophosphate dehydrogenase (mGPD), a component of the glycerophosphate shuttle, and causes elevations of intracellular NADH content. Inhibition of mGPD mimics phenformin action and promotes an association between corepressor CtBP2 and Gli1, thereby inhibiting Hh transcriptional output and tumor growth. Because ablation of CtBP2 abrogates the therapeutic effect of phenformin in mice, these data illustrate a biguanide-mediated redox/corepressor interplay, which may represent a relevant target for tumor therapy.
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Antineoplásicos/uso terapéutico , Proteínas Co-Represoras/efectos de los fármacos , Proteínas Hedgehog/efectos de los fármacos , Hipoglucemiantes/uso terapéutico , Neoplasias/tratamiento farmacológico , Fenformina/uso terapéutico , Animales , Antineoplásicos/farmacología , Humanos , Hipoglucemiantes/farmacología , Ratones , Fenformina/farmacologíaRESUMEN
The aberrant activation of hedgehog (HH) signaling is a leading cause of the development of medulloblastoma, a pediatric tumor of the cerebellum. The FDAapproved HH inhibitor, Vismodegib, which targets the transmembrane transducer SMO, has shown limited efficacy in patients with medulloblastoma, due to compensatory mechanisms that maintain an active HHGLI signaling status. Thus, the identification of novel actionable mechanisms, directly affecting the activity of the HHregulated GLI transcription factors is an important goal for these malignancies. In this study, using gene expression and reporter assays, combined with biochemical and cellular analyses, we demonstrate that mitogenactivated kinase kinase kinase 1 (MEKK1), the most upstream kinase of the mitogenactivated protein kinase (MAPK) phosphorylation modules, suppresses HH signaling by associating and phosphorylating GLI1, the most potent HHregulated transcription factor. Phosphorylation occurred at multiple residues in the Cterminal region of GLI1 and was followed by an increased association with the cytoplasmic proteins 1433. Of note, the enforced expression of MEKK1 or the exposure of medulloblastoma cells to the MEKK1 activator, Nocodazole, resulted in a marked inhibitory effect on GLI1 activity and tumor cell proliferation and viability. Taken together, the results of this study shed light on a novel regulatory mechanism of HH signaling, with potentially relevant implications in cancer therapy.
Asunto(s)
Proteínas Hedgehog/genética , Quinasa 1 de Quinasa de Quinasa MAP/genética , Meduloblastoma/genética , Proteína con Dedos de Zinc GLI1/genética , Anilidas/administración & dosificación , Animales , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Ratones , Células 3T3 NIH , Fosforilación/efectos de los fármacos , Piridinas/administración & dosificación , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
SHH Medulloblastoma (SHH-MB) is a pediatric brain tumor characterized by an inappropriate activation of the developmental Hedgehog (Hh) signaling. SHH-MB patients treated with the FDA-approved vismodegib, an Hh inhibitor that targets the transmembrane activator Smoothened (Smo), have shown the rapid development of drug resistance and tumor relapse due to novel Smo mutations. Moreover, a subset of patients did not respond to vismodegib because mutations were localized downstream of Smo. Thus, targeting downstream Hh components is now considered a preferable approach. We show here that selective inhibition of the downstream Hh effectors HDAC1 and HDAC2 robustly counteracts SHH-MB growth in mouse models. These two deacetylases are upregulated in tumor and their knockdown inhibits Hh signaling and decreases tumor growth. We demonstrate that mocetinostat (MGCD0103), a selective HDAC1/HDAC2 inhibitor, is a potent Hh inhibitor and that its effect is linked to Gli1 acetylation at K518. Of note, we demonstrate that administration of mocetinostat to mouse models of SHH-MB drastically reduces tumor growth, by reducing proliferation and increasing apoptosis of tumor cells and prolongs mouse survival rate. Collectively, these data demonstrate the preclinical efficacy of targeting the downstream HDAC1/2-Gli1 acetylation in the treatment of SHH-MB.
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Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Meduloblastoma/metabolismo , Neoplasias Experimentales/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Línea Celular Tumoral , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Ratones Transgénicos , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Proteínas Supresoras de Tumor/genética , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Hedgehog signaling controls proliferation of cerebellar granule cell precursors (GCPs) and its aberrant activation is a leading cause of Medulloblastoma, the most frequent pediatric brain tumor. We show here that the energy sensor AMPK inhibits Hh signaling by phosphorylating a single residue of human Gli1 that is not conserved in other species. Studies with selective agonists and genetic deletion have revealed that AMPK activation inhibits canonical Hh signaling in human, but not in mouse cells. Indeed we show that AMPK phosphorylates Gli1 at the unique residue Ser408, which is conserved only in primates but not in other species. Once phosphorylated, Gli1 is targeted for proteasomal degradation. Notably, we show that selective AMPK activation inhibits Gli1-driven proliferation and that this effect is linked to Ser408 phosphorylation, which represents a key metabolic checkpoint for Hh signaling. Collectively, this data unveil a novel mechanism of inhibition of Gli1 function, which is exclusive for human cells and may be exploited for the treatment of Medulloblastoma or other Gli1 driven tumors.