RESUMEN

The vacuolar H+-translocating ATPase (V-ATPase) is the major proton pump for intra-organellar acidification. Therefore, the integrity of the V-ATPase is closely associated with cellular homeostasis, and mutations in genes encoding V-ATPase components and assembly factors have been reported in certain types of diseases. For instance, the recurrent mutations of ATP6AP1, a gene encoding a V-ATPase accessory protein, have been associated with cancers and immunodeficiency. With the aim of studying V-ATPase-related mutations using the yeast model system, we report that Big1 is another homolog of ATP6AP1 in yeast cells, and we characterize the role of Big1 in maintaining a fully functional V-ATPase. In addition to its role in acidifying the vacuole or lysosome, our data support the concept that the V-ATPase may function as part of a signaling pathway to regulate macroautophagy/autophagy through a mechanism that is independent from Tor/MTOR.

RESUMEN

The recent discovery of recurrent gene mutations in chaperones or components of the vacuolar-type H+-translocating ATPase (V-ATPase) in follicular lymphoma (FL) was an unexpected finding. The application of whole exome sequencing and targeted gene re-sequencing has resulted in the identification of mutations in ATP6AP1, ATP6V1B2 and VMA21 in a combined 30% of FL, together constituting a major novel mutated pathway in this disease. Interestingly, no other human hematological malignancy carries these mutations at more than sporadic occurrences, implicating unique aspects of FL biology requiring these mutations. The mutations in ATP6V1B2 and VMA21 through separate mechanisms impair lysosomal V-ATPase activity resulting in an elevated lysosomal pH. The elevated lysosomal pH impairs protein and peptide hydrolysis and associates with reduced cytoplasmic amino acid concentrations resulting in compensatory activation of autophagic flux. The elevated autophagic flux constitutes a survival dependency for FL cells and can be targeted with inhibitors to ULK1 and multiple recently identified cyclin-dependent kinase inhibitors. Targeting autophagy alone or in combination with other targeted therapies constitutes a novel therapeutic opportunity for FL patients.

Asunto(s)

Linfoma Folicular , ATPasas de Translocación de Protón Vacuolares , Humanos , Autofagia/genética , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Mutación/genética , Vacuolas/metabolismo , Lisosomas/metabolismo

RESUMEN

The discovery of recurrent mutations in subunits and regulators of the vacuolar-type H+-translocating ATPase (V-ATPase) in follicular lymphoma (FL) highlights a role for macroautophagy/autophagy, amino-acid, and nutrient-sensing pathways in the pathogenesis of this disease. Here, we report on novel mutations in the ER-resident chaperone VMA21, which is involved in V-ATPase assembly in 12% of FL. Mutations in a novel VMA21 hotspot (p.93X) result in the removal of a C-terminal non-canonical ER retrieval signal thus causing VMA21 mislocalization to lysosomes. The resulting impairment in V-ATPase activity prevents full lysosomal acidification and function, including impaired pH-dependent protein degradation as shown via lysosomal metabolomics and ultimately causes a degree of amino acid depletion in the cytoplasm. These deficiencies result in compensatory autophagy activation, as measured using multiple complementary assays in human and yeast cells. Of translational significance, the compensatory activation of autophagy creates a dependency for survival for VMA21-mutated primary human FL as shown using inhibitors to ULK1, the proximal autophagy-regulating kinase. Using high-throughput microscopy-based screening assays for autophagy-inhibiting compounds, we identify multiple clinical grade cyclin-dependent kinase inhibitors as promising drugs and thus provide new rationale for innovative clinical trials in FL harboring aberrant V-ATPase.Abbreviations: ALs: autolysosomes; APs: autophagosomes; ER: endoplasmic reticulum; FL: follicular lymphoma; GFP: green fluorescent protein; IP: immunoprecipitation; LE/LY: late endosomes/lysosomes; Lyso-IP: lysosomal immunoprecipitation; OST: oligosaccharide transferase; prApe1: precursor aminopeptidase I; SEP: super ecliptic pHluorin; V-ATPase: vacuolar-type H+-translocating ATPase.

