ABSTRACT
BACKGROUND: 7,12-Dimethylbenzanthracene (DMBA) is a member of the polycyclic aromatic hydrocarbon family. It is a member of the polycyclic aromatic hydrocarbon family. It is a mutagenic, carcinogenic, and immunosuppressor agent. Cannabidiol (CBD) is a phytocannabinoid. It has anticonvulsant, anti-inflammatory, anti-anxiety, antioxidant, and anti-cancer properties. The purpose of this study was to investigate the possible protective and therapeutic benefits of CBD oil in DMBA-induced leukemia in rats. METHOD: Experimental animals were divided into six groups of five rats each. Group 1 (normal control) included healthy rats. Group 2 included normal rats that received olive oil. Group 3 included normal rats that received CBD. Group 4 included the DMBA-induced leukemic group. Group 5 (prophylactic group) included rats that received CBD as a prophylaxis before IV injection with DMBA. Group 6 (treated group) included DMBA-induced leukemic rats that received CBD as treatment. Liver functions (total, direct and indirect bilirubin, alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), albumin, globulin, and albumin globulin ratio) were measured. Superoxide dismutase (SOD) and catalase (CAT) were also measured. Total RNA extraction followed by-real time qRT-PCR gene expression of LC3-II, Beclin, mTOR, and P62 was performed. Histopathological examination of liver and spleen tissues was performed. RESULTS: Administration of CBD in groups 5 and 6 resulted in a significant improvement of the levels of liver functions compared to the leukemic untreated rats. Also, the levels of catalase and SOD significantly increased after treatment with CBD compared to the leukemic group. After treatment with CBD in groups 5 and 6, there were downregulations in the expression of all studied genes compared to leukemic untreated rats. Treatment with CBD was more statistically effective than prophylactic use. CONCLUSION: Administration of CBD resulted in a significant improvement in the biochemical, antioxidant status, morphological, and molecular measures in DMBA-induced leukemia in adult male rats. The therapeutic use was more effective than the prophylactic one.
Subject(s)
Cannabidiol , Globulins , Leukemia, Experimental , Rats , Male , Animals , Antioxidants/pharmacology , Catalase/metabolism , 9,10-Dimethyl-1,2-benzanthracene/metabolism , 9,10-Dimethyl-1,2-benzanthracene/pharmacology , Leukemia, Experimental/drug therapy , Leukemia, Experimental/metabolism , Leukemia, Experimental/pathology , Liver , Globulins/metabolism , Globulins/pharmacology , Superoxide Dismutase/metabolism , Albumins/metabolismABSTRACT
Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes. Here, we find that the RNA-binding protein IGF2BP3, which is overexpressed in MLL-translocated leukemia, strongly amplifies MLL-Af4-mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4-driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. At the molecular level, IGF2BP3 regulates a complex posttranscriptional operon governing leukemia cell survival and proliferation. IGF2BP3-targeted mRNA transcripts include important MLL-Af4-induced genes, such as those in the Hoxa locus, and the Ras signaling pathway. Targeting of transcripts by IGF2BP3 regulates both steady-state mRNA levels and, unexpectedly, pre-mRNA splicing. Together, our findings show that IGF2BP3 represents an attractive therapeutic target in this disease, providing important insights into mechanisms of posttranscriptional regulation in leukemia.
Subject(s)
Carcinogenesis/pathology , DNA-Binding Proteins/genetics , Gene Expression Regulation, Leukemic , Histone-Lysine N-Methyltransferase/genetics , Leukemia, Experimental/pathology , Myeloid-Lymphoid Leukemia Protein/genetics , Nuclear Proteins/genetics , Oncogene Proteins, Fusion/genetics , RNA-Binding Proteins/physiology , Animals , Apoptosis , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Proliferation , Female , Leukemia, Experimental/etiology , Leukemia, Experimental/metabolism , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
The existence of a dysfunctional CD8+ T cell state in cancer is well established. However, the degree to which CD8+ T cell fates are influenced by the context in which they encounter cognate tumor antigen is less clear. We previously demonstrated that CD8+ T cells reactive to a model leukemia antigen were deleted by antigen cross-presenting type 1 conventional dendritic cells (cDC1s). Here, through a study of T cell receptor (TCR) transgenic CD8+ T cells (TCRTg101) reactive to a native C1498 leukemia cell antigen, we uncover a different mode of T cell tolerance in which TCRTg101 undergo progressive expansion and differentiation into an exhausted state. Antigen encounter by TCRTg101 requires leukemia cell major histocompatibility complex (MHC)-I expression and is independent of DCs, implying that leukemia cells directly mediate the exhausted TCRTg101 phenotype. Collectively, our data reveal that leukemia antigens are presented to CD8+ T cells via discrete pathways, leading to distinct tolerant states.
