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1.
NPJ Precis Oncol ; 8(1): 124, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38822082

ABSTRACT

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and cell line-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, cell line-specific resistance programs.

2.
Res Sq ; 2023 Nov 02.
Article in English | MEDLINE | ID: mdl-37961649

ABSTRACT

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and cell line-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, cell line-specific resistance programs.

3.
Cell Rep ; 39(1): 110630, 2022 04 05.
Article in English | MEDLINE | ID: mdl-35385747

ABSTRACT

DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator, a 26-subunit complex that controls RNA polymerase II activity genome-wide. We sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterize a stapled peptide, with functional mimics of both p53 activation domains, that blocks p53-Mediator binding and selectively inhibits p53-dependent transcription in human cells; importantly, this "bivalent" peptide has negligible impact, genome-wide, on non-p53 target genes. Our proof-of-concept strategy circumvents the TF entirely and targets the TF-Mediator interface instead, with desired functional outcomes (i.e., selective inhibition of p53 activation). Furthermore, these results demonstrate that TF activation domains represent viable starting points for Mediator-targeting molecular probes, as an alternative to large compound libraries. Different TFs bind Mediator through different subunits, suggesting this strategy could be broadly applied to selectively alter gene expression programs.


Subject(s)
Transcription Factors , Tumor Suppressor Protein p53 , Humans , Molecular Probes , Peptides/metabolism , Protein Binding , Transcription Factors/metabolism , Tumor Suppressor Protein p53/metabolism
4.
PLoS Biol ; 19(8): e3001364, 2021 08.
Article in English | MEDLINE | ID: mdl-34351910

ABSTRACT

The naturally occurring Δ40p53 isoform heterotetramerizes with wild-type p53 (WTp53) to regulate development, aging, and stress responses. How Δ40p53 alters WTp53 function remains enigmatic because their co-expression causes tetramer heterogeneity. We circumvented this issue with a well-tested strategy that expressed Δ40p53:WTp53 as a single transcript, ensuring a 2:2 tetramer stoichiometry. Human MCF10A cell lines expressing Δ40p53:WTp53, WTp53, or WTp53:WTp53 (as controls) from the native TP53 locus were examined with transcriptomics (precision nuclear run-on sequencing [PRO-seq] and RNA sequencing [RNA-seq]), metabolomics, and other methods. Δ40p53:WTp53 was transcriptionally active, and, although phenotypically similar to WTp53 under normal conditions, it failed to induce growth arrest upon Nutlin-induced p53 activation. This occurred via Δ40p53:WTp53-dependent inhibition of enhancer RNA (eRNA) transcription and subsequent failure to induce mRNA biogenesis, despite similar genomic occupancy to WTp53. A different stimulus (5-fluorouracil [5FU]) also showed Δ40p53:WTp53-specific changes in mRNA induction; however, other transcription factors (TFs; e.g., E2F2) could then drive the response, yielding similar outcomes vs. WTp53. Our results establish that Δ40p53 tempers WTp53 function to enable compensatory responses by other stimulus-specific TFs. Such modulation of WTp53 activity may be an essential physiological function for Δ40p53. Moreover, Δ40p53:WTp53 functional distinctions uncovered herein suggest an eRNA requirement for mRNA biogenesis and that human p53 evolved as a tetramer to support eRNA transcription.


Subject(s)
Tumor Suppressor Protein p53/metabolism , Cell Line , Fluorouracil , Genes, p53 , Humans , Imidazoles , Piperazines , Protein Isoforms , Protein Structure, Quaternary , Transcription Factors/metabolism , Transcription, Genetic , Transcriptome
5.
Commun Biol ; 4(1): 661, 2021 06 02.
Article in English | MEDLINE | ID: mdl-34079046

ABSTRACT

Detecting changes in the activity of a transcription factor (TF) in response to a perturbation provides insights into the underlying cellular process. Transcription Factor Enrichment Analysis (TFEA) is a robust and reliable computational method that detects positional motif enrichment associated with changes in transcription observed in response to a perturbation. TFEA detects positional motif enrichment within a list of ranked regions of interest (ROIs), typically sites of RNA polymerase initiation inferred from regulatory data such as nascent transcription. Therefore, we also introduce muMerge, a statistically principled method of generating a consensus list of ROIs from multiple replicates and conditions. TFEA is broadly applicable to data that informs on transcriptional regulation including nascent transcription (eg. PRO-Seq), CAGE, histone ChIP-Seq, and accessibility data (e.g., ATAC-Seq). TFEA not only identifies the key regulators responding to a perturbation, but also temporally unravels regulatory networks with time series data. Consequently, TFEA serves as a hypothesis-generating tool that provides an easy, rigorous, and cost-effective means to broadly assess TF activity yielding new biological insights.


Subject(s)
Transcription Factors/metabolism , Breast/cytology , Breast/metabolism , Cell Line , Chromatin Immunoprecipitation Sequencing/statistics & numerical data , Computational Biology/methods , Computer Simulation , Dexamethasone/pharmacology , Epithelial Cells/metabolism , Female , Gene Expression Regulation , Genetic Techniques/statistics & numerical data , HCT116 Cells , Humans , Imidazoles/pharmacology , Piperazines/pharmacology , Receptors, Glucocorticoid/drug effects , Receptors, Glucocorticoid/metabolism , Transcription Factors/genetics , Transcription, Genetic , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
6.
Mol Cell ; 78(4): 785-793.e8, 2020 05 21.
Article in English | MEDLINE | ID: mdl-32229306

ABSTRACT

RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID.


