RESUMEN
B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), remain incurable, with MM particularly prone to relapse. Our study introduces a novel mouse model with active RANK signaling and the TCL1 oncogene, displaying both CLL and MM phenotypes. In younger mice, TCL1 and RANK expression expands CLL-like B1-lymphocytes, while MM originates from B2-cells, becoming predominant in later stages and leading to severe disease progression and mortality. The induced MM mimics human disease, exhibiting features like clonal plasma cell expansion, paraproteinemia, anemia, and kidney and bone failure, as well as critical immunosurveillance strategies that promote a tumor-supportive microenvironment. This research elucidates the differential impacts of RANK activation in B1- and B2-cells and underscores the distinct roles of single versus combined oncogenes in B-cell malignancies. We also demonstrate that human MM cells express RANK and that inhibiting RANK signaling can reduce MM progression in a xenotransplantation model. Our study provides a rationale for further investigating the effects of RANK signaling in B-cell transformation and the shaping of a tumor-promoting microenvironment.
Asunto(s)
Linfocitos B , Transformación Celular Neoplásica , Mieloma Múltiple , Proteínas Proto-Oncogénicas , Transducción de Señal , Animales , Humanos , Ratones , Linfocitos B/metabolismo , Linfocitos B/inmunología , Linaje de la Célula , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Microambiente TumoralRESUMEN
Nuclear factor kappa B (NFκB) is a pathogenic factor in chronic lymphocytic leukemia (CLL) that is not addressed specifically by current therapies. NFκB is activated by inflammatory factors that stimulate toll-like receptors (TLRs) and receptors for interleukin-1 (IL-1) family members. IL-1 is considered a master regulator of inflammation, and IL-1 receptor signaling is inhibited by the IL-1 receptor antagonist anakinra. These considerations suggested that anakinra might have a role in the treatment of CLL. Consistent with this idea, anakinra inhibited spontaneous and TLR7-mediated activation of the canonical NFκB pathway in CLL cells in vitro. However, CLL cells exhibited only weak signaling responses to IL-1 itself, and anakinra was found to inhibit NFκB along with oxidative stress in an IL-1 receptor-independent manner. Anakinra was then administered with minimal toxicity to 11 previously untreated CLL patients in a phase I dose-escalation trial (NCT04691765). A stereotyped clinical response was observed in all patients. Anakinra lowered blood lymphocytes and lymph node sizes within the first month that were associated with downregulation of NFκB and oxidative stress in the leukemia cells. However, inhibition of NFκB was accompanied by upregulation of type 1 interferon (IFN) signaling, c-MYC-regulated genes and proteins, and loss of the initial clinical response. Anakinra increased IFN signaling and survival of CLL cells in vitro that were, respectively, phenocopied by mitochondrial antioxidants and reversed by IFN receptor blocking antibodies. These observations suggest that anakinra has activity in CLL and may be a useful adjunct for conventional therapies as long as compensatory IFN signaling is blocked at the same time.
Asunto(s)
Proteína Antagonista del Receptor de Interleucina 1 , Leucemia Linfocítica Crónica de Células B , FN-kappa B , Transducción de Señal , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Interferones/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-1/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 7/antagonistas & inhibidoresRESUMEN
ABSTRACT: Autologous T-cell-based therapies, such as chimeric antigen receptor (CAR) T-cell therapy, exhibit low success rates in chronic lymphocytic leukemia (CLL) and correlate with a dysfunctional T-cell phenotype observed in patients. Despite various proposed mechanisms of T-cell dysfunction in CLL, the specific CLL-derived factors responsible remain unidentified. This study aimed to investigate the mechanisms through which CLL cells suppress CAR T-cell activation and function. We found that CLL-derived T cells get activated, albeit in a delayed fashion, and specifically that restimulation of CAR T cells in the presence of CLL cells causes impaired cytokine production and reduced proliferation. Notably, coculture of T cells with CD40-activated CLL cells did not lead to T-cell dysfunction, and this required direct cell contact between the CD40-stimulated CLL cells and T cells. Inhibition of kinases involved in the CD40 signaling cascade revealed that the Spare Respiratory Capacity (SRC) kinase inhibitor dasatinib prevented rescue of T-cell function independent of CD40-mediated increased levels of costimulatory and adhesion ligands on CLL cells. Transcriptome profiling of CD40-stimulated CLL cells with or without dasatinib identified widespread differential gene expression. Selecting for surface receptor genes revealed CD40-mediated downregulation of the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52, which was prevented by dasatinib, suggesting a role for these ligands in functional T-cell suppression in CLL. Indeed, blocking CD24 and/or CD52 markedly reduced CAR T-cell dysfunction upon coculture with resting CLL cells. These results demonstrated that T cells derived from CLL patients can be reinvigorated by manipulating CLL-T-cell interactions. Targeting CD24- and CD52-mediated CLL-T-cell interaction could be a promising therapeutic strategy to enhance T-cell function in CLL.
