RESUMEN
The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and outbreaks of new variants highlight the need for preventive treatments. Here, we identified heparan sulfate proteoglycans as attachment receptors for SARS-CoV-2. Notably, neutralizing antibodies against SARS-CoV-2 isolated from COVID-19 patients interfered with SARS-CoV-2 binding to heparan sulfate proteoglycans, which might be an additional mechanism of antibodies to neutralize infection. SARS-CoV-2 binding to and infection of epithelial cells was blocked by low molecular weight heparins (LMWH). Although dendritic cells (DCs) and mucosal Langerhans cells (LCs) were not infected by SARS-CoV-2, both DC subsets efficiently captured SARS-CoV-2 via heparan sulfate proteoglycans and transmitted the virus to ACE2-positive cells. Notably, human primary nasal cells were infected by SARS-CoV-2, and infection was blocked by pre-treatment with LMWH. These data strongly suggest that heparan sulfate proteoglycans are important attachment receptors facilitating infection and transmission, and support the use of LMWH as prophylaxis against SARS-CoV-2 infection.
Asunto(s)
COVID-19/transmisión , Proteoglicanos de Heparán Sulfato/metabolismo , Heparina de Bajo-Peso-Molecular/farmacología , SARS-CoV-2/patogenicidad , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/farmacología , Chlorocebus aethiops , Células Dendríticas/metabolismo , Células Dendríticas/virología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Interacciones Huésped-Patógeno , Humanos , Membrana Mucosa/citología , Membrana Mucosa/virología , SARS-CoV-2/metabolismo , Sindecano-1/metabolismo , Sindecano-4/metabolismo , Células Vero , Tratamiento Farmacológico de COVID-19RESUMEN
Complex karyotypes have been associated with inferior outcomes in chronic lymphocytic leukemia (CLL) treated with chemoimmunotherapy (CIT), whereas their prognostic impact in the context of venetoclax-based treatments is still debated. In this prospective analysis on karyotype complexity in CLL, we evaluated the impact of complex (≥3 chromosomal aberrations [CAs], CKTs) and highly complex karyotypes (≥5 CAs; hCKTs) as well as specific aberrations in previously untreated patients without TP53 aberrations undergoing either CIT or time-limited venetoclax-based therapies in the phase 3 GAIA/CLL13 trial. Karyotype analyses were available for 895 of 926 patients (96.7%), of whom 153 (17%) had a CKT and 43 (5%) hCKT. In the CIT arm, CKT was associated with shorter progression-free survival (PFS) (hazard ratio [HR] 2.58; 95% confidence interval [95% CI], 1.54-4.32; P < .001) and overall survival (HR, 3.25; 95% CI, 1.03-10.26; P = .044). In the pooled venetoclax arms, a multivariable analysis identified hCKTs (HR, 1.96; 95% CI, 1.03-3.72; P = .041), but not CKTs, as independent adverse prognosticators for PFS. The presence of translocations (unbalanced and/or balanced) was also independently associated with shorter PFSs in the venetoclax arms. CIT led to the acquisition of additional CAs (mean CAs, 2.0-3.4; from baseline to CLL progression), whereas karyotype complexity remained stable after venetoclax-based treatments (2.0, both time points). This analysis establishes highly complex karyotypes and translocations as adverse prognostic factors in the context of venetoclax-based combination treatments. The findings of this study support the incorporation of karyotyping into the standard diagnostic workup of CLL, because it identifies patients at high risk of poor treatment outcomes and thereby improves prognostication. This trial was registered at www.clinicaltrials.gov as #NCT02950051.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Humanos , Cariotipo Anormal , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Cariotipo , Cariotipificación , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , PronósticoRESUMEN
The endothelial lining of blood vessels is covered with a thin polysaccharide coat called the glycocalyx. This layer of polysaccharides contains hyaluronan that forms a protective coat on the endothelial surface. Upon inflammation, leukocytes leave the circulation and enter inflamed tissue by crossing inflamed endothelial cells, mediated by adhesion molecules such as ICAM-1/CD54. To what extent the glycocalyx participates in the regulation of leukocyte transmigration is not clear. During extravasation, leukocyte integrins cluster ICAM-1, resulting in the recruitment of a number of intracellular proteins and subsequent downstream effects in the endothelial cells. For our studies, we used primary human endothelial and immune cells. With an unbiased proteomics approach, we identified the full ICAM-1 adhesome and identified 93 (to our knowledge) new subunits of the ICAM-1 adhesome. Interestingly, we found the glycoprotein CD44 as part of the glycocalyx to be recruited to clustered ICAM-1 specifically. Our data demonstrate that CD44 binds hyaluronan to the endothelial surface, where it locally concentrates and presents chemokines that are essential for leukocytes to cross the endothelial lining. Taken together, we discover a link between ICAM-1 clustering and hyaluronan-mediated chemokine presentation by recruiting hyaluronan to sites of leukocyte adhesion via CD44.
