Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
1.
Cell ; 184(12): 3143-3162.e32, 2021 06 10.
Article in English | MEDLINE | ID: mdl-34004147

ABSTRACT

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.


Subject(s)
Cyclin-Dependent Kinase 9/metabolism , Molecular Targeted Therapy , Neoplasms/drug therapy , Neoplasms/genetics , Protein Phosphatase 2/metabolism , RNA-Binding Proteins/metabolism , Transcription, Genetic , Tumor Suppressor Proteins/metabolism , Animals , Cell Line, Tumor , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Humans , Mice, Inbred NOD , Phosphorylation , Protein Binding , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Substrate Specificity
2.
Mol Cell ; 81(10): 2183-2200.e13, 2021 05 20.
Article in English | MEDLINE | ID: mdl-34019788

ABSTRACT

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.


Subject(s)
Biocatalysis , Histones/metabolism , Oncogenes , Transcription, Genetic , p300-CBP Transcription Factors/metabolism , Acetylation , Cell Line , Chromatin/metabolism , Co-Repressor Proteins/metabolism , Conserved Sequence , Evolution, Molecular , Gene Regulatory Networks , Genome , Histone Deacetylases/metabolism , Humans , Kinetics , Methylation , Models, Biological , RNA Polymerase II/metabolism
3.
Mol Ther ; 28(11): 2379-2393, 2020 11 04.
Article in English | MEDLINE | ID: mdl-32735774

ABSTRACT

Chimeric antigen receptor (CAR) T cell therapy has been highly successful in hematological malignancies leading to their US Food and Drug Administration (FDA) approval. However, the efficacy of CAR T cells in solid tumors is limited by tumor-induced immunosuppression, leading to the development of combination approaches, such as adjuvant programmed cell death 1 (PD-1) blockade. Current FDA-approved methods for generating CAR T cells utilize either anti-CD3 and interleukin (IL)-2 or anti-CD3/CD28 beads, which can generate a T cell product with an effector/exhausted phenotype. Whereas different cytokine preconditioning milieu, such as IL-7/IL-15, have been shown to promote T cell engraftment, the impact of this approach on CAR T cell responses to adjuvant immune-checkpoint blockade has not been assessed. In the current study, we reveal that the preconditioning of CAR T cells with IL-7/IL-15 increased CAR T cell responses to anti-PD-1 adjuvant therapy. This was associated with the emergence of an intratumoral CD8+CD62L+TCF7+IRF4- population that was highly responsive to anti-PD-1 therapy and mediated the vast majority of transcriptional and epigenetic changes in vivo following PD-1 blockade. Our data indicate that preservation of CAR T cells in a TCF7+ phenotype is crucial for their responsiveness to adjuvant immunotherapy approaches and should be a key consideration when designing clinical protocols.


Subject(s)
Immune Checkpoint Inhibitors/therapeutic use , Immunotherapy, Adoptive , Interleukin-15/administration & dosage , Neoplasms/therapy , Biomarkers, Tumor , Combined Modality Therapy , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immune Checkpoint Proteins/metabolism , Immunotherapy, Adoptive/methods , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Neoplasms/etiology , Treatment Outcome
4.
NAR Cancer ; 6(4): zcae039, 2024 Dec.
Article in English | MEDLINE | ID: mdl-39372038

ABSTRACT

Therapeutic targeting of dysregulated transcription has emerged as a promising strategy for the treatment of cancers, such as leukaemias. The therapeutic response to small molecule inhibitors of Bromodomain-Containing Proteins (BRD), such as BRD2 and BRD4, P300/cAMP-response element binding protein (CBP) and Cyclin Dependent Kinases (CDKs), is generally attributed to the selective disruption of oncogenic gene expression driven by enhancers, super-enhancers (SEs) and lineage-specific transcription factors (TFs), including the c-MYC oncogene. The selectivity of compounds targeting the transcriptional machinery may be further shaped by post-transcriptional processes. To quantitatively assess the contribution of post-transcriptional regulation in responses to transcription inhibition, we performed multi-omics analyses to accurately measure mRNA production and decay kinetics. We demonstrate that it is not only the selective disruption of mRNA production, but rather mRNA decay rates that largely influence the selectivity associated with transcriptional inhibition. Accordingly, genes down-regulated with transcriptional inhibitors are largely characterized by extremely rapid mRNA production and turnover. In line with this notion, stabilization of the c-MYC transcript through swapping of its 3' untranslated region (UTR) rendered c-MYC insensitive to transcriptional targeting. This failed to negate the impact on c-MYC downstream targets and did not abrogate therapeutic responses. Finally, we provide evidence that modulating post-transcriptional pathways, such as through ELAVL1 targeting, can sensitize long-lived mRNAs to transcriptional inhibition and be considered as a combination therapy approach in leukaemia. Taken together, these data demonstrate that mRNA kinetics influence the therapeutic response to transcriptional perturbation and can be modulated for novel therapeutic outcomes using transcriptional agents in leukaemia.