Subject(s)
Antigen Presentation/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Immune Tolerance , Leukemia, Experimental/immunology , Receptors, Antigen, T-Cell/immunology , Animals , Cells, Cultured , Leukemia, Experimental/metabolism , Leukemia, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, TransgenicSubject(s)
Hematopoietic Stem Cells/pathology , Homeodomain Proteins/genetics , Karyopherins/metabolism , Leukemia, Experimental/pathology , Oncogene Proteins, Fusion/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Active Transport, Cell Nucleus , Animals , Apoptosis , Cell Proliferation , Cells, Cultured , Gene Expression Regulation, Leukemic , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/metabolism , Karyopherins/genetics , Leukemia, Experimental/etiology , Leukemia, Experimental/metabolism , Mice , Oncogene Proteins, Fusion/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/genetics , Exportin 1 ProteinABSTRACT
The pharmacologically active metabolite of cyclophosphamide is aldophosphamide. With cysteine, aldophosphamide forms stable aldophosphamide-thiazolidine which under physiological pH and temperature conditions hydrolyzes to aldophosphamide and cysteine. Aldophosphamide-thiazolidine was synthesized and tested for its ability as a cytostatic. The LD50 after a single intraperitoneal injection in mice was determined to be 2162 mg/kg, but after intravenous bolus administration of 500 mg/kg or in chronic toxicity tests with daily intraperitoneal injections, neurological side effects were observed. Antitumor activity was determined in therapy experiments in CD2F1 mice bearing subcutaneously transplanted P388 mouse leukemia cells. Administration of 100 mg/kg (less than 5% LD50) days 1-5 after tumor transplantation yielded an ILS of 100%. Organ distribution studies showed that aldophosphamide-thiazolidine is evenly distributed in all tissues examined, including brain tissue. The possibilities to increase the antitumor activity of aldophosphamide-thiazolidine by modulating the alkylating function are discussed.
Subject(s)
Blood-Brain Barrier/metabolism , Cytostatic Agents/pharmacology , Leukemia, Experimental/drug therapy , Nitrogen Mustard Compounds/pharmacology , Thiazolidines/pharmacology , Animals , Apoptosis , Blood-Brain Barrier/drug effects , Cell Proliferation , Cytostatic Agents/pharmacokinetics , Female , Leukemia, Experimental/metabolism , Leukemia, Experimental/pathology , Mice , Nitrogen Mustard Compounds/pharmacokinetics , Thiazolidines/pharmacokinetics , Tissue Distribution , Tumor Cells, CulturedSubject(s)
Enhancer Elements, Genetic , GATA2 Transcription Factor/physiology , Gene Expression Regulation, Neoplastic , Hematopoietic Stem Cells/cytology , Leukemia, Experimental/pathology , Animals , GATA2 Transcription Factor/genetics , Hematopoietic Stem Cells/metabolism , Leukemia, Experimental/genetics , Leukemia, Experimental/metabolism , Mice , Mice, KnockoutABSTRACT
Mixed lineage leukemia (MLL) arises from several KMT2A-gene chromosomal translocations. Shb gene deficiency has been found to exhibit pleiotropic effects in different models of leukemia, and consequently, this study aimed to investigate MLL-AF9-induced leukemia in Shb deficiency. Bone marrow cells from wild type and Shb knockout (KO) mice were transduced with the MLL-AF9 gene. Shb KO MLL-AF9 cells proliferated at an increased rate, exhibited altered expression of certain cytokine genes (Kitl, Csf3, IL6, IL1b) and higher expression of cell cycle genes (Ccnd2, Ccne1). Mice receiving Shb KO MLL-AF9 cells showed longer latency without displaying any difference in rates of leukemic cell proliferation, indicating a dichotomy between the in vitro and in vivo phenotypes. The mice with Shb deficient MLL-AF9 cells had a lower content of leukemic bone marrow cells allowing elevated normal hematopoiesis, explaining the longer latency. Finally, Shb knockout GFP-positive bone marrow cells showed a higher percentage of cells expressing myeloid markers. The result suggests a role of Shb in the progression of leukemia and that the relevance of the Shb gene is context-dependent as inferred from the differences between the in vivo and in vitro responses. These findings help to obtain an increased understanding of human MLL-AF9 leukemia.
Subject(s)
Cell Proliferation , Cell Transformation, Neoplastic/pathology , Gene Expression Regulation, Leukemic , Leukemia, Experimental/pathology , Myeloid-Lymphoid Leukemia Protein/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Proteins/physiology , Animals , Apoptosis , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Humans , Leukemia, Experimental/genetics , Leukemia, Experimental/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , Tumor Cells, CulturedABSTRACT
Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 µmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.
Subject(s)
Acetylcysteine/analogs & derivatives , Antiviral Agents/pharmacology , Cysteamine/analogs & derivatives , Immunoglobulins/metabolism , Plasma Cells/drug effects , Retroviridae Infections/drug therapy , Tumor Virus Infections/drug therapy , Unfolded Protein Response/drug effects , Acetylcysteine/administration & dosage , Acetylcysteine/pharmacology , Animals , Antiviral Agents/administration & dosage , Cysteamine/administration & dosage , Cysteamine/pharmacology , Disease Models, Animal , Female , Immunoglobulins/blood , Injections, Intraperitoneal , Leukemia, Experimental/drug therapy , Leukemia, Experimental/metabolism , Leukemia, Experimental/virology , Mice , Mice, Inbred C57BL , Plasma Cells/metabolism , Plasma Cells/virology , Protein Unfolding/drug effects , Retroviridae Infections/metabolism , Retroviridae Infections/virology , Tumor Virus Infections/metabolism , Tumor Virus Infections/virologyABSTRACT
Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) protein to counteract APOBEC3 proteins. Vif induces degradation of APOBEC3G and other APOBEC3 proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like protein. Here, we show that the MLV P50 protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus compared to lentiviruses, it does encode proteins generated from alternatively spliced RNAs. Here, we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.
Subject(s)
Cytidine Deaminase/genetics , Gene Expression Regulation, Viral , Gene Products, gag/genetics , Leukemia, Experimental/genetics , Moloney murine leukemia virus/genetics , Retroviridae Infections/genetics , Tumor Virus Infections/genetics , Alternative Splicing , Animals , Capsid/metabolism , Cytidine Deaminase/deficiency , Gene Products, gag/metabolism , HEK293 Cells , Host-Pathogen Interactions/genetics , Humans , Leukemia, Experimental/metabolism , Leukemia, Experimental/virology , Mice , Mice, Knockout , Moloney murine leukemia virus/metabolism , Moloney murine leukemia virus/pathogenicity , NIH 3T3 Cells , Retroviridae Infections/metabolism , Retroviridae Infections/virology , Signal Transduction , Tumor Virus Infections/metabolism , Tumor Virus Infections/virology , Virion/genetics , Virion/metabolism , Virion/pathogenicity , Virus ReplicationABSTRACT
Drug resistance is a common obstacle in leukemia treatment and failing to eradicate leukemia stem cells is the main cause of leukemia relapse. Previous studies have demonstrated that telomerase activity is associated with deregulated self-renewal of leukemia stem cells (LSCs). Here, we identified a novel compound IX, an imatinib derivative with a replacement fragment of a telomerase inhibitor, which can effectively eradicate LSCs but had no influence on normal hematopoietic stem cells (HSCs) survival. We showed that compound IX can decrease the viability of drug-resistant K562/G cells and blast crisis CML primary patient cells. Besides, IX can affect LSC survival, inhibit the colony-forming ability, and reduce LSC frequency. In vivo results showed that IX can relieve the tumor burden in patient-derived xenograft (PDX) model and prolong the lifespan. We observed that compound IX can not only decrease telomerase activity, but also affect the alternative lengthening of telomeres. In addition, IX can inhibit both the canonical and non-canonical Wnt pathways. Our data suggested this novel compound IX as a promising candidate for drug-resistant leukemia therapy.