Asunto(s)
Células Endoteliales , Ácido Hialurónico , Humanos , Células Endoteliales/metabolismo , Ácido Hialurónico/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Endotelio/metabolismo , Adhesión Celular/fisiología , Leucocitos/metabolismo , Receptores de Hialuranos/metabolismoRESUMEN
Plasma cells no longer express a B-cell antigen receptor and are hence deprived of signals crucial for survival throughout B-cell development. Instead, normal plasma cells, as well as their malignant myeloma counterparts, heavily rely on communication with the bone marrow (BM) microenvironment for survival. The plasma cell heparan sulfate proteoglycan (HSPG) syndecan-1 (CD138) and HSPGs in the BM microenvironment act as master regulators of this communication by co-opting specific growth and survival factors from the BM niche. This designates syndecan-1/HSPGs and their synthesis machinery as potential treatment targets in multiple myeloma.
Asunto(s)
Heparitina Sulfato/metabolismo , Mieloma Múltiple/patología , Células Plasmáticas/patología , Proteoglicanos/metabolismo , Sindecano-1/metabolismo , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Humanos , Mieloma Múltiple/metabolismo , Células Plasmáticas/metabolismo , Microambiente TumoralRESUMEN
BCL-2 family proteins are frequently aberrantly expressed in mantle cell lymphoma (MCL). Recently, the BCL-2-specific inhibitor venetoclax has been approved by the US Food and Drug Administration for chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). In MCL, venetoclax has shown promising efficacy in early clinical trials; however, a significant subset of patients is resistant. By conducting a kinome-centered CRISPR-Cas9 knockout sensitizer screen, we identified casein kinase 2 (CK2) as a major regulator of venetoclax resistance in MCL. Interestingly, CK2 is over-expressed in MCL and high CK2 expression is associated with poor patient survival. Targeting of CK2, either by inducible short hairpin RNA (shRNA)-mediated knockdown of CK2 or by the CK2-inhibitor silmitasertib, did not affect cell viability by itself, but strongly synergized with venetoclax in both MCL cell lines and primary samples, also if combined with ibrutinib. Furthermore, targeting of CK2 reduced MCL-1 levels, which involved impaired MCL-1 translation by inhibition of eIF4F complex assembly, without affecting BCL-2 and BCL-XL expression. Combined, this results in enhanced BCL-2 dependence and, consequently, venetoclax sensitization. In cocultures, targeting of CK2 overcame stroma-mediated venetoclax resistance of MCL cells. Taken together, our findings indicate that targeting of CK2 sensitizes MCL cells to venetoclax through downregulation of MCL-1. These novel insights provide a strong rationale for combining venetoclax with CK2 inhibition as therapeutic strategy for MCL patients.