Asunto(s)

Linfoma Folicular , ATPasas de Translocación de Protón Vacuolares , Autofagia/genética , Humanos , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Lisosomas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación/genética , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo

RESUMEN

Follicular lymphoma (FL) is the 2nd most common lymphoma in the USA/Western Europe. While incurable, the majority of patients are able to survive at least a decade with this disease. Response duration though varies, and subset of patients will relapse within 24 months of initial therapy (POD24). These patients have shortened survival compared to those who achieve more durable responses. Treatment interventions for patients are varied and include observation, radiation, or systemic therapies. Treatment outcomes have improved considerably over the last several decades with the introduction of new agents such as the CD 20 antibody rituximab and more recently with the advent of more targeted therapy. Most of the newer agents work differently from cytotoxic chemotherapy and either inhibit tumor-specific mutations, survival pathways, or harness the immune systems. While outcomes with traditional cytotoxic agents have been historically poor in certain subtypes such as POD24 and rituximab refractory disease, the reported outcomes with the newer agents have been encouraging as evident by several new drug approvals in FL. The biggest impact has been in the relapsed/refractory setting where we have approval of the immunomodulatory agent lenalidomide given in combination with rituximab. Based on the AUGMENT study, this agent has been approved for patients with R/R FL after one previous line of therapy. The EZH2 inhibitor, tazemetostat, was approved recently for patients with a known EZH2 mutation after one prior line of therapy or for FL patients who are deemed intolerant to other agents given the impressive safety profile in all patients. Finally, there is a plethora of agents that are designed to harness the immune system to combat this lymphoma. The data for these agents is still very early but nonetheless very impressive. In summary, FL is an incurable lymphoma without any standard of care options but has numerous treatments that have demonstrated some degree of efficacy. Recently we have made enormous strides in the understanding of some of the biological drivers of this disease which has allowed for refinement of treatment options. Moving forward, I would anticipate that we will continue to explore the use of agents that target specific mutations or utilize the immune system to hopefully one day achieve a cure.

Asunto(s)

Linfoma Folicular/terapia , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos/uso terapéutico , Humanos , Inmunoconjugados/uso terapéutico , Factores Inmunológicos/uso terapéutico , Inmunoterapia Adoptiva , Lenalidomida/uso terapéutico , Linfoma Folicular/tratamiento farmacológico , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/uso terapéutico

RESUMEN

PURPOSE: On the basis of the recent discovery of mutations in Bruton tyrosine kinase (BTK) in follicular lymphoma, we studied their functional properties. EXPERIMENTAL DESIGN: We identified novel somatic BTK mutations in 7% of a combined total of 139 follicular lymphoma and 11 transformed follicular lymphoma cases, none of which had received prior treatment with B-cell receptor (BCR) targeted drugs. We reconstituted wild-type (WT) and mutant BTK into various engineered lymphoma cell lines. We measured BCR-induced signal transduction events in engineered cell lines and primary human follicular lymphoma B cells. RESULTS: We uncovered that all BTK mutants destabilized the BTK protein and some created BTK kinase-dead mutants. The phospholipase C gamma 2 (PLCγ2) is a substrate of BTK but the BTK mutants did not alter PLCγ2 phosphorylation. Instead, we discovered that BTK mutants induced an exaggerated AKT phosphorylation phenotype in anti-Ig-treated recombinant lymphoma cell lines. The short hairpin RNA-mediated knockdown of BTK expression in primary human nonmalignant lymph node-derived B cells resulted in strong anti-Ig-induced AKT activation, as did the degradation of BTK protein in cell lines using ibrutinib-based proteolysis targeting chimera. Finally, through analyses of primary human follicular lymphoma B cells carrying WT or mutant BTK, we detected elevated AKT phosphorylation following surface Ig crosslinking in all follicular lymphoma B cells, including all BTK-mutant follicular lymphoma. The augmented AKT phosphorylation following BCR crosslinking could be abrogated by pretreatment with a PI3Kδ inhibitor. CONCLUSIONS: Altogether, our data uncover novel unexpected properties of follicular lymphoma-associated BTK mutations with direct implications for targeted therapy development in follicular lymphoma.See related commentary by Afaghani and Taylor, p. 2123.

