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BACKGROUND: Genetic alterations activating the MAPK pathway are common in non-small cell lung cancer (NSCLC). Patients with NSCLC may benefit from treatment with the pan-RAF inhibitor naporafenib (LXH254) plus the ERK1/2 inhibitor rineterkib (LTT462) or MEK1/2 inhibitor trametinib. METHODS: This first-in-human phase 1b dose-escalation/dose-expansion study investigated the combinations of naporafenib (50-350 mg once daily [QD] or 300-600 mg twice daily [BID]) with rineterkib (100-300 mg QD) in patients with KRAS-/BRAF-mutant NSCLC and naporafenib (200 mg BID or 400 mg BID) with trametinib (0.5 mg QD, 1 mg QD or 1 mg QD 2 weeks on/2 weeks off) in patients with KRAS-/BRAF-mutant NSCLC and NRAS-mutant melanoma. The primary objectives were to identify the recommended dose for expansion (RDE) and evaluate tolerability and safety. Secondary objectives included antitumor activity and pharmacodynamics. RESULTS: Overall, 216 patients were treated with naporafenib plus rineterkib (NSCLC: n = 101) or naporafenib plus trametinib (NSCLC: n = 79; melanoma: n = 36). In total, 10 of 62 (16%) patients experienced at least one dose-limiting toxicity. The RDEs were established as naporafenib 400 mg BID plus rineterkib 200 mg QD, naporafenib 200 mg BID plus trametinib 1 mg QD and naporafenib 400 mg BID plus trametinib 0.5 mg QD. The most frequent grade ≥ 3 treatment-related adverse event was increased lipase (8/101 [7.9%] patients) for naporafenib plus rineterkib and rash (22/115 [19.1%] patients) for naporafenib plus trametinib. Among patients with NSCLC, partial response was observed in three patients (one with KRAS-mutant, two with BRAFnon-V600-mutant NSCLC) treated with naporafenib plus rineterkib and two patients (both with KRAS-mutant NSCLC) treated with naporafenib plus trametinib. On-treatment median reductions in DUSP6 mRNA levels from baseline were 45.5% and 76.1% with naporafenib plus rineterkib or trametinib, respectively. CONCLUSIONS: Both naporafenib combinations had acceptable safety profiles. Antitumor activity was limited in patients with NSCLC, despite the observed on-target pharmacodynamic effect. CLINICALTRIALS: gov identifier: NCT02974725.
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ABSTRACT: The t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre-B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.
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Agammaglobulinemia Tirosina Quinasa , Dasatinib , Inhibidores de Proteínas Quinasas , Dasatinib/uso terapéutico , Dasatinib/farmacología , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/metabolismo , Humanos , Animales , Ratones , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacosRESUMEN
Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) is treated with ALK tyrosine kinase inhibitors (TKIs), but the lack of activity of immune checkpoint inhibitors (ICIs) is poorly understood. Here, we identified immunogenic ALK peptides to show that ICIs induced rejection of ALK+ tumors in the flank but not in the lung. A single-peptide vaccination restored priming of ALK-specific CD8+ T cells, eradicated lung tumors in combination with ALK TKIs and prevented metastatic dissemination of tumors to the brain. The poor response of ALK+ NSCLC to ICIs was due to ineffective CD8+ T cell priming against ALK antigens and is circumvented through specific vaccination. Finally, we identified human ALK peptides displayed by HLA-A*02:01 and HLA-B*07:02 molecules. These peptides were immunogenic in HLA-transgenic mice and were recognized by CD8+ T cells from individuals with NSCLC, paving the way for the development of a clinical vaccine to treat ALK+ NSCLC.
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Vacunas contra el Cáncer , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Ratones , Animales , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Quinasa de Linfoma Anaplásico/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Vacunas contra el Cáncer/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/uso terapéutico , Linfocitos T CD8-positivos/patología , Vacunas de Subunidad/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/uso terapéutico , Ratones Transgénicos , VacunaciónRESUMEN
Internal tandem duplications (ITD) of the FMS-like tyrosine kinase 3 (FLT3) predict poor prognosis in acute myeloid leukemia (AML) and often co-exist with inactivating DNMT3A mutations. In vitro studies implicated Grb2-associated binder 2 (GAB2) as FLT3-ITD effector. Utilizing a Flt3-ITD knock-in, Dnmt3a haploinsufficient mouse model, we demonstrate that Gab2 is essential for the development of Flt3-ITD driven AML in vivo, as Gab2 deficient mice displayed prolonged survival, presented with attenuated liver and spleen pathology and reduced blast counts. Furthermore, leukemic bone marrow from Gab2 deficient mice exhibited reduced colony-forming unit capacity and increased FLT3 inhibitor sensitivity. Using transcriptomics, we identify the genes encoding for Axl and the Ret co-receptor Gfra2 as targets of the Flt3-ITD/Gab2/Stat5 axis. We propose a pathomechanism in which Gab2 increases signaling of these receptors by inducing their expression and by serving as downstream effector. Thereby, Gab2 promotes AML aggressiveness and drug resistance as it incorporates these receptor tyrosine kinases into the Flt3-ITD signaling network. Consequently, our data identify GAB2 as a promising biomarker and therapeutic target in human AML.
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Proteínas Adaptadoras Transductoras de Señales , Leucemia Mieloide Aguda , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Mutación , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
FLT3-ITD is the most predominant mutation in AML being expressed in about one-third of AML patients and is associated with a poor prognosis. Efforts to better understand FLT3-ITD downstream signaling to possibly improve therapy response are needed. We have previously described FLT3-ITD-dependent phosphorylation of CSF2RB, the common receptor beta chain of IL-3, IL-5, and GM-CSF, and therefore examined its significance for FLT3-ITD-dependent oncogenic signaling and transformation. We discovered that FLT3-ITD directly binds to CSF2RB in AML cell lines and blasts isolated from AML patients. A knockdown of CSF2RB in FLT3-ITD positive AML cell lines as well as in a xenograft model decreased STAT5 phosphorylation, attenuated cell proliferation, and sensitized to FLT3 inhibition. Bone marrow from CSF2RB-deficient mice transfected with FLT3-ITD displayed decreased colony formation capacity and delayed disease onset together with increased survival upon transplantation into lethally irradiated mice. FLT3-ITD-dependent CSF2RB phosphorylation required phosphorylation of the FLT3 juxtamembrane domain at tyrosines 589 or 591, whereas the ITD insertion site and sequence were of no relevance. Our results demonstrate that CSF2RB participates in FLT3-ITD-dependent oncogenic signaling and transformation in vitro and in vivo. Thus, CSF2RB constitutes a rational treatment target in FLT3-ITD-positive AML.
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Subunidad beta Común de los Receptores de Citocinas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Línea Celular Tumoral , Subunidad beta Común de los Receptores de Citocinas/genética , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Mutación , Fosforilación , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
The expression of BCL6 in B-cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions, or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase that is involved in the degradation of BCL6, and it is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of patients with Burkitt lymphoma (BL). FBXO11 mutations impaired BCL6 degradation, and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of 1 or 2 copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B-cell lymphoma onset, providing experimental evidence that FBXO11 is a haploinsufficient oncosuppressor in B-cell lymphoma. In wild-type and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degraders or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL, we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches, such as BCL6 degraders and direct MYC inhibition, could be exploited as a targeted therapy for BL.
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Linfoma de Burkitt , Proteínas F-Box , Linfoma de Células B , Animales , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/genética , Proteínas F-Box/genética , Genes myc , Humanos , Linfoma de Células B/genética , Ratones , Mutación , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismoRESUMEN
Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function, and cancer susceptibility. Diagnosis of IBMFS presents a major challenge due to the large variety of associated phenotypes, and novel, clinically relevant biomarkers are urgently needed. Our study identified nuclear interaction partner of ALK (NIPA) as an IBMFS gene, as it is significantly downregulated in a distinct subset of myelodysplastic syndrome-type (MDS-type) refractory cytopenia in children. Mechanistically, we showed that NIPA is major player in the Fanconi anemia (FA) pathway, which binds FANCD2 and regulates its nuclear abundance, making it essential for a functional DNA repair/FA/BRCA pathway. In a knockout mouse model, Nipa deficiency led to major cell-intrinsic defects, including a premature aging phenotype, with accumulation of DNA damage in hematopoietic stem cells (HSCs). Induction of replication stress triggered a reduction in and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the knockout mice with 100% penetrance. Taken together, the results of our study add NIPA to the short list of FA-associated proteins, thereby highlighting its potential as a diagnostic marker and/or possible target in diseases characterized by hematopoietic failure.