Asunto(s)
Antineoplásicos , Linfoma de Células del Manto , Humanos , Adulto , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/metabolismo , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Regulación hacia Abajo , Línea Celular Tumoral , Proteínas Proto-Oncogénicas c-bcl-2 , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéuticoRESUMEN
Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells in the bone marrow (BM). Most MMs display aberrant Wnt/ß-catenin signaling, which drives proliferation; however, they lack oncogenic Wnt pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts from the BM microenvironment. Expression of the heparan sulfate (HS) proteoglycan syndecan-1 is a hallmark of MM. Syndecan-1 is a critical player in the complex reciprocal interaction between MM cells and their BM niche, mediating growth factor/cytokine binding and signaling by its HS chains. Here, by means of CRISPR/Cas9-mediated knockout and doxycycline-inducible short hairpin RNA-mediated knockdown of EXT1, a critical enzyme for HS polymerization, we demonstrate that the HS chains decorating syndecan-1 mediate aberrant Wnt pathway activation in MM. HS-deficient MM cells exhibited strongly decreased autocrine Wnt/ß-catenin pathway activity and reduced Wnt pathway-dependent proliferation. In addition, we demonstrate that Wnts bind to the HS side chains of syndecan-1 and that this binding contributes to paracrine Wnt pathway activation through the Wnt receptor Frizzled (Fzd). Furthermore, in an HS-dependent fashion, syndecan-1 also binds osteoblast-produced R-spondin, which represses Fzd degradation by activation of LGR4, an R-spondin receptor aberrantly expressed on MM cells. Costimulation with R-spondin and its binding to HS chains decorating syndecan-1 are indispensable for optimal stimulation of Wnt signaling in MM. Taken together, our results identify syndecan-1 as a crucial component of the Wnt signalosome in MM cells, binding Wnts and R-spondins to promote aberrant Wnt/ß-catenin signaling and cell growth, and suggest HS and its biosynthetic enzymes as potential targets in the treatment of MM.
Asunto(s)
Mieloma Múltiple/metabolismo , Proteínas de Neoplasias/metabolismo , Sindecano-1/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Línea Celular Tumoral , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Proteínas de Neoplasias/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sindecano-1/genética , Trombospondinas/genética , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
The 2016 World Health Organization classification defines diffuse large B-cell lymphoma (DLBCL) subtypes based on Epstein-Barr virus (EBV) infection and oncogenic rearrangements of MYC/BCL2/BCL6 as drivers of lymphomagenesis. A subset of DLBCL, however, is characterized by activating mutations in MYD88/CD79B We investigated whether MYD88/CD79B mutations could improve the classification and prognostication of DLBCL. In 250 primary DLBCL, MYD88/CD79B mutations were identified by allele-specific polymerase chain reaction or next-generation-sequencing, MYC/BCL2/BCL6 rearrangements were analyzed by fluorescence in situ hybridization, and EBV was studied by EBV-encoded RNA in situ hybridization. Associations of molecular features with clinicopathologic characteristics, outcome, and prognosis according to the International Prognostic Index (IPI) were investigated. MYD88 and CD79B mutations were identified in 29.6% and 12.3%, MYC, BCL2, and BCL6 rearrangements in 10.6%, 13.6%, and 20.3%, and EBV in 11.7% of DLBCL, respectively. Prominent mutual exclusivity between EBV positivity, rearrangements, and MYD88/CD79B mutations established the value of molecular markers for the recognition of biologically distinct DLBCL subtypes. MYD88-mutated DLBCL had a significantly inferior 5-year overall survival than wild-type MYD88 DLBCL (log-rank; P=0.019). DLBCL without any of the studied aberrations had superior overall survival compared to cases carrying ≥1 aberrancy (log-rank; P=0.010). MYD88 mutations retained their adverse prognostic impact upon adjustment for other genetic and clinical variables by multivariable analysis and improved the prognostic performance of the IPI. This study demonstrates the clinical utility of defining MYD88-mutated DLBCL as a distinct molecular subtype with adverse prognosis. Our data call for sequence analysis of MYD88 in routine diagnostics of DLBCL to optimize classification and prognostication, and to guide the development of improved treatment strategies.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Linfoma de Células B Grandes Difuso , Factor 88 de Diferenciación Mieloide/genética , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4 , Humanos , Hibridación Fluorescente in Situ , Linfoma de Células B Grandes Difuso/diagnóstico , Linfoma de Células B Grandes Difuso/genética , Mutación , PronósticoRESUMEN
