ABSTRACT
Recurrent loss-of-function mutations of spliceosome gene, ZRSR2, occur in myelodysplastic syndromes (MDS). Mutation/loss of ZRSR2 in human myeloid cells primarily causes impaired splicing of the U12-type introns. In order to further investigate the role of this splice factor in RNA splicing and hematopoietic development, we generated mice lacking ZRSR2. Unexpectedly, Zrsr2-deficient mice developed normal hematopoiesis with no abnormalities in myeloid differentiation evident in either young or ≥1-year old knockout mice. Repopulation ability of Zrsr2-deficient hematopoietic stem cells was also unaffected in both competitive and non-competitive reconstitution assays. Myeloid progenitors lacking ZRSR2 exhibited mis-splicing of U12-type introns, however, this phenotype was moderate compared to the ZRSR2-deficient human cells. Our investigations revealed that a closely related homolog, Zrsr1, expressed in the murine hematopoietic cells, but not in human cells contributes to splicing of U12-type introns. Depletion of Zrsr1 in Zrsr2 KO myeloid cells exacerbated retention of the U12-type introns, thus highlighting a collective role of ZRSR1 and ZRSR2 in murine U12-spliceosome. We also demonstrate that aberrant retention of U12-type introns of MAPK9 and MAPK14 leads to their reduced protein expression. Overall, our findings highlight that both ZRSR1 and ZRSR2 are functional components of the murine U12-spliceosome, and depletion of both proteins is required to accurately model ZRSR2-mutant MDS in mice.
Subject(s)
Myelodysplastic Syndromes , RNA Splicing , Ribonucleoproteins , Splicing Factor U2AF , Animals , Introns , Mice , Mutation , Myelodysplastic Syndromes/genetics , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Spliceosomes/genetics , Splicing Factor U2AF/genetics , Splicing Factor U2AF/metabolismABSTRACT
Liposarcoma, the most common soft tissue sarcoma, is a group of fat cell mesenchymal tumors with different histological subtypes. The dysregulation of long non-coding RNAs (lncRNAs) has been observed in human cancers including a few studies in sarcoma. However, the global transcriptome analysis and potential role of lncRNAs remain unexplored in liposarcoma. The present investigation uncovers the transcriptomic profile of liposarcoma by RNA sequencing to gain insight into the global transcriptional changes in liposarcoma. Our RNA sequencing analysis has identified that many oncogenic lncRNAs are differentially expressed in different subtypes of liposarcoma including MALAT1, PVT1, SNHG15, LINC00152, and MIR210HG. Importantly, we identified a highly overexpressed, unannotated, and novel lncRNA in dedifferentiated liposarcomas. We have named it TODL, transcript overexpressed in dedifferentiated liposarcoma. TODL lncRNA displayed significantly higher expression in dedifferentiated liposarcoma cell lines and patient samples. Interestingly, functional studies revealed that TODL lncRNA has an oncogenic function in liposarcoma cells by regulating proliferation, cell cycle, apoptosis, differentiation, and tumorigenesis in the murine model. Silencing of TODL lncRNA highlighted the enrichment of several key oncogenic signaling pathways including cell cycle, transcriptional misregulation, FOXM1 network, p53 signaling, PLK1 signaling, FoxO, and signaling Aurora signaling pathways. RNA pull-down assay revealed the binding of TODL lncRNA with FOXM1, an oncogenic transcription factor, and the key regulator of the cell cycle. Silencing of TODL lncRNA also induces adipogenesis in dedifferentiated liposarcomas. Altogether, our finding indicates that TODL could be utilized as a novel, specific diagnostic biomarker, and a pharmacological target for therapeutic development in controlling aggressive and metastatic dedifferentiated liposarcomas.
Subject(s)
Forkhead Box Protein M1 , Liposarcoma , RNA, Long Noncoding , Animals , Humans , Mice , Carcinogenesis/genetics , Cell Proliferation , Forkhead Box Protein M1/genetics , Forkhead Box Protein M1/metabolism , Gene Expression Profiling , Liposarcoma/genetics , Liposarcoma/metabolism , Liposarcoma/pathology , RNA, Long Noncoding/genetics , TranscriptomeABSTRACT
Proteasome inhibitors, such as bortezomib and carfilzomib, have shown efficacy in anti-cancer therapy in hematological diseases but not in solid cancers. Here, we found that liposarcomas (LPS) are susceptible to proteasome inhibition, and identified drugs that synergize with carfilzomib, such as selinexor, an inhibitor of XPO1-mediated nuclear export. Through quantitative nuclear protein profiling and phospho-kinase arrays, we identified potential mode of actions of this combination, including interference with ribosome biogenesis and inhibition of pro-survival kinase PRAS40. Furthermore, by assessing global protein levels changes, FADS2, a key enzyme regulating fatty acids synthesis, was found down-regulated after proteasome inhibition. Interestingly, SC26196, an inhibitor of FADS2, synergized with carfilzomib. Finally, to identify further combinational options, we performed high-throughput drug screening and uncovered novel drug interactions with carfilzomib. For instance, cyclosporin A, a known immunosuppressive agent, enhanced carfilzomib's efficacy in vitro and in vivo. Altogether, these results demonstrate that carfilzomib and its combinations could be repurposed for LPS clinical management.
Subject(s)
Fatty Acid Desaturases/genetics , Karyopherins/genetics , Liposarcoma/drug therapy , Oligopeptides/pharmacology , Receptors, Cytoplasmic and Nuclear/genetics , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bortezomib/pharmacology , Cell Line, Tumor , Cell Nucleus/drug effects , Drug Resistance, Neoplasm/genetics , Drug Synergism , Fatty Acid Desaturases/antagonists & inhibitors , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hydrazines/pharmacology , Liposarcoma/genetics , Liposarcoma/pathology , Piperazines/pharmacology , Proteasome Endopeptidase Complex/drug effects , Proteasome Inhibitors/pharmacology , Triazoles/pharmacology , Exportin 1 ProteinABSTRACT
Multiple myeloma (MM) is a hematological disease marked by abnormal growth of B cells in bone marrow. Inherent chromosomal instability and DNA damage are major hallmarks of MM, which implicates an aberrant DNA repair mechanism. Studies have implicated a role for CDK12 in the control of expression of DNA damage response genes. In this study, we examined the effect of a small molecule inhibitor of CDK12-THZ531 on MM cells. Treatment of MM cells with THZ531 led to heightened cell death accompanied by an extensive effect on gene expression changes. In particular, we observed downregulation of genes involved in DNA repair pathways. With this insight, we extended our study to identify synthetic lethal mechanisms that could be exploited for the treatment of MM cells. Combination of THZ531 with either DNA-PK inhibitor (KU-0060648) or PARP inhibitor (Olaparib) led to synergistic cell death. In addition, combination treatment of THZ531 with Olaparib significantly reduced tumor burden in animal models. Our findings suggest that using a CDK12 inhibitor in combination with other DNA repair inhibitors may establish an effective therapeutic regimen to benefit myeloma patients.
Subject(s)
Anilides/pharmacology , Biomarkers, Tumor/genetics , DNA Repair , Gene Expression Regulation, Neoplastic/drug effects , Multiple Myeloma/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Pyrimidines/pharmacology , Synthetic Lethal Mutations , Animals , Apoptosis , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Cell Proliferation , Drug Therapy, Combination , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Prognosis , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND & AIMS: We investigated the transcriptome of esophageal squamous cell carcinoma (ESCC) cells, activity of gene regulatory (enhancer and promoter regions), and the effects of blocking epigenetic regulatory proteins. METHODS: We performed chromatin immunoprecipitation sequencing with antibodies against H3K4me1, H3K4me3, and H3K27ac and an assay for transposase-accessible chromatin to map the enhancer regions and accessible chromatin in 8 ESCC cell lines. We used the CRC_Mapper algorithm to identify core regulatory circuitry transcription factors in ESCC cell lines, and determined genome occupancy profiles for 3 of these factors. In ESCC cell lines, expression of transcription factors was knocked down with small hairpin RNAs, promoter and enhancer regions were disrupted by CRISPR/Cas9 genome editing, or bromodomains and extraterminal (BET) family proteins and histone deacetylases (HDACs) were inhibited with ARV-771 and romidepsin, respectively. ESCC cell lines were then analyzed by whole-transcriptome sequencing, immunoprecipitation, immunoblots, immunohistochemistry, and viability assays. Interactions between distal enhancers and promoters were identified and verified with circular chromosome conformation capture sequencing. NOD-SCID mice were given injections of modified ESCC cells, some mice where given injections of HDAC or BET inhibitors, and growth of xenograft tumors was measured. RESULTS: We identified super-enhancer-regulated circuits and transcription factors TP63, SOX2, and KLF5 as core regulatory factors in ESCC cells. Super-enhancer regulation of ALDH3A1 mediated by core regulatory factors was required for ESCC viability. We observed direct interactions between the promoter region of TP63 and functional enhancers, mediated by the core regulatory circuitry transcription factors. Deletion of enhancer regions from ESCC cells decreased expression of the core regulatory circuitry transcription factors and reduced cell viability; these same results were observed with knockdown of each core regulatory circuitry transcription factor. Incubation of ESCC cells with BET and HDAC disrupted the core regulatory circuitry program and the epigenetic modifications observed in these cells; mice given injections of HDAC or BET inhibitors developed smaller xenograft tumors from the ESCC cell lines. Xenograft tumors grew more slowly in mice given the combination of ARV-771 and romidepsin than mice given either agent alone. CONCLUSIONS: In epigenetic and transcriptional analyses of ESCC cell lines, we found the transcription factors TP63, SOX2, and KLF5 to be part of a core regulatory network that determines chromatin accessibility, epigenetic modifications, and gene expression patterns in these cells. A combination of epigenetic inhibitors slowed growth of xenograft tumors derived from ESCC cells in mice.
Subject(s)
Epigenesis, Genetic , Esophageal Neoplasms/genetics , Esophageal Squamous Cell Carcinoma/genetics , Gene Expression Regulation, Neoplastic , Kruppel-Like Transcription Factors/genetics , SOXB1 Transcription Factors/genetics , Transcription Factors/genetics , Transcription, Genetic , Tumor Suppressor Proteins/genetics , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation , Chromatin Assembly and Disassembly , Epigenesis, Genetic/drug effects , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma/drug therapy , Esophageal Squamous Cell Carcinoma/metabolism , Esophageal Squamous Cell Carcinoma/pathology , Gene Expression Regulation, Neoplastic/drug effects , Histone Deacetylase Inhibitors/pharmacology , Humans , Kruppel-Like Transcription Factors/metabolism , Mice, Inbred NOD , Mice, SCID , Proteins/antagonists & inhibitors , Proteins/metabolism , SOXB1 Transcription Factors/metabolism , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Transcriptome , Tumor Burden , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
CCAAT/enhancer binding protein ε (CEBPE) is an essential transcription factor for granulocytic differentiation. Mutations of CEBPE occur in individuals with neutrophil-specific granule deficiency (SGD), which is characterized by defects in neutrophil maturation. Cebpe-knockout mice also exhibit defects in terminal differentiation of granulocytes, a phenotype reminiscent of SGD. Analysis of DNase I hypersensitive sites sequencing data revealed an open chromatin region 6 kb downstream of the transcriptional start site of Cebpe in murine myeloid cells. We identified an interaction between this +6-kb region and the core promoter of Cebpe using circular chromosome conformation capture sequencing (4C-seq). To understand the role of this putative enhancer in transcriptional regulation of Cebpe, we targeted it using catalytically inactive Cas9 fused to Krüppel-associated box (KRAB) domain and observed a significant downregulation of transcript and protein levels of CEBPE in cells expressing guide RNA targeting the +6-kb region. To further investigate the role of this novel enhancer further in myelopoiesis, we generated mice with deletion of this region using CRISPR/Cas9 technology. Germline deletion of the +6-kb enhancer resulted in reduced levels of CEBPE and its target genes and caused a severe block in granulocytic differentiation. We also identified binding of CEBPA and CEBPE to the +6-kb enhancer, which suggests their role in regulating the expression of Cebpe In summary, we have identified a novel enhancer crucial for regulating expression of Cebpe and required for normal granulocytic differentiation.
Subject(s)
CCAAT-Enhancer-Binding Proteins/biosynthesis , Cell Differentiation/genetics , Gene Expression Regulation/genetics , Granulocytes/metabolism , Myelopoiesis/genetics , Animals , CCAAT-Enhancer-Binding Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
Proteolysis targeting chimeric molecule ARV 825 causes ubiquitination of bromodomains resulting in their efficient degradation by proteasome activity. Bromodomain degradation down-regulates MYC transcription contributing to growth inhibition of various human cancers. We examined the therapeutic potential of ARV 825 against multiple myeloma (MM) cells both in vitro and in vivo In a dose-dependent manner, ARV 825 inhibited proliferation of 13 human MM cell lines and three fresh patient samples, and was associated with cell cycle arrest and apoptosis. ARV 825 rapidly and efficiently degraded BRD 2 and BRD 4. Sensitivity of MM cells to ARV 825 was positively correlated with cereblon levels. RNA sequencing analysis showed important genes such as CCR1, RGS, MYB and MYC were down-regulated by ARV 825. A total of 170 small molecule inhibitors were screened for synergy with ARV 825. Combination of ARV 825 with inhibitor of either dual PI3K/mTOR, CRM1, VEGFR, PDGFRα/b, FLT3, IGF-1R, protein kinase C, CBP-EP300 or JAK1/2 showed synergistic activity. Importantly, ARV 825 significantly inhibited the growth of MM xenografts and improved mice survival. Taken together, our results, in conjunction with recently published findings, provide a rationale for investigating the efficacy of ARV 825 for MM, use of cereblon as a biomarker for therapy of MM patients, and the combination of ARV 825 with small molecule inhibitors to improve the outcome of MM patients.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor , Molecular Targeted Therapy , Multiple Myeloma/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Azepines/pharmacology , Azepines/therapeutic use , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Gene Expression Profiling , Humans , Ikaros Transcription Factor/metabolism , Mice , Multiple Myeloma/etiology , Multiple Myeloma/pathology , Multiple Myeloma/therapy , Proteolysis/drug effects , Thalidomide/analogs & derivatives , Thalidomide/pharmacology , Thalidomide/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
Maturation of granulocytes is dependent on controlled gene expression by myeloid lineage restricted transcription factors. CEBPE is one of the essential transcription factors required for granulocytic differentiation. Identification of downstream targets of CEBPE is vital to understand better its role in terminal granulopoiesis. In this study, we have identified Card10 as a novel target of CEBPE. We show that CEBPE binds to regulatory elements upstream of the murine Card10 locus, and expression of CARD10 is significantly reduced in Cebpe knock-out mice. Silencing Card10 in a human cell line and in murine primary cells impaired granulopoiesis, affecting expression of genes involved in myeloid cell development and function. Taken together, our data demonstrate for the first time that Card10 is expressed in granulocytes and is a direct target of CEBPE with functions extending to myeloid differentiation.
Subject(s)
CARD Signaling Adaptor Proteins/metabolism , CCAAT-Enhancer-Binding Proteins/physiology , Cell Differentiation , Granulocytes/cytology , Animals , Binding Sites , Cell Line , Cells, Cultured , Gene Expression Regulation , Granulocytes/metabolism , Humans , Mice , Myeloid Cells , Protein Binding , Transcription Factors/geneticsABSTRACT
Chromosomal translocation t(8;21)(q22;q22) which leads to the generation of oncogenic RUNX1-RUNX1T1 (AML1-ETO) fusion is observed in approximately 10% of acute myelogenous leukemia (AML). To identify somatic mutations that co-operate with t(8;21)-driven leukemia, we performed whole and targeted exome sequencing of an Asian cohort at diagnosis and relapse. We identified high frequency of truncating alterations in ASXL2 along with recurrent mutations of KIT, TET2, MGA, FLT3, and DHX15 in this subtype of AML. To investigate in depth the role of ASXL2 in normal hematopoiesis, we utilized a mouse model of ASXL2 deficiency. Loss of ASXL2 caused progressive hematopoietic defects characterized by myeloid hyperplasia, splenomegaly, extramedullary hematopoiesis, and poor reconstitution ability in transplantation models. Parallel analyses of young and >1-year old Asxl2-deficient mice revealed age-dependent perturbations affecting, not only myeloid and erythroid differentiation, but also maturation of lymphoid cells. Overall, these findings establish a critical role for ASXL2 in maintaining steady state hematopoiesis, and provide insights into how its loss primes the expansion of myeloid cells.
Subject(s)
Cell Differentiation/genetics , Cell Proliferation/genetics , Hematopoiesis/genetics , Myeloid Cells/metabolism , Repressor Proteins/genetics , Acute Disease , Animals , Gene Expression Profiling/methods , Humans , Leukemia, Myeloid/genetics , Leukemia, Myeloid/pathology , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Myelopoiesis/geneticsABSTRACT
BACKGROUND: Coats plus syndrome is an autosomal recessive, pleiotropic, multisystem disorder characterized by retinal telangiectasia and exudates, intracranial calcification with leukoencephalopathy and brain cysts, osteopenia with predisposition to fractures, bone marrow suppression, gastrointestinal bleeding and portal hypertension. It is caused by compound heterozygous mutations in the CTC1 gene. CASE PRESENTATION: We encountered a case of an eight-year old boy from an Indian family with manifestations of Coats plus syndrome along with an unusual occurrence of dextrocardia and situs inversus. Targeted resequencing of the CTC1 gene as well as whole exome sequencing (WES) were conducted in this family to identify the causal variations. The identified candidate variations were screened in ethnicity matched healthy controls. The effect of CTC1 variation on telomere length was assessed using Southern blot. A novel homozygous missense mutation c.1451A > C (p.H484P) in exon 9 of the CTC1 gene and a rare 3'UTR known dbSNP variation (c.*556 T > C) in HES7 were identified as the plausible candidates associated with this complex phenotype of Coats plus and dextrocardia. This CTC1 variation was absent in the controls and we also observed a reduced telomere length in the affected individual's DNA, suggesting its likely pathogenic nature. The reported p.H484P mutation is located in the N-terminal 700 amino acid regionthat is important for the binding of CTC1 to ssDNA through its two OB domains. WES data also showed a rare homozygous missense variation in the TEK gene in the affected individual. Both HES7 and TEK are targets of the Notch signaling pathway. CONCLUSIONS: This is the first report of a genetically confirmed case of Coats plus syndrome from India. By means of WES, the genetic variations in this family with unique and rare complex phenotype could be traced effectively. We speculate the important role of Notch signaling in this complex phenotypic presentation of Coats plus syndrome and dextrocardia. The present finding will be useful for genetic diagnosis and carrier detection in the family and for other patients with similar disease manifestations.
Subject(s)
Ataxia/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Brain Neoplasms/genetics , Calcinosis/genetics , Central Nervous System Cysts/genetics , Dextrocardia/complications , Exome/genetics , Leukoencephalopathies/genetics , Muscle Spasticity/genetics , Mutation, Missense , Retinal Diseases/genetics , Seizures/genetics , Sequence Analysis, DNA , Telomere-Binding Proteins/genetics , Animals , Ataxia/complications , Ataxia/pathology , Base Sequence , Brain Neoplasms/complications , Brain Neoplasms/pathology , Calcinosis/complications , Calcinosis/pathology , Central Nervous System Cysts/complications , Central Nervous System Cysts/pathology , Child , Genomics , Homozygote , Humans , India , Leukoencephalopathies/complications , Leukoencephalopathies/pathology , Male , Muscle Spasticity/complications , Muscle Spasticity/pathology , Pedigree , Phenotype , Receptors, Notch/metabolism , Retinal Diseases/complications , Retinal Diseases/pathology , Seizures/complications , Seizures/pathology , Signal Transduction , Telomere/geneticsABSTRACT
The adenosine triphosphate (ATP)-dependent chromatin remodeling complex, SWItch/Sucrose Non-Fermentable (SWI/SNF), has been implicated in normal hematopoiesis. The AT-rich interaction domain 1B (ARID1B) and its paralog, ARID1A, are mutually exclusive, DNA-interacting subunits of the BRG1/BRM-associated factor (BAF) subclass of SWI/SNF complex. Although the role of several SWI/SNF components in hematopoietic differentiation and stem cell maintenance has been reported, the function of ARID1B in hematopoietic development has not been defined. To this end, we generated a mouse model of Arid1b deficiency specifically in the hematopoietic compartment. Unlike the extensive phenotype observed in mice deficient in its paralog, ARID1A, Arid1b knockout (KO) mice exhibited a modest effect on steady-state hematopoiesis. Nonetheless, transplantation experiments showed that the reconstitution of myeloid cells in irradiated recipient mice was dependent on ARID1B. Furthermore, to assess the effect of the complete loss of ARID1 proteins in the BAF complex, we generated mice lacking both ARID1A and ARID1B in the hematopoietic compartment. The double-KO mice succumbed to acute bone marrow failure resulting from complete loss of BAF-mediated chromatin remodeling activity. Our Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses revealed that >80% of loci regulated by ARID1B were distinct from those regulated by ARID1A; and ARID1B controlled expression of genes crucial in myelopoiesis. Overall, loss of ARID1B affected chromatin dynamics in murine hematopoietic stem and progenitor cells, albeit to a lesser extent than cells lacking ARID1A.
Subject(s)
Hematopoiesis , Nuclear Proteins , Animals , Mice , Cell Differentiation/genetics , Chromatin , Hematopoiesis/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolismABSTRACT
Alternative lengthening of telomeres (ALT) supports telomere maintenance in 10-15% of cancers, thus representing a compelling target for therapy. By performing anti-cancer compound library screen on isogenic cell lines and using extrachromosomal telomeric C-circles, as a bona fide marker of ALT activity, we identify a receptor tyrosine kinase inhibitor ponatinib that deregulates ALT mechanisms, induces telomeric dysfunction, reduced ALT-associated telomere synthesis, and targets, in vivo, ALT-positive cells. Using RNA-sequencing and quantitative phosphoproteomic analyses, combined with C-circle level assessment, we find an ABL1-JNK-JUN signalling circuit to be inhibited by ponatinib and to have a role in suppressing telomeric C-circles. Furthermore, transcriptome and interactome analyses suggest a role of JUN in DNA damage repair. These results are corroborated by synergistic drug interactions between ponatinib and either DNA synthesis or repair inhibitors, such as triciribine. Taken together, we describe here a signalling pathway impacting ALT which can be targeted by a clinically approved drug.
Subject(s)
Signal Transduction , Telomere , Cell Survival , Signal Transduction/drug effects , Gene Expression Regulation , DNA Repair , DNA Replication , JNK Mitogen-Activated Protein Kinases/metabolism , Humans , Animals , Mice , Cell Line, TumorABSTRACT
Despite the remarkable clinical responses achieved with immune checkpoint blockade therapy, the response rate is relatively low and only a subset of patients can benefit from the treatment. Aberrant RNA accumulation can mediate IFN signaling and stimulate an immune response, suggesting that targeting RNA decay machinery might sensitize tumor cells to immunotherapy. With this in mind, we identified an RNA exoribonuclease, XRN1, as a potential therapeutic target to suppress RNA decay and stimulate antitumor immunity. Silencing of XRN1 suppressed tumor growth in syngeneic immunocompetent mice and potentiated immunotherapy efficacy, while silencing of XRN1 alone did not affect tumor growth in immunodeficient mice. Mechanistically, XRN1 depletion activated IFN signaling and the viral defense pathway; both pathways play determinant roles in regulating immune evasion. Aberrant RNA-sensing signaling proteins (RIG-I/MAVS) mediated the expression of IFN genes, as depletion of each of them blunted the elevation of antiviral/IFN signaling in XRN1-silenced cells. Analysis of pan-cancer CRISPR-screening data indicated that IFN signaling triggered by XRN1 silencing is a common phenomenon, suggesting that the effect of XRN1 silencing may be extended to multiple types of cancers. Overall, XRN1 depletion triggers aberrant RNA-mediated IFN signaling, highlighting the importance of the aberrant RNA-sensing pathway in regulating immune responses. These findings provide the molecular rationale for developing XRN1 inhibitors and exploring their potential clinical application in combination with cancer immunotherapy. SIGNIFICANCE: Targeting XRN1 activates an intracellular innate immune response mediated by RNA-sensing signaling and potentiates cancer immunotherapy efficacy, suggesting inhibition of RNA decay machinery as a novel strategy for cancer treatment.
Subject(s)
Neoplasms , RNA , Animals , Mice , Exonucleases/metabolism , Exoribonucleases/genetics , Exoribonucleases/metabolism , Immunotherapy , Neoplasms/genetics , Neoplasms/therapy , RNA Stability , Signal TransductionABSTRACT
The differentiation of naive CD8+ T lymphocytes into cytotoxic effector and memory CTL results in large-scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organization underpin these transcriptional programs. We use Hi-C to map changes in the spatial organization of long-range genome contacts within naive, effector, and memory virus-specific CD8+ T cells. We observe that the architecture of the naive CD8+ T cell genome is distinct from effector and memory genome configurations, with extensive changes within discrete functional chromatin domains associated with effector/memory differentiation. Deletion of BACH2, or to a lesser extent, reducing SATB1 DNA binding, within naive CD8+ T cells results in a chromatin architecture more reminiscent of effector/memory states. This suggests that key transcription factors within naive CD8+ T cells act to restrain T cell differentiation by actively enforcing a unique naive chromatin state.
Subject(s)
CD8-Positive T-Lymphocytes , Chromatin , Cell Differentiation , Transcription Factors/genetics , Immunologic Memory/geneticsABSTRACT
Precision medicine aims to move from traditional reactive medicine to a system where risk groups can be identified before the disease occurs. However, phenotypic heterogeneity amongst the diseased and healthy poses a major challenge for identification markers for risk stratification and early actionable interventions. In Ayurveda, individuals are phenotypically stratified into seven constitution types based on multisystem phenotypes termed "Prakriti". It enables the prediction of health and disease trajectories and the selection of health interventions. We hypothesize that exome sequencing in healthy individuals of phenotypically homogeneous Prakriti types might enable the identification of functional variations associated with the constitution types. Exomes of 144 healthy Prakriti stratified individuals and controls from two genetically homogeneous cohorts (north and western India) revealed differential risk for diseases/traits like metabolic disorders, liver diseases, and body and hematological measurements amongst healthy individuals. These SNPs differ significantly from the Indo-European background control as well. Amongst these we highlight novel SNPs rs304447 (IFIT5) and rs941590 (SERPINA10) that could explain differential trajectories for immune response, bleeding or thrombosis. Our method demonstrates the requirement of a relatively smaller sample size for a well powered study. This study highlights the potential of integrating a unique phenotyping approach for the identification of predictive markers and the at-risk population amongst the healthy.
ABSTRACT
Soft tissue sarcoma (STS) is a heterogeneous disease that arises from connective tissues. Clinical outcome of patients with advanced tumors especially de-differentiated liposarcoma and uterine leiomyosarcoma remains unsatisfactory, despite intensive treatment regimens including maximal surgical resection, radiation, and chemotherapy. MAP kinase-interacting serine/threonine-protein kinase 1 and 2 (MNK1/2) have been shown to contribute to oncogenic translation via phosphorylation of eukaryotic translation initiation factor 4E (eIF4E). However, little is known about the role of MNK1/2 and their downstream targets in STS. In this study, we show that depletion of either MNK1 or MNK2 suppresses cell viability, anchorage-independent growth, and tumorigenicity of STS cells. We also identify a compelling antiproliferative efficacy of a novel, selective MNK inhibitor ETC-168. Cellular responsiveness of STS cells to ETC-168 correlates positively with that of phosphorylated ribosomal protein S6 (RPS6). Mirroring MNK1/2 silencing, ETC-168 treatment strongly blocks eIF4E phosphorylation and represses expression of sarcoma-driving onco-proteins including E2F1, FOXM1, and WEE1. Moreover, combination of ETC-168 and MCL1 inhibitor S63845 exerts a synergistic antiproliferative activity against STS cells. In summary, our study reveals crucial roles of MNK1/2 and their downstream targets in STS tumorigenesis. Our data encourage further clinical translation of MNK inhibitors for STS treatment.
Subject(s)
Cell Cycle Proteins/genetics , E2F1 Transcription Factor/genetics , Forkhead Box Protein M1/genetics , Intracellular Signaling Peptides and Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Sarcoma/drug therapy , Carcinogenesis/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Sarcoma/genetics , Sarcoma/pathology , Thiophenes/pharmacologyABSTRACT
Molecular profiling of the most aggressive brain tumor glioblastoma (GBM) on the basis of gene expression, DNA methylation, and genomic variations advances both cancer research and clinical diagnosis. The enhancer architectures and regulatory circuitries governing tumor-intrinsic transcriptional diversity and subtype identity are still elusive. Here, by mapping H3K27ac deposition, we analyze the active regulatory landscapes across 95 GBM biopsies, 12 normal brain tissues, and 38 cell line counterparts. Analyses of differentially regulated enhancers and super-enhancers uncovered previously unrecognized layers of intertumor heterogeneity. Integrative analysis of variant enhancer loci and transcriptome identified topographies of transcriptional enhancers and core regulatory circuitries in four molecular subtypes of primary tumors: AC1-mesenchymal, AC1-classical, AC2-proneural, and AC3-proneural. Moreover, this study reveals core oncogenic dependency on super-enhancer-driven transcriptional factors, long noncoding RNAs, and druggable targets in GBM. Through profiling of transcriptional enhancers, we provide clinically relevant insights into molecular classification, pathogenesis, and therapeutic intervention of GBM.
Subject(s)
Brain Neoplasms , Glioblastoma , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Chromatin/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Glioblastoma/pathology , HumansABSTRACT
ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved adenylate-uridylate-rich elements (ARE) located in 3'untranslated regions (UTR) to mediate mRNA decay. We hypothesized that ZFP36L1 is a negative regulator of a posttranscriptional hub involved in mRNA half-life regulation of cancer-related transcripts. Analysis of in silico data revealed that ZFP36L1 was significantly mutated, epigenetically silenced, and downregulated in a variety of cancers. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo, whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wild-type and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells {plus minus} tet-on ZFP36L1 was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1. These targets included a number of key oncogenic transcripts such as HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3'UTRs of these targets for mRNA degradation, thus suppressing their expression. Dual luciferase reporter assays and RNA electrophoretic mobility shift assays showed that wild-type, but not zinc-finger mutant ZFP36L1, bound to HIF1A 3'UTR and mediated HIF1A mRNA degradation, leading to reduced expression of HIF1A and its downstream targets. Collectively, our findings reveal an indispensable role of ZFP36L1 as a posttranscriptional safeguard against aberrant hypoxic signaling and abnormal cell-cycle progression. SIGNIFICANCE: RNA-binding protein ZFP36L1 functions as a tumor suppressor by regulating the mRNA stability of a number of mRNAs involved in hypoxia and cell-cycle signaling.
Subject(s)
Breast Neoplasms/genetics , Butyrate Response Factor 1/metabolism , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Urinary Bladder Neoplasms/genetics , 3' Untranslated Regions/genetics , Animals , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Butyrate Response Factor 1/genetics , Carcinogenesis/genetics , Cell Cycle/genetics , Cell Hypoxia/genetics , Cell Line, Tumor , Cyclin D1/genetics , E2F1 Transcription Factor/genetics , Epigenesis, Genetic , Female , Gene Knockdown Techniques , Humans , Mice , Mutation , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Urinary Bladder Neoplasms/pathology , Xenograft Model Antitumor Assays , Zinc Fingers/geneticsABSTRACT
Lipomas are benign fatty tumors with a high prevalence rate, mostly found in adults but have a good prognosis. Until now, reason for lipoma occurrence not been identified. We performed whole exome sequencing to define the mutational spectrum in ten lipoma patients along with their matching control samples. We presented genomic insight into the development of lipomas, the most common benign tumor of soft tissue. Our analysis identified 412 somatic variants including missense mutations, splice site variants, frameshift indels, and stop gain/lost. Copy number variation analysis highlighted minor aberrations in patients. Kinase genes and transcriptions factors were among the validated mutated genes critical for cell proliferation and survival. Pathway analysis revealed enrichment of calcium, Wnt and phospholipase D signaling in patients. In conclusion, whole exome sequencing in lipomas identified mutations in genes with a possible role in development and progression of lipomas.
Subject(s)
Cell Proliferation/genetics , DNA Copy Number Variations/genetics , Exome Sequencing , Lipoma/genetics , Adult , Aged , Codon, Nonsense/genetics , Exome/genetics , Female , Frameshift Mutation/genetics , Humans , INDEL Mutation/genetics , Lipoma/pathology , Male , Middle Aged , Mutation, Missense/genetics , Phosphotransferases/genetics , Transcription Factors/geneticsABSTRACT
Precise regulation of chromatin architecture is vital to physiological processes including hematopoiesis. ARID1A is a core component of the mammalian SWI/SNF complex, which is one of the ATP-dependent chromatin remodeling complexes. To uncover the role of ARID1A in hematopoietic development, we utilized hematopoietic cell-specific deletion of Arid1a in mice. We demonstrate that ARID1A is essential for maintaining the frequency and function of hematopoietic stem cells and its loss impairs the differentiation of both myeloid and lymphoid lineages. ARID1A deficiency led to a global reduction in open chromatin and ensuing transcriptional changes affected key genes involved in hematopoietic development. We also observed that silencing of ARID1A affected ATRA-induced differentiation of NB4 cells, suggesting its role in granulocytic differentiation of human leukemic cells. Overall, our study provides a comprehensive elucidation of the function of ARID1A in hematopoiesis and highlights the central role of ARID1A-containing SWI/SNF complex in maintaining chromatin dynamics in hematopoietic cells.