ABSTRACT
The Hippo tumor suppressor pathway controls transcription by regulating nuclear abundance of YAP and TAZ, which activate transcription with the TEAD1-TEAD4 DNA-binding proteins. Recently, several small-molecule inhibitors of YAP and TEADs have been reported, with some entering clinical trials for different cancers with Hippo pathway deregulation, most notably, mesothelioma. Using genome-wide CRISPR/Cas9 screens we reveal that mutations in genes from the Hippo, MAPK, and JAK-STAT signaling pathways all modulate the response of mesothelioma cell lines to TEAD palmitoylation inhibitors. By exploring gene expression programs of mutant cells, we find that MAPK pathway hyperactivation confers resistance to TEAD inhibition by reinstating expression of a subset of YAP/TAZ target genes. Consistent with this, combined inhibition of TEAD and the MAPK kinase MEK, synergistically blocks proliferation of multiple mesothelioma and lung cancer cell lines and more potently reduces the growth of patient-derived lung cancer xenografts in vivo. Collectively, we reveal mechanisms by which cells can overcome small-molecule inhibition of TEAD palmitoylation and potential strategies to enhance the anti-tumor activity of emerging Hippo pathway targeted therapies.
ABSTRACT
The liver exhibits a remarkable capacity to regenerate following injury. Despite this unique attribute, toxic injury is a leading cause of liver failure. The temporal processes by which the liver senses injury and initiates regeneration remain unclear. Here, we developed a transgenic zebrafish model wherein hepatocyte-specific expression of uracil phosphoribosyltransferase (UPRT) enabled the implementation of SLAM-ITseq to investigate the nascent transcriptome during initiation of liver injury and regeneration. Using this approach, we identified a rapid metabolic transition from the fed to the fasted state that was followed by induction of the nuclear erythroid 2-related factor (Nrf2) antioxidant program. We find that activation of Nrf2 in hepatocytes is required to induce the pentose phosphate pathway (PPP) and improve survival following liver injury. Mechanistically, we demonstrate that inhibition of the PPP disrupts nucleotide biosynthesis to prevent liver regeneration. Together, these studies provide fundamental insights into the mechanism by which early metabolic adaptation to injury facilitates tissue regeneration.
Subject(s)
Liver Regeneration , Pentose Phosphate Pathway , Animals , Pentose Phosphate Pathway/genetics , Liver Regeneration/genetics , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Liver/metabolismABSTRACT
Chromatin regulation involves the selective recruitment of chromatin factors to facilitate DNA repair, replication and transcription. Here we demonstrate the utility of coupling unbiased functional genomics with chromatin immunoprecipitation (CRISPR-ChIP) to identify the factors associated with active chromatin modifications in mammalian cells. Specifically, an integrated reporter containing a cis-regulatory element of interest and a single guide RNA provide a chromatinized template for a direct readout for regulators of histone modifications associated with actively transcribed genes such as H3K4me3 and H3K79me2. With CRISPR-ChIP, we identify all the nonredundant COMPASS complex members required for H3K4me3 and demonstrate that RNA polymerase II is dispensable for the maintenance of H3K4me3. As H3K79me2 has a putative oncogenic function in leukemia cells driven by MLL translocations, using CRISPR-ChIP we reveal a functional partitioning of H3K79 methylation into two distinct regulatory units: an oncogenic DOT1L complex directed by the MLL fusion protein in a Menin-dependent manner and a separate endogenous DOT1L complex, where catalytic activity is directed by MLLT10. Overall, CRISPR-ChIP provides a powerful tool for the unbiased interrogation of the mechanisms underpinning chromatin regulation.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Leukemia , Animals , Humans , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Transcription Factors/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Chromatin , Leukemia/genetics , Chromatin Immunoprecipitation , Mammals/geneticsABSTRACT
Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl-transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti-PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole-cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic. SIGNIFICANCE: This work demonstrates that METTL3 inhibition stimulates a cell-intrinsic interferon response through dsRNA formation. This immunomodulatory mechanism is distinct from current immunotherapeutic agents and provides the molecular rationale for combination with anti-PD-1 immune-checkpoint blockade to augment antitumor immunity. This article is featured in Selected Articles from This Issue, p. 2109.