The unrestrained growth of tumor cells is generally attributed to mutations in essential growth control genes, but tumor cells are also affected by, or even addicted to, signals from the microenvironment. As therapeutic targets, these extrinsic signals may be equally significant as mutated oncogenes. In multiple myeloma (MM), a plasma cell malignancy, most tumors display hallmarks of active Wnt signaling but lack activating Wnt-pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts emanating from the bone marrow (BM) niche. Here, we report a pivotal role for the R-spondin/leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) axis in driving aberrant Wnt/ß-catenin signaling in MM. We show that LGR4 is expressed by MM plasma cells, but not by normal plasma cells or B cells. This aberrant LGR4 expression is driven by IL-6/STAT3 signaling and allows MM cells to hijack R-spondins produced by (pre)osteoblasts in the BM niche, resulting in Wnt (co)receptor stabilization and a dramatically increased sensitivity to auto- and paracrine Wnts. Our study identifies aberrant R-spondin/LGR4 signaling with consequent deregulation of Wnt (co)receptor turnover as a driver of oncogenic Wnt/ß-catenin signaling in MM cells. These results advocate targeting of the LGR4/R-spondin interaction as a therapeutic strategy in MM.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Mieloma Múltiple/metabolismo , Osteoblastos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Interleucina-6/metabolismo , Ligandos , Ratones , Unión Proteica/fisiología , Factor de Transcripción STAT3/metabolismo , beta Catenina/metabolismoRESUMEN
BACKGROUND: The aggressive B-cell non-Hodgkin lymphomas diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are characterised by a high proliferation rate. The anaphase-promoting complex/cyclosome (APC/C) and its co-activators Cdc20 and Cdh1 represent an important checkpoint in mitosis. Here, the role of the APC/C and its co-activators is examined in DLBCL and MCL. METHODS: The expression and prognostic value of Cdc20 and Cdh1 was investigated using GEP data and immunohistochemistry. Moreover, the therapeutic potential of APC/C targeting was evaluated using the small-molecule inhibitor proTAME and the underlying mechanisms of action were investigated by western blot. RESULTS: We demonstrated that Cdc20 is highly expressed in DLBCL and aggressive MCL, correlating with a poor prognosis in DLBCL. ProTAME induced a prolonged metaphase, resulting in accumulation of the APC/C-Cdc20 substrate cyclin B1, inactivation/degradation of Bcl-2 and Bcl-xL and caspase-dependent apoptosis. In addition, proTAME strongly enhanced the anti-lymphoma effect of the clinically relevant agents doxorubicin and venetoclax. CONCLUSION: We identified for the first time APC/C as a new, promising target in DLBCL and MCL. Moreover, we provide evidence that Cdc20 might be a novel, independent prognostic factor in DLBCL and MCL.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células del Manto/tratamiento farmacológico , Profármacos/farmacología , Tosilarginina Metil Éster/farmacología , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Antígenos CD/biosíntesis , Antígenos CD/genética , Apoptosis/efectos de los fármacos , Cadherinas/biosíntesis , Cadherinas/genética , Proteínas Cdc20/biosíntesis , Proteínas Cdc20/genética , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células del Manto/metabolismo , Linfoma de Células del Manto/patología , Terapia Molecular Dirigida , Pronóstico , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND & AIMS: Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice. METHODS: We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (AhCre/Metfl/fl/LacZ) or ISC-specific disruption of MET (Lgr5Creert2/Metfl/fl/LacZ) and control mice (AhCre/Met+/+/LacZ, Lgr5Creert2/Met+/+/LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5Creert2/Metfl/fl/Apcfl/fl and Lgr5Creert2/Met+/+/Apcfl/fl mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in AhCre/Metfl/fl/Apcfl/+ mice compared with AhCre/Met+/+/Apcfl/+ control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44+/+, Cd44-/-, Cd44s/s, or Cd44v4-10/v4-10 mice). RESULTS: Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in AhCre/Metfl/fl/LacZ mice. Lgr5Creert2/Metfl/fl/LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5Creert2/Metfl/fl/Apcfl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (AhCre/Metfl/fl/Apcfl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44-/- mice did not expand to the same extent as crypts from Cd44+/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4-10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4-10. CONCLUSIONS: In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4-10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors.
Asunto(s)
Adenoma/metabolismo , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Receptores de Hialuranos/metabolismo , Neoplasias Intestinales/metabolismo , Intestinos/enzimología , Proteínas Proto-Oncogénicas c-met/metabolismo , Regeneración , Células Madre/enzimología , Adenoma/genética , Adenoma/patología , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Genotipo , Factor de Crecimiento de Hepatocito/farmacología , Homeostasis , Receptores de Hialuranos/genética , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Intestinos/efectos de los fármacos , Intestinos/patología , Intestinos/efectos de la radiación , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Proteínas Proto-Oncogénicas c-met/genética , Regeneración/efectos de los fármacos , Regeneración/efectos de la radiación , Transducción de Señal , Células Madre/efectos de los fármacos , Células Madre/patología , Células Madre/efectos de la radiación , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Carga TumoralAsunto(s)
Antígeno B7-H1/inmunología , Neoplasias del Sistema Nervioso Central/inmunología , Linfoma de Células B/inmunología , Complejo Mayor de Histocompatibilidad , Proteína 2 Ligando de Muerte Celular Programada 1/inmunología , Neoplasias Testiculares/inmunología , Anciano , Anciano de 80 o más Años , Antígeno B7-H1/genética , Neoplasias del Sistema Nervioso Central/genética , Femenino , Eliminación de Gen , Genes MHC Clase I , Genes MHC Clase II , Humanos , Evasión Inmune , Linfoma de Células B/genética , Masculino , Persona de Mediana Edad , Mutación , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Neoplasias Testiculares/genéticaRESUMEN
Expression of the forkhead transcription factor FOXP1 is essential for early B-cell development, whereas downregulation of FOXP1 at the germinal center (GC) stage is required for GC B-cell function. Aberrantly high FOXP1 expression is frequently observed in diffuse large B-cell lymphoma and mucosa-associated lymphoid tissue lymphoma, being associated with poor prognosis. Here, by gene expression analysis upon ectopic overexpression of FOXP1 in primary human memory B cells (MBCs) and B-cell lines, combined with chromatin immunoprecipitation and sequencing, we established that FOXP1 directly represses expression of PRDM1, IRF4, and XBP1, transcriptional master regulators of plasma cell (PC) differentiation. In accordance, FOXP1 is prominently expressed in primary human naive and MBCs, but expression strongly decreases during PC differentiation. Moreover, as compared with immunoglobulin (Ig) M(+) MBCs, IgG(+) MBCs combine lower expression of FOXP1 with an enhanced intrinsic PC differentiation propensity, and constitutive (over)expression of FOXP1 in B-cell lines and primary human MBCs represses their ability to differentiate into PCs. Taken together, our data indicate that proper control of FOXP1 expression plays a critical role in PC differentiation, whereas aberrant expression of FOXP1 might contribute to lymphomagenesis by blocking this terminal B-cell differentiation.
Asunto(s)
Linfocitos B/citología , Factores de Transcripción Forkhead/metabolismo , Células Plasmáticas/citología , Proteínas Represoras/metabolismo , Linfocitos B/metabolismo , Diferenciación Celular , Línea Celular , Células Cultivadas , Factores de Transcripción Forkhead/genética , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Células Plasmáticas/metabolismo , Proteínas Represoras/genética , Activación Transcripcional , Regulación hacia ArribaRESUMEN
The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 rather than full-length FOXP1, may possess this oncogenic activity. Corroborating those studies, we herein show that activated B cell-like diffuse large B-cell lymphoma cell lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express a small FOXP1 isoform, and that the 5'-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukemia virus- and transposon-mediated insertional mutagenesis screens. By combined mass spectrometry, (quantative) reverse transcription polymerase chain reaction/sequencing, and small interfering ribonucleic acid-mediated gene silencing, we determined that the small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma lacks the N-terminal 100 amino acids of full-length FOXP1. Aberrant overexpression of this FOXP1 isoform (ΔN100) in primary human B cells revealed its oncogenic capacity; it repressed apoptosis and plasma cell differentiation. However, no difference in potency was found between this small FOXP1 isoform and full-length FOXP1. Furthermore, overexpression of full-length FOXP1 or this small FOXP1 isoform in primary B cells and diffuse large B-cell lymphoma cell lines resulted in similar gene regulation. Taken together, our data indicate that this small FOXP1 isoform and full-length FOXP1 have comparable oncogenic and transcriptional activity in human B cells, suggesting that aberrant expression or overexpression of FOXP1, irrespective of the specific isoform, contributes to lymphomagenesis. These novel insights further enhance the value of FOXP1 for the diagnostics, prognostics, and treatment of diffuse large B-cell lymphoma patients.
