ABSTRACT
The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.
Subject(s)
Hematopoietic Stem Cells/cytology , Immunotherapy/methods , Leukemia, Myeloid, Acute/therapy , RNA, Guide, Kinetoplastida/genetics , Sialic Acid Binding Ig-like Lectin 3/genetics , T-Lymphocytes/immunology , Animals , Cell Differentiation , Cell Line, Tumor , Cell Lineage , Electroporation , Female , Hematopoiesis , Humans , Leukemia, Myeloid, Acute/immunology , Macaca mulatta , Male , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Neoplasm Transplantation , Reactive Oxygen Species , T-Lymphocytes/cytologyABSTRACT
According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34-CD117+ ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34-CD117+ ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.
Subject(s)
Killer Cells, Natural/immunology , Lymphocytes/immunology , Palatine Tonsil/immunology , Animals , Antigens, CD34/metabolism , CD56 Antigen/metabolism , Cell Differentiation , Cell Lineage , Cells, Cultured , Gene Expression Profiling , Humans , Immunity, Innate , Lymphocyte Activation , Mice , Proto-Oncogene Proteins c-kit/metabolismABSTRACT
Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. In this study, we found that the silencing of protein phosphatase 2A (PP2A) directly blocks differentiation in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling revealed that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent, OSU-2S, in parallel with genetic approaches, we discovered that PP2A enforced c-Myc and p21 dependent terminal differentiation, proliferation arrest, and apoptosis in AML. Finally, we demonstrated that PP2A activation decreased leukemia-initiating stem cells, increased leukemic blast maturation, and improved overall survival in murine Tet2-/-Flt3ITD/WT and human cell-line derived xenograft AML models in vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Leukemia, Myeloid, Acute/metabolism , Protein Phosphatase 2/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Animals , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Knockout , Protein Phosphatase 2/genetics , Proto-Oncogene Proteins c-myc/geneticsABSTRACT
Natural killer (NK)-cells have potent anti-tumor effects, yet it remains unclear if they are effective for patients with relapsed acute myeloid leukemia (AML). In a phase I clinical trial, we treated 12 patients (median age 60 years) with refractory AML (median 5 lines of prior therapy, median bone marrow blast count of 47%) with fludarabine/cytarabine followed by 6 infusions of NK-cells expanded from haploidentical donors using K562 feeder cells expressing membrane-bound IL21 and 4-1BBL. Patients received 106-107/kg/dose. No toxicity or graft-versus-host disease (GVHD) was observed and MTD was not reached. Seven patients (58.3%) responded and achieved a complete remission (CR) with/without count recovery. Median time to best response was 48 days. Five responding patients proceeded to a haploidentical transplant from the same donor. After a median follow-up of 52 months, 1-year overall survival (OS) for the entire group was 41.7%, better for patients who responded with CR/CRi (57.14%), and for patients who responded and underwent transplantation (60%). Persistence and expansion of donor-derived NK-cells were identified in patients' blood, and serum IFNγ levels rose concurrently with NK cell infusions. A higher count-functional inhibitory KIR was associated with higher likelihood of achieving CR/CRi. In conclusion, we observed a significant response to ex vivo expanded NK-cell administration in refractory AML patients without adverse effects.
Subject(s)
Graft vs Host Disease , Leukemia, Myeloid, Acute , Humans , Middle Aged , Killer Cells, Natural/pathology , Graft vs Host Disease/etiology , Cytarabine , HaplotypesABSTRACT
BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 virus-specific cytotoxic T-cell lymphocytes (vCTLs) could provide a promising modality in COVID-19 treatment. We aimed to screen, manufacture, and characterize SARS-CoV-2-vCTLs generated from convalescent COVID-19 donors using the CliniMACS Cytokine Capture System (CCS). METHODS: Donor screening was done by stimulation of convalescent COVID-19 donor peripheral blood mononuclear cells with viral peptides and identification of interferonγ (IFN-γ)+ CD4 and CD8 T cells using flow cytometry. Clinical-grade SARS-CoV-2-vCTLs were manufactured using the CliniMACS CCS. The enriched SARS-CoV-2-vCTLs were characterized by T-cell receptor sequencing, mass cytometry, and transcriptome analysis. RESULTS: Of the convalescent donor blood samples, 93% passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T cells that were 79% (standard error of the mean 21%) IFN-γ+ T cells. SARS-CoV-2-vCTLs displayed a highly diverse T-cell receptor repertoire with enhancement of both memory CD8 and CD4 T cells, especially in CD8 TEM, CD4 TCM, and CD4 TEMRA cell subsets. SARS-CoV-2-vCTLs were polyfunctional with increased gene expression in T-cell function, interleukin, pathogen defense, and tumor necrosis factor superfamily pathways. CONCLUSIONS: Highly functional SARS-CoV-2-vCTLs can be rapidly generated by direct cytokine enrichment (12 hours) from convalescent donors. CLINICAL TRIALS REGISTRATION: NCT04896606.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , T-Lymphocytes, Cytotoxic , Leukocytes, Mononuclear , COVID-19 Drug Treatment , CD8-Positive T-Lymphocytes , CD4-Positive T-Lymphocytes , Cytokines , Interferon-gammaABSTRACT
The neonatal Fc receptor (FcRn) is responsible for recycling of IgG antibodies and albumin throughout the body. This mechanism has been exploited for pharmaceutic delivery across an array of diseases to either enhance or diminish this function. Monoclonal antibodies and albumin-bound nanoparticles are examples of FcRn-dependent anti-cancer therapeutics. Despite its importance in drug delivery, little is known about FcRn expression in circulating immune cells. Through time-of-flight mass cytometry (CyTOF) we were able to characterize FcRn expression in peripheral blood mononuclear cell (PBMC) populations of pancreatic ductal adenocarcinoma (PDAC) patients and non-cancer donors. Furthermore, we were able to replicate these findings in an orthotopic murine model of PDAC. Altogether, we found that in both patients and mice with PDAC, FcRn was elevated in migratory and resident classical dendritic cell type 2 (cDC2) as well as monocytic and granulocytic myeloid-derived suppressor cell (MDSC) populations compared to tumor-free controls. Furthermore, PBMCs from PDAC patients had elevated monocyte, dendritic cells and MDSCs relative to non-cancer donor PBMCs. Future investigations into FcRn activity may further elucidate possible mechanisms of poor efficacy of antibody immunotherapies in patients with PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Albumins , Animals , Histocompatibility Antigens Class I , Leukocytes, Mononuclear/metabolism , Mice , Monocytes/metabolism , Receptors, Fc , Pancreatic NeoplasmsABSTRACT
The identification and discrimination of viable cells is important to understand how experimental variables may influence biochemical processes such as cell metabolism, cell cycle, and signaling pathways. Cisplatin is commonly used as a viability stain in mass cytometry studies, however, recent work by Mei et al. has demonstrated that cisplatin can also be used to label antibodies, complicating the simultaneous use of the platinum measurement channels for both antibody and viability staining. This study demonstrates that other metal salts (hafnium chloride, niobium chloride, and zirconium chloride) can serve as substitutes for cisplatin in viability staining. These stains yield similar fractions of live and dead cells and stain the same dead cells in parallel high parameter analyses. In addition, this study demonstrates how a variety of protein antigen viability markers (pRb, Ki-67, Histone H1, cleaved PARP, and GAPDH) can be used to discriminate live and dead cell populations, without the need for a separate viability staining step. As few as two of these protein antigen viability markers can help identify live and dead cell populations in fixed samples and can identify the same viable cells in high dimensional analyses with or without use of viability stain information. This study demonstrates several alternative approaches to mass cytometry viability assessment that can facilitate use of platinum isotopes for antibody staining and enables identification of live and dead cell populations in samples for which a separate viability stain is not practical.
Subject(s)
Coloring Agents , Cell Count , Staining and LabelingABSTRACT
Activating FLT3 internal tandem duplication (FLT3-ITD) mutations in acute myeloid leukemia (AML) associate with inferior outcomes. We determined that pacritinib, a JAK2/FLT3 inhibitor, has in vitro activity against FLT3-ITD and tyrosine kinase domain (TKD) mutations. Therefore, we conducted a phase I study of pacritinib in combination with chemotherapy in AML patients with FLT3 mutations to determine the pharmacokinetics and preliminary toxicity and clinical activity. Pacritinib was administered at a dose of 100 mg or 200 mg twice daily following a 3 + 3 dose-escalation in combination with cytarabine and daunorubicin (cohort A) or with decitabine induction (cohort B). A total of thirteen patients were enrolled (five in cohort A; eight in cohort B). Dose limiting toxicities include hemolytic anemia and grade 3 QTc prolongation in two patients who received 100 mg. Complete remission was achieved in two patients in cohort A, one of whom had a minor D835Y clone at baseline. One patient in cohort B achieved morphologic leukemia free state. Seven patients (two in cohort A; five in cohort B) had stable disease. In conclusion, pacritinib, an inhibitor of FLT3-ITD and resistant-conferring TKD mutations, was well tolerated and demonstrated preliminary anti-leukemic activity in combination with chemotherapy in patients with FLT3 mutations.
Subject(s)
Antineoplastic Agents/therapeutic use , Bridged-Ring Compounds/therapeutic use , Janus Kinase 2/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Adult , Aged , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bridged-Ring Compounds/adverse effects , Bridged-Ring Compounds/pharmacokinetics , Bridged-Ring Compounds/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cytarabine/adverse effects , Cytarabine/therapeutic use , Daunorubicin/adverse effects , Daunorubicin/therapeutic use , Decitabine/adverse effects , Decitabine/therapeutic use , Drug Resistance, Neoplasm/drug effects , Female , Humans , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/mortality , Male , Middle Aged , Mutation , Pilot Projects , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Pyrimidines/adverse effects , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/metabolismABSTRACT
Epithelial growth factor-like 7 (EGFL7) is a protein that is secreted by endothelial cells and plays an important role in angiogenesis. Although EGFL7 is aberrantly overexpressed in solid tumors, its role in leukemia has not been evaluated. Here, we report that levels of both EGFL7 mRNA and EGFL7 protein are increased in blasts of patients with acute myeloid leukemia (AML) compared with normal bone marrow cells. High EGFL7 mRNA expression associates with lower complete remission rates, and shorter event-free and overall survival in older (age ≥60 y) and younger (age <60 y) patients with cytogenetically normal AML. We further show that AML blasts secrete EGFL7 protein and that higher levels of EGFL7 protein are found in the sera from AML patients than in sera from healthy controls. Treatment of patient AML blasts with recombinant EGFL7 in vitro leads to increases in leukemic blast cell growth and levels of phosphorylated AKT. EGFL7 blockade with an anti-EGFL7 antibody reduced the growth potential and viability of AML cells. Our findings demonstrate that increased EGFL7 expression and secretion is an autocrine mechanism supporting growth of leukemic blasts in patients with AML.
Subject(s)
Endothelial Growth Factors/genetics , Endothelial Growth Factors/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Adult , Age Factors , Aged , Aged, 80 and over , Angiogenic Proteins/antagonists & inhibitors , Angiogenic Proteins/genetics , Angiogenic Proteins/metabolism , Animals , Antibodies, Blocking/pharmacology , Calcium-Binding Proteins , Case-Control Studies , Cell Line, Tumor , Cell Proliferation , Disease-Free Survival , EGF Family of Proteins , Endothelial Growth Factors/antagonists & inhibitors , Female , Humans , Leukemia, Myeloid, Acute/therapy , Male , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Middle Aged , Prognosis , Proteins/metabolism , Proteins/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Risk Factors , Up-Regulation , Young AdultABSTRACT
Mass cytometry (MCM; CyTOF) utilizes isotopically purified metal-tagged antibodies for single-cell analysis and can analyze more than 40 parameters simultaneously with minimum signal spillover to other mass channels as compared to fluorescent flow cytometry. In spite of this improvement, various factors such as metal oxidation, abundance sensitivity related spillover, and metal impurities can cause measurable amounts of spillover in MCM that can potentially lead to misinterpretation of data. Linear spillover can be corrected by applying compensation; however, we demonstrate that at high signal intensities, MCM channel spillovers are frequently nonlinear. This report describes a simple method to correct for nonlinear signal spillover (due to abundance sensitivity, isotopic contamination, or oxide formation) that can occur at high signal intensity through the use of unlabeled competitor antibodies to the specific metal-tagged antibodies causing spillover. This method significantly decreased high signal intensity and nonlinear spillover to other mass channels while maintaining saturating antibody concentrations, thereby facilitating accurate staining and compensation. In contrast, the common method of using under-titrated antibodies to overcome spillover lead to staining intensity that varied with cell numbers and antigen abundance. We demonstrate that this technique reduces total signal without significantly altering immunophenotypic or functional measurement of relative antigen levels and could be used to enable improved linear compensation of signal spillovers from high abundance antigens. STATEMENT OF SIGNIFICANCE: Mass cytometry is becoming a well-established technology for comprehensive analysis of complex biological samples, due to its ability to enable measurement of more than 40 simultaneous parameters. Due to the use of isotopically pure metal-tagged antibodies, measurement channel spillover in mass cytometry is drastically lower than in fluorescent cytometry but can still occur due to metal oxidation, isotopic impurities, or abundance sensitivity when mass signals have high intensity. We show in this report that high abundance antigens with high signal intensity exhibit non-linear mass channel spillovers that cannot be easily compensated. We also demonstrate a simple method for the use of unlabeled competitor antibody to decrease antigen signal intensity while maintaining antigen abundance to allow for more accurate linear compensation. This method performs more consistently than the commonly used approach of using under-titrated antibodies. We believe that this report has immediate practical utility for researchers using mass cytometry and can be broadly utilized to enable compensation of mass cytometry data when needed. We thus feel that this article merits publication as a Brief Report in Cytometry Part A. © 2019 International Society for Advancement of Cytometry.
Subject(s)
Antibodies/immunology , Flow Cytometry/methods , Immunophenotyping/methods , Single-Cell Analysis/methods , Antibodies/pharmacology , Humans , Immunoconjugates/pharmacology , Mass Spectrometry/methodsABSTRACT
Cell cycle analysis is a recognized and important application of flow cytometry and, more recently, mass cytometry (MCM). Both technologies have been utilized for analysis of the cell cycle state of ex vivo samples from patients with hematologic malignancies. Clinical samples are frequently stored for hours at room temperature or cryogenically frozen before processing and analysis; however, how these processing methods alter cell cycle state is not well described. To understand how storage time and temperature affect the analysis of cell cycle distribution by MCM, two leukemia cell lines, HL-60 and MOLM13, and primary human cells from three human bone marrow aspirates were stored and frozen under a variety of conditions that are likely to be encountered in a clinical setting. Our findings indicate that short delays in sample processing (less than 1 h), have little to no effect on cell cycle distribution, while longer delays or cryopreservation cause significant disruptions to the cell cycle fraction characterized by consistent reductions in IdU incorporation and variable alterations in other cell cycle phases. Analysis of the recovery of cryopreserved leukemia cell lines and marrow cells demonstrated that cell cycle alterations persist for at least 48 h after thawing. Our findings demonstrate that accurate cell cycle analysis requires that samples be processed rapidly after collection, and that cryopreservation significantly alters cell cycle fractions. Measurement of IdU incorporation was the most sensitive to both delays in processing and cryopreservation, while estimation of the total cycling cell fraction using Ki-67 or phosphorylated retinoblastoma protein were least altered by the conditions tested. These findings provide guidance for the ideal approach to collection of samples for cell cycle analysis and can aid interpretation of cell cycle data from samples that cannot be collected under ideal circumstances. © 2018 International Society for Advancement of Cytometry.
Subject(s)
Cell Cycle/physiology , Flow Cytometry/methods , Cell Line, Tumor , Cryopreservation/methods , HL-60 Cells , Humans , Ki-67 Antigen/metabolism , Leukemia/metabolism , Leukemia/pathology , Retinoblastoma Protein/metabolism , TemperatureSubject(s)
Leukemia, Myeloid/genetics , Neoplasms, Second Primary/genetics , Antineoplastic Agents/adverse effects , DNA Damage/drug effects , Humans , Leukemia, Myeloid/chemically induced , Leukemia, Myeloid/etiology , Mutation/drug effects , Mutation Rate , Neoplasms, Second Primary/chemically induced , Neoplasms, Second Primary/etiology , Risk Factors , Smoking/adverse effectsABSTRACT
Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. (3)H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS.
Subject(s)
RNA Polymerase I/metabolism , RNA, Ribosomal/genetics , RecQ Helicases/metabolism , Transcription, Genetic , Cell Line , Cell Line, Tumor , Cell Nucleolus/metabolism , Cell Nucleus/metabolism , DNA/chemistry , DNA/metabolism , DNA, Ribosomal/chemistry , DNA, Ribosomal/metabolism , Dactinomycin/pharmacology , Humans , Nucleic Acid Conformation , Protein Binding , Protein Subunits/metabolism , RNA Polymerase I/antagonists & inhibitors , RecQ Helicases/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression.
Subject(s)
Cytophotometry/methods , High-Throughput Screening Assays/methods , Optical Imaging/methods , Saponins , Humans , Jurkat Cells , U937 CellsABSTRACT
Low-grade glioma (LGG) is the most common brain tumor affecting pediatric patients (pLGG) and BRAF mutations constitute the most frequent genetic alterations. Within the spectrum of pLGGs, approximately 70%-80% of pediatric patients diagnosed with transforming pleomorphic xanthoastrocytoma (PXA) harbor the BRAF V600E mutation. However, the impact of glioma BRAF V600E cell regulation of tumor-infiltrating immune cells and their contribution to tumor progression remains unclear. Moreover, the efficacy of BRAF inhibitors in treating pLGGs is limited compared with their impact on BRAF-mutated melanoma. Here we report a novel immunocompetent RCAS-BRAF V600E murine glioma model. Pathological assessment indicates this model seems to be consistent with diffuse gliomas and morphological features of PXA. Our investigations revealed distinct immune cell signatures associated with increased trafficking and activation within the tumor microenvironment (TME). Intriguingly, immune system activation within the TME also generated a pronounced inflammatory response associated with dysfunctional CD8+ T cells, increased presence of immunosuppressive myeloid cells and regulatory T cells. Further, our data suggests tumor-induced inflammatory processes, such as cytokine storm. These findings suggest a complex interplay between tumor progression and the robust inflammatory response within the TME in preclinical BRAF V600E LGGs, which may significantly influence animal survival.
ABSTRACT
Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD 1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.
ABSTRACT
The prognosis for children with recurrent and/or refractory neuroblastoma (NB) is dismal. The receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is highly expressed on the surface of NB cells, provides a potential target for novel immunotherapeutics. Anti-ROR1 chimeric antigen receptor engineered ex vivo expanded peripheral blood natural killer (anti-ROR1 CAR exPBNK) cells represent this approach. N-803 is an IL-15 superagonist with enhanced biological activity. In this study, we investigated the in vitro and in vivo anti-tumor effects of anti-ROR1 CAR exPBNK cells with or without N-803 against ROR1+ NB models. Compared to mock exPBNK cells, anti-ROR1 CAR exPBNK cells had significantly enhanced cytotoxicity against ROR1+ NB cells, and N-803 further increased cytotoxicity. High-dimensional analysis revealed that N-803 enhanced Stat5 phosphorylation and Ki67 levels in both exPBNK and anti-ROR1 CAR exPBNK cells with or without NB cells. In vivo, anti-ROR1 CAR exPBNK plus N-803 significantly (p < 0.05) enhanced survival in human ROR1+ NB xenografted NSG mice compared to anti-ROR1 CAR exPBNK alone. Our results provide the rationale for further development of anti-ROR1 CAR exPBNK cells plus N-803 as a novel combination immunotherapeutic for patients with recurrent and/or refractory ROR1+ NB.
ABSTRACT
Cellular senescence is a durable cell cycle arrest as a result of the finite proliferative capacity of cells. Senescence responds to both intrinsic and extrinsic cellular stresses, such as aging, mitochondrial dysfunction, irradiation, and chemotherapy. Here, we report on the use of mass cytometry (MC) to analyze multiple model systems and demonstrate MC as a platform for senescence analysis at the single-cell level. We demonstrate changes to p16 expression, cell cycling fraction, and histone tail modifications in several established senescent model systems and using isolated human T cells. In bone marrow mesenchymal stromal cells (BMSCs), we show increased p16 expression with subsequent passage as well as a reduction in cycling cells and open chromatin marks. In WI-38 cells, we demonstrate increased p16 expression with both culture-induced senescence and oxidative stress-induced senescence (OSIS). We also use Wanderlust, a trajectory analysis tool, to demonstrate how p16 expression changes with histone tail modifications and cell cycle proteins. Finally, we demonstrate that repetitive stimulation of human T cells with CD3/CD28 beads induces an exhausted phenotype with increased p16 expression. This p16-expressing population exhibited higher expression of exhaustion markers such as EOMES and TOX. This work demonstrates that MC is a useful platform for studying senescence at a single-cell protein level, and is capable of measuring multiple markers of senescence at once with high confidence, thereby improving our understanding of senescent pathways.
Subject(s)
Histones , Research , Humans , Aging , CD28 Antigens , Cell CycleABSTRACT
Gliomas are one of the leading causes of cancer-related death in the adolescent and young adult (AYA) population. Two-thirds of AYA glioma patients are affected by low-grade gliomas (LGGs), but there are no specific treatments. Malignant progression is supported by the immunosuppressive stromal component of the tumor microenvironment (TME) exacerbated by M2 macrophages and a paucity of cytotoxic T cells. A single intravenous dose of engineered bone-marrow-derived myeloid cells that release interleukin-2 (GEMys-IL2) was used to treat mice with LGGs. Our results demonstrate that GEMys-IL2 crossed the blood-brain barrier, infiltrated the TME, and reprogrammed the immune cell composition and transcriptome. Moreover, GEMys-IL2 extended survival in an LGG immunocompetent mouse model. Here, we report the efficacy of an in vivo approach that demonstrates the potential for a cell-mediated innate immunotherapy designed to enhance the recruitment of activated effector T and natural killer cells within the glioma TME.