Subject(s)
Interferons , Methyltransferases , Animals , Mice , Interferons/genetics , Methyltransferases/genetics , Methyltransferases/metabolism , RNA, Double-StrandedABSTRACT
Precise control of activating H3K4me3 and repressive H3K27me3 histone modifications at bivalent promoters is essential for normal development and frequently corrupted in cancer. By coupling a cell surface readout of bivalent MHC class I gene expression with whole-genome CRISPR-Cas9 screens, we identify specific roles for MTF2-PRC2.1, PCGF1-PRC1.1 and Menin-KMT2A/B complexes in maintaining bivalency. Genetic loss or pharmacological inhibition of Menin unexpectedly phenocopies the effects of polycomb disruption, resulting in derepression of bivalent genes in both cancer cells and pluripotent stem cells. While Menin and KMT2A/B contribute to H3K4me3 at active genes, a separate Menin-independent function of KMT2A/B maintains H3K4me3 and opposes polycomb-mediated repression at bivalent genes. Release of KMT2A from active genes following Menin targeting alters the balance of polycomb and KMT2A at bivalent genes, facilitating gene activation. This functional partitioning of Menin-KMT2A/B complex components reveals therapeutic opportunities that can be leveraged through inhibition of Menin.
Subject(s)
Pluripotent Stem Cells , Transcription Factors , Polycomb-Group Proteins/genetics , Transcription Factors/genetics , Genome , Promoter Regions, GeneticABSTRACT
There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.
Subject(s)
F-Box Proteins , Leukemia, Myeloid, Acute , Alcohol Oxidoreductases , DNA-Binding Proteins , F-Box Proteins/genetics , HLA Antigens/genetics , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/therapy , Lymphocyte Activation , Protein-Arginine N-Methyltransferases/metabolism , Recurrence , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.
Subject(s)
Cell Competition , Clone Cells/pathology , Leukemia, Myeloid, Acute/pathology , Single-Cell Analysis , Animals , Cell Competition/drug effects , Cell Line , Cell Lineage/drug effects , Clone Cells/drug effects , Clone Cells/metabolism , Female , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Inbred C57BL , Secretory Leukocyte Peptidase Inhibitor/metabolismABSTRACT
Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples. SIGNIFICANCE: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587.
Subject(s)
Iron , bcl-2 Homologous Antagonist-Killer Protein , Apoptosis , Cell Death , Humans , Iron/metabolism , Mitochondria/metabolism , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
Angiogenesis underlies development, physiology and pathogenesis of cancer, eye and cardiovascular diseases. Inhibiting aberrant angiogenesis using anti-angiogenic therapy (AAT) has been successful in the clinical treatment of cancer and eye diseases. However, resistance to AAT inevitably occurs and its molecular basis remains poorly understood. Here, we uncover molecular modifiers of the blood endothelial cell (EC) response to a widely used AAT bevacizumab by performing a pooled genetic screen using three-dimensional microcarrier-based cell culture and CRISPR-Cas9. Functional inhibition of the epigenetic reader BET family of proteins BRD2/3/4 shows unexpected mitigating effects on EC survival and/or proliferation upon VEGFA blockade. Moreover, transcriptomic and pathway analyses reveal an interaction between epigenetic regulation and anti-angiogenesis, which may affect chromosomal structure and activity in ECs via the cell cycle regulator CDC25B phosphatase. Collectively, our findings provide insight into epigenetic regulation of the EC response to VEGFA blockade and may facilitate development of quality biomarkers and strategies for overcoming resistance to AAT.