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Síndromes Congénitos de Insuficiencia de la Médula Ósea , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Células Madre Hematopoyéticas/metabolismo , Proteínas Nucleares , Animales , Síndromes Congénitos de Insuficiencia de la Médula Ósea/genética , Síndromes Congénitos de Insuficiencia de la Médula Ósea/metabolismo , Síndromes Congénitos de Insuficiencia de la Médula Ósea/patología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Células Madre Hematopoyéticas/patología , Ratones , Ratones Noqueados , Proteínas Nucleares/deficiencia , Proteínas Nucleares/metabolismo , Unión ProteicaRESUMEN
While cancer stem cells are well established in certain hematologic and solid malignancies, their existence in T cell lymphoma is unclear and the origin of disease is not fully understood. To examine the existence of lymphoma stem cells, we utilized a mouse model of anaplastic large cell lymphoma. Established NPM-ALK+ lymphomas contained heterogeneous cell populations ranging from mature T cells to undifferentiated hematopoietic stem cells. Interestingly, CD4-/CD8- double negative (DN) lymphoma cells aberrantly expressed the T cell receptor α/ß chain. Serial transplantation of sorted CD4/CD8 and DN lymphoma subpopulations identified lymphoma stem cells within the DN3/DN4 T cell population, whereas all other subpopulations failed to establish serial lymphomas. Moreover, transplanted lymphoma DN3/DN4 T cells were able to differentiate and gave rise to mature lymphoma T cells. Gene expression analyses unmasked stem-cell-like transcriptional regulation of the identified lymphoma stem cell population. Furthermore, these lymphoma stem cells are characterized by low CD30 expression levels, which might contribute to limited long-term therapeutic success in patients treated with anti-CD30-targeted therapies. In summary, our results highlight the existence of a lymphoma stem cell population in a NPM-ALK-driven CD30+ mouse model, thereby giving the opportunity to test innovative treatment strategies developed to eradicate the origin of disease.
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Linfoma Anaplásico de Células Grandes/inmunología , Linfoma de Células T/inmunología , Células Madre/inmunología , Quinasa de Linfoma Anaplásico/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Modelos Animales de Enfermedad , Femenino , Expresión Génica/inmunología , Antígeno Ki-1/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Translocación Genética/inmunologíaRESUMEN
Targeted expression of transgenes is essential for the accurate representation of human disease in in vivo models. Current approaches to generate conditional transgenic mouse models are cumbersome and not amenable to high-throughput analysis since they require de novo generation and characterization of genetically modified mice. Here we describe a new system for lineage-restricted expression of transgenes based on a retroviral vector incorporating a translational stop cassette flanked by loxP recombination sites. Conditional transgene expression in chimeric mice is achieved by retroviral infection and transplantation of hematopoietic stem cells (HSC) derived from transgenic mice expressing Cre-recombinase from a lineage-specific promoter. For validation, we directed expression of NPM-ALK, the fusion oncogene driving a subset of anaplastic large cell lymphoma (ALCL), to T-cells by infecting hematopoietic stem cells from Lck-Cre-transgenic mice with a retroviral construct containing the NPM-ALK cDNA preceded by a translational stop cassette. These mice developed T-cell lymphomas within 12-16 weeks, featuring increased expression of the ALCL hallmark antigen CD30 as well as other cytotoxic T-cell markers, similar to the human disease. The new model represents a versatile tool for the rapid analysis of gene function in a defined lineage or in a developmental stage in vivo.
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Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/patología , Linfoma de Células T/patología , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/metabolismo , Animales , Apoptosis , Proliferación Celular , Femenino , Humanos , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Tirosina Quinasas/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Anaplastic large-cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma that shows in 60% of cases a translocation t(2;5)(p23;q35), which leads to the expression of the oncogenic kinase NPM-ALK. The nuclear interaction partner of ALK (NIPA) defines an E3-SCF ligase that contributes to the timing of mitotic entry. It has been shown that co-expression of NIPA and NPM-ALK results in constitutive NIPA phosphorylation. By mass spectrometry-based proteomics we identified nine serine/threonine residues to be significantly upregulated in NIPA upon NPM-ALK expression. Generation of phospho-deficient mutants of the respective phospho-residues specified five serine/threonine residues (Ser-338, Ser-344, Ser-370, Ser-381 and Thr-387) as key phosphorylation sites involved in NPM-ALK-directed phosphorylation of NIPA. Analysis of the biological impact of NIPA phosphorylation by NPM-ALK demonstrated that the ALK-induced phosphorylation does not change the SCFNIPA-complex formation but may influence the localization of NIPA and NPM-ALK. Biochemical analyses with phospho-deficient mutants elucidated the importance of NIPA phosphorylation by NPM-ALK for the interaction of the two proteins and proliferation potential of respective cells: Silencing of the five crucial NIPA serine/threonine residues led to a highly enhanced NIPA-NPM-ALK binding capacity as well as a slightly reduced proliferation in Ba/F3 cells.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteómica/métodos , Serina/química , Treonina/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Citometría de Flujo , Humanos , Inmunoprecipitación , Linfoma Anaplásico de Células Grandes/metabolismo , Microscopía Fluorescente , Proteínas Nucleares/genética , Fosfoproteínas/metabolismo , Fosforilación , Transducción de SeñalRESUMEN
Activating mutations in FMS-like tyrosine kinase receptor-3 (FLT3) and Nucleophosmin-1 (NPM1) are most frequent alterations in acute myeloid leukemia (AML), and are often coincidental. The mutational status of NPM1 has strong prognostic relevance to patients with point mutations of the FLT3 tyrosine kinase domain (TKD), but the biological mechanism underlying this effect remains unclear. In the present study, we investigated the effect of the coincidence of NPM1c and FLT3-TKD. Although expression of FLT3-TKD is not sufficient to induce a disease in mice, coexpression with NPM1c rapidly leads to an aggressive myeloproliferative disease in mice with a latency of 31.5 days. Mechanistically, we could show that FLT3-TKD is able to activate the downstream effector molecule signal transducer and activator of transcription 5 (STAT5) exclusively in the presence of mutated NPM1c. Moreover, NPM1c alters the cellular localization of FLT3-TKD from the cell surface to the endoplasmic reticulum, which might thereby lead to the aberrant STAT5 activation. Importantly, aberrant STAT5 activation occurs not only in primary murine cells but also in patients with AML with combined FLT3-TKD and NPM1c mutations. Thus, our data indicate a new mechanism, how NPM1c mislocalizes FLT3-TKD and changes its signal transduction ability.
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Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutación , Proteínas Nucleares/genética , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo , Sustitución de Aminoácidos , Animales , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Duplicación de Gen , Regulación Leucémica de la Expresión Génica , Humanos , Ratones , Proteínas Nucleares/metabolismo , Nucleofosmina , Transporte de Proteínas , Factor de Transcripción STAT5/metabolismo , Secuencias Repetidas en TándemRESUMEN
In T lymphocytes, the Wiskott-Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In anaplastic lymphoma kinase-positive (ALK+) ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-ß. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased mitogen-activated protein kinase (MAPK) pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MAP-kinase kinase (MEK) inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL.
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Linfoma de Células T/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Quinasa de Linfoma Anaplásico/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Proteínas del Citoesqueleto/metabolismo , Regulación hacia Abajo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Estimación de Kaplan-Meier , Linfoma de Células T/enzimología , Linfoma de Células T/patología , Sistema de Señalización de MAP Quinasas , Ratones , Unión Proteica , Factor de Transcripción STAT3/metabolismo , Linfocitos T/inmunología , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
T cell non-Hodgkin lymphoma (T-NHL) is a rare and heterogeneous group of neoplasms of the lymphoid system. With the exception of a few relatively indolent entities, T-NHL is typically aggressive, treatment resistant, and associated with poor prognosis. Relatively few options with proven clinical benefit are available for patients with relapsed or refractory disease. Immunotherapy has emerged as a promising treatment for the management of patients with hematological malignancies. The identification of tumor antigens has provided a large number of potential targets. Therefore, several monoclonal antibodies (alemtuzumab, SGN-30, brentuximab vedotin, and mogamulizumab), directed against tumor antigens, have been investigated in different subtypes of T-NHL. In addition to targeting antigens involved in cancer cell physiology, antibodies can stimulate immune effector functions or counteract immunosuppressive mechanisms. Chimeric antigen receptor (CAR)-T cells directed against CD30 and immune checkpoint inhibitors are currently being investigated in clinical trials. In this review, we summarize the currently available clinical evidence for immunotherapy in T-NHL, focusing on the results of clinical trials using first generation monoclonal antibodies, new immunotherapeutic agents, immune checkpoint inhibitors, and CAR-T cell therapies.
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Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F-myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient-derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1-mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.
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Antígeno B7-H1/metabolismo , Neoplasias Hematológicas/metabolismo , Janus Quinasa 2/metabolismo , Trastornos Mieloproliferativos/metabolismo , Animales , Antígeno B7-H1/genética , Proliferación Celular/genética , Proliferación Celular/fisiología , Transformación Celular Neoplásica , Neoplasias Hematológicas/genética , Humanos , Janus Quinasa 2/genética , Células K562 , Ratones , Trastornos Mieloproliferativos/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
Activation-induced cytidine deaminase (AID) is a B-cell-specific enzyme that targets immunoglobulin genes to initiate class switch recombination and somatic hypermutation. In addition, through off-target activity, AID has a much broader effect on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in the development and progression of lymphoma. AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation. The phosphatidylinositol 3-kinase δ (PI3Kδ) pathway regulates AID by suppressing its expression in B cells. Drugs for leukaemia or lymphoma therapy such as idelalisib, duvelisib and ibrutinib block PI3Kδ activity directly or indirectly, potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both of these effects were completely abrogated in AID-deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumours in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IGH and AID off-target sites in human chronic lymphocytic leukaemia and mantle cell lymphoma cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased somatic hypermutation in AID off-targets. In summary, we show that PI3Kδ or Bruton's tyrosine kinase inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism. This effect should be carefully considered, as such inhibitors can be administered to patients for years.
Asunto(s)
Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Inestabilidad Genómica/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Linfocitos B/enzimología , Linfocitos B/patología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Citidina Desaminasa/metabolismo , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Cambio de Clase de Inmunoglobulina/efectos de los fármacos , Cadenas Pesadas de Inmunoglobulina/genética , Isoquinolinas/efectos adversos , Isoquinolinas/farmacología , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/patología , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Piperidinas , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Purinas/efectos adversos , Purinas/farmacología , Pirazoles/efectos adversos , Pirazoles/farmacología , Pirimidinas/efectos adversos , Pirimidinas/farmacología , Quinazolinonas/efectos adversos , Quinazolinonas/farmacología , Recombinación Genética/efectos de los fármacos , Hipermutación Somática de Inmunoglobulina/efectos de los fármacos , Translocación Genética/efectos de los fármacosRESUMEN
A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Transición Epitelial-Mesenquimal , Neoplasias Pulmonares/enzimología , Proteínas de Unión al ARN/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Quinasa de Linfoma Anaplásico , Animales , Antígenos CD , Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Cadherinas/genética , Cadherinas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones Endogámicos NOD , Ratones SCID , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fenotipo , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Vimentina/genética , Vimentina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A 'drug holiday' where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.