Asunto(s)
Linfocitos B/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Activación Transcripcional , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Ciclo Celular/genética , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/química , Humanos , Memoria Inmunológica , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Mutagénesis Insercional , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Isoformas de Proteínas , Proteínas Represoras/químicaRESUMEN
The forkhead transcription factor FOXP1 is involved in B-cell development and function and is generally regarded as an oncogene in activated B-cell-like subtype of diffuse large B-cell lymphoma (DLBCL) and mucosa-associated lymphoid tissue lymphoma, lymphomas relying on constitutive nuclear factor κB (NF-κB) activity for survival. However, the mechanism underlying its putative oncogenic activity has not been established. By gene expression microarray, upon overexpression or silencing of FOXP1 in primary human B cells and DLBCL cell lines, combined with chromatin immunoprecipitation followed by next-generation sequencing, we established that FOXP1 directly represses a set of 7 proapoptotic genes. Low expression of these genes, encoding the BH3-only proteins BIK and Harakiri, the p53-regulatory proteins TP63, RASSF6, and TP53INP1, and AIM2 and EAF2, is associated with poor survival in DLBCL patients. In line with these findings, we demonstrated that FOXP1 promotes the expansion of primary mature human B cells by inhibiting caspase-dependent apoptosis, without affecting B-cell proliferation. Furthermore, FOXP1 is dependent upon, and cooperates with, NF-κB signaling to promote B-cell expansion and survival. Taken together, our data indicate that, through direct repression of proapoptotic genes, (aberrant) expression of FOXP1 complements (constitutive) NF-κB activity to promote B-cell survival and can thereby contribute to B-cell homeostasis and lymphomagenesis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Linfocitos B/fisiología , Factores de Transcripción Forkhead/fisiología , Regulación Leucémica de la Expresión Génica , FN-kappa B/fisiología , Proteínas Represoras/fisiología , Transcripción Genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Células Cultivadas , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Análisis por Micromatrices , Regulación hacia ArribaAsunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Resistencia a Antineoplásicos , Gefitinib/farmacología , Mucosa Intestinal/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genes APC , Humanos , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Mutación , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/genética , Pirazinas/farmacología , Transducción de Señal , Triazinas/farmacologíaRESUMEN
Ibrutinib (PCI-32765) is a highly potent oral Bruton tyrosine kinase (BTK) inhibitor in clinical development for treating B-cell lymphoproliferative diseases. Patients with chronic lymphocytic leukemia (CLL) often show marked, transient increases of circulating CLL cells following ibrutinib treatments, as seen with other inhibitors of the B-cell receptor (BCR) pathway. In a phase 1 study of ibrutinib, we noted similar effects in patients with mantle cell lymphoma (MCL). Here, we characterize the patterns and phenotypes of cells mobilized among patients with MCL and further investigate the mechanism of this effect. Peripheral blood CD19(+)CD5(+) cells from MCL patients were found to have significant reduction in the expression of CXCR4, CD38, and Ki67 after 7 days of treatment. In addition, plasma chemokines such as CCL22, CCL4, and CXCL13 were reduced 40% to 60% after treatment. Mechanistically, ibrutinib inhibited BCR- and chemokine-mediated adhesion and chemotaxis of MCL cell lines and dose-dependently inhibited BCR, stromal cell, and CXCL12/CXCL13 stimulations of pBTK, pPLCγ2, pERK, or pAKT. Importantly, ibrutinib inhibited migration of MCL cells beneath stromal cells in coculture. We propose that BTK is essential for the homing of MCL cells into lymphoid tissues, and its inhibition results in an egress of malignant cells into peripheral blood. This trial was registered at www.clinicaltrials.gov as #NCT00114738.