Subject(s)
Angiogenesis Inhibitors/genetics , Bevacizumab/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Epigenesis, Genetic , Blood , Endothelial Cells/drug effectsABSTRACT
B lymphoid development is initiated by the differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating mature B cells. This highly regulated process generates clonal immunological diversity via recombination of immunoglobulin V, D and J gene segments. While several transcription factors that control B cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for early B lymphoid differentiation. Erg initiates a transcriptional network involving the B cell lineage defining genes, Ebf1 and Pax5, which directly promotes expression of key genes involved in V(D)J recombination and formation of the B cell receptor. Complementation of Erg deficiency with a productively rearranged immunoglobulin gene rescued B lineage development, demonstrating that Erg is an essential and stage-specific regulator of the gene regulatory network controlling B lymphopoiesis.
Subject(s)
B-Lymphocytes/metabolism , Cell Differentiation/genetics , Hematopoietic Stem Cells/metabolism , Lymphopoiesis/genetics , Oncogene Proteins/genetics , Transcription, Genetic , Transcriptional Regulator ERG/genetics , Animals , B-Lymphocytes/cytology , Cell Lineage/genetics , Cells, Cultured , Gene Regulatory Networks/genetics , Hematopoietic Stem Cells/cytology , Mice, Inbred C57BL , Mice, Knockout , Oncogene Proteins/metabolism , PAX5 Transcription Factor/genetics , PAX5 Transcription Factor/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Regulator ERG/metabolism , V(D)J Recombination/geneticsABSTRACT
Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.
Subject(s)
Histone Acetyltransferases/metabolism , Leukemia, Myeloid, Acute/metabolism , Neoplastic Stem Cells/metabolism , Animals , Cell Line, Tumor , Histone Acetyltransferases/chemistry , Histone Acetyltransferases/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Inbred C57BL , Models, Molecular , Protein Structure, TertiaryABSTRACT
Loss of MHC class I (MHC-I) antigen presentation in cancer cells can elicit immunotherapy resistance. A genome-wide CRISPR/Cas9 screen identified an evolutionarily conserved function of polycomb repressive complex 2 (PRC2) that mediates coordinated transcriptional silencing of the MHC-I antigen processing pathway (MHC-I APP), promoting evasion of T cell-mediated immunity. MHC-I APP gene promoters in MHC-I low cancers harbor bivalent activating H3K4me3 and repressive H3K27me3 histone modifications, silencing basal MHC-I expression and restricting cytokine-induced upregulation. Bivalent chromatin at MHC-I APP genes is a normal developmental process active in embryonic stem cells and maintained during neural progenitor differentiation. This physiological MHC-I silencing highlights a conserved mechanism by which cancers arising from these primitive tissues exploit PRC2 activity to enable immune evasion.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Gene Expression Regulation, Neoplastic/immunology , Histocompatibility Antigens Class I/genetics , Neoplasms/immunology , Polycomb Repressive Complex 2/metabolism , Tumor Escape/genetics , Animals , Antigen Presentation/drug effects , Antigen Presentation/immunology , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , DNA Methylation/immunology , Down-Regulation/drug effects , Down-Regulation/genetics , Down-Regulation/immunology , Drug Resistance, Neoplasm/genetics , Epigenetic Repression/drug effects , Epigenetic Repression/immunology , Female , Gene Expression Regulation, Neoplastic/drug effects , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Histone Code/drug effects , Humans , Mice , Middle Aged , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Polycomb Repressive Complex 2/antagonists & inhibitors , T-Lymphocytes/immunology , Tumor Escape/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Leukemic/drug effects , Leukemia, Myeloid, Acute/drug therapy , Trans-Activators/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Bone Marrow/pathology , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Epigenesis, Genetic/drug effects , Female , HEK293 Cells , Humans , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred C57BL , Sequence Analysis, RNA , Single-Cell Analysis , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription, Genetic/drug effects , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Ibrutinib plus venetoclax is a highly effective combination in mantle cell lymphoma. However, strategies to enable the evaluation of therapeutic response are required. Our prospective analyses of patients within the AIM study revealed genomic profiles that clearly dichotomized responders and nonresponders. Mutations in ATM were present in most patients who achieved a complete response, while chromosome 9p21.1-p24.3 loss and/or mutations in components of the SWI-SNF chromatin-remodeling complex were present in all patients with primary resistance and two-thirds of patients with relapsed disease. Circulating tumor DNA analysis revealed that these alterations could be dynamically monitored, providing concurrent information on treatment response and tumor evolution. Functional modeling demonstrated that compromise of the SWI-SNF complex facilitated transcriptional upregulation of BCL2L1 (Bcl-xL) providing a selective advantage against ibrutinib plus venetoclax. Together these data highlight important insights into the molecular basis of therapeutic response and provide a model for real-time assessment of innovative targeted therapies.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Chromosomal Proteins, Non-Histone/genetics , Drug Resistance, Neoplasm/genetics , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/genetics , Mutation/genetics , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Sulfonamides/therapeutic use , Transcription Factors/genetics , Activating Transcription Factor 3/metabolism , Adenine/analogs & derivatives , Cell Line, Tumor , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Circulating Tumor DNA/genetics , Cohort Studies , DNA Helicases/metabolism , Genome, Human , Humans , Models, Biological , Nuclear Proteins/metabolism , Piperidines , Prognosis , Transcription Factors/metabolism , Treatment Outcome , bcl-X Protein/metabolismABSTRACT
Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR-Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.
Subject(s)
B7-H1 Antigen/biosynthesis , B7-H1 Antigen/metabolism , Membrane Proteins/metabolism , Neoplasms/immunology , Neoplasms/metabolism , Animals , B7-H1 Antigen/immunology , CRISPR-Cas Systems , Cell Line , Cell Membrane/metabolism , Endosomes/metabolism , Female , Histocompatibility Antigens Class I/immunology , Humans , Lysosomes/metabolism , Mice , Proteolysis , Proteome/metabolism , Substrate Specificity , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Escape/immunologyABSTRACT
The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.
Subject(s)
Benzodiazepines/therapeutic use , Click Chemistry , Drug Delivery Systems , Epigenomics , Leukemia/drug therapy , Animals , Benzodiazepines/pharmacology , Cells, Cultured , Disease Models, Animal , Leukemia/pathology , Mice , Precision Medicine , Tissue Distribution , Transcription Factors/antagonists & inhibitorsABSTRACT
Several novel therapeutics are poised to change the natural history of chronic lymphocytic leukaemia (CLL) and the increasing use of these therapies has highlighted limitations of traditional disease monitoring methods. Here we demonstrate that circulating tumour DNA (ctDNA) is readily detectable in patients with CLL. Importantly, ctDNA does not simply mirror the genomic information contained within circulating malignant lymphocytes but instead parallels changes across different disease compartments following treatment with novel therapies. Serial ctDNA analysis allows clonal dynamics to be monitored over time and identifies the emergence of genomic changes associated with Richter's syndrome (RS). In addition to conventional disease monitoring, ctDNA provides a unique opportunity for non-invasive serial analysis of CLL for molecular disease monitoring.