5.
Clin Epigenetics ; 14(1): 96, 2022 07 28.
Article in English | MEDLINE | ID: mdl-35902886

ABSTRACT

BACKGROUND: Interferon gamma (IFNγ) is a pro-inflammatory cytokine that directly activates the JAK/STAT pathway. However, the temporal dynamics of chromatin remodeling and transcriptional activation initiated by IFNγ have not been systematically profiled in an unbiased manner. Herein, we integrated transcriptomic and epigenomic profiling to characterize the acute epigenetic changes induced by IFNγ stimulation in a murine breast cancer model. RESULTS: We identified de novo activation of cis-regulatory elements bound by Irf1 that were characterized by increased chromatin accessibility, differential usage of pro-inflammatory enhancers, and downstream recruitment of BET proteins and RNA polymerase II. To functionally validate this hierarchical model of IFNγ-driven transcription, we applied selective antagonists of histone acetyltransferases P300/CBP or acetyl-lysine readers of the BET family. This highlighted that histone acetylation is an antecedent event in IFNγ-driven transcription, whereby targeting of P300/CBP acetyltransferase activity but not BET inhibition could curtail the epigenetic remodeling induced by IFNγ through suppression of Irf1 transactivation. CONCLUSIONS: These data highlight the ability for epigenetic therapies to reprogram pro-inflammatory gene expression, which may have therapeutic implications for anti-tumor immunity and inflammatory diseases.


Subject(s)
Breast Neoplasms , Interferon-gamma , Acetylation , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , DNA Methylation , E1A-Associated p300 Protein , Female , Interferon-gamma/pharmacology , Janus Kinases , Membrane Proteins , Mice , Phosphoproteins , STAT Transcription Factors , Signal Transduction
6.
Cell Death Differ ; 29(12): 2519-2530, 2022 12.
Article in English | MEDLINE | ID: mdl-35831623

ABSTRACT

High-throughput methodologies are the cornerstone of screening approaches to identify novel compounds that regulate immune cell function. To identify novel targeted therapeutics to treat immune disorders and haematological malignancies, there is a need to integrate functional cellular information with the molecular mechanisms that regulate changes in immune cell phenotype. We facilitate this goal by combining quantitative methods for dissecting complex simultaneous cell phenotypic effects with genomic analysis. This combination strategy we term Multiplexed Analysis of Cells sequencing (MAC-seq), a modified version of Digital RNA with perturbation of Genes (DRUGseq). We applied MAC-seq to screen compounds that target the epigenetic machinery of B cells and assess altered humoral immunity by measuring changes in proliferation, survival, differentiation and transcription. This approach revealed that polycomb repressive complex 2 (PRC2) inhibitors promote antibody secreting cell (ASC) differentiation in both murine and human B cells in vitro. This is further validated using T cell-dependent immunization in mice. Functional dissection of downstream effectors of PRC2 using arrayed CRISPR screening uncovered novel regulators of B cell differentiation, including Mybl1, Myof, Gas7 and Atoh8. Together, our findings demonstrate that integrated phenotype-transcriptome analyses can be effectively combined with drug screening approaches to uncover the molecular circuitry that drives lymphocyte fate decisions.


Subject(s)
B-Lymphocytes , Epigenesis, Genetic , Animals , Humans , Mice , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/genetics , Gene Expression Profiling , Phenotype , Polycomb Repressive Complex 2/metabolism
7.
Cancer Discov ; 12(6): 1560-1579, 2022 06 02.
Article in English | MEDLINE | ID: mdl-35311997

ABSTRACT

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. SIGNIFICANCE: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397.


Subject(s)
Leukemia, Myeloid, Acute , Cell Differentiation , Dendritic Cells , Epigenesis, Genetic , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Histone Deacetylases/genetics , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Panobinostat/pharmacology
8.
Sci Adv ; 7(8)2021 02.
Article in English | MEDLINE | ID: mdl-33608275

ABSTRACT

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

9.
Cancer Discov ; 11(6): 1582-1599, 2021 06.
Article in English | MEDLINE | ID: mdl-33436370

ABSTRACT

Internal tandem duplication of the FMS-like tyrosine kinase 3 gene (FLT3-ITD) occurs in 30% of all acute myeloid leukemias (AML). Limited clinical efficacy of FLT3 inhibitors highlights the need for alternative therapeutic modalities in this subset of disease. Using human and murine models of FLT3-ITD-driven AML, we demonstrate that FLT3-ITD promotes serine synthesis and uptake via ATF4-dependent transcriptional regulation of genes in the de novo serine biosynthesis pathway and neutral amino acid transport. Genetic or pharmacologic inhibition of PHGDH, the rate-limiting enzyme of de novo serine biosynthesis, selectively inhibited proliferation of FLT3-ITD AMLs in vitro and in vivo. Moreover, pharmacologic inhibition of PHGDH sensitized FLT3-ITD AMLs to the standard-of-care chemotherapeutic cytarabine. Collectively, these data reveal novel insights into FLT3-ITD-induced metabolic reprogramming and reveal a targetable vulnerability in FLT3-ITD AML. SIGNIFICANCE: FLT3-ITD mutations are common in AML and are associated with poor prognosis. We show that FLT3-ITD stimulates serine biosynthesis, thereby rendering FLT3-ITD-driven leukemias dependent upon serine for proliferation and survival. This metabolic dependency can be exploited pharmacologically to sensitize FLT3-ITD-driven AMLs to chemotherapy.This article is highlighted in the In This Issue feature, p. 1307.


Subject(s)
Leukemia, Myeloid, Acute/metabolism , Serine/metabolism , fms-Like Tyrosine Kinase 3/metabolism , Animals , Cell Line, Tumor/metabolism , Disease Models, Animal , Humans , Mice , Protein Kinase Inhibitors
10.
Cancer Discov ; 11(10): 2582-2601, 2021 10.
Article in English | MEDLINE | ID: mdl-33990344

ABSTRACT

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are an approved treatment for hormone receptor-positive breast cancer and are currently under evaluation across hundreds of clinical trials for other cancer types. The clinical success of these inhibitors is largely attributed to well-defined tumor-intrinsic cytostatic mechanisms, whereas their emerging role as immunomodulatory agents is less understood. Using integrated epigenomic, transcriptomic, and proteomic analyses, we demonstrated a novel action of CDK4/6 inhibitors in promoting the phenotypic and functional acquisition of immunologic T-cell memory. Short-term priming with a CDK4/6 inhibitor promoted long-term endogenous antitumor T-cell immunity in mice, enhanced the persistence and therapeutic efficacy of chimeric antigen receptor T cells, and induced a retinoblastoma-dependent T-cell phenotype supportive of favorable responses to immune checkpoint blockade in patients with melanoma. Together, these mechanistic insights significantly broaden the prospective utility of CDK4/6 inhibitors as clinical tools to boost antitumor T-cell immunity. SIGNIFICANCE: Immunologic memory is critical for sustained antitumor immunity. Our discovery that CDK4/6 inhibitors drive T-cell memory fate commitment sheds new light on their clinical activity, which is essential for the design of clinical trial protocols incorporating these agents, particularly in combination with immunotherapy, for the treatment of cancer.This article is highlighted in the In This Issue feature, p. 2355.


Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Piperazines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyridines/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Breast Neoplasms/pathology , Cell Line, Tumor , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Female , Humans , Memory T Cells/drug effects , Mice , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Xenograft Model Antitumor Assays
11.
Cell Rep ; 33(3): 108290, 2020 10 20.
Article in English | MEDLINE | ID: mdl-33086063

ABSTRACT

JQ1 is a BET-bromodomain inhibitor that has immunomodulatory effects. However, the precise molecular mechanism that JQ1 targets to elicit changes in antibody production is not understood. Our results show that JQ1 induces apoptosis, reduces cell proliferation, and as a consequence, inhibits antibody-secreting cell differentiation. ChIP-sequencing reveals a selective displacement of Brd4 in response to acute JQ1 treatment (<2 h), resulting in specific transcriptional repression. After 8 h, subsequent alterations in gene expression arise as a result of the global loss of Brd4 occupancy. We demonstrate that apoptosis induced by JQ1 is solely attributed to the pro-apoptotic protein Bim (Bcl2l11). Conversely, cell-cycle regulation by JQ1 is associated with multiple Myc-associated gene targets. Our results demonstrate that JQ1 drives temporal changes in Brd4 displacement that results in a specific transcriptional profile that directly affects B cell survival and proliferation to modulate the humoral immune response.


Subject(s)
Bcl-2-Like Protein 11/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Apoptosis/drug effects , Azepines/pharmacology , B-Lymphocytes/metabolism , Bcl-2-Like Protein 11/physiology , Cell Cycle Checkpoints/drug effects , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Proto-Oncogene Proteins c-myc/metabolism , Transcription Factors/genetics , Transcription Factors/physiology , Triazoles/pharmacology , Xenograft Model Antitumor Assays
12.
Sci Adv ; 6(18)2020 05.
Article in English | MEDLINE | ID: mdl-32917631

ABSTRACT

The RNA polymerase II (POLII)-driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3' polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation.

13.
Nat Commun ; 8: 14581, 2017 03 06.
Article in English | MEDLINE | ID: mdl-28262675

ABSTRACT

The Eµ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eµ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eµ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.


Subject(s)
B-Lymphocytes/metabolism , Gene Expression Regulation, Neoplastic , Lymphoma, B-Cell/genetics , Mutation , Proto-Oncogene Proteins c-myc/genetics , Repressor Proteins/genetics , Alleles , Animals , B-Lymphocytes/immunology , B-Lymphocytes/pathology , CRISPR-Cas Systems , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/immunology , Disease Models, Animal , Gene Editing , Gene Frequency , Janus Kinase 2/genetics , Janus Kinase 2/immunology , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/pathology , Mice , Mice, Transgenic , Proto-Oncogene Proteins c-myc/immunology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/immunology , Repressor Proteins/immunology , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/immunology , Transcriptome , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/immunology , Whole Genome Sequencing
SELECTION OF CITATIONS
SEARCH DETAIL