Asunto(s)

Agammaglobulinemia Tirosina Quinasa/genética , Linfoma Folicular/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Agammaglobulinemia Tirosina Quinasa/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Análisis Mutacional de ADN , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Mutación con Pérdida de Función , Linfoma Folicular/patología , Mutagénesis Sitio-Dirigida , Fosfolipasa C gamma/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Cultivo Primario de Células , Estabilidad Proteica

RESUMEN

Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are characterized by a progressive accumulation of leukemic cells in the peripheral blood, bone marrow, and lymphoid tissues. Treatment of CLL/SLL has evolved significantly in recent years because of the improved understanding of the disease biology and the development of novel targeted therapies. In patients with indications for initiating treatment, the selection of treatment should be based on the disease stage, patient's age and overall fitness (performance status and comorbid conditions), and cytogenetic abnormalities. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with CLL/SLL.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/normas , Trasplante de Células Madre Hematopoyéticas/normas , Leucemia Linfocítica Crónica de Células B/terapia , Oncología Médica/normas , Recurrencia Local de Neoplasia/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Médula Ósea/patología , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos/genética , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Inmunofenotipificación , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/mortalidad , Ganglios Linfáticos/citología , Ganglios Linfáticos/patología , Linfocitos/patología , Oncología Médica/métodos , Mutación , Recurrencia Local de Neoplasia/epidemiología , Estadificación de Neoplasias , Organizaciones sin Fines de Lucro/normas , Pronóstico , Inducción de Remisión/métodos , Trasplante Homólogo/normas , Estados Unidos/epidemiología

RESUMEN

TP53 mutation in acute myeloid leukemia (AML) is associated with poor prognosis. Since no targeted therapy is available to restore p53 function, it is of great interest to test whether other pathways activated by TP53 mutations can be therapeutically targeted. Here, we showed HIF-1α target genes are enriched in TP53-mutated versus TP53-wild-type AML. To determine the role of this activation, we tested efficacy of HIF-1α inhibitor echinomycin in TP53-mutated AML samples in vitro and in vivo. Echinomycin was broadly effective against a panel of primary AML blast cells, with low nanomolar IC50s and, based on colony-forming unit assay, was tenfold more effective in eliminating AML stem cells. Echinomycin selectively eliminated CD34+CD38- AML cells. To test the therapeutic efficacy of echinomycin, we established a xenograft model of TP53-mutated AML. Echinomycin was broadly effective against xenografts from multiple AML samples in vivo, and more effective than cytarabine + daunorubicin chemotherapy. Importantly, while cytarabine + daunorubicin enriched for AML stem cells, echinomycin nearly eliminated this population. Using TP53-mutated AML cell line THP1 and patient-derived AML cells, we tested a new echinomycin formulation with longer half-life and significantly improved therapeutic effect. Our data suggest a novel approach to treat AML with TP53 mutations.

Asunto(s)

Equinomicina/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Mutación/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Ratones , Mutación/genética

RESUMEN

Centromere genomics remain poorly characterized in cancer, due to technologic limitations in sequencing and bioinformatics methodologies that make high-resolution delineation of centromeric loci difficult to achieve. We here leverage a highly specific and targeted rapid PCR methodology to quantitatively assess the genomic landscape of centromeres in cancer cell lines and primary tissue. PCR-based profiling of centromeres revealed widespread heterogeneity of centromeric and pericentromeric sequences in cancer cells and tissues as compared to healthy counterparts. Quantitative reductions in centromeric core and pericentromeric markers (α-satellite units and HERV-K copies) were observed in neoplastic samples as compared to healthy counterparts. Subsequent phylogenetic analysis of a pericentromeric endogenous retrovirus amplified by PCR revealed possible gene conversion events occurring at numerous pericentromeric loci in the setting of malignancy. Our findings collectively represent a more comprehensive evaluation of centromere genetics in the setting of malignancy, providing valuable insight into the evolution and reshuffling of centromeric sequences in cancer development and progression.