Subject(s)
Circulating Tumor DNA/genetics , Clonal Evolution/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Adenine/analogs & derivatives , Aged , Aged, 80 and over , Antineoplastic Agents/therapeutic use , Ataxia Telangiectasia Mutated Proteins/genetics , Baculoviral IAP Repeat-Containing 3 Protein/genetics , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Circulating Tumor DNA/blood , Disease Progression , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Male , Middle Aged , Myeloid Differentiation Factor 88/genetics , Phosphoproteins/genetics , Piperidines , Proto-Oncogene Proteins p21(ras)/genetics , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , RNA Splicing Factors/genetics , Receptor, Notch1/genetics , Sulfonamides/therapeutic use , Treatment Outcome , Tumor Suppressor Protein p53/geneticsABSTRACT
BACKGROUND: Chronic rhinosinusitis with nasal polyps is associated with local immunoglobulin hyperproduction and the presence of IgE antibodies against Staphylococcus aureus enterotoxins (SAEs). Aspirin-exacerbated respiratory disease is a severe form of chronic rhinosinusitis with nasal polyps in which nearly all patients express anti-SAEs. OBJECTIVES: We aimed to understand antibodies reactive to SAEs and determine whether they recognize SAEs through their complementarity-determining regions (CDRs) or framework regions. METHODS: Labeled staphylococcal enterotoxin (SE) A, SED, and SEE were used to isolate single SAE-specific B cells from the nasal polyps of 3 patients with aspirin-exacerbated respiratory disease by using fluorescence-activated cell sorting. Recombinant antibodies with "matched" heavy and light chains were cloned as IgG1, and those of high affinity for specific SAEs, assayed by means of ELISA and surface plasmon resonance, were recloned as IgE and antigen-binding fragments. IgE activities were tested in basophil degranulation assays. RESULTS: Thirty-seven SAE-specific, IgG- or IgA-expressing B cells were isolated and yielded 6 anti-SAE clones, 2 each for SEA, SED, and SEE. Competition binding assays revealed that the anti-SEE antibodies recognize nonoverlapping epitopes in SEE. Unexpectedly, each anti-SEE mediated SEE-induced basophil degranulation, and IgG1 or antigen-binding fragments of each anti-SEE enhanced degranulation by the other anti-SEE. CONCLUSIONS: SEEs can activate basophils by simultaneously binding as antigens in the conventional manner to CDRs and as superantigens to framework regions of anti-SEE IgE in anti-SEE IgE-FcεRI complexes. Anti-SEE IgG1s can enhance the activity of anti-SEE IgEs as conventional antibodies through CDRs or simultaneously as conventional antibodies and as "superantibodies" through CDRs and framework regions to SEEs in SEE-anti-SEE IgE-FcεRI complexes.
Subject(s)
Enterotoxins/immunology , Nasal Polyps/immunology , Rhinitis/immunology , Sinusitis/immunology , Asthma, Aspirin-Induced/immunology , Basophil Degranulation Test , Basophils/immunology , Cell Separation , Chronic Disease , Complementarity Determining Regions , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Immunoglobulin E/immunology , Immunoglobulin G/immunology , Male , Middle Aged , Staphylococcus aureus/immunology , Superantigens/immunology , Surface Plasmon ResonanceABSTRACT
Omalizumab therapy of non-atopic asthmatics reduces bronchial mucosal IgE and inflammation and preserves/improves lung function when disease is destabilised by staged withdrawal of therapy.18 symptomatic, non-atopic asthmatics were randomised (1:1) to receive omalizumab or identical placebo treatment in addition to existing therapy for 20â weeks. Bronchial biopsies were collected before and after 12-14â weeks of treatment, then the patients destabilised by substantial, supervised reduction of their regular therapy. Primary outcome measures were changes in bronchial mucosal IgE+ cells at 12-14â weeks, prior to regular therapy reduction, and changes in lung function (forced expiratory volume in 1â s) after destabilisation at 20â weeks. Quality of life was also monitored.Omalizumab but not placebo therapy significantly reduced median total bronchial mucosal IgE+ cells (p<0.01) but did not significantly alter median total mast cells, plasma cells, B lymphocytes, eosinophils and plasmablasts, although the latter were difficult to enumerate, being distributed as disperse clusters. By 20â weeks, lung function declined in the placebo-treated patients but improved in the omalizumab treated patients, with significant differences in absolute (p=0.04) and % predicted forced expiratory volume in 1â s (p=0.015).Omalizumab therapy of non-atopic asthmatics reduces bronchial mucosal IgE+ mast cells and improves lung function despite withdrawal of conventional therapy.
Subject(s)
Anti-Asthmatic Agents/therapeutic use , Asthma/drug therapy , Bronchi/pathology , Immunoglobulin E/blood , Omalizumab/therapeutic use , Adult , Aged , Bronchoscopy , Double-Blind Method , Female , Forced Expiratory Volume , Humans , Leukocyte Count , Male , Middle Aged , Proof of Concept Study , Quality of Life , Treatment Outcome , United Kingdom , Young AdultABSTRACT
Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis.