ABSTRACT
Clinical trials of CD19-specific chimeric antigen receptor (CAR19) T cells have demonstrated remarkable efficacy against relapsed and refractory B cell malignancies. The piggyBac transposon system offers a less complex and more economical means for generating CAR19 T cells compared to viral vectors. We have previously optimized a protocol for the generation of CAR19 T cells using the piggyBac system, but we found that CAR19 T cells had poor in vivo efficacy and persistence, probably due to deleterious FcγR interactions with the CAR's IgG1 Fc-containing spacer domain. We therefore designed three CD19-specifc CARs that lacked the IgG1 Fc region, and we incorporated combinations of CD28 or 4-1BB transmembrane and co-stimulatory domains. PiggyBac-generated CAR19 T cells expressing these re-designed constructs all demonstrated reactivity in vitro specifically against CD19+ cell lines. However, those combining CD28 transmembrane and co-stimulatory domains showed CD4 predominance and inferior cytotoxicity. At high doses, CAR19 T cells were effective against B-ALL in a xenograft mouse model, regardless of co-stimulatory domain. At diminishing doses, 4-1BB co-stimulation led to greater potency and persistence of CAR19 T cells, and it provided protection against B-ALL re-challenge. Production of potent CAR T cells using piggyBac is simple and cost-effective, and it may enable wider access to CAR T cell therapy.
Subject(s)
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Receptors, Antigen, T-Cell/metabolism , Receptors, IgG/genetics , T-Lymphocytes/transplantation , Animals , Cell Line, Tumor , DNA Transposable Elements , Humans , Immunotherapy, Adoptive/methods , Jurkat Cells , K562 Cells , Mice , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
High-risk childhood leukemia has a poor prognosis because of treatment failure and toxic side effects of therapy. Drug encapsulation into liposomal nanocarriers has shown clinical success at improving biodistribution and tolerability of chemotherapy. However, enhancements in drug efficacy have been limited because of a lack of selectivity of the liposomal formulations for the cancer cells. Here, we report on the generation of bispecific antibodies (BsAbs) with dual binding to a leukemic cell receptor, such as CD19, CD20, CD22, or CD38, and methoxy polyethylene glycol (PEG) for the targeted delivery of PEGylated liposomal drugs to leukemia cells. This liposome targeting system follows a "mix-and-match" principle where BsAbs were selected on the specific receptors expressed on leukemia cells. BsAbs improved the targeting and cytotoxic activity of a clinically approved and low-toxic PEGylated liposomal formulation of doxorubicin (Caelyx) toward leukemia cell lines and patient-derived samples that are immunophenotypically heterogeneous and representative of high-risk subtypes of childhood leukemia. BsAb-assisted improvements in leukemia cell targeting and cytotoxic potency of Caelyx correlated with receptor expression and were minimally detrimental in vitro and in vivo toward expansion and functionality of normal peripheral blood mononuclear cells and hematopoietic progenitors. Targeted delivery of Caelyx using BsAbs further enhanced leukemia suppression while reducing drug accumulation in the heart and kidneys and extended overall survival in patient-derived xenograft models of high-risk childhood leukemia. Our methodology using BsAbs therefore represents an attractive targeting platform to potentiate the therapeutic efficacy and safety of liposomal drugs for improved treatment of high-risk leukemia.
Subject(s)
Antibodies, Bispecific , Antineoplastic Agents , Leukemia , Humans , Antibodies, Bispecific/therapeutic use , Tissue Distribution , Leukocytes, Mononuclear , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Antineoplastic Agents/therapeutic use , Polyethylene Glycols , Liposomes , Leukemia/drug therapyABSTRACT
Glycogen synthase kinase-3ß (GSK-3ß) has been identified as an important regulator of stem cell function acting through activation of the wingless (Wnt) pathway. Here, we report that treatment with an inhibitor of GSK-3ß, 6-bromoindirubin 3'-oxime (BIO) delayed cell cycle progression by increasing cell cycle time. BIO treatment resulted in the accumulation of late dividing cells enriched with primitive progenitor cells retaining the ability for sustained proliferation. In vivo analysis using a Non-obese diabetic/severe combined immunodeficient (NOD/SCID) transplantation model has demonstrated that pretreatment with BIO promotes engraftment of ex vivo-expanded hematopoietic stem cells. BIO enhanced the engraftment of myeloid, lymphoid and primitive stem cell compartments. Limiting dilution analysis of SCID repopulating cells (SRC) revealed that BIO treatment increased human chimerism without increasing SRC frequency. Clonogenic analysis of human cells derived from the bone marrow of transplant recipient mice demonstrated that a higher level of human chimerism and cellularity was related to increased regeneration per SRC unit. Gene expression analysis showed that treatment with BIO did not modulate the expression of canonical Wnt target genes upregulated during cytokine-induced cell proliferation. BIO increased the expression of several genes regulating Notch and Tie2 signaling downregulated during ex vivo expansion, suggesting a role in improving stem cell engraftment. In addition, treatment with BIO upregulated CDK inhibitor p57 and downregulated cyclin D1, providing a possible mechanism for the delay seen in cell cycle progression. We conclude that transient, pharmacologic inhibition of GSK-3ß provides a novel approach to improve engraftment of expanded HSC after stem cell transplantation.
Subject(s)
Gene Expression/drug effects , Glycogen Synthase Kinase 3/antagonists & inhibitors , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/drug effects , Indoles/pharmacology , Oximes/pharmacology , Wnt Proteins/metabolism , Animals , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Proliferation/drug effects , Cells, Cultured , Chimerism/drug effects , Cyclin D1/metabolism , Cyclin-Dependent Kinase Inhibitor p57/metabolism , Fetal Blood/cytology , Flow Cytometry , Glycogen Synthase Kinase 3 beta , Hematopoietic Stem Cells/enzymology , Hematopoietic Stem Cells/physiology , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Signal Transduction/geneticsABSTRACT
Adoptive cell therapy using patient-derived chimeric receptor antigen (CAR) T cells redirected against tumor cells has shown remarkable success in treating hematologic cancers. However, wider accessibility of cellular therapies for all patients is needed. Manufacture of patient-derived CAR T cells is limited by prolonged lymphopenia in heavily pre-treated patients and risk of contamination with tumor cells when isolating T cells from patient blood rich in malignant blasts. Donor T cells provide a good source of immune cells for adoptive immunotherapy and can be used to generate universal off-the-shelf CAR T cells that are readily available for administration into patients as required. Genome editing tools such as TALENs and CRISPR-Cas9 and non-gene editing methods such as short hairpin RNA and blockade of protein expression are currently used to enhance CAR T cell safety and efficacy by abrogating non-specific toxicity in the form of graft versus host disease (GVHD) and preventing CAR T cell rejection by the host.
ABSTRACT
Chimeric antigen receptor (CAR) T cells targeting CD19 have demonstrated remarkable efficacy in the treatment of B cell malignancies. Current CAR T cell manufacturing protocols are complex and costly due to their reliance on viral vectors. Non-viral systems of genetic modification, such as with transposase and transposon systems, offer a potential streamlined alternative for CAR T cell manufacture and are currently being evaluated in clinical trials. In this study, we utilized the previously described transposase from the little brown bat, designated piggyBat, for production of CD19-specific CAR T cells. PiggyBat demonstrates efficient CAR transgene delivery, with a relatively low variability in integration copy number across a range of manufacturing conditions as well as a similar integration site profile to super-piggyBac transposon and viral vectors. PiggyBat-generated CAR T cells demonstrate CD19-specific cytotoxic efficacy in vitro and in vivo. These data demonstrate that alternative, naturally occurring DNA transposons can be efficiently re-tooled to be exploited in real-world applications.