Asunto(s)

Biomarcadores de Tumor/genética , Carcinogénesis/genética , Centrómero/genética , Evolución Molecular , Neoplasias/genética , Biomarcadores de Tumor/aislamiento & purificación , Línea Celular Tumoral , ADN Satélite/genética , ADN Satélite/aislamiento & purificación , ADN Viral/genética , ADN Viral/aislamiento & purificación , Progresión de la Enfermedad , Retrovirus Endógenos/genética , Genómica , Humanos , Neoplasias/patología , Filogenia , Reacción en Cadena de la Polimerasa

RESUMEN

The discovery of recurrent mutations in subunits of the vacuolar-type H+-translocating ATPase (v-ATPase) in follicular lymphoma (FL) highlights a role for the amino acid- and energy-sensing pathway to mTOR in the pathogenesis of this disease. Here, through the use of complementary experimental approaches involving mammalian cells and Saccharomyces cerevisiae, we have demonstrated that mutations in the human v-ATPase subunit ATP6V1B2 (also known as Vma2 in yeast) activate autophagic flux and maintain mTOR/TOR in an active state. Engineered lymphoma cell lines and primary FL B cells carrying mutated ATP6V1B2 demonstrated a remarkable ability to survive low leucine concentrations. The treatment of primary FL B cells with inhibitors of autophagy uncovered an addiction for survival for FL B cells harboring ATP6V1B2 mutations. These data support the idea of mutational activation of autophagic flux by recurrent hotspot mutations in ATP6V1B2 as an adaptive mechanism in FL pathogenesis and as a possible new therapeutically targetable pathway.

Asunto(s)

Muerte Celular Autofágica , Linfoma Folicular/enzimología , Mutación , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Humanos , Linfoma Folicular/genética , Linfoma Folicular/patología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina-Treonina Quinasas TOR/genética , ATPasas de Translocación de Protón Vacuolares/genética

RESUMEN

Chronic lymphocytic leukemia (CLL) is generally characterized by an indolent disease course. Histologic transformation (also known as Richter's transformation) to more aggressive lymphomas, such as diffuse large B-cell lymphoma or Hodgkin lymphoma, occurs in approximately 2% to 10% of patients and is associated with a poor prognosis. These NCCN Guidelines Insights discuss the recommendations for the diagnosis and management of patients with histologic transformation.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Oncología Médica/normas , Sociedades Médicas/normas , Protocolos de Quimioterapia Combinada Antineoplásica/normas , Ensayos Clínicos como Asunto , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/etiología , Leucemia Linfocítica Crónica de Células B/mortalidad , Oncología Médica/métodos , Supervivencia sin Progresión , Estados Unidos

RESUMEN

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based technology enables efficient and precise perturbations of target genomic sites. Combining the endonuclease Cas9 and a pooled guide RNA library allows for systematic screenings of genes associated with a growth disadvantage or lethal phenotype under various conditions in organisms and tissues. Here, we describe a complete protocol for scalable CRISPR/Cas9-based dropout screening for essential genes from focused genomic regions to whole genomes.

Asunto(s)

Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Marcación de Gen/métodos , Genómica/métodos , Edición Génica/instrumentación , Biblioteca de Genes , Marcación de Gen/instrumentación , Genómica/instrumentación , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , ARN Guía de Kinetoplastida/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Análisis de Secuencia de ADN/instrumentación , Análisis de Secuencia de ADN/métodos

RESUMEN

The emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology provides tools for researchers to modify genomes in a specific and efficient manner. The Type II CRISPR-Cas9 system enables gene editing by directed DNA cleavage followed by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Here, we described the use of the Type II CRISPR-Cas9 system in detail from designing the guides to analyzing the desired gene disruption events.

Asunto(s)

Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Marcación de Gen/métodos , Reparación del ADN por Unión de Extremidades/genética , Edición Génica/instrumentación , Marcación de Gen/instrumentación , Vectores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , ARN Guía de Kinetoplastida/genética , Reparación del ADN por Recombinación/genética , Transducción Genética/instrumentación , Transducción Genética/métodos

RESUMEN

Hairy cell leukemia (HCL) is a rare type of indolent B-cell leukemia, characterized by symptoms of fatigue and weakness, organomegaly, pancytopenia, and recurrent opportunistic infections. Classic HCL should be considered a distinct clinical entity separate from HCLvariant (HCLv), which is associated with a more aggressive disease course and may not respond to standard HCL therapies. Somatic hypermutation in the IGHV gene is present in most patients with HCL. The BRAF V600E mutation has been reported in most patients with classic HCL but not in those with other B-cell leukemias or lymphomas. Therefore, it is necessary to distinguish HCLv from classic HCL. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of classic HCL.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/normas , Leucemia de Células B/diagnóstico , Leucemia de Células Pilosas/diagnóstico , Leucemia de Células Pilosas/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Citodiagnóstico/métodos , Citodiagnóstico/normas , Diagnóstico Diferencial , Reordenamiento Génico , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Inmunofenotipificación/métodos , Inmunofenotipificación/normas , Leucemia de Células B/genética , Leucemia de Células Pilosas/genética , Leucemia de Células Pilosas/patología , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Resultado del Tratamiento

RESUMEN

Monitoring the snow pack is crucial for many stakeholders, whether for hydro-power optimization, water management or flood control. Traditional forecasting relies on regression methods, which often results in snow melt runoff predictions of low accuracy in non-average years. Existing ground-based real-time measurement systems do not cover enough physiographic variability and are mostly installed at low elevations. We present the hardware and software design of a state-of-the-art distributed Wireless Sensor Network (WSN)-based autonomous measurement system with real-time remote data transmission that gathers data of snow depth, air temperature, air relative humidity, soil moisture, soil temperature, and solar radiation in physiographically representative locations. Elevation, aspect, slope and vegetation are used to select network locations, and distribute sensors throughout a given network location, since they govern snow pack variability at various scales. Three WSNs were installed in the Sierra Nevada of Northern California throughout the North Fork of the Feather River, upstream of the Oroville dam and multiple powerhouses along the river. The WSNs gathered hydrologic variables and network health statistics throughout the 2017 water year, one of northern Sierra's wettest years on record. These networks leverage an ultra-low-power wireless technology to interconnect their components and offer recovery features, resilience to data loss due to weather and wildlife disturbances and real-time topological visualizations of the network health. Data show considerable spatial variability of snow depth, even within a 1 km 2 network location. Combined with existing systems, these WSNs can better detect precipitation timing and phase in, monitor sub-daily dynamics of infiltration and surface runoff during precipitation or snow melt, and inform hydro power managers about actual ablation and end-of-season date across the landscape.

RESUMEN

Acute myelogenous leukemia (AML) frequently relapses after complete remission (CR), necessitating improved detection and phenotypic characterization of treatment-resistant residual disease. In this work, we have optimized droplet digital PCR to broadly measure mutated alleles of recurrently mutated genes in CR marrows of AML patients at levels as low as 0.002% variant allele frequency. Most gene mutations persisted in CR, albeit at highly variable and gene-dependent levels. The majority of AML cases demonstrated residual aberrant oligoclonal hematopoiesis. Importantly, we detected very rare cells (as few as 1 in 15,000) that were genomically similar to the dominant blast populations at diagnosis and were fully clonally represented at relapse, identifying these rare cells as one common source of AML relapse. Clinically, the mutant allele burden was associated with overall survival in AML, and our findings narrow the repertoire of gene mutations useful in minimal residual disease-based prognostication in AML. Overall, this work delineates rare cell populations that cause AML relapse, with direct implications for AML research directions and strategies to improve AML therapies and outcome.

Asunto(s)

Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Mutación , Recurrencia Local de Neoplasia , Neoplasia Residual/genética , Adulto , Anciano , Alelos , Antraciclinas/administración & dosificación , Médula Ósea/patología , Citarabina/administración & dosificación , Exoma , Femenino , Hematopoyesis , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Fenotipo , Pronóstico , Inducción de Remisión , Trasplante de Células Madre , Factores de Tiempo , Trasplante Homólogo , Resultado del Tratamiento , Adulto Joven

RESUMEN

Purpose: Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed chronic lymphocytic leukemia (CLL) and CLL with del17p. Mechanistically, ibrutinib interferes with B-cell receptor (BCR) signaling as well as multiple CLL cell-to-microenvironment interactions. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance is warranted.Experimental Design: We studied 48 longitudinally sampled paired CLL samples, 42 of which were procured before and after standard CLL chemotherapies, and characterized them for well-studied CLL molecular traits as well as by whole-exome sequencing and SNP 6.0 array profiling. We exposed these samples to 0.25 to 5 µmol/L of ibrutinib ex vivo and measured apoptosis fractions as well as BCR signaling by immunoblotting. We disrupted TP53 in HG3, PGA1, and PG-EBV cell lines and measured BCR signaling and ibrutinib responses.Results: CLL samples demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib, with IC50 values ranging from 0.4 to 9.7 µmol/L. Unmutated IGVH status, elevated ZAP70 expression, and trisomy 12 were associated with heightened sensitivity to ibrutinib treatment. Five CLL samples were substantially more resistant to ibrutinib following relapse from chemotherapy; of these, three had acquired a del17p/TP53-mutated status. A validation sample of 15 CLL carrying TP53 mutations, of which 13 carried both del17p and a TP53 mutation, confirmed substantially less sensitivity to ibrutinib-induced apoptosis.Conclusions: This study identifies that CLL harboring del17p/TP53-mutated cells are substantially less sensitive to ibrutinib-induced apoptosis than del17p/TP53 wild-type cells. Clin Cancer Res; 23(4); 1049-59. ©2016 AACR.

