RESUMO
Pancreatic cancer is an aggressive disease with a 5 year survival rate of 13%. This poor survival is attributed, in part, to limited and ineffective treatments for patients with metastatic disease, highlighting a need to identify molecular drivers of pancreatic cancer to target for more effective treatment. CD200 is a glycoprotein that interacts with the receptor CD200R and elicits an immunosuppressive response. Overexpression of CD200 has been associated with differential outcomes, depending on the tumor type. In the context of pancreatic cancer, we have previously reported that CD200 is expressed in the pancreatic tumor microenvironment (TME), and that targeting CD200 in murine tumor models reduces tumor burden. We hypothesized that CD200 is overexpressed on tumor and stromal populations in the pancreatic TME and that circulating levels of soluble CD200 (sCD200) have prognostic value for overall survival. We discovered that CD200 was overexpressed on immune, stromal, and tumor populations in the pancreatic TME. Particularly, single-cell RNA-sequencing indicated that CD200 was upregulated on inflammatory cancer-associated fibroblasts. Cytometry by time of flight analysis of PBMCs indicated that CD200 was overexpressed on innate immune populations, including monocytes, dendritic cells, and monocytic myeloid-derived suppressor cells. High sCD200 levels in plasma correlated with significantly worse overall and progression-free survival. Additionally, sCD200 correlated with the ratio of circulating matrix metalloproteinase (MMP) 3: tissue inhibitor of metalloproteinase (TIMP) 3 and MMP11/TIMP3. This study highlights the importance of CD200 expression in pancreatic cancer and provides the rationale for designing novel therapeutic strategies that target this protein.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Pancreáticas , Humanos , Imunossupressores , Pâncreas , Microambiente TumoralRESUMO
Generating a hematopoietic stem cell (HSC) in vitro from nonhematopoietic tissue has been a goal of experimental hematologists for decades. Until recently, no in vitro-derived cell has closely demonstrated the full lineage potential and self-renewal capacity of a true HSC. Studies revealing stem cell ontogeny from embryonic mesoderm to hemogenic endothelium to HSC provided the key to inducing HSC-like cells in vitro from a variety of cell types. Here we review the path to this discovery and discuss the future of autologous transplantation with in vitro-derived HSCs as a therapeutic modality.
Assuntos
Hemangioblastos/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem da Célula , Técnicas de Reprogramação Celular/métodos , Transplante de Células-Tronco Hematopoéticas , HumanosRESUMO
Myeloid-derived suppressor cell (MDSC) levels are elevated in patients with cancer and contribute to reduced efficacy of immune checkpoint therapy. MDSC express Bruton's tyrosine kinase (BTK) and BTK inhibition with ibrutinib, an FDA-approved irreversible inhibitor of BTK, leads to reduced MDSC expansion/function in mice and significantly improves the antitumor activity of anti-PD-1 antibody treatments. Single-cell RNA sequencing (scRNA-seq) was used to characterize the effect of ibrutinib on gene expression of fluorescence-activated cell sorting-enriched MDSC from patients with different cancer types [breast, melanoma, head and neck squamous cell cancer (HNSCC)]. Melanoma patient MDSC were treated in vitro for 4 hours with 5 µmol/L ibrutinib or DMSO, processed for scRNA-seq using the Chromium 10× Genomics platform, and analyzed via the Seurat v4 standard integrative workflow. Baseline gene expression of MDSC from patients with breast, melanoma, and HNSCC cancer revealed similarities among the top expressed genes. In vitro ibrutinib treatment of MDSC from patients with melanoma resulted in significant changes in gene expression. GBP1, IL-1ß, and CXCL8 were among the top downregulated genes whereas RGS2 and ABHD5 were among the top upregulated genes (P < 0.001). Double positive CD14+CD15+ MDSC and PMN-MDSC responded similarly to BTK inhibition and exhibited more pronounced gene changes compared with early MDSC and M-MDSC. Pathway analysis revealed significantly downregulated pathways including TREM1, nitric oxide signaling, and IL-6 signaling (P < 0.004). IMPLICATIONS: scRNA-seq revealed characteristic gene expression patterns for MDSC from different patients with cancer and BTK inhibition led to the downregulation of multiple genes and pathways important to MDSC function and migration.
Assuntos
Neoplasias de Cabeça e Pescoço , Melanoma , Células Supressoras Mieloides , Animais , Humanos , Camundongos , 1-Acilglicerol-3-Fosfato O-Aciltransferase , Tirosina Quinase da Agamaglobulinemia , Análise da Expressão Gênica de Célula Única , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
ABSTRACT: Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma, and patients who relapse on targeted therapies have poor prognosis. Protein arginine methyltransferase 5 (PRMT5), an enzyme essential for B-cell transformation, drives multiple oncogenic pathways and is overexpressed in MCL. Despite the antitumor activity of PRMT5 inhibition (PRT-382/PRT-808), drug resistance was observed in a patient-derived xenograft (PDX) MCL model. Decreased survival of mice engrafted with these PRMT5 inhibitor-resistant cells vs treatment-naive cells was observed (P = .005). MCL cell lines showed variable sensitivity to PRMT5 inhibition. Using PRT-382, cell lines were classified as sensitive (n = 4; 50% inhibitory concentration [IC50], 20-140 nM) or primary resistant (n = 4; 340-1650 nM). Prolonged culture of sensitive MCL lines with drug escalation produced PRMT5 inhibitor-resistant cell lines (n = 4; 200-500 nM). This resistant phenotype persisted after prolonged culture in the absence of drug and was observed with PRT-808. In the resistant PDX and cell line models, symmetric dimethylarginine reduction was achieved at the original PRMT5 inhibitor IC50, suggesting activation of alternative resistance pathways. Bulk RNA sequencing of resistant cell lines and PDX relative to sensitive or short-term-treated cells, respectively, highlighted shared upregulation of multiple pathways including mechanistic target of rapamycin kinase [mTOR] signaling (P < 10-5 and z score > 0.3 or < 0.3). Single-cell RNA sequencing analysis demonstrated a strong shift in global gene expression, with upregulation of mTOR signaling in resistant PDX MCL samples. Targeted blockade of mTORC1 with temsirolimus overcame the PRMT5 inhibitor-resistant phenotype, displayed therapeutic synergy in resistant MCL cell lines, and improved survival of a resistant PDX.
Assuntos
Linfoma de Célula do Manto , Humanos , Camundongos , Animais , Adulto , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Linhagem Celular Tumoral , Recidiva Local de Neoplasia , Transdução de Sinais , Inibidores Enzimáticos/uso terapêutico , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Monokines (i.e., interleukin [IL]-12, -18, and -15) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma), which is a critical factor for immune surveillance of cancer and monocyte clearance of infection. We show that SET, which is a potent inhibitor of protein phosphatase type 2A (PP2A) activity, is highly expressed in human CD56bright NK cells, which produce more IFN-gamma than CD56dim NK cells. SET was up-regulated upon monokine stimulation of primary human NK cells. Furthermore, ectopic overexpression of SET significantly enhanced IFN-gamma gene expression in monokine-stimulated NK cells. In contrast, RNAi-mediated suppression of SET expression renders NK cells inefficient in producing high levels of IFN-gamma in response to monokine costimulation. Mechanistically, suppression of PP2A activity by SET is important for IFN-gamma gene expression in NK cells. In fact, treatment of primary human NK cells with the PP2A activator 1,9-dideoxy-forskolin, as well as administration of the drug to C57BL/6 mice, significantly reduced NK-dependent IFN-gamma production in response to monokine treatment. Further, SET knockdown or pharmacologic activation of PP2A diminished extracellular signal-regulated kinase 1/2, p65RelA, signal transducer and activator of transduction 4 (STAT4), and STAT5 activity in monokine-stimulated NK cells, potentially contributing to the reduction in IFN-gamma gene expression. Thus, SET expression is essential for suppressing PP2A phosphatase activity that would otherwise limit NK cell antitumoral and/or antiinflammatory functions by impairing NK cell production of IFN-gamma.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Interferon gama/biossíntese , Células Matadoras Naturais/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA , Ativação Enzimática , Regulação da Expressão Gênica , Chaperonas de Histonas , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Monocinas/farmacologia , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The oncogenic BCR/ABL kinase activity induces and maintains chronic myelogenous leukemia (CML). We show here that, in BCR/ABL-transformed cells and CML blast crisis (CML-BC) progenitors, the phosphatase activity of the tumor suppressor PP2A is inhibited by the BCR/ABL-induced expression of the PP2A inhibitor SET. In imatinib-sensitive and -resistant (T315I included) BCR/ABL+ cell lines and CML-BC progenitors, molecular and/or pharmacological activation of PP2A promotes dephosphorylation of key regulators of cell proliferation and survival, suppresses BCR/ABL activity, and induces BCR/ABL degradation. Furthermore, PP2A activation results in growth suppression, enhanced apoptosis, restored differentiation, impaired clonogenic potential, and decreased in vivo leukemogenesis of imatinib-sensitive and -resistant BCR/ABL+ cells. Thus, functional inactivation of PP2A is essential for BCR/ABL leukemogenesis and, perhaps, required for blastic transformation.
Assuntos
Crise Blástica/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Fusão bcr-abl/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Fatores de Transcrição/fisiologia , Animais , Antineoplásicos/farmacologia , Benzamidas , Linhagem Celular Transformada , Colforsina/farmacologia , Proteínas de Ligação a DNA , Inibidores Enzimáticos/metabolismo , Chaperonas de Histonas , Humanos , Mesilato de Imatinib , Técnicas In Vitro , Células K562 , Leucemia/prevenção & controle , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos SCID , Transplante de Neoplasias , Fosfoproteínas Fosfatases/antagonistas & inibidores , Piperazinas/farmacologia , Proteína Fosfatase 2 , Pirimidinas/farmacologia , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Altered hematopoietic stem cell (HSC) fate underlies primary blood disorders but microenvironmental factors controlling this are poorly understood. Genetically barcoded genome editing of synthetic target arrays for lineage tracing (GESTALT) zebrafish were used to screen for factors expressed by the sinusoidal vascular niche that alter the phylogenetic distribution of the HSC pool under native conditions. Dysregulated expression of protein kinase C delta (PKC-δ, encoded by prkcda) increases the number of HSC clones by up to 80% and expands polyclonal populations of immature neutrophil and erythroid precursors. PKC agonists such as cxcl8 augment HSC competition for residency within the niche and expand defined niche populations. CXCL8 induces association of PKC-δ with the focal adhesion complex, activating extracellular signal-regulated kinase (ERK) signaling and expression of niche factors in human endothelial cells. Our findings demonstrate the existence of reserve capacity within the niche that is controlled by CXCL8 and PKC and has significant impact on HSC phylogenetic and phenotypic fate.
Assuntos
Células Endoteliais , Peixe-Zebra , Animais , Humanos , Células Endoteliais/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Filogenia , Proteína Quinase C-delta/metabolismo , Nicho de Células-Tronco , Interleucina-8/metabolismoRESUMO
Richter's Transformation (RT) is a poorly understood and fatal progression of chronic lymphocytic leukemia (CLL) manifesting histologically as diffuse large B-cell lymphoma. Protein arginine methyltransferase 5 (PRMT5) is implicated in lymphomagenesis, but its role in CLL or RT progression is unknown. We demonstrate herein that tumors uniformly overexpress PRMT5 in patients with progression to RT. Furthermore, mice with B-specific overexpression of hPRMT5 develop a B-lymphoid expansion with increased risk of death, and Eµ-PRMT5/TCL1 double transgenic mice develop a highly aggressive disease with transformation that histologically resembles RT; where large-scale transcriptional profiling identifies oncogenic pathways mediating PRMT5-driven disease progression. Lastly, we report the development of a SAM-competitive PRMT5 inhibitor, PRT382, with exclusive selectivity and optimal in vitro and in vivo activity compared to available PRMT5 inhibitors. Taken together, the discovery that PRMT5 drives oncogenic pathways promoting RT provides a compelling rationale for clinical investigation of PRMT5 inhibitors such as PRT382 in aggressive CLL/RT cases.
Assuntos
Leucemia Linfocítica Crônica de Células B , Linfoma Difuso de Grandes Células B , Animais , Camundongos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma Difuso de Grandes Células B/patologiaRESUMO
An increased incidence of cardiovascular complications has been documented in recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Despite this, little is known about the risk factors for hyperlipidemia or the role of lipid-lowering therapy early after transplantation. We performed a retrospective analysis of all patients who underwent allogeneic HSCT at the Dana-Farber Cancer Institute from 1998 to 2008 and who survived more than 100 days. The incidence of hypercholesterolemia and hypertriglyceridemia in the first 2 years after transplantation was 73.4% and 72.5%, respectively. In multivariable analysis, the development of acute graft-versus-host disease was independently associated with both hypercholesterolemia (odds ratio [OR] = 1.62) and hypertriglyceridemia (OR = 1.54) after transplantation. Statin use was instituted in 29% of patients and was associated with a significant net reduction in total cholesterol (65 mg/dL, P < .0001), triglyceride (118 mg/dL P < .0001), and LDL levels (59 mg/dL P < .0001) without any significant adverse effects. These data suggest that hyperlipidemia is common in the first 2 years after allogeneic transplantation when most patients remain under the care of the transplantation physician and lipid-lowering therapy may be underutilized. Given the cardiovascular risk associated with hyperlipidemia and the tolerability of statins, further prospective evaluation of lipid abnormalities and their treatment seems well warranted.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Hipertrigliceridemia/tratamento farmacológico , Hipolipemiantes/uso terapêutico , Condicionamento Pré-Transplante , Adolescente , Adulto , Idoso , Doenças Cardiovasculares/prevenção & controle , Feminino , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Hipercolesterolemia/sangue , Hipercolesterolemia/etiologia , Hipertrigliceridemia/sangue , Hipertrigliceridemia/etiologia , Hipolipemiantes/administração & dosagem , Masculino , Pessoa de Meia-Idade , Agonistas Mieloablativos/administração & dosagem , Agonistas Mieloablativos/efeitos adversos , Estudos Retrospectivos , Risco , Transplante HomólogoRESUMO
IL-15 is required for NK cell development and homeostasis in vivo. Because IL-15 is presented in trans via its high-affinity IL-15Ralpha-chain to cells expressing the IL-15Rbetagamma complex, we postulated that certain IL-15-bearing cells must be required for NK cell homeostasis. Using IL-15(WT/WT) and IL-15(-/-) mice, bone marrow chimeras with normal cellularity, and a selective depletion of CD11c(hi) dendritic cells (DCs), we demonstrate that ablation of the resting CD11c(hi) DC population results in a highly significant decrease in the absolute number of mature NK cells. In contrast, administration of Flt3 ligand increases the CD11c(hi) DC population, which, when expressing IL-15, significantly expands mature NK cells via enhanced survival and proliferation. In summary, a CD11c(hi) DC population expressing IL-15 is required to maintain NK cell homeostasis under conditions of normal cellularity and also is required to mediate Flt3 ligand-induced NK cell expansion in vivo.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Homeostase/imunologia , Células Matadoras Naturais/citologia , Proteínas de Membrana/fisiologia , Animais , Antígeno CD11c/biossíntese , Diferenciação Celular/imunologia , Proliferação de Células , Sobrevivência Celular/imunologia , Feminino , Humanos , Interleucina-15/deficiência , Interleucina-15/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , Ligantes , Proteínas de Membrana/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Recombinantes/administração & dosagemRESUMO
Despite the clinical benefit associated with gilteritinib in relapsed/refractory acute myeloid leukemia (AML), most patients eventually develop resistance through unknown mechanisms. To delineate the mechanistic basis of resistance to gilteritinib, we performed targeted sequencing and scRNASeq on primary FLT3-ITD-mutated AML samples. Co-occurring mutations in RAS pathway genes were the most common genetic abnormalities, and unresponsiveness to gilteritinib was associated with increased expression of bone marrow-derived hematopoietic cytokines and chemokines. In particular, we found elevated expression of the TEK-family kinase, BMX, in gilteritinib-unresponsive patients pre- and post-treatment. BMX contributed to gilteritinib resistance in FLT3-mutant cell lines in a hypoxia-dependent manner by promoting pSTAT5 signaling, and these phenotypes could be reversed with pharmacological inhibition and genetic knockout. We also observed that inhibition of BMX in primary FLT3-mutated AML samples decreased chemokine secretion and enhanced the activity of gilteritinib. Collectively, these findings indicate a crucial role for microenvironment-mediated factors modulated by BMX in the escape from targeted therapy and have implications for the development of novel therapeutic interventions to restore sensitivity to gilteritinib.
Assuntos
Compostos de Anilina , Leucemia Mieloide Aguda , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Proteínas Tirosina Quinases/genética , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Microambiente Tumoral , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/uso terapêuticoRESUMO
BACKGROUND: Mantle cell lymphoma (MCL) is a rare, highly heterogeneous type of B-cell non-Hodgkin's lymphoma. The sumoylation pathway is known to be upregulated in many cancers including lymphoid malignancies. However, little is known about its oncogenic role in MCL. METHODS: Levels of sumoylation enzymes and sumoylated proteins were quantified in MCL cell lines and primary MCL patient samples by scRNA sequencing and immunoblotting. The sumoylation enzyme SAE2 was genetically and pharmacologically targeted with shRNA and TAK-981 (subasumstat). The effects of SAE2 inhibition on MCL proliferation and cell cycle were evaluated using confocal microscopy, live-cell microscopy, and flow cytometry. Immunoprecipitation and orbitrap mass spectrometry were used to identify proteins targeted by sumoylation in MCL cells. RESULTS: MCL cells have significant upregulation of the sumoylation pathway at the level of the enzymes SAE1 and SAE2 which correlated with poor prognosis and induction of mitosis associated genes. Selective inhibition of SAE2 with TAK-981 results in significant MCL cell death in vitro and in vivo with mitotic dysregulation being an important mechanism of action. We uncovered a sumoylation program in mitotic MCL cells comprised of multiple pathways which could be directly targeted with TAK-981. Centromeric localization of topoisomerase 2A, a gene highly upregulated in SAE1 and SAE2 overexpressing MCL cells, was lost with TAK-981 treatment likely contributing to the mitotic dysregulation seen in MCL cells. CONCLUSIONS: This study not only validates SAE2 as a therapeutic target in MCL but also opens the door to further mechanistic work to uncover how to best use desumoylation therapy to treat MCL and other lymphoid malignancies.
RESUMO
Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell-mediated killing of HLA class I-expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell-mediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3-positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Células Matadoras Naturais/efeitos dos fármacos , Neoplasias/terapia , Receptores KIR/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Células Cultivadas , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias/imunologia , Neoplasias/patologia , Receptores KIR/antagonistas & inibidores , Receptores KIR2DL1/química , Receptores KIR2DL1/genética , Receptores KIR2DL1/imunologia , Receptores KIR2DL2/química , Receptores KIR2DL2/genética , Receptores KIR2DL2/imunologia , Receptores KIR2DL3/genética , Receptores KIR2DL3/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Setting nutritional standards for larval zebrafish (Danio rerio) that maximize growth, survival, and reproductive success is challenging. We evaluated the effects of different feeding regimens on larval zebrafish by comparing Gemma Micro 75 pelleted diet and live-type L rotifers (Brachionus plicatilis) in 3 feeding regimens starting at 9 days postfertilization (dpf): bolus feeding of live diet (BL), continuous feeding of live diet (CL), and pelleted diet (PD). Animals in the PD and CL groups were longer than the BL group at 4-5 weeks postfertilization. The PD group was also greater in body depth than both live diet groups. There was no significant difference in weight between the groups. There were also no significant differences in fecundity or sex ratios indicating that all feeding methods successfully promote growth of a useful breeding stock of fish. In addition, we quantified the equipment, consumable, and labor costs associated with these methods, and found that the PD regimen was superior to both live diet regimens. These data suggest that providing a high nutrient-density pelleted diet to larval and juvenile zebrafish is an effective means to increase early growth and to decrease cost and labor associated with nursery care.
Assuntos
Rotíferos , Peixe-Zebra , Ração Animal/análise , Animais , Dieta/veterinária , LarvaRESUMO
Acute myeloid leukemia patients refractory to induction therapy or relapsed within one year have poor outcomes. Autocrine production of hepatocyte growth factor by myeloid blasts drives leukemogenesis in pre-clinical models. A phase Ib trial evaluated ficlatuzumab, a first-in-class anti-HGF antibody, in combination with cytarabine in this high-risk population. Dose-limiting toxicities were not observed, and 20 mg/kg was established as the recommended phase II dose. The most frequent treatment-related adverse event was febrile neutropenia. Among 17 evaluable patients, the overall response rate was 53%, all complete remissions. Phospho-proteomic mass cytometry showed potent on-target suppression of p-MET after ficlatuzumab treatment and that attenuation of p-S6 was associated with clinical response. Multiplexed single cell RNA sequencing using prospectively acquired patient specimens identified interferon response genes as adverse predictive factors. The ficlatuzumab and cytarabine combination is well-tolerated with favorable efficacy. High-dimensional analyses at single-cell resolution represent promising approaches for identifying biomarkers of response and mechanisms of resistance in prospective clinical studies.
Assuntos
Leucemia Mieloide Aguda , Proteômica , Anticorpos Monoclonais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Estudos ProspectivosRESUMO
Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190(BCR/ABL) myeloid and lymphoid cell lines and CML-BC(CD34+) and Ph1 ALL(CD34+/CD19+) progenitors but not of normal CD34+ and CD34+/CD19+ bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190(BCR/ABL)-driven [including p210/p190(BCR/ABL)(T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients.
Assuntos
Crise Blástica/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Propilenoglicóis/uso terapêutico , Esfingosina/análogos & derivados , Animais , Benzamidas , Crise Blástica/genética , Crise Blástica/metabolismo , Crise Blástica/patologia , Sobrevivência Celular/efeitos dos fármacos , Dasatinibe , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Cloridrato de Fingolimode , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Estrutura Molecular , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Piperazinas/farmacologia , Propilenoglicóis/química , Proteína Fosfatase 2 , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Esfingosina/química , Esfingosina/uso terapêutico , Tiazóis/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Hematopoietic stem cells (HSCs) are functionally and genetically diverse and this diversity decreases with age and disease. Numerous systems have been developed to quantify HSC diversity by genetic barcoding, but no framework has been established to empirically validate barcode sequences. Here we have developed an analytical framework, Selection of informative Amplicon Barcodes from Experimental Replicates (SABER), that identifies barcodes that are unique among a large set of experimental replicates. Amplicon barcodes were sequenced from the blood of 56 adult zebrafish divided into training and validation sets. Informative barcodes were identified and samples with a high fraction of informative barcodes were chosen by bootstrapping. There were 4.2 ± 1.8 barcoded HSC clones per sample in the training set and 3.5 ± 2.1 in the validation set (p = 0.3). SABER reproducibly quantifies functional HSCs and can accommodate a wide range of experimental group sizes. Future large-scale studies aiming to understand the mechanisms of HSC clonal evolution will benefit from this new approach to identifying informative amplicon barcodes.
Assuntos
Evolução Clonal , Técnicas de Genotipagem/métodos , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Mutação , Algoritmos , Animais , Técnicas de Genotipagem/normas , Células-Tronco Hematopoéticas/citologia , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas , Peixe-ZebraRESUMO
BACKGROUND: A significant challenge to overcome in pancreatic ductal adenocarcinoma (PDAC) is the profound systemic immunosuppression that renders this disease non-responsive to immunotherapy. Our supporting data provide evidence that CD200, a regulator of myeloid cell activity, is expressed in the PDAC microenvironment. Additionally, myeloid-derived suppressor cells (MDSC) isolated from patients with PDAC express elevated levels of the CD200 receptor (CD200R). Thus, we hypothesize that CD200 expression in the PDAC microenvironment limits responses to immunotherapy by promoting expansion and activity of MDSC. METHODS: Immunofluorescent staining was used to determine expression of CD200 in murine and human PDAC tissue. Flow cytometry was utilized to test for CD200R expression by immune populations in patient blood samples. In vivo antibody blocking of CD200 was conducted in subcutaneous MT-5 tumor-bearing mice and in a genetically engineered PDAC model (KPC-Brca2 mice). Peripheral blood mononuclear cells (PBMC) from patients with PDAC were analyzed by single-cell RNA sequencing. MDSC expansion assays were completed using healthy donor PBMC stimulated with IL-6/GM-CSF in the presence of recombinant CD200 protein. RESULTS: We found expression of CD200 by human pancreatic cell lines (BxPC3, MiaPaca2, and PANC-1) as well as on primary epithelial pancreatic tumor cells and smooth muscle actin+ stromal cells. CD200R expression was found to be elevated on CD11b+CD33+HLA-DRlo/- MDSC immune populations from patients with PDAC (p=0.0106). Higher expression levels of CD200R were observed in CD15+ MDSC compared with CD14+ MDSC (p<0.001). In vivo studies demonstrated that CD200 antibody blockade limited tumor progression in MT-5 subcutaneous tumor-bearing and in KPC-Brca2 mice (p<0.05). The percentage of intratumoral MDSC was significantly reduced in anti-CD200 treated mice compared with controls. Additionally, in vivo blockade of CD200 can also significantly enhance the efficacy of PD-1 checkpoint antibodies compared with single antibody therapies (p<0.05). Single-cell RNA sequencing of PBMC from patients revealed that CD200R+ MDSC expressed genes involved in cytokine signaling and MDSC expansion. Further, in vitro cytokine-driven expansion and the suppressive activity of human MDSC was enhanced when cocultured with recombinant CD200 protein. CONCLUSIONS: These results indicate that CD200 expression in the PDAC microenvironment may regulate MDSC expansion and that targeting CD200 may enhance activity of checkpoint immunotherapy.
Assuntos
Antígenos CD/metabolismo , Carcinoma Ductal Pancreático/imunologia , Terapia de Imunossupressão , Leucócitos Mononucleares/imunologia , Células Supressoras Mieloides/imunologia , Neoplasias Pancreáticas/imunologia , Microambiente Tumoral/imunologia , Animais , Antígenos CD/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Modelos Animais de Doenças , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Camundongos , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias PancreáticasRESUMO
AIM: There are limited data describing incidence of symptomatic venous thromboembolism (VTE) in adolescent and young adult (AYA) acute lymphoblastic leukemia (ALL) patients receiving peg-asparaginase. MATERIALS & METHODS: Single-institution retrospective analysis of 44 AYA ALL patients treated with peg-asparaginase. Rates of VTE and proposed risk factors were assessed. RESULTS: 18 patients (41%) had a symptomatic VTE following peg-asparaginase. The cumulative incidence rate was 25% (95% CI: 13-38%) within 30 days of the initial dose. Personal history of thrombosis was statistically significantly associated with an increased risk of VTE with HR of 2.73 (95% CI: 1.40-5.33, p = 0.003) after adjusting for gender. CONCLUSION: These data indicate a high rate of VTE in the AYA ALL population following treatment with peg-asparaginase.
RESUMO
PURPOSE: Expression of the FHIT protein is lost or reduced in most solid tumors and a significant fraction of hematopoietic malignancies. Adenovirus 5 (Ad5) virus or adeno-associated viral vectors have been used to study the tumor suppressor function of FHIT in solid tumors, but these tools have not been effective in leukemias. We have generated a chimeric FHIT-containing adenovirus composed of Ad5 and the group B adenovirus called F35 with which we have been able to efficiently infect hematopoietic cells. EXPERIMENTAL DESIGN: Infection efficiency of Ad5/F35-FHIT and Ad5/F35-GFP viruses was tested in leukemia cell lines that lacked FHIT expression, and biological effects of successful infection were assessed. An acute myelogenous leukemia, a chronic myelogenous leukemia, and four acute lymphoblastic leukemia human cell lines were examined as well as two EBV-transformed B lymphoblastoid cell lines that expressed endogenous FHIT. RESULTS: Two of four acute lymphoblastic leukemia cell lines, Jurkat and MV4;11, which were efficiently infected with Ad5/F35-FHIT, underwent growth suppression and massive induction of apoptosis without apparent activation of caspase-8 or caspase-2 and late activation of caspase-3. Treatment of infected cells with caspase-9 and caspase-3 inhibitors partially blocked FHIT-induced apoptosis. The two remaining infected acute lymphoblastic leukemia cell lines, Molt-3 and RS4;11, were apparently unaffected. Restoration of FHIT expression in the chronic myelogenous leukemia K562 cell line and the acute myelogenous leukemia KG1a cell line also induced apoptosis but at later time points than seen in the acute lymphoblastic leukemia Jurkat and MV4;11 cell lines. I.v. injection of Ad5/F35-FHIT-infected Jurkat cells resulted in abrogation of tumorigenicity in the NOD/SCID xenogeneic engraftment model. CONCLUSION: FHIT restoration in some FHIT-deficient leukemia cells induces both antiproliferative and proapoptotic effects involving the intrinsic caspase apoptotic pathway.