RESUMEN
Diffuse large B-cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease with at least 5 recognized molecular subtypes. Cluster 5 (C5)/MCD tumors frequently exhibit concurrent alterations in the toll-like receptor (TLR) and B-cell receptor (BCR) pathway members, MYD88L265P and CD79B, and have a less favorable prognosis. In healthy B cells, the synergy between TLR and BCR signaling pathways integrates innate and adaptive immune responses and augments downstream NF-κB activation. In addition, physiologic TLR9 pathway engagement via MYD88, protein tyrosine kinase 2 (PYK2), and dedicator of cytokinesis 8 (DOCK8) increases proximal BCR signaling in healthy murine B cells. Although C5/MCD DLBCLs are selectively sensitive to Bruton tyrosine kinase (BTK) inhibition in in vitro studies and certain clinical trials, the role of mutated MYD88 in proximal BCR signaling remains undefined. Using engineered DLBCL cell line models, we found that concurrent MYD88L265P and CD79B alterations significantly increased the magnitude and duration of proximal BCR signaling, at the level of spleen tyrosine kinase and BTK, and augmented PYK2-dependent DOCK8 phosphorylation. MYD88L265P DLBCLs have significantly increased colocalization of DOCK8 with both MYD88 and the proximal BCR-associated Src kinase, LYN, in comparison with MYD88WT DLBCLs, implicating DOCK8 in MYD88L265P/proximal BCR cross talk. Additionally, DOCK8 depletion selectively decreased proximal BCR signaling, cellular proliferation, and viability of DLBCLs with endogenous MYD88L265P/CD79BY196F alterations and increased the efficacy of BTK blockade in these lymphomas. Therefore, MYD88L265P/DOCK8-enhanced proximal BCR signaling is a likely mechanism for the increased sensitivity of C5/MCD DLBCLs to BTK blockade.
Asunto(s)
Linfoma de Células B Grandes Difuso , Factor 88 de Diferenciación Mieloide , Animales , Humanos , Ratones , Quinasa 2 de Adhesión Focal/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Linfoma de Células B Grandes Difuso/patología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Receptores Toll-LikeRESUMEN
Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/metabolismo , Glicina Hidroximetiltransferasa/antagonistas & inhibidores , Glicina Hidroximetiltransferasa/metabolismo , Animales , Linfoma de Burkitt/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Descubrimiento de Drogas , Formiatos/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glicina/metabolismo , Glicina Hidroximetiltransferasa/genética , Humanos , Ratones , Terapia Molecular Dirigida , Proteolisis/efectos de los fármacosRESUMEN
The family of PIM serine/threonine kinases includes three highly conserved oncogenes, PIM1, PIM2, and PIM3, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as MYC. Recent genomic CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase-associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition-induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including PLK1. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab in vitro, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies. SIGNIFICANCE: These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Rituximab/farmacología , Animales , Antígenos CD20 , Antineoplásicos Inmunológicos/farmacología , Apoptosis , Proliferación Celular , Femenino , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones SCID , Fosforilación , Proteínas Proto-Oncogénicas c-myc/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Large-scale genomic profiling of pancreatic cancer (PDAC) has revealed two distinct subtypes: 'classical' and 'basal-like'. Their variable coexistence within the stromal immune microenvironment is linked to differential prognosis; however, the extent to which these neoplastic subtypes shape the stromal immune landscape and impact clinical outcome remains unclear. By combining preclinical models, patient-derived xenografts, as well as FACS-sorted PDAC patient biopsies, we show that the basal-like neoplastic state is sustained via BRD4-mediated cJUN/AP1 expression, which induces CCL2 to recruit tumor necrosis factor (TNF)-α-secreting macrophages. TNF-α+ macrophages force classical neoplastic cells into an aggressive phenotypic state via lineage reprogramming. Integration of ATAC-, ChIP- and RNA-seq data revealed distinct JUNB/AP1 (classical) and cJUN/AP1 (basal-like)-driven regulation of PDAC subtype identity. Pharmacological inhibition of BRD4 led to suppression of the BRD4-cJUN-CCL2-TNF-α axis, restoration of classical subtype identity and a favorable prognosis. Hence, patient-tailored therapy for a cJUNhigh/TNF-αhigh subtype is paramount in overcoming highly inflamed and aggressive PDAC states.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/genética , Proteínas de Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Macrófagos/metabolismo , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Pronóstico , Factores de Transcripción/genética , Microambiente Tumoral/genética , Factor de Necrosis Tumoral alfa/genética , Neoplasias PancreáticasRESUMEN
Large B-cell lymphomas (LBCLs) represent a frequent but clinically and morphologically heterogeneous group of tumors. Technological advances over the last 2 decades prompted the development of new classification schemas to sharpen diagnoses, dissect molecular heterogeneity, and identify rational treatment targets. Despite increased molecular understanding of these lymphomas, the clinical perspectives of patients largely remain unchanged. Recently finished comprehensive genomic studies discovered genetically defined LBCL subtypes that predict outcome, provide insight into lymphomagenesis, and suggest rational therapies with the hope of generating patient-tailored treatments with increased perspective for patients in greatest need. Here, we summarize notable examples of how high-throughput technologies aided in better molecular understanding of LBCLs and provided examples of rationally designed targeted treatments.
Asunto(s)
Linfoma de Células B Grandes Difuso/clasificación , Linfoma no Hodgkin/clasificación , HumanosRESUMEN
Tightly regulated activity of the transcription factor MYC is essential for orderly cell proliferation. Upon deregulation, MYC elicits and promotes cancer progression. Proteasomal degradation is an essential element of MYC regulation, initiated by phosphorylation at Serine62 (Ser62) of the MB1 region. Here, we found that Ser62 phosphorylation peaks in mitosis, but that a fraction of nonphosphorylated MYC binds to the microtubules of the mitotic spindle. Consequently, the microtubule-destabilizing drug vincristine decreases wild-type MYC stability, whereas phosphorylation-deficient MYC is more stable, contributing to vincristine resistance and induction of polyploidy. PI3K inhibition attenuates postmitotic MYC formation and augments the cytotoxic effect of vincristine. IMPLICATIONS: The spindle's function as a docking site for MYC during mitosis may constitute a window of specific vulnerability to be exploited for cancer treatment.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica , Microtúbulos/metabolismo , Mitosis , Neoplasias/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Vincristina/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Ciclo Celular , Proliferación Celular , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Células Tumorales CultivadasRESUMEN
B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell malignancies including diffuse large B-cell lymphoma (DLBCL). In in vitro and in vivo model systems, a subset of DLBCLs depend upon BCR survival signals and respond to proximal BCR/phosphoinositide 3 kinase (PI3K) blockade. However, single-agent BCR pathway inhibitors have had more limited activity in patients with DLBCL, underscoring the need for indicators of sensitivity to BCR blockade and insights into potential resistance mechanisms. Here, we report highly significant transcriptional upregulation of C-X-C chemokine receptor 4 (CXCR4) in BCR-dependent DLBCL cell lines and primary tumors following chemical spleen tyrosine kinase (SYK) inhibition, molecular SYK depletion or chemical PI3K blockade. SYK or PI3K inhibition also selectively upregulated cell surface CXCR4 protein expression in BCR-dependent DLBCLs. CXCR4 expression was directly modulated by fork-head box O1 via the PI3K/protein kinase B/forkhead box O1 signaling axis. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased stromal cell-derived factor-1α (SDF-1α) induced chemotaxis, consistent with the role of CXCR4 signaling in B-cell migration. Select PI3K isoform inhibitors also augmented SDF-1α induced chemotaxis. These data define CXCR4 upregulation as an indicator of sensitivity to BCR/PI3K blockade and identify CXCR4 signaling as a potential resistance mechanism in BCR-dependent DLBCLs.
Asunto(s)
Linfoma de Células B Grandes Difuso , Fosfatidilinositol 3-Quinasas , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Fosfatidilinositol 3-Quinasa , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Regulación hacia ArribaRESUMEN
Classical Hodgkin lymphoma (cHL) is composed of rare malignant Hodgkin Reed-Sternberg (HRS) cells within an extensive, but ineffective, inflammatory/immune cell infiltrate. HRS cells exhibit near-universal somatic copy gains of chromosome 9p/9p24.1, which increase expression of the programmed cell death protein 1 (PD-1) ligands. To define genetic mechanisms of response and resistance to PD-1 blockade and identify complementary treatment targets, we performed whole-exome sequencing of flow cytometry-sorted HRS cells from 23 excisional biopsies of newly diagnosed cHLs, including 8 Epstein-Barr virus-positive (EBV+) tumors. We identified significantly mutated cancer candidate genes (CCGs) as well as somatic copy number alterations and structural variations and characterized their contribution to disease-defining immune evasion mechanisms and nuclear factor κB (NF-κB), JAK/STAT, and PI3K signaling pathways. EBV- cHLs had a higher prevalence of genetic alterations in the NF-κB and major histocompatibility complex class I antigen presentation pathways. In this young cHL cohort (median age, 26 years), we identified a predominant mutational signature of spontaneous deamination of cytosine- phosphate-guanines ("Aging"), in addition to apolipoprotein B mRNA editing catalytic polypeptide-like, activation-induced cytidine deaminase, and microsatellite instability (MSI)-associated hypermutation. In particular, the mutational burden in EBV- cHLs was among the highest reported, similar to that of carcinogen-induced tumors. Together, the overall high mutational burden, MSI-associated hypermutation, and newly identified genetic alterations represent additional potential bases for the efficacy of PD-1 blockade in cHL. Of note, recurrent cHL alterations, including B2M, TNFAIP3, STAT6, GNA13, and XPO1 mutations and 2p/2p15, 6p21.32, 6q23.3, and 9p/9p24.1 copy number alterations, were also identified in >20% of primary mediastinal B-cell lymphomas, highlighting shared pathogenetic mechanisms in these diseases.
Asunto(s)
Genómica/métodos , Células de Reed-Sternberg/inmunología , Adulto , Humanos , Evasión InmuneRESUMEN
Inhibition of the B-cell receptor (BCR) signaling pathway is a promising treatment strategy in multiple B-cell malignancies. However, the role of BCR blockade in diffuse large B-cell lymphoma (DLBCL) remains undefined. We recently characterized primary DLBCL subsets with distinct genetic bases for perturbed BCR/phosphoinositide 3-kinase (PI3K) signaling and dysregulated B-cell lymphoma 2 (BCL-2) expression. Herein, we explore the activity of PI3K inhibitors and BCL-2 blockade in a panel of functionally and genetically characterized DLBCL cell line models. A PI3K inhibitor with predominant α/δ activity, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. The proapoptotic effect of copanlisib was associated with DLBCL subtype-specific dysregulated expression of BCL-2 family members including harakiri (HRK) and its antiapoptotic partner BCL extra large (BCL-xL), BCL2 related protein A1, myeloid cell leukemia 1 (MCL-1), and BCL2 interacting mediator of cell death. Using functional BH3 profiling, we found that the cytotoxic activity of copanlisib was primarily mediated through BCL-xL and MCL-1-dependent mechanisms that might complement BCL-2 blockade. For these reasons, we evaluated single-agent activity of venetoclax in the DLBCLs and identified a subset with limited sensitivity to BCL-2 blockade despite having genetic bases of BCL-2 dysregulation. As these were largely BCR-dependent DLBCLs, we hypothesized that combined inhibition of PI3Kα/δ and BCL-2 would perturb BCR-dependent and BCL-2-mediated survival pathways. Indeed, we observed synergistic activity of copanlisib/venetoclax in BCR-dependent DLBCLs with genetic bases for BCL-2 dysregulation in vitro and confirmed these findings in a xenograft model. These results provide preclinical evidence for the rational combination of PI3Kα/δ and BCL-2 blockade in genetically defined DLBCLs.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Sinergismo Farmacológico , Linfoma de Células B Grandes Difuso/patología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Pirimidinas/farmacología , Quinazolinas/farmacología , Sulfonamidas/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Femenino , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Diminished overall survival rate of non-Hodgkin lymphoma (NHL) patients treated with a combination regimen of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) has been recently linked to recurrent somatic mutations activating FOXO1. Despite of the clinical relevance of this finding, the molecular mechanism driving resistance to R-CHOP therapy remains largely unknown. Herein, we investigated the potential role of FOXO1 in the therapeutic efficacy of rituximab, the only targeted therapy included in the R-CHOP regimen. We found CD20 transcription is negatively regulated by FOXO1 in NHL cell lines and in human lymphoma specimens carrying activating mutations of FOXO1. Furthermore, both the expression of exogenous mutants of FOXO1 and the inhibition of AKT led to FOXO1 activation in lymphoma cells, increased binding to MS4A1 promoter and diminished CD20 expression levels. In contrast, a disruption of FOXO1 with CRISPR/Cas9 genome-editing (sgFOXO1) resulted in CD20 upregulation, improved the cytotoxicity induced by rituximab and the survival of mice with sgFOXO1 tumors. Accordingly, pharmacological inhibition of FOXO1 activity in primary samples upregulated surface CD20 levels. Importantly, FOXO1 was required for the downregulation of CD20 levels by the clinically tested inhibitors of BTK, SYK, PI3K and AKT. Taken together, these results indicate for the first time that the AKT-unresponsive mutants of FOXO1 are important determinant of cell response to rituximab-induced cytotoxicity, and suggest that the genetic status of FOXO1 together with its transcriptional activity need further attention while designing anti-CD20 antibodies based regimens for the therapy of pre-selected lymphomas.
RESUMEN
Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.
Asunto(s)
Antígenos CD20/genética , Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma no Hodgkin/tratamiento farmacológico , Rituximab/farmacología , Animales , Antígenos CD20/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Desacetilasa 6 , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/patología , Ratones Endogámicos BALB C , Ratones SCID , ARN Mensajero/genética , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Both myoblasts and mesenchymal stem cells (MSC) take part in the muscle tissue regeneration and have been used as experimental cellular therapy in muscular disorders treatment. It is possible that co-transplantation approach could improve the efficacy of this treatment. However, the relations between those two cell types are not clearly defined. The aim of this study was to determine the reciprocal interactions between myoblasts and MSC in vitro in terms of the features important for the muscle regeneration process. Primary caprine muscle-derived cells (MDC) and bone marrow-derived MSC were analysed in autologous settings. We found that MSC contribute to myotubes formation by fusion with MDC when co-cultured directly, but do not acquire myogenic phenotype if exposed to MDC-derived soluble factors only. Experiments with exposure to hydrogen peroxide showed that MSC are significantly more resistant to oxidative stress than MDC, but a direct co-culture with MSC does not diminish the cytotoxic effect of H2O2 on MDC. Cell migration assay demonstrated that MSC possess significantly greater migration ability than MDC which is further enhanced by MDC-derived soluble factors, whereas the opposite effect was not found. MSC-derived soluble factors significantly enhanced the proliferation of MDC, whereas MDC inhibited the division rate of MSC. To conclude, presented results suggest that myogenic precursors and MSC support each other during muscle regeneration and therefore myoblasts-MSC co-transplantation could be an attractive approach in the treatment of muscular disorders.
Asunto(s)
Comunicación Celular , Células Madre Mesenquimatosas/metabolismo , Mioblastos/metabolismo , Animales , Biomarcadores , Diferenciación Celular , Fusión Celular , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Técnicas de Cocultivo , Femenino , Cabras , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Células Madre Mesenquimatosas/citología , Desarrollo de Músculos , Mioblastos/citologíaRESUMEN
The Bcl-2 antagonist ABT-199 (venetoclax) has demonstrated promising clinical activity in patients with chronic lymphocytic leukemia (CLL). ABT-199 is strongly cytotoxic against unstimulated peripheral blood CLL cells in vitro but is much less effective against CLL cells that have received survival signals from the microenvironment. In particular, stimulation of CLL cells with CD40L results in substantial resistance mediated by induction of the antiapoptotic Bcl-2 family proteins Bcl-xL and Bfl-1. In this study, we investigated whether resistance to ABT-199 can be conferred by B-cell receptor (BCR) stimulation, which is another important survival signal from the leukemic microenvironment. We show that sustained BCR stimulation results in significant ABT-199 resistance, which correlates with induction of the antiapoptotic protein Mcl-1 and less consistently with downregulation of proapoptotic Bmf, Hrk, and BimEL A major role for Mcl-1 in conferring ABT-199 resistance is additionally supported by knockdown and enforced expression experiments with primary CLL cells. We further show that SYK, BTK, and phosphatidylinositol 3-kinase δ (PI3Kδ) inhibitors significantly downregulate Mcl-1, but with different efficacy. Complete Mcl-1 downregulation was consistently achieved only with SYK inhibitors R406 and GS-9973 (entospletinib), whereas the BTK inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib in more than half of the cases had only a partial effect. The greater ability of SYK inhibitors to downregulate Mcl-1 correlated with their greater capacity to block BCR-mediated inactivation of GSK-3, a major negative regulator of Mcl-1. The finding that BCR signaling inhibitors differ in their ability to target Mcl-1 is relevant for the design of clinical trials combining these agents with ABT-199.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Receptores de Antígenos de Linfocitos B/antagonistas & inhibidores , Sulfonamidas/uso terapéutico , Adenina/análogos & derivados , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Oxazinas/farmacología , Piperidinas , Purinas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Quinazolinonas/farmacologíaRESUMEN
B-cell receptor (BCR) signaling pathway plays a central role in B-lymphocyte development and initiation of humoral immunity. Recently, BCR signaling pathway has been shown as a major driver in the pathogenesis of B-cell malignancies. As a result, a vast array of BCR-associated kinases has emerged as rational therapeutic targets changing treatment paradigms in B cell malignancies. Based on high efficacy in early-stage clinical trials, there is rapid clinical development of inhibitors targeting BCR signaling pathway. Here, we describe the essential components of BCR signaling, their function in normal and pathogenic signaling and molecular effects of their inhibition in vitro and in vivo.
Asunto(s)
Leucemia Linfoide/etiología , Receptores de Antígenos de Linfocitos B/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Humanos , Leucemia Linfoide/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de SeñalRESUMEN
Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD20/inmunología , Neoplasias/inmunología , Inhibidores de Proteínas Quinasas/inmunología , Familia-src Quinasas/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos CD20/genética , Antígenos CD20/metabolismo , Western Blotting , Línea Celular Tumoral , Dasatinib , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/inmunología , Células HEK293 , Humanos , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Ratones Endogámicos C57BL , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinas/inmunología , Pirimidinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rituximab , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Tiazoles/inmunología , Tiazoles/farmacología , Transcriptoma/efectos de los fármacos , Transcriptoma/inmunología , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
Primary graft non-function (PNF) is a rare, life-threatening complication of liver transplantation. Increasing use of extended criteria donor pools and high-risk recipients seem to influence the incidence of PNF. Primary failure is associated with high patient morbidity and inferior graft survival. The only available treatment for PNF is emergency hepatic retransplantation, which is also correlated with significant morbidity and mortality. Therefore, researchers are working to identify risk factors of diagnostic value to prevent PNF. The current study attempted to explore liver proteomic patterns in patients with PNF. Using two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry (LC-MS), we compared liver protein homogenates from 3 patients with PNF to those obtained from 6 healthy liver samples to identify potential new biomarkers of PNF. Our comparisons revealed 21 proteins with differential expression (13 upregulated and 8 downregulated). Most of these proteins are involved in energy metabolism, lipid metabolism, peptide cleavage, cell differentiation, and apoptosis. Although none of these proteins appeared more than once in separate analyses, this preliminary study shows that two-dimensional gel electrophoresis and LC-MS may allow identification of characteristic proteins to be used as biomarkers of a life-threatening complication of liver transplantation. Larger-scale analyses could improve patient care by finding suitable prognostic and therapeutic options. These data represent the first global proteomic approach to study PNF.
Asunto(s)
Funcionamiento Retardado del Injerto/diagnóstico , Funcionamiento Retardado del Injerto/fisiopatología , Trasplante de Hígado/patología , Hígado/fisiopatología , Proteoma , Proteómica/métodos , Anciano , Cromatografía Liquida/métodos , Funcionamiento Retardado del Injerto/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Hígado/metabolismo , Masculino , Espectrometría de Masas/métodos , Proteoma/análisis , Proteoma/metabolismoRESUMEN
Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos CD20/biosíntesis , Proteínas del Sistema Complemento/química , Dimetilaliltranstransferasa/fisiología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Citometría de Flujo/métodos , Células HEK293 , Humanos , Linfoma de Células B/metabolismo , Metionina/análogos & derivados , Metionina/farmacología , Regiones Promotoras GenéticasRESUMEN
Photodynamic therapy (PDT) is a clinically approved method of tumor treatment. Its unique mechanism of action results from minimal invasiveness and high selectivity towards transformed cells. However, visible light used to excite most photosensitizers has rather limited ability to penetrate tissues resulting in insufficient destruction of deeply seated malignant cells. Therefore, novel strategies for further potentiation of the anticancer effectiveness of PDT have been developed. These include combined treatments with surgery, chemo- and radiotherapy, strategies targeting cytoprotective mechanisms induced in PDT-treated cells, as well as attempts aimed at enhancement of PDT-mediated antitumor immune response. Moreover, new photosensitizers and novel light sources are being developed. Impressive progress in nanotechnology and understanding of tumor cell biology rise hopes for further improvements in this elegant and promising method of cancer treatment.
Asunto(s)
Neoplasias/tratamiento farmacológico , Fotoquimioterapia/métodos , Inmunidad Adaptativa/efectos de los fármacos , Atención Ambulatoria , Animales , Antineoplásicos/uso terapéutico , Terapia Combinada , Humanos , Nanotecnología , Estrés Oxidativo/efectos de los fármacos , Fármacos Fotosensibilizantes/metabolismo , Fármacos Fotosensibilizantes/farmacocinética , Fármacos Fotosensibilizantes/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/tratamiento farmacológicoRESUMEN
Histone deacetylases inhibitors (HDACi) have recently emerged as potent antitumor treatment modality. They are currently tested in many phase I, II and III clinical trials as single agents as wells as in combination schemes. They have demonstrated promising antitumor activity and favorable clinical outcome. Histone deacetylases (HDACs) are involved in the process of epigenetic regulation of gene expression. Epigenetic changes are believed to be crucial for the onset and progression of cancer and have recently gained remarkable attention. Since epigenetic regulation of gene expression is a reversible process, targeting histone deacetylases provides a good rationale for anticancer therapy. The acetylation status of histones regulates the organization of chromatin and the access of transcription factors. Moreover, functions of many non-histone proteins are controlled by acetylation. The broad and complicated influences of HDACi on various molecular processes may account for the observed pleiotropic effects. In this review we summarize recent advances in the understanding of biology of HDACs and mechanism of action of their inhibitors.