Asunto(s)

Biomarcadores Farmacológicos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Proteína p53 Supresora de Tumor/genética , Adenina/análogos & derivados , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Piperidinas , Polimorfismo de Nucleótido Simple , Pirazoles/efectos adversos , Pirimidinas/efectos adversos , Secuenciación del Exoma

RESUMEN

The HVEM (TNFRSF14) receptor gene is among the most frequently mutated genes in germinal center lymphomas. We report that loss of HVEM leads to cell-autonomous activation of B cell proliferation and drives the development of GC lymphomas in vivo. HVEM-deficient lymphoma B cells also induce a tumor-supportive microenvironment marked by exacerbated lymphoid stroma activation and increased recruitment of T follicular helper (TFH) cells. These changes result from the disruption of inhibitory cell-cell interactions between the HVEM and BTLA (B and T lymphocyte attenuator) receptors. Accordingly, administration of the HVEM ectodomain protein (solHVEM(P37-V202)) binds BTLA and restores tumor suppression. To deliver solHVEM to lymphomas in vivo, we engineered CD19-targeted chimeric antigen receptor (CAR) T cells that produce solHVEM locally and continuously. These modified CAR-T cells show enhanced therapeutic activity against xenografted lymphomas. Hence, the HVEM-BTLA axis opposes lymphoma development, and our study illustrates the use of CAR-T cells as "micro-pharmacies" able to deliver an anti-cancer protein.

Asunto(s)

Traslado Adoptivo/métodos , Linfoma Folicular/terapia , Receptores Inmunológicos/metabolismo , Miembro 14 de Receptores del Factor de Necrosis Tumoral/genética , Linfocitos T/inmunología , Proteínas Supresoras de Tumor/genética , Animales , Antígenos CD19/inmunología , Linfocitos B/inmunología , Proliferación Celular , Humanos , Activación de Linfocitos , Linfoma Folicular/genética , Ratones , Neoplasias Experimentales/genética , Neoplasias Experimentales/terapia , Dominios Proteicos , Ingeniería de Proteínas , Miembro 14 de Receptores del Factor de Necrosis Tumoral/química , Miembro 14 de Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Microambiente Tumoral , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Although over expression of chimeric FGFR1 kinase consistently leads to the development of AML in the rare Stem Cell Leukemia and Lymphoma syndrome, we now show that overexpression of FGFR1 is also seen in up to 20% of non-syndromic, de novo AML. To determine whether targeting FGFR1 in both of these AML subtypes can suppress leukemogenesis, we evaluated the effects of different FGFR1 inhibitors in a side-by-side comparison for their ability to affect in vitro proliferation in FGFR1 overexpressing murine and human cells lines. Three newly developed pan-FGFR inhibitors, AZD4547, BGJ398 and JNJ42756493, show a significantly improved efficacy over the more established FGFR inhibitors, PD173074 and TKI258. To examine whether targeting FGFR1 suppresses leukemogenesis in de novo AML in vivo, we created xenografts in immunocompromized mice from primary, de novo AML that showed > 3-fold increased expression of FGFR1. Using BGJ398, the most potent inhibitor identified in the in vitro studies, AML progression in these mice was significantly suppressed compared with vehicle treated animals and overall survival improved. Importantly, no difference in disease course or survival was seen in AML xenografts that did not show overexpression of FGFR1. These observations support the idea that FGFR1 is a driver oncogene in de novo, FGFR1-overexpressing AML and that molecularly targeted therapies using FGFR1 inhibitors may provide a valuable therapeutic regimen for all FGFR1-overexpressing AML.

Asunto(s)

Leucemia/metabolismo , Terapia Molecular Dirigida , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Benzamidas/farmacología , Bencimidazoles/farmacología , Carcinogénesis , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Ratones , Trasplante de Neoplasias , Compuestos de Fenilurea/farmacología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Quinolonas/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

PURPOSE: Monocytes and their progeny are abundant constituents of the tumor microenvironment in lymphoproliferative disorders, including chronic lymphocytic leukemia (CLL). Monocyte-derived cells, including nurse-like cells (NLC) in CLL, promote lymphocyte proliferation and survival, confer resistance to chemotherapy, and are associated with more rapid disease progression. Colony-stimulating factor-1 receptor (CSF-1R) regulates the homeostatic survival of tissue-resident macrophages. Therefore, we sought to determine whether CSF-1R is similarly required for NLC survival. EXPERIMENTAL DESIGN: CSF-1R expression by NLC was examined by flow cytometry and IHC. CSF-1R blocking studies were performed using an antagonistic mAb to examine its role in NLC generation and in CLL survival. A rational search strategy was performed to identify a novel tyrosine kinase inhibitor (TKI) targeting CSF-1R. The influence of TKI-mediated CSF-1R inhibition on NLC and CLL viability was examined. RESULTS: We demonstrated that the generation and survival of NLC in CLL is dependent upon CSF-1R signaling. CSF-1R blockade is associated with significant depletion of NLC and consequently inhibits CLL B-cell survival. We found that the JAK2/FLT3 inhibitor pacritinib suppresses CSF-1R signaling, thereby preventing the generation and survival of NLC and impairs CLL B-cell viability. CONCLUSIONS: CSF-1R is a novel therapeutic target that may be exploited in lymphoproliferative disorders, like CLL, that are dependent upon lymphoma-associated macrophages. Clin Cancer Res; 22(24); 6118-28. ©2016 AACR.

Asunto(s)

Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología

RESUMEN

PURPOSE: This study was performed to further our understanding of the biological and genetic basis of follicular lymphoma and to identify potential novel therapy targets. EXPERIMENTAL DESIGN: We analyzed previously generated whole exome sequencing data of 23 follicular lymphoma cases and one transformed follicular lymphoma case and expanded findings to a combined total of 125 follicular lymphoma/3 transformed follicular lymphoma. We modeled the three-dimensional location of RRAGC-associated hotspot mutations. We performed functional studies on novel RRAGC mutants in stable retrovirally transduced HEK293T cells, stable lentivirally transduced lymphoma cell lines, and in Saccharomyces cerevisiae RESULTS: We report recurrent mutations, including multiple amino acid hotspots, in the small G-protein RRAGC, which is part of a protein complex that signals intracellular amino acid concentrations to MTOR, in 9.4% of follicular lymphoma cases. Mutations in RRAGC distinctly clustered on one protein surface area surrounding the GTP/GDP-binding sites. Mutated RRAGC proteins demonstrated increased binding to RPTOR (raptor) and substantially decreased interactions with the product of the tumor suppressor gene FLCN (folliculin). In stable retrovirally transfected 293T cells, cultured in the presence or absence of leucine, multiple RRAGC mutations demonstrated elevated MTOR activation as evidenced by increased RPS6KB/S6-kinase phosphorylation. Similar activation phenotypes were uncovered in yeast engineered to express mutations in the RRAGC homolog Gtr2 and in multiple lymphoma cell lines expressing HA-tagged RRAGC-mutant proteins. CONCLUSIONS: Our discovery of activating mutations in RRAGC in approximately 10% of follicular lymphoma provides the mechanistic rationale to study mutational MTOR activation and MTOR inhibition as a potential novel actionable therapeutic target in follicular lymphoma. Clin Cancer Res; 22(21); 5383-93. ©2016 AACR.

Asunto(s)

Linfoma Folicular/genética , Proteínas de Unión al GTP Monoméricas/genética , Mutación/genética , Recurrencia Local de Neoplasia/genética , Serina-Treonina Quinasas TOR/genética , Aminoácidos/genética , Sitios de Unión/genética , Línea Celular , Genes Supresores de Tumor/fisiología , Guanosina Difosfato/genética , Guanosina Trifosfato/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Fosforilación/genética , Proteína Reguladora Asociada a mTOR/genética , Transducción de Señal/genética