Subject(s)
Drosophila Proteins/metabolism , Drosophila/metabolism , Promoter Regions, Genetic , RNA Polymerase II/genetics , Transcription Factor TFIID/metabolism , Transcription, Genetic , Animals , Drosophila/genetics , Drosophila Proteins/genetics , HCT116 Cells , Humans , Protein Binding , RNA Polymerase II/metabolism , Transcription Factor TFIID/genetics
7.
Mol Cell ; 76(3): 485-499.e8, 2019 11 07.
Article in English | MEDLINE | ID: mdl-31495563

ABSTRACT

Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.


Subject(s)
Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinases/metabolism , Fibroblasts/drug effects , Interferon-gamma/pharmacology , Transcription, Genetic/drug effects , Animals , Cyclin-Dependent Kinase 8/genetics , Cyclin-Dependent Kinases/genetics , Fibroblasts/enzymology , Fibroblasts/virology , HCT116 Cells , Host-Pathogen Interactions , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , RNA Polymerase II/metabolism , STAT1 Transcription Factor/metabolism , Signal Transduction , Vesiculovirus/pathogenicity
8.
Wilderness Environ Med ; 28(2): 127-138, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28602271

ABSTRACT

Mountains are home to numerous organisms known to cause skin disease. Bites, stings, poisons, chemicals, toxins, trauma, and infections all contribute to this end. Numerous plants, animals, fungi, bacteria, viruses, and protozoa are responsible. This paper aims to review skin illness and injury sustained from organisms in the mountains of North America. Other factors such as increased ultraviolet radiation, temperature extremes, and decreasing atmospheric pressure along with human physiologic parameters, which contribute to disease severity, will also be discussed. After reading this review, one should feel more comfortable identifying potentially harmful organisms, as well as diagnosing, treating, and preventing organism-inflicted skin pathology sustained in the high country.


Subject(s)
Bites and Stings , Skin Diseases/chemically induced , Skin Diseases/microbiology , Skin/injuries , Animals , Bacteria , Dermatitis/etiology , Fungi , Humans , North America , Skin/radiation effects , Viruses , Wilderness
9.
Dermatol Online J ; 23(11)2017 Nov 15.
Article in English | MEDLINE | ID: mdl-29447629

ABSTRACT

Exploring the mountains is a highly rewarding past time; however, certain high-altitude exposures can lead to dermatologic manifestations. In this review article, the authors will describe cold, solar, and severe weather that one may experience when spending time outdoors. Factors such as increased ultraviolet radiation, temperature extremes, and low partial pressure of oxygen, along with human physiologic parameters also contribute to disease severity and presentation. This review article will address the diagnosis, treatment, and prevention of high-altitude dermatology exposures.


Subject(s)
Cold Injury , Mountaineering , Ultraviolet Rays/adverse effects , Weather , Altitude , Cold Injury/diagnosis , Cold Injury/therapy , Dermatology , Humans , Skin Diseases/diagnosis , Skin Diseases/etiology , Skin Diseases/therapy
11.
Proc Natl Acad Sci U S A ; 110(8): 2952-6, 2013 Feb 19.
Article in English | MEDLINE | ID: mdl-23382179

ABSTRACT

Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a key regulator of the innate immune system, particularly in the skin where, in response to molecular triggers such as pathogen-associated or damage-associated molecular patterns, the NLRP1 inflammasome promotes caspase-1-dependent processing of bioactive interleukin-1ß (IL-1ß), resulting in IL-1ß secretion and downstream inflammatory responses. NLRP1 is genetically associated with risk of several autoimmune diseases including generalized vitiligo, Addison disease, type 1 diabetes, rheumatoid arthritis, and others. Here we identify a repertoire of variation in NLRP1 by deep DNA resequencing. Predicted functional variations in NLRP1 reside in several common high-risk haplotypes that differ from the reference by multiple nonsynonymous substitutions. The haplotypes that are high risk for disease share two substitutions, L155H and M1184V, and are inherited largely intact due to extensive linkage disequilibrium across the region. Functionally, we found that peripheral blood monocytes from healthy subjects homozygous for the predominant high-risk haplotype 2A processed significantly greater (P < 0.0001) amounts of the IL-1ß precursor to mature bioactive IL-1ß under basal (resting) conditions and in response to Toll-like receptor (TLR) agonists (TLR2 and TLR4) compared with monocytes from subjects homozygous for the reference haplotype 1. The increase in basal release was 1.8-fold greater in haplotype 2A monocytes, and these differences between the two haplotypes were consistently observed three times over a 3-mo period; no differences were observed for IL-1α or TNFα. NLRP1 RNA and protein levels were not altered by the predominant high-risk haplotype, indicating that altered function of the corresponding multivariant NLRP1 polypeptide predisposes to autoimmune diseases by activation of the NLRP1 inflammasome.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Apoptosis Regulatory Proteins/immunology , Autoimmunity/genetics , Haplotypes , Inflammasomes/immunology , Interleukin-1beta/metabolism , Vitiligo/immunology , Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins/genetics , Humans , Monocytes/immunology , Monocytes/metabolism , NLR Proteins
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