Asunto(s)
Antígeno CD24 , Antígeno CD52 , Leucemia Linfocítica Crónica de Células B , Linfocitos T , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/terapia , Antígeno CD52/metabolismo , Linfocitos T/metabolismo , Linfocitos T/inmunología , Antígeno CD24/metabolismo , Activación de Linfocitos/inmunología , Ligandos , Receptores Quiméricos de Antígenos/metabolismoRESUMEN
ABSTRACT: Covalent Bruton tyrosine kinase inhibitors (cBTKis), which bind to the BTK C481 residue, are now primary therapeutics for chronic lymphocytic leukemia (CLL). Alterations at C481, primarily C481S, prevent cBTKi binding and lead to the emergence of resistant clones. Pirtobrutinib is a noncovalent BTKi that binds to both wild-type (WT) and C481S-mutated BTK and has shown efficacy in BTK-WT and -mutated CLL patient groups. To compare baseline clinical, transcriptomic, and proteomic characteristics and their changes during treatment in these 2 groups, we used 67 longitudinal peripheral blood samples obtained during the first 3 cycles of treatment with pirtobrutinib from 18 patients with CLL (11 BTK-mutated, 7 BTK-WT) enrolled in the BRUIN (pirtobrutinib in relapsed or refractory B-cell malignancies) trial. Eastern Cooperative Oncology Group performance status, age, and Rai stage were similar in both groups. At baseline, lymph nodes were larger in the BTK-mutated cohort. All patients achieved partial remission within 4 cycles of pirtobrutinib. Lactate dehydrogenase and ß2-microglobulin levels decreased in both cohorts after 1 treatment cycle. Expression analysis demonstrated upregulation of 35 genes and downregulation of 6 in the BTK-mutated group. Gene set enrichment analysis revealed that the primary pathways enriched in BTK-mutated cells were involved in cell proliferation, metabolism, and stress response. Pathways associated with metabolism and proliferation were downregulated in both groups during pirtobrutinib treatment. Proteomic data corroborated transcriptomic findings. Our data identified inherent differences between BTK-mutated and -WT CLL and demonstrated molecular normalization of plasma and omics parameters with pirtobrutinib treatment in both groups.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa , Leucemia Linfocítica Crónica de Células B , Mutación , Piperidinas , Inhibidores de Proteínas Quinasas , Proteómica , Pirimidinas , Transcriptoma , Humanos , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/metabolismo , Agammaglobulinemia Tirosina Quinasa/genética , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Persona de Mediana Edad , Pirimidinas/uso terapéutico , Pirimidinas/farmacología , Proteómica/métodos , Femenino , Masculino , Anciano , Piperidinas/uso terapéutico , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Proteoma , Adenina/análogos & derivados , Adenina/uso terapéutico , Pirazoles/uso terapéutico , Pirazoles/farmacología , Anciano de 80 o más AñosRESUMEN
Morphologic features of aggressive/ "accelerated" chronic lymphocytic leukemia/small lymphocytic lymphoma (aCLL/SLL) have been described. Richter transformation (RT) also occurs in a subset of CLL/SLL cases. This case series examined inter-observer variability when assessing for aCLL/SLL and RT, with attention to how immunohistochemical (IHC) markers may assist in this evaluation. Twelve cases of CLL/SLL with available FFPE tissue were identified. H&E staining and IHC (CD3, CD20, CD5, CD23, LEF1, LAG3, C-MYC, PD-1, MUM1, Cyclin D1, BCL-6, p53, and Ki-67) were performed. Three hematopathologists reviewed each case. The pathologists provided a final interpretation of (1) CLL/SLL, (2) CLL/SLL with expanded and/or confluent proliferation centers or increased Ki-67 (aCLL/SLL), or (3) large cell transformation/DLBCL. The pathologists lacked consensus in the diagnosis in 6/12 cases (50%). The reviewers disagreed on the presence of expanded/confluent proliferation centers in 8/12 cases (67%). With the exception of Ki-67, no IHC marker showed a difference in the staining profile in aCLL/SLL or RT compared to low-grade cases. This series showed inter-observer variability in the evaluation for aCLL/SLL and RT. A study that serially examines genetic alterations in FFPE tissue and correlates the features with histology and IHC, at diagnosis and throughout the disease course, may help refine indicators of aggressive disease.
Asunto(s)
Inmunohistoquímica , Leucemia Linfocítica Crónica de Células B , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/diagnóstico , Masculino , Femenino , Anciano , Persona de Mediana Edad , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Transformación Celular Neoplásica/patología , Anciano de 80 o más Años , Variaciones Dependientes del ObservadorRESUMEN
PURPOSE: Pirtobrutinib, a non-covalent Bruton's tyrosine kinase (BTK) inhibitor, has been approved as the first agent to overcome resistance to covalent BTK inhibitors (such as ibrutinib, acalabrutinib, and zanubrutinib). However, the mechanisms of pirtobrutinib resistance in chronic lymphocytic leukemia (CLL) remain poorly understood. METHODS: To investigate pirtobrutinib resistance, we established resistant cell models using BTK knock-out via CRISPR-Cas9 or chronic exposure to pirtobrutinib in MEC-1 cells. These models mimicked intrinsic or acquired resistance, respectively. We then analyzed differential protein expression between wild-type (WT) and resistant MEC-1 cells using Revers Phase Protein microArray (RPPA) and confirmed the findings through Western Blot. Additionally, we evaluated potential drugs to overcome pirtobrutinib resistance by conducting cell proliferation assays, apoptosis studies, and animal experiments using both sensitive and resistant cells. RESULTS: MEC-1 cells developed resistance to pirtobrutinib either through BTK knock-out or prolonged drug exposure over three months. RPPA analysis revealed significant activation of proteins related to the PI3K/AKT pathway, including AKT and S6, in the resistant cells. Western Blot confirmed increased phosphorylation of AKT and S6 in pirtobrutinib-resistant MEC-1 cells. Notably, both the PI3K inhibitor (CAL101) and the AKT inhibitor (MK2206) effectively reduced cell proliferation and induced apoptosis in the resistant cells. The anti-tumor efficacy of these drugs was mediated by inhibiting the PI3K/AKT pathway. In vivo animal studies further supported the potential of targeting PI3K/AKT to overcome both intrinsic and acquired resistance to pirtobrutinib. CONCLUSION: The PI3K/AKT pathway plays a crucial role in both intrinsic and acquired resistance to pirtobrutinib in CLL. Therapeutically targeting this pathway may offer a promising strategy to overcome pirtobrutinib resistance.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa , Resistencia a Antineoplásicos , Leucemia Linfocítica Crónica de Células B , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Pirimidinas , Humanos , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/metabolismo , Pirimidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Piperidinas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Adenina/análogos & derivados , Adenina/farmacología , Transducción de Señal/efectos de los fármacos , Pirazoles/farmacologíaRESUMEN
Chronic lymphocytic leukemia (CLL) is the most common leukemia in western societies, recognized by clinical and molecular heterogeneity. Despite the success of targeted therapies, acquired resistance remains a challenge for relapsed and refractory CLL, as a consequence of mutations in the target or the upregulation of other survival pathways leading to the progression of the disease. Research on proteins that can trigger such pathways may define novel therapies for a successful outcome in CLL such as the receptor tyrosine kinase-like orphan receptor 1 (ROR1). ROR1 is a signaling receptor for Wnt5a, with an important role during embryogenesis. The aberrant expression on CLL cells and several types of tumors, is involved in cell proliferation, survival, migration as well as drug resistance. Antibody-based immunotherapies and small-molecule compounds emerged to target ROR1 in preclinical and clinical studies. Efforts have been made to identify new prognostic markers having predictive value to refine and increase the detection and management of CLL. ROR1 can be considered as an attractive target for CLL diagnosis, prognosis, and treatment. It can be clinically effective alone and/or in combination with current approved agents. In this review, we summarize the scientific achievements in targeting ROR1 for CLL diagnosis, prognosis, and treatment.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Humanos , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Pronóstico , Terapia Molecular Dirigida , Animales , Biomarcadores de Tumor/metabolismoRESUMEN
Elevated levels of circulating C16:0 glucosylceramides (GluCer) and increased mRNA expression of UDP-glucose ceramide glycosyltransferase (UGCG), the enzyme responsible for converting ceramides (Cer) to GluCer, represent unfavorable prognostic markers in chronic lymphocytic leukemia (CLL) patients. To evaluate the therapeutic potential of inhibiting GluCer synthesis, we genetically repressed the UGCG pathway using in vitro models of leukemic B cells, in addition to UGCG pharmacological inhibition with approved drugs such as eliglustat and ibiglustat, both individually and in combination with ibrutinib, assessed in cell models and primary CLL patient cells. Cell viability, apoptosis, and proliferation were evaluated in vitro, and survival and apoptosis were examined ex vivo. UGCG inhibition efficacy was confirmed by quantifying intracellular sphingolipid levels through targeted lipidomics using mass spectrometry. Other inhibitors of sphingolipid biosynthesis pathways were similarly assessed. Blocking UGCG significantly decreased cell viability and proliferation, highlighting the oncogenic role of UGCG in CLL. The efficient inhibition of UGCG was confirmed by a significant reduction in GluCer intracellular levels. The combination of UGCG inhibitors with ibrutinib demonstrated synergistic effect. Inhibitors that target alternative pathways within sphingolipid metabolism, like sphingosine kinases inhibitor SKI-II, also demonstrated promising therapeutic effects both alone and when used in combination with ibrutinib, reinforcing the oncogenic impact of sphingolipids in CLL cells. Targeting sphingolipid metabolism, especially the UGCG pathway, represents a promising therapeutic strategy and as a combination therapy for potential treatment of CLL patients, warranting further investigation.
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Supervivencia Celular , Leucemia Linfocítica Crónica de Células B , Esfingolípidos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Humanos , Esfingolípidos/metabolismo , Supervivencia Celular/efectos de los fármacos , Glucosiltransferasas/antagonistas & inhibidores , Glucosiltransferasas/metabolismo , Glucosiltransferasas/genética , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Piperidinas/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Glucosilceramidas/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacologíaRESUMEN
Richter's transformation (RT) is a progression of chronic lymphocytic leukemia (CLL) to aggressive lymphoma. MGA (Max gene associated), a functional MYC suppressor, is mutated at 3% in CLL and 36% in RT. However, genetic models and molecular mechanisms of MGA deletion that drive CLL to RT remain elusive. We established an RT mouse model by knockout of Mga in the Sf3b1/Mdr CLL model using CRISPR-Cas9 to determine the role of Mga in RT. Murine RT cells exhibited mitochondrial aberrations with elevated oxidative phosphorylation (OXPHOS). Through RNA sequencing and functional characterization, we identified Nme1 (nucleoside diphosphate kinase) as an Mga target, which drives RT by modulating OXPHOS. Given that NME1 is also a known MYC target without targetable compounds, we found that concurrent inhibition of MYC and electron transport chain complex II substantially prolongs the survival of RT mice in vivo. Our results suggest that the Mga-Nme1 axis drives murine CLL-to-RT transition via modulating OXPHOS, highlighting a potential therapeutic avenue for RT.
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Leucemia Linfocítica Crónica de Células B , Mitocondrias , Fosforilación Oxidativa , Animales , Mitocondrias/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/metabolismo , Ratones , Eliminación de Gen , Humanos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Modelos Animales de EnfermedadRESUMEN
The resistance of Chronic Lymphocytic Leukemia (CLL) B-cells to cell death is mainly attributed to interactions within their microenvironment, where they interact with various types of cells. Within this microenvironment, CLL-B-cells produce and bind cytokines, growth factors, and extracellular vesicles (EVs). In the present study, EVs purified from nurse-like cells and M2-polarized THP1 cell (M2-THP1) cultures were added to CLL-B-cells cultures. EVs were rapidly internalized by B-cells, leading to a decrease in apoptosis (P = 0.0162 and 0.0469, respectively) and an increased proliferation (P = 0.0335 and 0.0109). Additionally, they induced an increase in the resistance of CLL-B-cells to Ibrutinib, the Bruton kinase inhibitor in vitro (P = 0.0344). A transcriptomic analysis showed an increase in the expression of anti-apoptotic gene BCL-2 (P = 0.0286) but not MCL-1 and an increase in the expression of proliferation-inducing gene APRIL (P = 0.0286) following treatment with EVs. Meanwhile, an analysis of apoptotic protein markers revealed increased amounts of IGFBP-2 (P = 0.0338), CD40 (P = 0.0338), p53 (P = 0.0219) and BCL-2 (P = 0.0338). Finally, exploration of EVs protein content by mass spectrometry revealed they carry various proteins involved in known oncogenic pathways and the RNAseq analysis of CLL-B-cells treated or not with NLCs EVs show various differentially expressed genes.
Asunto(s)
Vesículas Extracelulares , Leucemia Linfocítica Crónica de Células B , Macrófagos , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/genética , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Apoptosis , Supervivencia Celular , Proliferación CelularRESUMEN
CD180 is a toll-like receptor that is highly expressed in complex with the MD-1 satellite molecule on the surface of B cells. In chronic lymphocytic leukaemia (CLL) however, the expression of CD180 is highly variable and overall, significantly reduced when compared to normal B cells. We have recently shown that reduced CD180 expression in CLL lymph nodes is associated with inferior overall survival. It was therefore important to better understand the causes of this downregulation through investigation of CD180 at the transcriptional and protein expression levels. Unexpectedly, we found CD180 RNA levels in CLL cells (n = 26) were comparable to those of normal B cells (n = 13), despite heterogeneously low expression of CD180 on the cell surface. We confirmed that CD180 RNA is translated into CD180 protein since cell surface CD180-negative cases presented with high levels of intracellular CD180 expression. Levels of MD-1 RNA were, however, significantly downregulated in CLL compared to normal controls. Together, these data suggest that changes in CD180 cell surface expression in CLL are not due to transcriptional downregulation, but defective post-translational stabilisation of the receptor due to MD-1 downregulation.
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Antígenos CD , Regulación hacia Abajo , Leucemia Linfocítica Crónica de Células B , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/inmunología , Antígenos CD/metabolismo , Antígenos CD/genética , Membrana Celular/metabolismo , Regulación Leucémica de la Expresión GénicaRESUMEN
BACKGROUND: Occurrence of squamous cell carcinoma (SCC) even in early-stage, untreated chronic lymphocytic leukemia (CLL) patients can be a significant morbidity issue with occasional transformation into metastatic skin lesions. METHODS: CLL cells and extracellular vesicles (EVs) from CLL patients' blood/plasma were purified and used. Expression/activation of AXL and its functions in normal keratinocytes (HEKa) were assessed in vitro co-culture system and in SCC tissues. RESULTS: We detected aberrant activation of AXL, AKT and ERK-1/2 in SCC cell lines compared to HEKa. We also detected increased expression of AXL in primary SCC tissues obtained from CLL patients. Increased activation of AXL, AKT, ERK-1/2 and Src was discernible in HEKa upon co-culturing with CLL cells. Further analysis suggests that Gas6, a ligand of AXL, regulates AXL activation in co-cultured HEKa. Interestingly, exposure of HEKa cells to CLL plasma-derived EVs induced expression of AXL, P-AKT, and EMT-associated markers leading to migration of the cells. Finally, pharmacologic inhibition of AXL induced cell death in SCC lines in a dose dependent manner. CONCLUSIONS: Our findings that CLL cells likely are involved in driving SCC progression, at least in part, via activation of the AXL signaling axis, indicating that AXL inhibition may be beneficial for our CLL patients with SCC.
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Tirosina Quinasa del Receptor Axl , Carcinoma de Células Escamosas , Progresión de la Enfermedad , Vesículas Extracelulares , Leucemia Linfocítica Crónica de Células B , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras , Humanos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Proteínas Proto-Oncogénicas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Vesículas Extracelulares/metabolismo , Queratinocitos/metabolismo , Queratinocitos/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/genéticaRESUMEN
Introduction: Therapeutic antibodies have become a major strategy to treat oncologic diseases. For chronic lymphocytic leukemia, antibodies against CD20 are used to target and elicit cytotoxic responses against malignant B cells. However, efficacy is often compromised due to a suppressive microenvironment that interferes with cellular immune responses. To overcome this suppression, agonists of pattern recognition receptors have been studied which promote direct cytotoxicity or elicit anti-tumoral immune responses. NOD2 is an intracellular pattern recognition receptor that participates in the detection of peptidoglycan, a key component of bacterial cell walls. This detection then mediates the activation of multiple signaling pathways in myeloid cells. Although several NOD2 agonists are being used worldwide, the potential benefit of these agents in the context of antibody therapy has not been explored. Methods: Primary cells from healthy-donor volunteers (PBMCs, monocytes) or CLL patients (monocytes) were treated with versus without the NOD2 agonist L18-MDP, then antibody-mediated responses were assessed. In vivo, the Eµ-TCL1 mouse model of CLL was used to test the effects of L18-MDP treatment alone and in combination with anti-CD20 antibody. Results: Treatment of peripheral blood mononuclear cells with L18-MDP led to activation of monocytes from both healthy donors and CLL patients. In addition, there was an upregulation of activating FcγR in monocytes and a subsequent increase in antibody-mediated phagocytosis. This effect required the NF-κB and p38 signaling pathways. Treatment with L18-MDP plus anti-CD20 antibody in the Eµ-TCL model of CLL led to a significant reduction of CLL load, as well as to phenotypic changes in splenic monocytes and macrophages. Conclusions: Taken together, these results suggest that NOD2 agonists help overturn the suppression of myeloid cells, and may improve the efficacy of antibody therapy for CLL.
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Leucemia Linfocítica Crónica de Células B , Macrófagos , Proteína Adaptadora de Señalización NOD2 , Receptores de IgG , Proteína Adaptadora de Señalización NOD2/agonistas , Proteína Adaptadora de Señalización NOD2/metabolismo , Proteína Adaptadora de Señalización NOD2/inmunología , Animales , Humanos , Receptores de IgG/metabolismo , Receptores de IgG/inmunología , Ratones , Macrófagos/inmunología , Macrófagos/metabolismo , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacología , Femenino , Ratones Endogámicos C57BL , Transducción de Señal , Fagocitosis , Rituximab/farmacología , Rituximab/uso terapéuticoRESUMEN
Venetoclax and obinutuzumab are becoming frontline therapies for chronic lymphocytic leukemia (CLL) patients. Unfortunately, drug resistance still occurs, and the combination could be immunosuppressive. Lysosomes have previously been identified as a target for obinutuzumab cytotoxicity in CLL cells, but the mechanism remains unclear. In addition, studies have shown that lysosomotropic agents can cause synergistic cell death in vitro when combined with the BTK inhibitor, ibrutinib, in primary CLL cells. This indicates that targeting lysosomes could be a treatment strategy for CLL. In this study, we have shown that obinutuzumab induces lysosome membrane permeabilization (LMP) and cathepsin D release in CLL cells. Inhibition of cathepsins reduced obinutuzumab-induced cell death in CLL cells. We further determined that the lysosomotropic agent siramesine in combination with venetoclax increased cell death in primary CLL cells through an increase in reactive oxygen species (ROS) and cathepsin release. Siramesine treatment also induced synergistic cytotoxicity when combined with venetoclax. Microenvironmental factors IL4 and CD40L or incubation with HS-5 stromal cells failed to significantly protect CLL cells from siramesine- and venetoclax-induced apoptosis. We also found that siramesine treatment inhibited autophagy through reduced autolysosomes. Finally, the autophagy inhibitor chloroquine failed to further increase siramesine-induced cell death. Taken together, lysosome-targeting drugs could be an effective strategy in combination with venetoclax to overcome drug resistance in CLL.
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Apoptosis , Autofagia , Compuestos Bicíclicos Heterocíclicos con Puentes , Catepsina D , Leucemia Linfocítica Crónica de Células B , Lisosomas , Sulfonamidas , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Sulfonamidas/farmacología , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Catepsina D/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sinergismo Farmacológico , Línea Celular TumoralRESUMEN
Siglec-6 is a lectin receptor with restricted expression in the placenta, mast cells and memory B-cells. Although Siglec-6 is expressed in patients with chronic lymphocytic leukemia (CLL), its pathophysiological role has not been elucidated. We describe here a role for Siglec-6 in migration and adhesion of CLL B cells to CLL- bone marrow stromal cells (BMSCs) in vitro and compromised migration to bone marrow and spleen in vivo. Mass spectrometry analysis revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of MEC1-002 CLL cells with a Siglec-6 ligand, sTn, results in Cdc42 activation, WASP protein recruitment and F-actin polymerization, which are all associated with cell migration. Therapeutically, a Siglec-6/CD3-bispecific T-cell-recruiting antibody (T-biAb) improves overall survival in an immunocompetent mouse model and eliminates CLL cells in a patient derived xenograft model. Our findings thus reveal a migratory role for Siglec-6 in CLL, which can be therapeutically targeted using a Siglec-6 specific T-biAb.
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Adhesión Celular , Movimiento Celular , Lectinas , Leucemia Linfocítica Crónica de Células B , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Humanos , Animales , Lectinas/metabolismo , Ratones , Antígenos CD/metabolismo , Antígenos CD/genética , Femenino , Linfocitos B/metabolismo , Linfocitos B/inmunología , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Línea Celular Tumoral , Células Madre Mesenquimatosas/metabolismo , Masculino , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Eµ-TCL1 mouse model has been used for over 20 years to study the pathobiology of chronic lymphocytic leukemia (CLL) and for preclinical testing of novel therapies. A CLL-like disease develops with increasing age in these mice due to a B cell specific overexpression of human TCL1. The reliability of this model to mirror human CLL is controversially discussed, as none of the known driver mutations identified in patients are found in Eµ-TCL1 mice. It has to be acknowledged that this mouse model was key to develop targeted therapies that aim at inhibiting the constitutive B cell receptor (BCR) signaling, a main driver of CLL. Inhibitors of BCR signaling became standard-of-care for a large proportion of patients with CLL as they are highly effective. The Eµ-TCL1 model further advanced our understanding of CLL biology owed to studies that crossed this mouse line with various transgenic mouse models and demonstrated the relevance of CLL-cell intrinsic and -extrinsic drivers of disease. These studies were instrumental in showing the relevance of the tumor microenvironment in the lymphoid tissues for disease progression and immune escape in CLL. It became clear that CLL cells shape and rely on stromal and immune cells, and that immune suppressive mechanisms and T cell exhaustion contribute to CLL progression. Based on this knowledge, new immunotherapy strategies were clinically tested for CLL, but so far with disappointing results. As some of these therapies were effective in the Eµ-TCL1 mouse model, the question arose concerning the translatability of preclinical studies in these mice. The aim of this review is to summarize lessons we have learnt over the last decades by studying CLL-like disease in the Eµ-TCL1 mouse model. The article focuses on pitfalls and limitations of the model, as well as the gained knowledge and potential of using this model for the development of novel treatment strategies to achieve the goal of curing patients with CLL.
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Modelos Animales de Enfermedad , Leucemia Linfocítica Crónica de Células B , Ratones Transgénicos , Proteínas Proto-Oncogénicas , Animales , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Ratones , Humanos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Microambiente Tumoral/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
BACKGROUND AND AIMS: Modern mass spectrometry imaging (MSI) enables single cells' metabolism exploration. Aims of this study were development of the single-cell MSI of human CD19+ lymphocytes and metabolic profiling of chronic lymphocytic leukemia (CLL). MATERIALS AND METHODS: Blood donor (BD) samples were used for the optimization of CD19+ lymphocyte isolation and single-cell matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) MSI. Independent set of 200 CD19+ lymphocytes coming from 5 CLL patients and 5 BD was used for the CD19+ lymphocytes classification assessment and the untargeted metabolic profiling. CLL vs BD lymphocyte classification was performed using partial least squares-discriminant analysis (PLS-DA) using normalized single-cell mass spectra recorded in 300-600 and 600-950 Da ranges was applied. RESULTS: Accuracy assessed by 10-fold cross-validation of CD19+ lymphocyte PLS-DA classification reached >90.0 %. Volcano plots showed 106 significantly altered m/z signals in CLL of which 9 were tentatively annotated. Among tentatively annotated m/z signals formaldehyde and glutathione metabolites and tetrahydrofolate stand out. CONCLUSION: A method for single-cell MALDI TOF MSI of CD19+ lymphocytes was successfully developed. The method confirmed the significance of oxidative stress and single-carbon metabolism, pyruvate and fatty acid metabolism and apoptosis in CLL and it provided metabolic candidates for diagnostic applications.
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Leucemia Linfocítica Crónica de Células B , Análisis de la Célula Individual , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Humanos , Antígenos CD19/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/patología , Linfocitos/metabolismo , Metabolómica/métodosRESUMEN
Chronic inflammation has been identified in leukemias as an essential regulator of angiogenesis. B-chronic lymphocytic leukemia (CLL) cells secrete high levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 alpha (HIF1α). The aim was to assess the role of inflammation in activation of angiogenic factors: endothelial nitric oxide synthase (eNOS), HIF1α and VEGF via proliferation related signaling pathways and VEGF autocrine control. We isolated mononuclear cells (MNC) and CD19+ cells from peripheral blood of 60 patients with CLL. MNC were treated with pro-inflammatory interleukin-6 (IL-6) and VEGF, in combination with inhibitors of JAK1/2 (Ruxolitinib), mTOR (Rapamycin), NF-κB (JSH23), SMAD (LDN-193189) and PI3K/AKT (Ly294002) signaling pathways, to evaluate eNOS, VEGF and HIF1α expression by immunoblotting, immunocytochemistry and RT-qPCR. Also, we investigated IL-6 dependent neovascularization in human microvascular endothelial cells (HMEC-1) in co-culture with MNC of CLL. The angiogenic factors eNOS, VEGF and HIF1α had significantly higher frequencies in MNC of CLL in comparison to healthy controls (p < 0.001) and CD19+ cells of CLL. IL-6 increased the quantity of HIF1α (p < 0.05) and VEGF positive cells in the presence of JSH23 (p < 0.01). VEGF increased HIF1α (p < 0.05), and decreased eNOS gene expression (p < 0.01) in MNC of CLL. VEGF significantly (p < 0.001) increased the number of HIF1α positive MNC of CLL, prevented by inhibitors of JAK1/2, PI3K and mTOR signaling pathways. VEGF stimulation of SMAD (p < 0.05) and STAT5 (p < 0.01) signaling has been prevented by inhibitors of JAK1/2, mTOR, PI3K and SMAD signaling, individually (p < 0.01) or mutually (p < 0.001). Also, we showed that MNC of CLL and IL-6 individually stimulate neovascularization in co-culture with HMEC-1, without a cumulative effect. We demonstrated elevated angiogenic factors in CLL, while VEGF and IL-6 independently stimulated HIF1α. VEGF stimulation of HIF1α was mostly mTOR dependent, while IL-6 stimulation was NF-κB dependent.