ABSTRACT
PURPOSE: Despite the success of chimeric antigen receptor (CAR) T cells in clinical studies, a significant proportion of responding patients eventually relapsed, with the latter correlating with low CAR T cell expansion and persistence. METHODS AND RESULTS: Using patient-derived xenograft (PDX) mouse models of CD19+ B cell acute lymphoblastic leukemia (B-ALL), we show that priming leukemia-bearing mice with 5-azacytidine (AZA) enhances CAR T cell therapy. AZA given 1 day prior to CAR T cell infusion delayed leukemia growth and promoted CAR T cell expansion and effector function. Priming leukemia cells with AZA increased CAR T cell/target cell conjugation and target cell killing, promoted CAR T cell divisions and expanded IFNγ+ effector T cells in co-cultures with CD19+ leukemia Nalm-6 and Raji cells. Transcriptome analysis revealed activation of diverse immune pathways in leukemia cells isolated from mice treated with AZA. We propose that epigenetic priming with AZA induces transcriptional changes that sensitize tumor cells to subsequent CAR T cell treatment. Among the candidate genes up-regulated by AZA is TNFSF4 which encodes OX40L, one of the strongest T cell co-stimulatory ligands. OX40L binds OX40, the TNF receptor superfamily member highly specific for activated T cells. TNFSF4 is heterogeneously expressed in a panel of pediatric PDXs, and high TNFSF4 expression correlated with increased CAR T cell numbers identified in co-cultures with individual PDXs. High OX40L expression in Nalm-6 cells increased their susceptibility to CAR T cell killing while OX40L blockade reduced leukemia cell killing. CONCLUSION: We propose that treatment with AZA activates OX40L/OX40 co-stimulatory signaling in CAR T cells. Our data suggest that the clinical use of AZA before CAR T cells could be considered.
ABSTRACT
PURPOSE: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. EXPERIMENTAL DESIGN: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. RESULTS: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. CONCLUSIONS: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.
Subject(s)
Acetanilides/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Gene Rearrangement , Heterocyclic Compounds, 3-Ring/pharmacology , Molecular Targeted Therapy/methods , Neuroblastoma/drug therapy , Oligopeptides/pharmacology , Telomerase/genetics , Transcription Factors/antagonists & inhibitors , Animals , Apoptosis , Cell Proliferation , Drug Therapy, Combination , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neuroblastoma/metabolism , Neuroblastoma/pathology , Proteasome Inhibitors/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Ex vivo expansion of cord blood cells generally results in reduced stem cell activity in vivo. Glycogen synthase kinase-3beta (GSK-3beta) regulates the degradation of beta-catenin, a critical regulator of hematopoietic stem cells (HSCs). Here we show that GSK-3beta inhibition activates beta-catenin in cord blood CD34(+) cells and upregulates beta-catenin transcriptional targets c-myc and HoxB4, both known to regulate HSC self-renewal. GSK-3beta inhibition resulted in delayed ex vivo expansion of CD34(+) cells, yet enhanced the preservation of stem cell activity as tested in long-term culture with bone marrow stroma. Delayed cell cycling, reduced apoptosis, and increased adherence of hematopoietic progenitor cells to bone marrow stroma were observed in these long-term cultures treated with GSK-3beta inhibitor. This improved adherence to stroma was mediated via upregulation of CXCR4. In addition, GSK-3beta inhibition preserved severe combined immunodeficiency (SCID) repopulating cells as tested in the nonobese diabetic/SCID mouse model. Our data suggest the involvement of GSK-3beta inhibition in the preservation of HSC and their interaction with the bone marrow environment. Methods for the inhibition of GSK-3beta may be developed for clinical ex vivo expansion of HSC for transplantation. In addition, GSK-3beta inhibition suppressed leukemic cell growth via the induction of apoptosis mediated by the downregulation of survivin. Modulators of GSK-3beta may increase the range of novel drugs that specifically kill leukemic cells while sparing normal stem cells.
Subject(s)
Glycogen Synthase Kinase 3/antagonists & inhibitors , Hematopoietic Stem Cells/enzymology , Leukemia/enzymology , Leukemia/pathology , Animals , Antigens, CD34/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Coculture Techniques , Cord Blood Stem Cell Transplantation , Disease Models, Animal , Glycogen Synthase Kinase 3 beta , Hematopoietic Stem Cells/drug effects , Humans , Indoles/pharmacology , Mice , Mice, SCID , Oximes/pharmacology , Stromal Cells/drug effects , Stromal Cells/metabolism , Time Factors , Umbilical Cord/cytology , Umbilical Cord/drug effects , Umbilical Cord/enzymology , beta Catenin/metabolismABSTRACT
Interferon regulatory factor (IRF) 1 and its functional antagonist IRF2 were originally discovered as transcription factors that regulate the interferon-beta gene. Control of cell growth has led to the definition of IRF1 as a tumour suppressor gene and IRF2 as an oncogene. Clinically, approximately 70% of cases of acute myeloid leukaemia demonstrate dysregulated expression of IRF1 and/or IRF2. Our previous studies have shown that human leukaemic TF-1 cells exhibit abnormally high expression of both IRF1 and IRF2, the latter acting to abrogate IRF1 tumour suppression, making these cells ideal for analysis of down-regulation of IRF2 expression. A novel G418 screening protocol was developed and used for identifying effective siRNA that targets IRF2 (siIRF2). Using optimized siIRF2 in leukaemic TF-1 cells, IRF2 was down-regulated by approximately 70% at both mRNA and protein levels. Phenotypically, this resulted in growth inhibition associated with G2/M arrest as well as induction of polyploidy, differentiation and apoptosis. In contrast to these results, siIRF2 targeting did not affect normal haematopoietic stem/progenitor cell growth. These results indicate the potential utility of IRF2 inhibition as a therapeutic approach to cancer.
Subject(s)
Interferon Regulatory Factor-2/antagonists & inhibitors , Leukemia/therapy , RNA, Small Interfering/genetics , Antigens, CD34/analysis , Cell Cycle , Cell Line, Tumor , Hematopoiesis , Humans , Interferon Regulatory Factor-2/genetics , Leukemia/pathology , Lipopolysaccharide Receptors/analysisABSTRACT
OBJECTIVES: Mutations in ras oncogenes occur at high frequency in acute myeloid leukemia and myelodysplastic syndromes; however, the role of ras genes in leukemogenesis has not been clearly defined. Our previous studies have shown that expression of mutant N-ras (N-rasG13R, G to C transversion) in human hematopoietic progenitor cells (HPC) promotes myeloid differentiation and proliferation both in vitro and in a NOD/SCID mouse model. In the present study, we performed expression profiling to identify the transcriptome induced by N-rasG13R in human HPC, and analyzed the effect of mutant N-ras in sorted specific subpopulations of HPC. METHODS: cDNA microarray analysis was performed on cord blood CD34(+) cells transduced with a retrovirus containing GFP alone or in combination with mutant N-ras. Transduced cells were also sorted into factorial subpopulations according to CD34 and transgene expression, and analyzed in suspension or semi-solid methylcellulose culture. RESULTS: Among a variety of changes, including upregulation of cytokine genes, we found that N-rasG13R induced expression of the cyclin-dependent kinase inhibitors p16(INK4a) and p21(CIP1/WAF1). Analysis by RT-PCR revealed that increased p16(INK4a) and p21(CIP1/WAF1) occurred in the most primitive, CD34(+)/Ras(+) population but not in the more mature CD34(-)/Ras(+) cells or in the CD34(+)/Ras(-) cells. Moreover, N-rasG13R inhibited the proliferation of the primitive CD34(+)/Ras(+) cells, both in liquid culture and in colony assays. This growth suppression correlated with an increased proportion of myelomonocytic colonies and a decrease of erythroid colonies. In contrast, the growth of CD34(-)/Ras(+) cells and CD34(+)/Ras(-) HPC was not inhibited. CONCLUSIONS: These findings demonstrated the mutant N-ras induced transcriptome, and that this is associated with HPC growth suppression/myelomonocytic differentiation, and identify upregulation of cyclin inhibitors as key events in this process. The results indicate that ras mutation alone is not sufficient to induce leukemogenesis; collaborative secondary event(s) are involved in the process.
Subject(s)
Cell Differentiation/genetics , Cell Transformation, Neoplastic/genetics , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Genes, ras/genetics , Myeloid Progenitor Cells/metabolism , Up-Regulation/genetics , Animals , Antigens, CD34/biosynthesis , Cell Proliferation , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cytokines/biosynthesis , Cytokines/genetics , Gene Expression Regulation, Leukemic/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Mutation, Missense , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Myelodysplastic Syndromes/pathology , Myeloid Progenitor Cells/pathologyABSTRACT
Activating mutations in ras oncogenes occur at high frequency in human malignancies and expression of activated ras in immortalized cells lines is generally transforming. However, somewhat paradoxically, ectopic expression of ras in some myeloid cell lines has been shown to induce growth suppression associated with up-regulation of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in a p16(INK4a), p15(INK4b), and p53 independent fashion. We have used cDNA array technology to compare the expression profile induced by activated N-ras (N-rasG13R) in growth-suppressed myeloid cells with that induced in myeloid cells, which are transformed by N-rasG13R. The expression profile induced in growth suppressed cells was consistent with differentiation and included the up-regulation of the transcription factor IFN regulatory factor-1 (IRF-1), a known transcriptional activator of p21(CIP/WAF1) expression and a target of oncogenic mutations associated with myeloid leukemia. Antisense suppression of IRF-1 prevented N-rasG13R-associated growth arrest and up-regulation of p21(CIP1/WAF1). These results define a novel tumor suppressive response to oncogenic signaling and provide a mechanistic link between growth suppression and differentiation in myeloid cells.
Subject(s)
DNA-Binding Proteins/physiology , Genes, ras/physiology , Myeloid Cells/physiology , Phosphoproteins/physiology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/genetics , Cell Growth Processes/physiology , Cyclin-Dependent Kinase Inhibitor p21 , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Disease Progression , Gene Expression Profiling , Humans , Interferon Regulatory Factor-1 , K562 Cells , Myeloid Cells/cytology , Oligonucleotide Array Sequence Analysis , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , U937 Cells , Up-RegulationABSTRACT
Umbilical cord blood (UCB) transplantation can provide a successful therapeutic option for patients that have no suitable related donor. UCB transplantation is often limited by the relatively small hematopoietic stem cell (HSC) numbers in UCB especially for adult recipients. Early neutrophil and platelet engraftment correlates with the stem cell numbers in UCB transplant. Compared to other HSC sources, immune reconstitution following UCB transplant is slower and complicated by increased frequency of opportunistic infections. The effect of HSC numbers in UCB transplant on immune reconstitution was not thoroughly examined. Using immunocompromised mice transplanted with purified UCB CD34+ stem cells, we have demonstrated that increasing the numbers of CD34+ cells in the transplant promotes hematopoietic and immune reconstitution. At early stages posttransplant, high stem cell dose generated relatively more B cells, while lower dose generated more myeloid and T cells. Thus, the size of the stem cell graft appears to modulate the differentiation potential of infused stem cells. In addition, increasing stem cell dose in the transplant improved CD8+ T cell development and delayed late memory T cell skewing in expense of naive T cells highlighting the importance of HSC dose to maintain the pool of naive T cells able to develop strong immune responses. Transplantation of ex vivo expanded CD34+ cells did not promote, but rather delayed immune reconstitution suggesting the loss of primitive lymphoid precursor cells during ex vivo expansion.
Subject(s)
Cell Differentiation/physiology , Fetal Blood/cytology , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Animals , Antigens, CD34/immunology , Cell Culture Techniques/methods , Cord Blood Stem Cell Transplantation , Hematopoietic Stem Cell Transplantation/methods , Humans , Mice , T-Lymphocytes/cytology , Transplantation, Homologous/methodsABSTRACT
The efficient use of hematopoietic stem cells (HSC) for transplantation is often limited by the relatively low numbers of HSC collected. The ex vivo expansion of HSC for clinical use is a potentially valuable and safe approach to increase HSC numbers thereby increasing engraftment and reducing the risk of morbidity from infection. Here, we describe a protocol for the robust ex vivo expansion of human CD34(+) HSC isolated from umbilical cord blood. The protocol described can efficiently generate large numbers of HSC. We also describe a flow cytometry-based method using high-resolution division tracking to characterize the kinetics of HSC growth and differentiation. Utilizing the guidelines discussed, it is possible for investigators to use this protocol as presented or to modify it for their specific needs.
Subject(s)
Cell Culture Techniques/methods , Hematopoietic Stem Cell Transplantation/methods , Animals , Antigens, CD34/metabolism , Cell Differentiation/genetics , Cell Differentiation/physiology , Fetal Blood/cytology , Flow Cytometry , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/physiology , HumansABSTRACT
Activating mutations of the N-ras gene occur at relatively high frequency in acute myeloid leukemia and myelodysplastic syndrome. Somewhat paradoxically, ectopic expression of activated N-ras in primary hematopoietic cells and myeloid cell lines (in some cases) can lead to inhibition of proliferation. Expression of mutant N-ras in murine hematopoietic stem/progenitor cells is sufficient to induce myeloid malignancies, but these pathologies occur with long latency. This suggests that mutations that disable the growth suppressive properties of N-ras in hematopoietic cells are required for the development of frank malignancy. In the present work, the growth suppression induced by a mutant N-ras gene in U937 myeloid cells was used as the basis to screen a retroviral cDNA library for genes that prevent mutant N-ras-induced growth suppression (i.e., putative cooperating oncogenes). This screen identified the gene for the transcription factor interferon regulatory factor-2 (IRF-2), and as confirmation of the screen, overexpression of this gene in U937 cells was shown to inhibit mutant N-ras-induced growth suppression. Also recovered from the screen were two truncated clones of an uncharacterized gene (interim official symbol: PP2135). Overexpression of this truncated PP2135 gene in U937 cells did not appear to abrogate mutant N-ras-induced growth suppression, but rather appeared to confer an increased sensitivity of U937 cells to retroviral infection, accounting for the recovery of this gene from the genetic screen.
Subject(s)
Cell Proliferation , Genes, ras/physiology , Interferon Regulatory Factor-2/physiology , Mutation/genetics , Retroviridae/genetics , Blotting, Northern , Gene Expression , Gene Library , Humans , Polymerase Chain Reaction , U937 Cells/cytology , U937 Cells/metabolismABSTRACT
Acute myeloid leukaemia (AML) is the most common form of leukaemia in adults. Although of the order of 75-85% of patients will achieve complete remission after induction chemotherapy, long-term survival is still relatively low. Despite the progress in the rational design of drugs in disorders such as chronic myeloid leukaemia, AML lacks a single specific pathogenomic event to act as a drug target. Interferon regulatory factor 1 (IRF1) is a member of a family of related proteins that act as transcriptional activators or repressors. IRF1 and its functional antagonist IRF2 originally discovered as transcription factors regulating the interferon-beta (IFN-beta) gene, are involved in the regulation of normal haematopoiesis and leukaemogenesis. IRF1 appears to act as a tumour suppressor gene and IRF2 as an oncogene. IRF1 acts to repress IRF2 function through the repression of cyclin-dependent kinase (CDK) inhibitor p21WAF1 critical for cell growth control. It appears that the tumour suppression function of IRF1 is abolished by IRF2. This review focuses on the interaction between IRF1 and IRF2 in myeloid development and leukaemogenesis, particularly in relation to the Ras signalling pathway. IRF2 may be a viable and specific therapeutic target in human leukaemia.
Subject(s)
Interferon Regulatory Factor-1/physiology , Interferon Regulatory Factor-2/physiology , Leukemia, Myeloid/therapy , Acute Disease , Animals , Humans , Interferon Regulatory Factor-1/genetics , Interferon Regulatory Factor-2/genetics , Leukemia, Myeloid/genetics , Leukemia, Myeloid/metabolism , Mice , Models, Animal , Phenotype , Signal Transduction , ras Proteins/genetics , ras Proteins/metabolismABSTRACT
OBJECTIVES: Ras oncogene mutations are the most frequently observed genetic abnormality (20-40% of patients) in acute myeloid leukemia (AML), and in the preleukemic conditions myelodysplastic syndrome (MDS) and myeloproliferative disorder (MPD). We have previously shown that mutant N-ras (N-rasm) can induce myeloproliferative disorders and apoptosis in a murine reconstitution system. In the present study we investigated the effect of N-rasm in human primary hematopoietic progenitor cells (HPC). METHODS: Cord blood CD34+ hematopoietic progenitor cells (HPC) were transduced with retroviral vectors containing green fluorescence protein (GFP) alone, or in combination with N-rasm. Cells were then cultured in vitro with a cytokine supplement or cocultured with murine stroma MS-5 cells. The in vivo behavior of transduced cells was examined in the NOD/SCID mouse model. RESULTS: N-rasm-transduced cells exhibited greater proliferative capacity; a higher frequency of granulocyte-macrophage colony-forming unit (CFU-GM); and an increase in myelomonocytic lineage cells with a concomitant decrease in lymphoid and erythroid cells. Analysis of transduced HPC in NOD/SCID mice revealed higher bone marrow engraftment by N-rasm HPC and increased numbers of myeloid lineage cells. CONCLUSIONS: The results demonstrate that N-rasm in HPC induces myeloproliferation both in vitro and in the NOD/SCID mouse model as a primary event that does not appear to be dependent on cooperating transforming events.
Subject(s)
Antigens, CD34/analysis , Genes, ras/physiology , Hematopoietic Stem Cells/cytology , Myeloid Cells/cytology , Animals , Cell Differentiation , Cell Division , Cell Lineage , Humans , Mice , Mice, Inbred NOD , Mice, SCIDABSTRACT
Small-molecule inhibitors of glycogen synthase kinase 3ß (GSK3ß) have demonstrated strong anti-leukemia effects in preclinical studies. Here, we investigated the effect of GSK3ß inhibitor 6-Bromoindirubin-3-oxime (BIO) previously shown to inhibit leukemia cell growth in vitro and of animal models on hematopoietic regeneration in recipients of stem cell transplant. BIO administered to immunocompromised mice transplanted with human hematopoietic stem cells inhibited human stem cell engraftment in the bone marrow (BM) and peripheral blood. BIO reduced CD34(+) progenitor cells in the BM, and primitive lymphoid progenitors re-populated host thymus at later stages post-transplant. The development of all T-cell subsets in the thymus was suppressed in BIO-treated mice. Human cell engraftment was gradually restored after discontinuation of BIO treatment; however, T-cell depletion remained until the end of experiment, which correlated with the attenuated thymic function in the host. BIO delayed CD34(+) cell expansion in stroma-supported or cytokine-only cultures. BIO treatment delayed progenitor cell divisions and induced apoptosis in cultures with sub-optimal cytokine support. In addition, BIO inhibited B- and T-cell development in co-cultures with MS5 and OP9-DL1 BM stroma cells, respectively. These data suggest that administration of GKS3ß inhibitors may act to delay hematopoietic regeneration in patients who received stem cell transplant.
Subject(s)
Glycogen Synthase Kinase 3/antagonists & inhibitors , Hematopoiesis , Hematopoietic Stem Cells/drug effects , Indoles/pharmacology , Oximes/pharmacology , Animals , Apoptosis , Cells, Cultured , Glycogen Synthase Kinase 3 beta , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Humans , Mice , Mice, Inbred NOD , Mice, SCIDABSTRACT
Adoptive therapy with chimeric antigen receptor (CAR) T cells (CART cells) has exhibited great promise in clinical trials, with efficient response correlated with CART-cell expansion and persistence. Despite extensive clinical use, the mechanisms regulating CART-cell expansion and persistence have not been completely elucidated. We have examined the antileukemia potency of CART cells targeting CD19 antigen using second-generation CAR containing a CD28 co-stimulatory domain cloned into piggyBac-transposon vector and patient-derived chemoresistant pediatric acute lymphoblastic leukemia samples. In the presence of large numbers of target cells characteristic of patients with high leukemia burden, excessive proliferation of CART cells leads to differentiation into short-lived effector cells. Transient leukemia growth delay was induced by CART-cell infusion in mice xenografted with rapidly growing CD19+ acute lymphoblastic leukemia cells and was followed by rapid CART-cell extinction. Conditioning with the hypomethylating agent 5-aza-2'-deoxycytidine-activating caspase 3 and promotion of apoptosis in leukemia cells maximized the effect of CART cells and improved CART-cell persistence. These data suggest that the clinical use of 5-aza-2'-deoxycytidine before CART cells could be considered. Coculture of leukemia cells with bone marrow stroma cells reduced target cell loss, suggesting that leukemia cell mobilization into circulation may help to remove the protective effect of bone marrow stroma and increase the efficacy of CART-cell therapy.
Subject(s)
Antigens, CD19/immunology , Drug Resistance, Neoplasm/immunology , Immunotherapy, Adoptive , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes , Animals , Child , Child, Preschool , Female , Heterografts , Humans , Male , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Neoplasm Transplantation , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , T-Lymphocytes/immunology , T-Lymphocytes/pathology , T-Lymphocytes/transplantation , Xenograft Model Antitumor AssaysABSTRACT
We have developed a novel dual-fluorescence reporter system incorporating green (GFP) and red (RFP) fluorescent proteins to monitor expression of the N-ras(m) gene and an N-ras(m) suppressor, respectively. Retroviral vectors were produced in which human N-ras(m) (codon 13 mutation) was coexpressed with GFP, and a ribozyme specifically targeting N-ras(m) was coexpressed with RFP. N-Ras(m) suppression was monitored by measurement of GFP fluorescence in dual-fluorescent (GFP and RFP) cells. We demonstrated that the degree of N-ras(m) suppression was dependent on the ribozyme dose, proportional to red fluorescence, in dual-fluorescent cells. We further showed that ribozyme-mediated N-ras(m)suppression inhibited growth of NIH3T3 and CD34-positive TF-1 cells. In these cultures, ras suppressor activity resulted in the depletion of suppressor-positive cells due to inhibition of cell growth. In contrast, N-ras(m) suppression produced a growth advantage to human leukemic K562 cells, presumably by inhibiting N-ras(m)-induced apoptosis. In K562 cells, ras suppression resulted in the outgrowth of suppressor-positive cells. This provides a platform to identify suppressors of ras that is based on function.
Subject(s)
Apoptosis , Genes, Reporter/genetics , RNA, Catalytic/metabolism , ras Proteins/antagonists & inhibitors , ras Proteins/genetics , Animals , Butadienes/pharmacology , Cell Line, Tumor , Chromones/pharmacology , Flow Cytometry , Fluorescence , Genetic Vectors/genetics , Humans , K562 Cells , MAP Kinase Signaling System , Mice , Morpholines/pharmacology , Mutation/genetics , NIH 3T3 Cells , Nitriles/pharmacology , RNA, Catalytic/genetics , ras Proteins/metabolismABSTRACT
The majority of patients diagnosed with neuroblastoma present with aggressive disease. Improved detection of neuroblastoma cancer cells following initial therapy may help in stratifying patient outcome and monitoring for relapse. To identify potential plasma biomarkers, we utilised a liquid chromatography-tandem mass spectrometry-based proteomics approach to detect differentially-expressed proteins in serum from TH-MYCN mice. TH-MYCN mice carry multiple copies of the human MYCN oncogene in the germline and homozygous mice for the transgene develop neuroblastoma in a manner resembling the human disease. The abundance of plasma proteins was measured over the course of disease initiation and progression. A list of 86 candidate plasma biomarkers was generated. Pathway analysis identified significant association of these proteins with genes involved in the complement system. One candidate, complement C3 protein, was significantly enriched in the plasma of TH-MYCN(+/+) mice at both 4 and 6weeks of age, and was found to be elevated in a cohort of human neuroblastoma plasma samples, compared to healthy subjects. In conclusion, we have demonstrated the suitability of the TH-MYCN(+/+) mouse model of neuroblastoma for identification of novel disease biomarkers in humans, and have identified Complement C3 as a candidate plasma biomarker for measuring disease state in neuroblastoma patients. BIOLOGICAL SIGNIFICANCE: This study has utilised a unique murine model which develops neuroblastoma tumours that are biologically indistinguishable from human neuroblastoma. This animal model has effectively allowed the identification of plasma proteins which may serve as potential biomarkers of neuroblastoma. Furthermore, the label-free ion count quantitation technique which was used displays significant benefits as it is less labour intensive, feasible and accurate. We have been able to successfully validate this approach by confirming the differential abundance of two different plasma proteins. In addition, we have been able to confirm that the candidate biomarker Complement C3, is more abundant in the plasma of human neuroblastoma patient plasma samples when compared to healthy counterparts. Overall we have demonstrated that this approach can be potentially useful in the identification of biomarker candidates, and that further validation of the candidates may lead to the discovery of novel, clinically useful diagnostic tools in the detection of sub-clinical neuroblastoma.