Asunto(s)
Antineoplásicos/uso terapéutico , Linfocitos B/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Linfoma de Células del Manto/sangre , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Adenina/análogos & derivados , Antígenos CD19/biosíntesis , Linfocitos B/metabolismo , Western Blotting , Antígenos CD5/biosíntesis , Adhesión Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Linfoma de Células del Manto/tratamiento farmacológico , Piperidinas , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Small-molecule drugs that target the B-cell antigen receptor (BCR) signalosome show clinical efficacy in the treatment of B-cell non-Hodgkin lymphoma. These agents, including the Bruton tyrosine kinase (BTK) inhibitor PCI-32765, display an unexpected response in patients with chronic lymphocytic leukemia (CLL): a rapid and sustained reduction of lymphadenopathy accompanied by transient lymphocytosis, which is reversible upon temporary drug deprivation. We hypothesized that this clinical response reflects impaired integrin-mediated adhesion and/or migration. Here, we show that PCI-32765 strongly inhibits BCR-controlled signaling and integrin α(4)ß(1)-mediated adhesion to fibronectin and VCAM-1 of lymphoma cell lines and primary CLL cells. Furthermore, PCI-32765 also inhibits CXCL12-, CXCL13-, and CCL19-induced signaling, adhesion, and migration of primary CLL cells. Our data indicate that inhibition of BTK by PCI-32765 overcomes BCR- and chemokine-controlled integrin-mediated retention and homing of malignant B cells in their growth- and survival-supporting lymph node and bone marrow microenvironment, which results in clinically evident CLL regression.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocinas/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Receptores de Antígenos de Linfocitos B/metabolismo , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Fibronectinas/metabolismo , Citometría de Flujo , Humanos , Integrina alfa4beta1/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/metabolismo , Piperidinas , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Although Bruton's tyrosine kinase (BTK) inhibitors (BTKi) have significantly improved patient prognosis, mantle cell lymphoma (MCL) is still considered incurable due to primary and acquired resistance. We have recently shown that aberrant expression of the Src-family tyrosine kinase hematopoietic cell kinase (HCK) in MCL correlates with poor prognosis, and that genetic HCK perturbation impairs growth and integrin-mediated adhesion of MCL cells. Here, we show that KIN-8194, a dual inhibitor of BTK and HCK with in vivo activity against Myd88-L265P-driven diffuse large B-cell lymphoma and Waldenström Macroglobulinemia, has a potent growth inhibitory effect in MCL cell lines and primary MCL cells, irrespective of their sensitivity to BTKi (ibrutinib and acalabrutinib). In BTKi-resistant cells this is mediated by inhibition of HCK, which results in repression of AKT-S6 signaling. In addition, KIN-8194 inhibits integrin-mediated adhesion of BTKi-sensitive and insensitive MCL cells to fibronectin and stromal cells in an HCK-dependent manner. Finally, we show that MCL cells with acquired BTKi resistance retain their sensitivity to KIN-8194. Taken together, our data demonstrate that KIN-8194 inhibits growth and integrin-mediated adhesion of BTKi-sensitive MCL cells, as well as MCL cells with primary or acquired BTKi resistance. This renders KIN-8194 a promising novel treatment for MCL patients.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa , Adhesión Celular , Proliferación Celular , Resistencia a Antineoplásicos , Integrinas , Linfoma de Células del Manto , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-hck , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/patología , Linfoma de Células del Manto/metabolismo , Linfoma de Células del Manto/genética , Humanos , Adhesión Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-hck/metabolismo , Proteínas Proto-Oncogénicas c-hck/genética , Proteínas Proto-Oncogénicas c-hck/antagonistas & inhibidores , Integrinas/metabolismo , Proliferación Celular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Pirimidinas/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
The development and antigen-dependent differentiation of B lymphocytes are orchestrated by an array of growth factors, cytokines, and chemokines that require tight spatiotemporal regulation. Heparan sulfate proteoglycans specifically bind and regulate the bioavailability of soluble protein ligands, but their role in the immune system has remained largely unexplored. Modification of heparan sulfate by glucuronyl C5-epimerase (Glce) controls heparan sulfate-chain flexibility and thereby affects ligand binding. Here we show that Glce deficiency impairs B-cell maturation, resulting in decreased plasma cell numbers and immunoglobulin levels. We demonstrate that C5-epimerase modification of heparan sulfate is critical for binding of a proliferation inducing ligand (APRIL) and that Glce-deficient plasma cells fail to respond to APRIL-mediated survival signals. Our results identify heparan sulfate proteoglycans as novel players in B-cell maturation and differentiation and suggest that heparan sulfate conformation is crucial for recruitment of factors that control plasma cell survival.