Asunto(s)
Apoptosis , Linfoma Anaplásico de Células Grandes/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Animales , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Crizotinib , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Histonas/metabolismo , Humanos , Hidrazinas/farmacología , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patología , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Microscopía Confocal , Nucleofosmina , Proteínas de Fusión Oncogénica/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/genética , Pirazoles/farmacología , Piridinas/farmacología , Interferencia de ARN , Trasplante Heterólogo , Triazoles/farmacologíaRESUMEN
Non-small cell lung cancer (NSCLC) harboring chromosomal rearrangements of the anaplastic lymphoma kinase (ALK) gene is treated with ALK tyrosine kinase inhibitors (TKI), but the treatment is successful for only a limited amount of time; most patients experience a relapse due to the development of drug resistance. Here, we show that a vaccine against ALK induced a strong and specific immune response that both prophylactically and therapeutically impaired the growth of ALK-positive lung tumors in mouse models. The ALK vaccine was efficacious also in combination with ALK TKI treatment and significantly delayed tumor relapses after TKI suspension. We found that lung tumors containing ALK rearrangements induced an immunosuppressive microenvironment, regulating the expression of PD-L1 on the surface of lung tumor cells. High PD-L1 expression reduced ALK vaccine efficacy, which could be restored by administration of anti-PD-1 immunotherapy. Thus, combinations of ALK vaccine with TKIs and immune checkpoint blockade therapies might represent a powerful strategy for the treatment of ALK-driven NSCLC.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Neoplasias Pulmonares/terapia , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/inmunología , Quinasa de Linfoma Anaplásico , Animales , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/inmunología , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Crizotinib , Humanos , Neoplasias Pulmonares/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Receptor de Muerte Celular Programada 1/inmunología , Pirazoles/inmunología , Pirazoles/uso terapéutico , Piridinas/inmunología , Piridinas/uso terapéutico , Microambiente Tumoral/inmunología , Vacunación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer evolution is a process that is still poorly understood because of the lack of versatile in vivo longitudinal studies. By generating murine non-small cell lung cancer (NSCLC) orthoallobanks and paired primary cell lines, we provide a detailed description of an in vivo, time-dependent cancer malignization process. We identify the acquisition of metastatic dissemination potential, the selection of co-driver mutations, and the appearance of naturally occurring intratumor heterogeneity, thus recapitulating the stochastic nature of human cancer development. This approach combines the robustness of genetically engineered cancer models with the flexibility of allograft methodology. We have applied this tool for the preclinical evaluation of therapeutic approaches. This system can be implemented to improve the design of future treatments for patients with NSCLC.
Asunto(s)
Evolución Biológica , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Neoplasias Experimentales/genética , Aloinjertos/efectos de los fármacos , Aloinjertos/patología , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patologíaRESUMEN
Rearrangements involving the anaplastic lymphoma kinase (ALK) gene are defining events in several tumors, including anaplastic large-cell lymphoma (ALCL) and non-small cell lung carcinoma (NSCLC). In such cancers, the oncogenic activity of ALK stimulates signaling pathways that induce cell transformation and promote tumor growth. In search for common pathways activated by oncogenic ALK across different tumors types, we found that hypoxia pathways were significantly enriched in ALK-rearranged ALCL and NSCLC, as compared with other types of T-cell lymphoma or EGFR- and K-RAS-mutated NSCLC, respectively. Consistently, in both ALCL and NSCLC, we found that under hypoxic conditions, ALK directly regulated the abundance of hypoxia-inducible factors (HIF), which are key players of the hypoxia response in normal tissues and cancers. In ALCL, the upregulation of HIF1α and HIF2α in hypoxic conditions required ALK activity and its downstream signaling proteins STAT3 and C/EBPß. In vivo, ALK regulated VEGFA production and tumor angiogenesis in ALCL and NSCLC, and the treatment with the anti-VEGFA antibody bevacizumab strongly impaired ALCL growth in mouse xenografts. Finally, HIF2α, but not HIF1α, was required for ALCL growth in vivo whereas the growth and metastasis potential of ALK-rearranged NSCLC required both HIF1α and HIF2α. In conclusion, we uncovered an ALK-specific regulation of the hypoxia response across different ALK(+) tumor types and propose HIFs as a powerful specific therapeutic target in ALK-rearranged ALCL and NSCLC.