Subject(s)
Carcinogenesis/drug effects , Drug Resistance, Neoplasm , Leukemia, Experimental/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myeloid, Acute/drug therapy , Small Molecule Libraries/pharmacology , Telomere/drug effects , Apoptosis , Carcinogenesis/metabolism , Carcinogenesis/pathology , Cell Cycle , Cell Movement , Cell Proliferation , Humans , Leukemia, Experimental/metabolism , Leukemia, Experimental/pathology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Pharmaceutical Preparations/administration & dosage , Telomere/metabolism , Tumor Cells, CulturedABSTRACT
Primary and acquired drug resistance imposes a major threat to achieving optimized clinical outcomes during cancer treatment. Aberrant changes in epigenetic modifications are closely involved in drug resistance of tumor cells. Using BET inhibitor (BETi) resistant leukemia cells as a model system, we demonstrated herein that genome-wide enhancer remodeling played a pivotal role in driving therapeutic resistance via compensational re-expression of pro-survival genes. Capitalizing on the CRISPR interference technology, we identified the second intron of IncRNA, PVT1, as a unique bona fide gained enhancer that restored MYC transcription independent of BRD4 recruitment in leukemia. A combined BETi and CDK7 inhibitor treatment abolished MYC transcription by impeding RNAPII loading without affecting PVT1-mediated chromatin looping at the MYC locus in BETi-resistant leukemia cells. Together, our findings have established the feasibility of targeting enhancer plasticity to overcome drug resistance associated with epigenetic therapies.
Subject(s)
Leukemia, Experimental/drug therapy , Leukemia, Experimental/genetics , Nuclear Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Animals , Cell Cycle Proteins/antagonists & inhibitors , Cell Line, Tumor , Cyclin-Dependent Kinases/antagonists & inhibitors , Drug Resistance, Neoplasm/genetics , Drug Synergism , Enhancer Elements, Genetic , Female , Genes, myc/drug effects , Heterocyclic Compounds, 4 or More Rings/administration & dosage , Humans , Jurkat Cells , K562 Cells , Leukemia, Experimental/metabolism , Mice , Models, Genetic , Phenylenediamines/administration & dosage , Pyrimidines/administration & dosage , RNA Polymerase II/metabolism , RNA, Long Noncoding/genetics , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
AIMS: Berberine (BBR) is reported to induce apoptosis and inhibit migration of leukemic cells, but the underlying pharmacological mechanisms have not been fully revealed. This study aims to investigate the possible mechanisms from the perspective of autophagy. MAIN METHODS: P-53-null leukemic cell lines Jurkat and U937 were used for the in vitro study. MDC staining was used for observation of autophagy in leukemic cells, and Western blot analysis was for determination of the expression levels of autophagy-associated proteins. Apoptosis of the leukemic cells was detected by flow cytometry. Cellular location of MDM2 was observed with immunofluorescence staining. Ubiquitination of MDM2 was assessed by immunoprecipitation. Male 6-8-week-old NOD/SCID mice were used for evaluating the effect of BBR on chemotherapy sensitivity in vivo. KEY FINDINGS: BBR induced autophagy in p53-null leukemic cells, which was inhibited by autophagy inhibitors 3-methyladenine. 3-methyladenine also inhibited BBR-induced apoptosis in leukemic cells. In addition, BBR not only decreased MDM2 mRNA expression, but also enhanced MDM2 self-ubiquitination in leukemic cells. Forced overexpression of MDM2 reversed the effect of BBR on autophagy and apoptosis. Furthermore, BBR promoted doxorubicin-induced autophagy and cell death in the leukemic cells and overexpression of MDM2 suppressed these effects. In vivo, BBR combined with doxorubicin achieved better therapeutic effect than doxorubicin alone. SIGNIFICANCE: MDM2 inhibits autophagy and apoptosis in leukemic cells in a p53-independent manner. BBR induces autophagy in p53-null leukemic cells through downregulating MDM2 expression at both transcriptional and post-transcriptional levels, which may contribute to the anti-cancer effect of BBR in leukemia.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Berberine/pharmacology , Jurkat Cells/drug effects , Leukemia, Experimental/drug therapy , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , U937 Cells/drug effects , Animals , Blotting, Western , Flow Cytometry , Fluorescent Antibody Technique , Humans , Jurkat Cells/metabolism , Leukemia, Experimental/metabolism , Male , Mice, Inbred NOD , Mice, SCID , Neoplasm Transplantation , Real-Time Polymerase Chain Reaction , U937 Cells/metabolism , UbiquitinationABSTRACT
ATP is one of the main components of the tumor microenvironment, where it affects cell growth, tumor progression and antitumor immune response. The development of the pmeLUC probe, a luciferase engineered to be expressed on the outer facet of the plasma membrane, allowed real-time measurement of extracellular ATP in vitro and in vivo systems, among which the tumor microenvironment. Here we describe the experimental procedures to measure extracellular ATP levels in the tumor microenvironment of three different cancer models generated by the implant of pmeLUC-expressing tumor cells into the appropriate mice strain: ACN human neuroblastoma (nude/nude mice host), WEHI-3B murine leukemia (BALB/c host), and B16F10 murine melanoma (C57Bl/6 host). The procedure to obtain stable expression of pmeLUC in different cell types and methods for the measurement of extracellular ATP with pmeLUC in vitro are also described.
Subject(s)
Adenosine Triphosphate/metabolism , Biosensing Techniques/methods , Leukemia, Experimental/metabolism , Luciferases/metabolism , Melanoma, Experimental/metabolism , Neuroblastoma/metabolism , Tumor Microenvironment/physiology , Animals , Cell Membrane/metabolism , Humans , Leukemia, Experimental/pathology , Melanoma, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Neuroblastoma/pathology , Tumor Cells, CulturedABSTRACT
Mouse APOBEC3 (mA3) inhibits murine leukemia virus (MuLV) replication by a deamination-independent mechanism in which the reverse transcription is considered the main target process. However, other steps in virus replication that can be targeted by mA3 have not been examined. We have investigated the possible effect of mA3 on MuLV protease-mediated processes and found that mA3 binds both mature viral protease and Pr180gag-pol precursor polyprotein. Using replication-competent MuLVs, we also show that mA3 inhibits the processing of Pr65 Gag precursor. Furthermore, we demonstrate that the autoprocessing of Pr180gag-pol is impeded by mA3, resulting in reduced production of mature viral protease. This reduction appears to link with the above inefficient Pr65gag processing in the presence of mA3. Two major isoforms of mA3, exon 5-containing and -lacking ones, equally exhibit this antiviral activity. Importantly, physiologically expressed levels of mA3 impedes both Pr180gag-pol autocatalysis and Pr65gag processing. This blockade is independent of the deaminase activity and requires the C-terminal region of mA3. These results suggest that the above impairment of Pr180gag-pol autoprocessing may significantly contribute to the deaminase-independent antiretroviral activity exerted by mA3.
Subject(s)
Cytidine Deaminase/metabolism , Fusion Proteins, gag-pol/metabolism , Leukemia Virus, Murine/metabolism , Retroviridae Infections/metabolism , Virus Replication/physiology , Animals , Gene Products, gag/metabolism , Leukemia, Experimental/metabolism , Mice , Mice, Inbred C57BL , Tumor Virus Infections/metabolismABSTRACT
Exposure to extremely low-frequency magnetic fields (ELF-MFs) has been classified by the International Agency for Research on Cancer (IARC) as "possibly carcinogenic to humans," based on limited scientific evidence concerning childhood leukemia. This assessment emphasized the lack of appropriate animal models recapitulating the natural history of this disease. Childhood B-cell acute lymphoblastic leukemia (B-ALL) is the result of complex interactions between genetic susceptibility and exposure to exogenous agents. The most common chromosomal alteration is the ETV6-RUNX1 fusion gene, which confers a low risk of developing the malignancy by originating a preleukemic clone requiring secondary hits for full-blown disease to appear. To develop potential prophylactic interventions, we need to identify the environmental triggers of the second hit. Recently, we generated a B-ALL mouse model of the human ETV6-RUNX1+ preleukemic state. Here, we present the results from the ARIMMORA pilot study, obtained by exposing 34 Sca1-ETV6-RUNX1 mice (vs. 27 unexposed) to a 50 Hz magnetic field of 1.5 mT with both fundamental and harmonic content, with an on/off cycle of 10 min/5 min, for 20 h/day, from conception until 3 months of age. Mice were monitored until 2 years of age and peripheral blood was periodically analyzed by flow cytometry. One of the exposed mice developed B-ALL while none of the non-exposed did. Although the results are statistically non-significant due to the limited number of mice used in this pilot experiment, overall, the results show that the newly developed Sca1-ETV6-RUNX1 mouse can be successfully used for ELF-MF exposure studies about the etiology of childhood B-ALL. Bioelectromagnetics. 2019;40:343-353. © 2019 Bioelectromagnetics Society.
Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Disease Models, Animal , Electromagnetic Fields/adverse effects , Leukemia, Experimental , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Proto-Oncogene Proteins c-ets/genetics , Radio Waves/adverse effects , Repressor Proteins/genetics , Animals , Core Binding Factor Alpha 2 Subunit/metabolism , Female , Humans , Leukemia, Experimental/genetics , Leukemia, Experimental/metabolism , Male , Mice , Mice, Inbred C57BL , Pilot Projects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/etiology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Repressor Proteins/metabolism , ETS Translocation Variant 6 ProteinABSTRACT
Leukemogenic potential of MLL fusion with the coiled-coil domain-containing partner genes and the downstream target genes of this type of MLL fusion have not been clearly investigated. In this study, we demonstrated that the coiled-coil-four-helix bundle structure of EB1 that participated in the MLL/EB1 was required for immortalizing mouse bone marrow (BM) cells and producing myeloid, but not lymphoid, cell lines. Compared to MLL/AF10, MLL/EB1 had low leukemogenic ability. The MLL/EB1 cells grew more slowly owing to increased apoptosis in vitro and induced acute monocytic leukemia with an incomplete penetrance and longer survival in vivo. A comparative analysis of transcriptome profiling between MLL/EB1 and MLL/AF10 cell lines revealed that there was an at least two-fold difference in the induction of 318 genes; overall, 51.3% (163/318) of the genes were known to be bound by MLL, while 15.4% (49/318) were bound by both MLL and MLL/AF9. Analysis of the 318 genes using Gene Ontology-PANTHER overrepresentation test revealed significant differences in several biological processes, including cell differentiation, proliferation/programmed cell death, and cell homing/recruitment. The Ets1 gene, bound by MLL and MLL/AF9, was involved in several biological processes. We demonstrated that Ets1 was selectively upregulated by MLL/EB1. Short hairpin RNA knockdown of Ets1 in MLL/EB1 cells reduced the expression of CD115, apoptosis rate, competitive engraftment to BM and spleen, and incidence of leukemia and prolonged the survival of the diseased mice. Our results demonstrated that MLL/EB1 upregulated Ets1, which controlled the balance of leukemia cells between apoptosis and BM engraftment/clonal expansion. Novelty and impact of this study The leukemogenic potential of MLL fusion with cytoplasmic proteins containing coiled-coil dimerization domains and the downstream target genes of this type of MLL fusion remain largely unknown. Using a retroviral transduction/transplantation mouse model, we demonstrated that MLL fusion with the coiled-coil-four-helix bundle structure of EB1 has low leukemogenic ability; Ets1, which is upregulated by MLL/EB1, plays a critical role in leukemic transformation by balance between apoptosis and BM engraftment/clonal expansion.
Subject(s)
Bone Marrow Transplantation , Cell Transformation, Neoplastic/pathology , Histone-Lysine N-Methyltransferase/metabolism , Leukemia, Experimental/pathology , Leukemia, Monocytic, Acute/pathology , Microtubule-Associated Proteins/metabolism , Myeloid-Lymphoid Leukemia Protein/metabolism , Proto-Oncogene Protein c-ets-1/metabolism , Animals , Apoptosis , Cell Differentiation , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Disease Models, Animal , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Histone-Lysine N-Methyltransferase/genetics , Leukemia, Experimental/genetics , Leukemia, Experimental/metabolism , Leukemia, Monocytic, Acute/genetics , Leukemia, Monocytic, Acute/metabolism , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , NIH 3T3 Cells , Oncogene Proteins, Fusion , Proto-Oncogene Protein c-ets-1/geneticsABSTRACT
Acute lymphoblastic leukemia (ALL) is the most common cancer in children. The highest rates of treatment failure occur in specific genetic subsets of ALL, including hypodiploid B-cell ALL (B-ALL), for which effective alternative therapies to current intensive chemotherapy treatments have yet to be developed. Here, we integrated biochemical and genomic profiling with functional drug assays to select effective agents with therapeutic potential against hypodiploid B-ALL. ABT-199, a selective Bcl-2 inhibitor, was effective in reducing leukemic burden in vitro and in vivo in patient-derived xenograft models of hypodiploid B-ALL. Daily oral treatment with ABT-199 significantly increased survival in xenografted mice. The unexpected efficacy of ABT-199 observed in hypodiploid leukemias lacking BIM expression (the major reported mediator of ABT-199-induced apoptosis) led us to investigate the mechanism of action of ABT-199 in the absence of BIM. Treatment with ABT-199 elicited responses in a dose-dependent manner, from cell-cycle arrest at low nanomolar concentrations to cell death at concentrations above 100 nmol/L. Collectively, these results demonstrate the efficacy of Bcl-2 inhibition and potential therapeutic strategy in hypodiploid B-ALL. SIGNIFICANCE: These results demonstrate the efficacy of ABT-199 in vivo and provide encouraging preclinical data of Bcl-2 as a potential target for the treatment of hypodiploid B-ALL.
