RESUMEN
Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.
Asunto(s)
Alelos , Síndrome de Bernard-Soulier/diagnóstico , Síndrome de Bernard-Soulier/genética , Predisposición Genética a la Enfermedad , Variación Genética , Fenotipo , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Síndrome de Bernard-Soulier/sangre , Plaquetas/metabolismo , Plaquetas/ultraestructura , República Checa , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Humanos , Inmunofenotipificación , Masculino , Linaje , Recuento de Plaquetas , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Trombocitopenia/sangre , Trombocitopenia/diagnósticoRESUMEN
Germline mutations in ETV6 gene cause inherited thrombocytopenia with leukemia predisposition. Here, we report on functional validation of ETV6 W380R mutation segregating with thrombocytopenia in a family where two family members also suffered from acute lymphoblastic leukemia (ALL) or essential thrombocythemia (ET). In-silico analysis predicted impaired DNA binding due to W380R mutation. Functional analysis showed that this mutation prevents the ETV6 protein from localizing into the cell nucleus and impairs the transcriptional repression activity of ETV6. Based on the germline ETV6 mutation, ET probably started with somatic JAK2 V617F mutation, whereas ALL could be caused by diverse mechanisms: high-hyperdiploidity; somatic deletion of exon 1 IKZF1 gene; or somatic mutations of other genes found by exome sequencing of the ALL sample taken at the diagnosis.
Asunto(s)
Mutación de Línea Germinal/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Represoras/metabolismo , Trombocitemia Esencial/genética , Trombocitopenia/metabolismo , Humanos , Proteína ETS de Variante de Translocación 6RESUMEN
Chimeric antigen receptor (CAR) T-cell therapy has already achieved remarkable remissions in some difficult-to-treat patients with B-cell malignancies. Although the clinical experience in chronic lymphocytic leukemia (CLL) patients is limited, the proportion of remissions reached in this disease is clearly the lowest from the spectrum of B-cell tumors. In this review, we discuss the antigenic targets exploited in CLL CAR-T therapy, the determinants of favorable responses, as well as the mechanisms of treatment failure specific to this disease.
Asunto(s)
Antígenos CD19/inmunología , Inmunoterapia Adoptiva/métodos , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/terapia , Receptores Quiméricos de Antígenos/inmunología , Linfocitos B/inmunología , Humanos , Inducción de Remisión , Linfocitos T/inmunologíaRESUMEN
Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-ß, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.
Asunto(s)
Biblioteca de Genes , Haploidia , Mutagénesis Insercional/métodos , Genética Inversa/métodos , Línea Celular Tumoral , Genoma Humano , Humanos , Datos de Secuencia MolecularRESUMEN
Rituximab (RTX) plus chemotherapy (R-CHOP) applied as a first-line therapy for lymphoma leads to a relapse in approximately 40% of the patients. Therefore, novel approaches to treat aggressive lymphomas are being intensively investigated. Several RTX-resistant (RR) cell lines have been established as surrogate models to study resistance to R-CHOP. Our study reveals that RR cells are characterized by a major downregulation of CD37, a molecule currently explored as a target for immunotherapy. Using CD20 knockout (KO) cell lines, we demonstrate that CD20 and CD37 form a complex, and hypothesize that the presence of CD20 stabilizes CD37 in the cell membrane. Consequently, we observe a diminished cytotoxicity of anti-CD37 monoclonal antibody (mAb) in complement-dependent cytotoxicity in both RR and CD20 KO cells that can be partially restored upon lysosome inhibition. On the other hand, the internalization rate of anti-CD37 mAb in CD20 KO cells is increased when compared to controls, suggesting unhampered efficacy of antibody drug conjugates (ADCs). Importantly, even a major downregulation in CD37 levels does not hamper the efficacy of CD37-directed chimeric antigen receptor (CAR) T cells. In summary, we present here a novel mechanism of CD37 regulation with further implications for the use of anti-CD37 immunotherapies.
Asunto(s)
Antígenos CD20 , Inmunoterapia , Linfoma de Células B , Rituximab , Tetraspaninas , Humanos , Antígenos CD20/inmunología , Antígenos CD20/metabolismo , Antígenos CD20/genética , Rituximab/farmacología , Rituximab/uso terapéutico , Tetraspaninas/genética , Tetraspaninas/metabolismo , Línea Celular Tumoral , Linfoma de Células B/inmunología , Linfoma de Células B/terapia , Linfoma de Células B/genética , Linfoma de Células B/tratamiento farmacológico , Inmunoterapia/métodos , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Doxorrubicina/farmacología , Doxorrubicina/administración & dosificación , Ciclofosfamida/farmacología , Ciclofosfamida/uso terapéutico , Vincristina/farmacología , Vincristina/uso terapéutico , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Regulación Neoplásica de la Expresión GénicaRESUMEN
Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. Here we establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to phosphoinositide 3-kinase (PI3K) inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC over-rode the dependency of cells on the PI3K-mTOR pathway for proliferation. These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: PAG/Cbp represents a ubiquitous mechanism for regulating Src family kinases by recruiting Csk to the plasma membrane, thereby controlling cellular activation. Since Src kinases are known oncogenes, we used RNA interference in primary human T cells to test whether the loss of PAG resulted in lymphocyte transformation. RESULTS: PAG-depletion enhanced Src kinase activity and augmented proximal T-cell receptor signaling; exactly the phenotype expected for loss of this negative regulator. Surprisingly, rather than becoming hyper-proliferative, PAG-suppressed T cells became unresponsive. This was mediated by a Fyn-dependent hyper-phosphorylation of the inhibitory receptor CTLA-4, which recruited the protein tyrosine phosphatase Shp-1 to lipid rafts. Co-suppression of CTLA-4 abrogates this inhibition and restores proliferation to T cells. CONCLUSION: We have identified a fail-safe mechanism as well as a novel contribution of CTLA-4 to setting the activation threshold in T cells.
RESUMEN
T cells orchestrate the adaptive immune response, making them targets for immunotherapy. Although immunosuppressive therapies prevent disease progression, they also leave patients susceptible to opportunistic infections. To identify novel drug targets, we established a logical model describing T-cell receptor (TCR) signaling. However, to have a model that is able to predict new therapeutic approaches, the current drug targets must be included. Therefore, as a next step we generated the interleukin-2 receptor (IL-2R) signaling network and developed a tool to merge logical models. For IL-2R signaling, we show that STAT activation is independent of both Src- and PI3-kinases, while ERK activation depends upon both kinases and additionally requires novel PKCs. In addition, our merged model correctly predicted TCR-induced STAT activation. The combined network also allows information transfer from one receptor to add detail to another, thereby predicting that LAT mediates JNK activation in IL-2R signaling. In summary, the merged model not only enables us to unravel potential cross-talk, but it also suggests new experimental designs and provides a critical step towards designing strategies to reprogram T cells.
Asunto(s)
Receptor Cross-Talk/fisiología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Interleucina-2/metabolismo , Linfocitos T/metabolismo , Células Cultivadas , Humanos , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Reproducibilidad de los Resultados , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
Signals emanating from antigen receptors critically regulate immune cell activation, survival, and differentiation. Transmembrane adapter proteins (TRAPs), a group of molecules that organize signaling complexes at the plasma membrane, play a pivotal role in propagating and fine-tuning antigen receptor-mediated signaling. During the last years, it has been demonstrated that most of the TRAPs possess inhibitory functions, including linker for activation of T cells (LAT), the best characterized adapter that links the T-cell receptor (TCR) to Ca(2+) flux and mitogen-activated protein kinase activation. Indeed, it appears that LAT may assemble inhibitory complexes that trigger negative feedback loops, thus terminating T-cell activation. Additionally, recent data demonstrate that SIT [Src homology 2 domain-containing phosphatase 2 (SHP2)-interacting TRAP] fine-tunes TCR-mediated signaling events and negatively regulates T-cell development and homeostasis. The experimental evidence suggests that TRAPs play a crucial role also in establishing tolerance. In fact, loss of SIT, LAX, or NTAL (non-T cell activation linker)/linker for activation of B cells (LAB) resulted in the spontaneous development of autoimmune diseases. Moreover, we recently showed that in addition to the inhibition of Src-family kinases, PAG (phosphoprotein associated with glycosphingolipid-enriched domains) is also involved in the negative regulation of Ras activation. Collectively, these data demonstrate that TRAPs are important modulators of immune cell activation and function. Finally, it appears that TRAPs possess redundant yet not completely overlapping functions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos de Diferenciación de Linfocitos T/inmunología , Retroalimentación Fisiológica/inmunología , Sistema Inmunológico/inmunología , Linfocitos/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Enfermedades Autoinmunes/inmunología , Diferenciación Celular/inmunología , Humanos , Sistema Inmunológico/citología , Sistema Inmunológico/crecimiento & desarrollo , Sistema Inmunológico/metabolismo , Activación de Linfocitos/inmunología , Linfocitos/citología , Linfocitos/inmunología , Proteínas de la Membrana/inmunología , Dominios Homologos srcRESUMEN
Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are malignancies characterized by the dependence on B-cell receptor (BCR) signaling and by the high expression of ROR1, the cell surface receptor for Wnt-5a. Both, BCR and ROR1 are therapeutic targets in these diseases and the understanding of their mutual cross talk is thus of direct therapeutic relevance. In this study we analyzed the role of Lyn, a kinase from the Src family participating in BCR signaling, as a mediator of the BCR-ROR1 crosstalk. We confirm the functional interaction between Lyn and ROR1 and demonstrate that Lyn kinase efficiently phosphorylates ROR1 in its kinase domain and aids the recruitment of the E3 ligase c-CBL. We show that ROR1 surface dynamics in migrating primary CLL cells as well as chemotactic properties of CLL cells were inhibited by Lyn inhibitor dasatinib. Our data establish Lyn-mediated phosphorylation of ROR1 as a point of crosstalk between BCR and ROR1 signaling pathways.
RESUMEN
Opioids are widely used for the treatment of severe pain. However, it is also known that opioids, in particular morphine, cause immunosuppression. Therefore, their use may complicate treatment of persons with an already impaired immune system, e.g., patients suffering from cancer or AIDS. We investigated the mechanisms of opioid-induced immunosuppression in primary human T lymphocytes and the human T cell line Jurkat. We demonstrated that morphine and the endogenous opioid beta-endorphin inhibited the transcription of IL-2 in activated human T lymphocytes as well as the activation of the transcription factors AP-1, NFAT, and NF-kappaB, which transactivate IL-2. In addition, the TCR-induced calcium flux and MAPK activation were inhibited by the opioids, as well as proximal signaling events, such as the phosphorylation of the linker for activation of T cells and Zap70. A more detailed characterization of the mechanism revealed that incubation of T cells with the opioids caused a marked increase in cAMP. This in turn activated protein kinase A, which augmented the kinase activity of C-terminal Src kinase bound to phosphoprotein associated with glycosphingolipid-enrich microdomains, resulting in a further enhancement of the tonic inhibition of the leukocyte-specific protein tyrosine kinase Lck, thereby blocking the initiation of TCR signaling. These effects were mediated by mu opioid receptors. Our findings contribute to the understanding of immunosuppressive side effects of morphine. Since beta-endorphin is expressed and secreted by immune effector cells, including T cells, and up-regulated in these cells by various stimuli, our data also suggest an inhibitory role for beta-endorphin in the physiological regulation of T cell activation.
Asunto(s)
Morfina/farmacología , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , betaendorfina/fisiología , Células Cultivadas , Humanos , Interleucina-2/genética , Células Jurkat , Activación de Linfocitos , Fosforilación , Receptores Opioides mu/metabolismo , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Anti-CD19 chimeric antigen receptor T cells (CART-19) frequently induce remissions in hemato-oncological patients with recurred and/or refractory B-cell tumors. However, malignant cells sometimes escape the immunotherapeutic targeting by CD19 gene mutations, alternative splicing or lineage switch, commonly causing lack of CD19 expression on the surface of neoplastic cells. We assumed that, in addition to the known mechanisms, other means could act on CD19 to drive antigen-negative relapse. METHODS: Herein, we studied the mechanism of antigen loss in an in vivo CD19-negative recurrence model of chronic lymphocytic leukemia (CLL) to CART-19, established using NOD-scid IL2Rgnull mice and HG3 cell line. We validated our findings in vitro in immortalized B-cell lines and primary CLL cells. RESULTS: In our in vivo CLL recurrence model, up to 70% of CART-19-treated mice eventually recurred with CD19-negative disease weeks after initial positive response. We found that the lack of CD19 expression was caused by promoter DNA hypermethylation. Importantly, the expression loss was partially reversible by treatment with a demethylating agent. Moreover, this escape mechanism was common for 3 B-cell immortalized lines as well as primary CLL cells, as assessed by in vitro coculture experiments. CONCLUSIONS: Epigenetically driven antigen escape could represent a novel, yet at least partially reversible, means of CD19 loss to CART-19 in B-cell tumors.
Asunto(s)
Metilación de ADN/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Antígenos CD19/inmunología , Femenino , Humanos , Masculino , RatonesRESUMEN
The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to G(i/o) proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Delta9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the zeta-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.
Asunto(s)
Cannabinoides/farmacología , Activación de Linfocitos/efectos de los fármacos , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/metabolismo , Ácidos Araquidónicos/antagonistas & inhibidores , Ácidos Araquidónicos/farmacología , Proteína Tirosina Quinasa CSK , Cannabinoides/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico , Dronabinol/antagonistas & inhibidores , Dronabinol/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/inmunología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Indoles/farmacología , Interleucina-2/inmunología , Interleucina-2/metabolismo , Células Jurkat , Activación de Linfocitos/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Morfolinas/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Proteínas Tirosina Quinasas , Psicotrópicos/análisis , Psicotrópicos/antagonistas & inhibidores , Psicotrópicos/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/inmunología , Receptor Cannabinoide CB2/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Familia-src QuinasasRESUMEN
Surface protein CD20 serves as the critical target of immunotherapy in various B-cell malignancies for decades, however its biological function and regulation remain largely elusive. Better understanding of CD20 function may help to design improved rational therapies to prevent development of resistance. Using CRISPR/Cas9 technique, we have abrogated CD20 expression in five different malignant B-cell lines. We show that CD20 deletion has no effect upon B-cell receptor signaling or calcium flux. Also B-cell survival and proliferation is unaffected in the absence of CD20. On the contrary, we found a strong defect in actin cytoskeleton polymerization and, consequently, defective cell adhesion and migration in response to homeostatic chemokines SDF1α, CCL19 and CCL21. Mechanistically, we could identify a reduction in chemokine-triggered PYK2 activation, a calcium-activated signaling protein involved in activation of MAP kinases and cytoskeleton regulation. These cellular defects in consequence result in a severely disturbed homing of B cells in vivo.
Asunto(s)
Actinas/metabolismo , Antígenos CD20/fisiología , Linfocitos B/fisiología , Leucemia de Células B/patología , Linfoma de Células B/patología , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Antígenos CD20/genética , Antígenos CD20/metabolismo , Linfocitos B/patología , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Leucemia de Células B/metabolismo , Linfoma de Células B/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Polimerizacion , Multimerización de Proteína/fisiología , Transducción de Señal/inmunologíaRESUMEN
BACKGROUND: While achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence of genetics on CLL response to anti-CD19 CAR T-cell therapy. METHODS: First, we studied 32 primary CLL samples composed of 26 immunoglobulin heavy-chain gene variable (IGHV)-unmutated (9 ATM-mutated, 8 TP53-mutated, and 9 without mutations in ATM, TP53, NOTCH1 or SF3B1) and 6 IGHV-mutated samples without mutations in the above-mentioned genes. Then, we mimicked the leukemic microenvironment in the primary cells by '2S stimulation' through interleukin-2 and nuclear factor kappa B. Finally, CRISPR/Cas9-generated ATM-knockout and TP53-knockout clones (four and seven, respectively) from CLL-derived cell lines MEC1 and HG3 were used. All these samples were exposed to CAR T cells. In vivo survival study in NSG mice using HG3 wild-type (WT), ATM-knockout or TP53-knockout cells was also performed. RESULTS: Primary unstimulated CLL cells were specifically eliminated after >24 hours of coculture with CAR T cells. '2S' stimulated cells showed increased survival when exposed to CAR T cells compared with unstimulated ones, confirming the positive effect of this stimulation on CLL cells' in vitro fitness. After 96 hours of coculture, there was no difference in survival among the genetic classes. Finally, CAR T cells were specifically activated in vitro in the presence of target knockout cell lines as shown by the production of interferon-γ when compared with control (CTRL) T cells (p=0.0020), but there was no difference in knockout cells' survival. In vivo, CAR T cells prolonged the survival of mice injected with WT, TP53-knockout and ATM-knockout HG3 tumor cells as compared with CTRL T cells (p=0.0485, 0.0204 and <0.0001, respectively). When compared with ATM-knockout, TP53-knockout disease was associated with an earlier time of onset (p<0.0001), higher tumor burden (p=0.0002) and inefficient T-cell engraftment (p=0.0012). CONCLUSIONS: While in vitro no differences in survival of CLL cells of various genetic backgrounds were observed, CAR T cells showed a different effectiveness at eradicating tumor cells in vivo depending on the driver mutation. Early disease onset, high-tumor burden and inefficient T-cell engraftment, associated with TP53-knockout tumors in our experimental setting, ultimately led to inferior performance of CAR T cells.
Asunto(s)
Antígenos CD19/uso terapéutico , Leucemia Linfocítica Crónica de Células B/genética , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Animales , Voluntarios Sanos , Humanos , RatonesRESUMEN
Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/prevención & control , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Benzamidas/farmacología , Línea Celular Tumoral , Proliferación Celular/genética , Difenilamina/análogos & derivados , Difenilamina/farmacología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones Desnudos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridonas/farmacología , Pirimidinonas/farmacología , Interferencia de ARN , Tiofenos/farmacología , Urea/análogos & derivados , Urea/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in the western world and monoclonal antibodies (mAbs) are important part of CLL treatment. The goal of this article was to summarize current literature on the position of mAbs in CLL treatment and to mention factors influencing effectiveness of mAbs in CLL. Several new mAbs have been developed and investigated in CLL over the past few years. Mainly anti-CD20 monoclonal antibodies are still used routinely in CLL therapy. Unfortunately, the clinical application of mAbs needs to be further improved. Novel combinations and sequences of mAbs with other compounds need to be studied in clinical trials in order to increase overall response rate and prolong remission duration. Mechanisms of action of mAbs or mechanisms of resistance to mAbs have to be also investigated to predict effectiveness of mAb in particular patient.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , HumanosRESUMEN
Despite the dawn of the genomic information era, the challenges of cancer treatment remain formidable. Particularly for the most prevalent cancer types, including lung cancer, successful treatment of metastatic disease is rare and escalating costs for modern targeted drugs place an increasing strain on healthcare systems. Although powerful diagnostic tools to characterize individual tumor samples in great molecular detail are becoming rapidly available, the transformation of this information into therapy provides a major challenge. A fundamental difficulty is the molecular complexity of cancer cells that often causes drug resistance, but can also render tumors exquisitely sensitive to targeted agents. By using lung cancer as an example, we outline the principles that govern drug sensitivity and resistance from a genetic perspective and discuss how in vitro chemical-genetic screens can impact on patient stratification in the clinic.
Asunto(s)
Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Terapia Molecular Dirigida/métodos , Protocolos de Quimioterapia Combinada Antineoplásica , Biomarcadores de Tumor/genética , Humanos , Medicina de Precisión/métodosRESUMEN
PAG, the phosphoprotein associated with glycosphingolipid-enriched microdomains (GEM), negatively regulates Src family kinases by recruiting C-terminal Src kinase (Csk) to the membrane, where Csk phosphorylates the inhibitory tyrosine of the Src kinases. S-acylation of a CxxC motif juxtaposed to the transmembrane domain within PAG has been proposed to be responsible for targeting PAG to the lipid rafts. Here, we present the characterization of a mutant PAG molecule lacking the palmitoylation motif. We demonstrate that the mutant protein is expressed at the plasma membrane, but does not localize within the GEM. Despite being displaced, the mutant PAG molecule still binds the Src kinase Fyn and the cytoskeletal adaptor ezrin-radixin-moesin-binding phosphoprotein of 50 kDa, becomes tyrosine-phosphorylated, and recruits Csk to the membrane. Functional characterization of the mutant shows that, unlike WT PAG, it does not block proximal TCR signaling, and surprisingly enhances stromal cell-derived factor 1 (CXCL12)-induced migration. The mutant functions by depleting Csk from the GEM fractions, as apparent by changes in the phosphorylation of the inhibitory tyrosines within the Src kinases. Indeed this mechanism is supported by RNA interference of PAG, which results in enhanced migration and Src kinase activity. Our results therefore support a functional role for the compartmentalization of Src kinases within the membrane.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular/fisiología , Quimiocina CXCL12/metabolismo , Microdominios de Membrana/metabolismo , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Western Blotting , Quimiocina CXCL12/inmunología , Humanos , Inmunoprecipitación , Células Jurkat , Activación de Linfocitos/inmunología , Datos de Secuencia Molecular , Mutación , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , TransfecciónRESUMEN
In primary human T cells, anergy induction results in enhanced p59Fyn activity. Because Fyn is the kinase primarily responsible for the phosphorylation of PAG (the phosphoprotein associated with glycosphingolipid-enriched microdomains), which negatively regulates Src-kinase activity by recruiting Csk (the C-terminal Src kinase) to the membrane, we investigated whether anergy induction also affects PAG. Analysis of anergic T cells revealed that PAG is hyperphosphorylated at the Csk binding site, leading to enhanced Csk recruitment and inhibitory tyrosine phosphorylation within Fyn. This together with enhanced phosphorylation of a tyrosine within the SH2 domain of Fyn leads to the formation of a hyperactive conformation, thus explaining the enhanced Fyn kinase activity. In addition, we have also identified the formation of a multiprotein complex containing PAG, Fyn, Sam68, and RasGAP in stimulated T cells. We demonstrate that PAG-Fyn overexpression is sufficient to suppress Ras activation in Jurkat T cells and show that this activity is independent of Csk binding. Thus, in addition to negatively regulating Src family kinases by recruiting Csk, PAG also negatively regulates Ras by recruiting RasGAP to the membrane. Finally, by knocking down PAG, we demonstrate both enhanced Src kinase activity and Ras activation, thereby establishing PAG as an important negative regulator of T-cell activation.