Subject(s)
Antineoplastic Agents/pharmacology , Diploidy , Leukemia, Experimental/drug therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Animals , Apoptosis , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Lineage , Cell Proliferation , Humans , Leukemia, Experimental/metabolism , Leukemia, Experimental/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Sulfonamides/pharmacology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Cancer sequencing studies have implicated regulators of pre-mRNA splicing as important disease determinants in acute myeloid leukemia (AML), but the underlying mechanisms have remained elusive. We hypothesized that "non-mutated" splicing regulators may also play a role in AML biology and therefore conducted an in vivo shRNA screen in a mouse model of CEBPA mutant AML. This has led to the identification of the splicing regulator RBM25 as a novel tumor suppressor. In multiple human leukemic cell lines, knockdown of RBM25 promotes proliferation and decreases apoptosis. Mechanistically, we show that RBM25 controls the splicing of key genes, including those encoding the apoptotic regulator BCL-X and the MYC inhibitor BIN1. This mechanism is also operative in human AML patients where low RBM25 levels are associated with high MYC activity and poor outcome. Thus, we demonstrate that RBM25 acts as a regulator of MYC activity and sensitizes cells to increased MYC levels.
Subject(s)
Gene Expression Regulation, Leukemic , Leukemia, Experimental/metabolism , Leukemia, Myeloid, Acute/metabolism , Proto-Oncogene Proteins c-myc/metabolism , RNA Recognition Motif Proteins/metabolism , RNA Splicing Factors/metabolism , RNA-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line, Tumor , Female , Humans , Leukemia, Myeloid, Acute/mortality , Mice , Nerve Tissue Proteins/metabolism , Nuclear Proteins , RNA Splicing , Tumor Suppressor Proteins/metabolismABSTRACT
Leukemias exhibit a dysregulated developmental program mediated through both genetic and epigenetic mechanisms. Although IKZF2 is expressed in hematopoietic stem cells (HSCs), we found that it is dispensable for mouse and human HSC function. In contrast to its role as a tumor suppressor in hypodiploid B-acute lymphoblastic leukemia, we found that IKZF2 is required for myeloid leukemia. IKZF2 is highly expressed in leukemic stem cells (LSCs), and its deficiency results in defective LSC function. IKZF2 depletion in acute myeloid leukemia (AML) cells reduced colony formation, increased differentiation and apoptosis, and delayed leukemogenesis. Gene expression, chromatin accessibility, and direct IKZF2 binding in MLL-AF9 LSCs demonstrate that IKZF2 regulates a HOXA9 self-renewal gene expression program and inhibits a C/EBP-driven differentiation program. Ectopic HOXA9 expression and CEBPE depletion rescued the effects of IKZF2 depletion. Thus, our study shows that IKZF2 regulates the AML LSC program and provides a rationale to therapeutically target IKZF2 in myeloid leukemia.
Subject(s)
Cell Differentiation , Cell Self Renewal , DNA-Binding Proteins/physiology , Gene Expression Regulation, Leukemic , Leukemia, Experimental/pathology , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/pathology , Transcription Factors/physiology , Animals , Chromatin/genetics , Chromatin/metabolism , Female , Hematopoiesis , Leukemia, Experimental/genetics , Leukemia, Experimental/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplastic Stem Cells/metabolismABSTRACT
Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity.