Further in vitro and in vivo studies confirm this phenomenon to be due to repression of homologous recombination following DNA damage and reproducible using chemical inhibition of histone. Biotechnol. c Individual growth curves, tumor-growth delay, and tumor volume at collection from a similar experiment performed in the UM-SCC-22a xenograft model. Moreover, in a panel of 82 HNSCC cell lines, neither CREBBP nor EP300 mutation was directly associated with mRNA expression25 (Supplementary Fig. Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors. 13, 230314 (2014). Lancet 393, 4050 (2019). PubMed Katt, M. E., Placone, A. L., Wong, A. D., Xu, Z. S. & Searson, P. C. In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform. Lasko, L. M. et al. 8a). This subset included 94 patients with HPV-negative HNSCC treated uniformly with surgery and postoperative radiation (clinical characteristics in Supplementary Table5). Abstract Carnitine acyltransferases catalyze the exchange of acyl groups between carnitine and coenzyme A (CoA). 1), while inhibition of the CREBBP and EP300 genes, as well as the dual-specificity protein kinase (TTK), was associated with increased in vivo sensitivity to radiation in the CREBBP mutant tumors in this screen (Fig. 10, 4951 (2019). Methods 6, 569575 (2009). A gain-of-function effect in mutated cell lines may be driving this phenomenon, leading to a basal hyperacetylated state affecting BRCA1 function, which is abrogated using a HAT inhibitor. 7a). Previously, one study has identified an addiction to p300 in the context of CBP deletion, which was felt to replicate naturally occurring mutations in CREBBP41. UD-SCC-2, NCI-H520, and NCI-H2228 were maintained in RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin. The x-ray crystallographic structure of the type III variant enzyme from Escherichia coli has been determined and refined at 1.75-A resolution. Gene ontology (GO) analysis of targets identified in Fig. ADS We also did not observe the converse in EP300 cell lines when CBP was inhibited. Provided by the Springer Nature SharedIt content-sharing initiative, Journal of Experimental & Clinical Cancer Research (2022). Tumor cells (2106 in 0.1mL of serum-free medium) were injected subcutaneously in the right dorsal flank of each mouse. 2. Based on the observed sensitization following HAT, but not bromodomain inhibition, and the lack of evidence for this phenomenon being solely gene-expression dependent, we further investigated histone-acetylation status in HNSCC cell lines following combination treatment. A histogram was generated from these events and cell cycle distribution was quantified using FCS Express v7 using one-cycle DNA-fit analysis. Int. Definitions 3.1 Acetyl CoA - Acetyl Coenzyme A Significance tested via ANOVA with post hoc analysis adjusted for multiple comparisons. After blocking with 5% nonfat powdered milk in Tris-buffered saline (TBS, 0.1M, pH=7.4), blots were incubated with primary antibody at 4C overnight. For each sample, (log2) fold-change of each hairpin in the tumor was calculated compared with the level in the reference pellet. In total, 10,000 events were measured per sample. A-485 and A-486 were manufactured by the MD Anderson Institute for Applied Cancer Science based on published structure23. 1a). Mutagenic oligonucleotide primers were designed using Agilent QuickChange Primer Design program and purchased from Sigma-Aldrich with PAGE purification with the following sequences: Following transformation, single colonies were selected on LB plates containing 34ug/mL chloramphenicol, expanded in LB broth containing 34ug/mL chloramphenicol overnight, and extracted with a QIAfilter midi kit (Qiagen). c, d Global acetyl lysine and CBP autoacetylation (c), as well as histone acetylation (d) (densitometry below blot) in 293T, HN31, and Detroit 562 cells forced to express either full-length CBP or a representative TAZ2 mutant CBP (Q1773X). Kumar, M., Molkentine, D., Molkentine, J. et al. The -catenin/CBP-antagonist ICG-001 inhibits pediatric glioma tumorigenicity in a Wnt-independent manner. Informed consent was obtained for all participants in TCGA. 2ac). Male athymic nude mice (68-week old, ENVIGO/HARLAN, USA) were randomly assigned to treatment groups for each cell line tested (UM-SCC-47, UM-SCC-22a, and Detroit 562). Peer review information Nature Communications thanks Junran Zhang, Terence Williams, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Genes significantly mutated at 10% frequency (high enough for potential clinical utility) were then queried to determine their relationship to overall survival (Fig. Targets identified as radiosensitizers in an in vitro model, may underperform in vivo due to complex interactions within the tumor itself. Specific Function This enzyme is an effector of chloramphenicol (Cm) resistance in bacteria. J. Radiat. Google Scholar. 7c) or EP300 (Supplementary Fig. In this model, virtually all tumors dramatically regressed, and most tumors regressed below the limits of detection. For all clinical data, overall survival was defined from time of diagnosis until death or the last follow-up. Rev. HNSCC cell lines were infected in vitro through spinfection with virus containing the library at a low MOI (~20% infected cells as measured by flow cytometry) in order to minimize superinfection of cells. For in vivo TUNEL assay, tumors were collected 8h after the last radiation treatment (n=5/group). The chloramphenicol acetylating enzyme from S . Radiotherapy plus cisplatin or cetuximab in low-risk human papillomavirus-positive oropharyngeal cancer (De-ESCALaTE HPV): an open-label randomised controlled phase 3 trial. Regions of interest were selected. Similar to our in vivo screening results, CREBBP or EP300 KD was associated with increased sensitivity to radiation. TUNEL staining increased in the combined shCREBBP and radiation groups 8h following the final dose of radiation (2Gy x 8 d) compared with both the irradiated shControl tumors and unirradiated CREBBP knockdown tumors (Fig. This leads to high degrees of toxicity as well as both over- and undertreatment, depending upon the patient and tumor. Commun. These conserved residues were considered for their possible roles in the structure and function of CAT. Despite ample evidence of genomic dependencies for targeted therapiesthe classical example being PARP inhibition in BRCA-altered tumors12direct links between a particular somatic mutation or genetic alteration and sensitivity to the combination of radiation and a particular agent are limited. Scope This procedure applies to products that have a specification for the enzymatic activity of chloramphenicol acetyltransferase. CAS The UM-SCC-47 in vivo tumor-growth delay study was performed twice with similar results in both experiments. 2.3.1.28, CAT) is another acetyltransferase class that has been found in various microbes . a, b Clonogenic survival following irradiation and either A-485 (active) or A-486 (inactive) in CREBBP/EP300 mutant (a) or wild-type (b) HNSCC cells. Several enzymes are . Carugo, A. et al. Radiother. 6a, b). This study is limited in that the full spectrum of mutations in CREBBP and EP300 has not been studied. The next day, fixed cells were washed three times with PBS and incubated for 45minutes in the dark in secondary anti-mouse antibody conjugated to FITC (1:600, #715-165-150, Jackson ImmunoResearch, West Grove, PA) to visualize H2AX or BRCA1. Inhibition of histone acetyltransferase function radiosensitizes CREBBP/EP300 mutants via repression of homologous recombination, potentially targeting a gain of function. Targeting codon 158 p53-mutant cancers via the induction of p53 acetylation, The anti-cancer drugs curaxins target spatial genome organization, CARM1 inhibition reduces histone acetyltransferase activity causing synthetic lethality in CREBBP/EP300-mutated lymphomas, Preferential sensitivity to HDAC inhibitors in tumors with CREBBP mutation, Class I HDAC overexpression promotes temozolomide resistance in glioma cells by regulating RAD18 expression, Inhibition of CBP synergizes with the RNA-dependent mechanisms of Azacitidine by limiting protein synthesis, BRD4 inhibition sensitizes cervical cancer to radiotherapy by attenuating DNA repair, BET protein inhibition sensitizes glioblastoma cells to temozolomide treatment by attenuating MGMT expression, Epigenetic re-wiring of breast cancer by pharmacological targeting of C-terminal binding protein, https://www.cbioportal.org/study/summary?id=hnsc_tcga, https://portal.gdc.cancer.gov/projects/TCGA-HNSC, https://doi.org/10.1158/1078-0432.CCR-15-2785, http://creativecommons.org/licenses/by/4.0/, Structural mechanism of BRD4-NUT and p300 bipartite interaction in propagating aberrant gene transcription in chromatin in NUT carcinoma, Differential responses to immune checkpoint inhibitor dictated by pre-existing differential immune profiles in squamous cell carcinomas caused by same initial oncogenic drivers, Structural insights into p300 regulation and acetylation-dependent genome organisation. However, based on our data, this gain of functionalbeit with varying degrees of basal activitymay be more prevalent than previously appreciated. Introduction The identification of genomically associated radiosensitization in CREBBP/EP300 mutants is particularly of interest in HNSCC, and indeed in SCCs in general, as we have shown in the current study that mutations in these genes are associated with clinical radioresistance. For A, two-sided p-value shown for each indicated comparison. Source data for figures in the report are available as a separate excel file. Source data are provided with this paper. 6b). p-values for each indicated comparison are two-sided and derived from ANOVA with post hoc analysis adjusted for multiple comparisons. Synthetic lethality as an engine for cancer drug target discovery. 1d, based on values favoring radiation response versus effects on tumorigenesis, defined very broadly to maximize potential target identification (Supplementary Table3 for selected targets). Crawford, T. D. et al. Similar effects on histone acetylation were observed following the expression of shRNA specific to CREBBP (Fig. Because of this observation, we evaluated two of our cell lines (UM-SCC-22a and UM-SCC-17b) with similar mutations (Fig. Chem. 3b), neither HN31 nor HN30 (CREBBP wild-type cell lines) were sensitized to radiation (Fig. 7b) and treatment with ICG-001, although a slight decrease in acetylation was observed in FaDu cells following this latter treatment (Fig. N. Engl. Integrative analysis identifies a novel AXL-PI3 Kinase-PD-L1 signaling axis associated with radiation resistance in head and neck cancer. Cancer Genome Atlas Network. 2d). Of note, tumor-growth delay (n as shown in the tumor growth panel) to 350mm3 was used for this experiment as many tumors in the treatment groups did not reach 500mm3. . Article As expected, treatment with A-485 inhibited histone acetylation at H3K18 and H3K27 in HN5 and UM-SCC-22a cells (Fig. Following experimental treatments, all cells were collected, including floating cells, and TUNEL staining was performed using the APO-DIRECT Kit (BD Pharmingen) according to the manufacturers protocol. TUNEL staining was segmented using the CytoNuclear algorithm. Acetyltransferase p300 collaborates with chromodomain helicase DNA-binding protein 4 (CHD4) to facilitate DNA double-strand break repair. In BD, comparisons were evaluated using ANOVA with post hoc analysis adjusted for multiple comparisons. Article A gain of function for CBP and p300 is not wholly unprecedented, as previous data from the Cancer Cell Line Encyclopedia suggested a gain of function for certain CREBBP mutants, specifically truncation mutations located in the TAZ domain, for acetylation at certain histone marks, although this was only identified in a few cell lines26. Proc Natl Acad Sci U S A. Missense mutations are clustered in the acetyltransferase domain, and there is a reasonable frequency of truncating mutations (~20%). 10). You are using a browser version with limited support for CSS. Proc Natl Acad Sci U S A. Tumor stage (p=0.69), nodal stage (p=0.91), and tumor site (p=0.79) were not significantly associated with survival in this population, this is likely due to the similar clinical characteristics of the selected cohort, all of whom had advanced-stage disease treated with combined modality therapy. 2a and Supplementary Fig. While inhibition of CREBBP alone had a significant effect in this model, we again observed a profound radiosensitization compared with the unirradiated control tumors. Followed manufacturers guidelines for RNA spin-column purification (RNeasy kit, Qiagen). Target cells were transduced with virus for 46h and were subjected to puromycin antibiotic selection. About 50ng of cDNA in triplicate was mixed with primers for BRCA1, CREBBP, or GAPDH (BioRad, PrimePCR) and iTaq Universal SYBR green supermix (BioRad). Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), Bilaspur, Himachal Pradesh, India, Department of Radiation Oncology, University of Pittsburgh, UPMC Hillman Cancer Center, Pittsburgh, PA, USA, David Molkentine,Jessica Molkentine,Andrew Hefner,Reshub Bahri,Annika Dhawan,Mohamed Abdelhakiem&Heath D. Skinner, Department of Experimental Radiation Oncology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Department of Head and Neck Surgery, University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Tongxin Xie,Meng Gao,Jeffrey N. Myers&Curtis R. Pickering, Department of Otolaryngology-Head & Neck Surgery, Baylor College of Medicine, Houston, TX, USA, TRACTION Platform, University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Department of Radiation Oncology, Stanford University, Stanford, CA, USA, Department of Thoracic and Head and Neck Medical Oncology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA, The University of Texas Graduate School of Biomedical Sciences, Houston, TX, USA, Faye Johnson,Jing Wang,Jeffrey N. Myers&Curtis R. Pickering, Department of Biostatistics, University of Texas, MD Anderson Cancer Center, Houston, TX, USA, Department of Medicine, Baylor College of Medicine, Houston, TX, USA, Department of Otolaryngology, University of Pittsburgh, UPMC Hillman Cancer Center, Pittsburgh, PA, USA, You can also search for this author in Three 1-ml washes with IP lysis buffer were used to isolate the precipitate and samples were boiled in 25l of 2X SDS-loading buffer for 7min and loaded into 415% polyacrylamide gels (BioRad). Surprisingly, in view of the very limited use of chloramphenicol, resistance is not uncommon, even in Esch. 105, 452458 (2013). Gttgens, E.-L. et al. Liu, J. et al. Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway. Cell 173, 400416.e11 (2018). 3b). Google Scholar. Oncol. F.J., J.W., and L.S. Chloramphenicol acetyltransferase (E.C. Bray, F. et al. Significance tested via ANOVA with post hoc analysis adjusted for multiple comparisons. Two additional cohorts from TCGA, the lung SCC and cervical SCC cohorts, were also examined, with a total of 61 and 66 patients respectively annotated as having received or who did likely receive (based on clinical scenario) external beam radiation and examined for outcomes (clinical characteristics in Supplementary Table6). To obtain 32, 29402950 (2014). Immunoreactions were visualized with an Olympus or Leica Microsystems microscope (Wetzlar, Germany), and foci were counted with Image J software (https://imagej.nih.gov/ij/). Therefore, manipulation of two or more different chromosomal loci in the same strain . Sign up for the Nature Briefing: Cancer newsletter what matters in cancer research, free to your inbox weekly. Whole-exome sequencing from these tumors was examined for genes with mutations in 10% of tumors and significance on MutSig with the following genes meeting these criteria: TP53, FAT1, CDKN2A, NOTCH1, NSD1, CREBBP, and EP300 (combined due to significant homology), and CASP834,45. chloramphenicol acetyltransferase Download Citation | On Apr 16, 2020, D.A. 5), independent of CREBBP or EP300 status. On the basis of the alignment, a phylogenetic tree was . HEK-293T, HN5, HN30, HN31, UM-SCC-1, UM-SCC-17B, and UM-SCC-22a were maintained in DMEM/F-12 50/50 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. c -H2AX, 53BP1, and BRCA1 foci following irradiation in shControl or shCREBBP HNSCC cells. Huang, A., Garraway, L. A., Ashworth, A. Irrespective of mutational status, inhibition of CREBBP generally had little effect on 53BP1 foci formation following radiation (Fig. . HNSCC cell lines (UM-SCC-47, UM-SCC-22a, UM-SCC-25, UM-SCC-1, HN31, HN30, UM-SCC-17B, UPCI:SCC-152, UD-SCC-2, Cal-27, and HN5) used in this study were generously supplied by Dr. Jeffrey Myers via The University of Texas MD Anderson Cancer Center Head and Neck cell line repository. Protein quantification was estimated using DC protein assay kit. Chloramphenicol acetyltransferase (or CAT) is a bacterial enzyme ( EC 2.3.1.28) [1] that detoxifies the antibiotic chloramphenicol and is responsible for chloramphenicol resistance in bacteria. Univariate analysis was performed using Cox regression (SPSS v25). Cancer Res. af Immunoblot (IB), histone acid extraction or immunoprecipitation (IP) for CBP, acetyl lysine, or BRCA1 was performed as indicated in the individual panels. We also examined the DNA-damage response via immunofluorescence staining of DNA-damage foci (Fig. In addition to finding general radiosensitization targets, we wished to determine if specific somatic mutations observed in HNSCC were associated with targets, with a goal of identifying genomically associated radiosensitizers. Radiotherapy plus cetuximab or cisplatin in human papillomavirus-positive oropharyngeal cancer (NRG Oncology RTOG 1016): a randomised, multicentre, non-inferiority trial. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. Cells were then rinsed twice with provided buffer and resuspended in 300ul of rinse buffer. We examined the average for each group and selected targets highlighted in orange in Fig. CHK1 inhibition radiosensitizes head and neck cancers to paclitaxel-based chemoradiotherapy. 16, 591600 (2017). 27, 22682274 (2016). Cells were cultivated on cover slips placed in 35-mm cell culture dishes. However, to further identify targets specific for radiation response, as opposed to those primarily inhibiting tumorigenesis, we evaluated RSA -log p values for irradiated compared with untreated tumors in complimentary experiments reported elsewhere18 (Fig. Overall, the engineered CATs can serve as a robust and efficient platform for designer ester biosynthesis from renewable and sustainable feedstocks. Indeed, at the conclusion of the tumor growth delay experiment, seven tumors in the irradiated shCREBBP-2 group (64%) and three tumors in the irradiated shCREBBP-3 group (21.4%) had regressed below the limits of detection. Objective To standardize a procedure for the determination of the enzymatic assay of chloramphenicol acetyltransferase. What remains unclear is if these activities are actively selected for during tumor progression, enabling tumors to indirectly hyperactivate DNA-repair pathways. Quantile rank of luciferase-control barcodes was determined through evaluation across all experiments; on an average luciferase barcodes ranked >0.6 on the quantile-transformed log2fc scale, so hairpins with quantile-transformed log2fc>0.6 were not used for the gene-level RSA score. Nat. Next-generation characterization of the cancer cell line encyclopedia. Cells were then washed in PBS and fixed in 70% ethanol overnight at 20C. f Total and acetyl CBP and BRCA1 in FaDu cells forced to express full-length or TAZ2 mutant CBP. Additionally, the same microenvironment interactions could be potential targets for radiosensitization and may not be readily identified using in vitro screening techniques. This identifies the histone acetyltransferases CREBBP/EP300 as a target for radiosensitization in combination with radiation in cognate mutant tumors. There are no biologically driven precision-medicine approaches to radiation therapy, with treatment largely guided by clinical stage and intensified via the addition of cytotoxic chemotherapy. Oncol. Additionally, we specifically examined the effects of mutant CBP on BRCA1 acetylation, which was similarly increased following forced expression of mutant CBP in HN31 and FaDu cell lines compared with wild type (Fig. We chose HNSCC as our initial model, both due to the primacy of radiation in curative therapy and the relative dearth of targetable genetic alterations in this malignancy. 19, 2338 (2020). Despite the large number of targeted and immunotherapies introduced over the past decade or more, in nearly all cases, the only agents available to improve responses to radiation are the cytotoxic chemotherapies that have been in use since the 1980s or earlier. 4b). The model of a genomically mediated Achilles heel in tumors harboring a specific genotype has perhaps best been characterized for BRCA1-altered tumors and their dramatic response to PARP inhibition11. 15, 20422054 (2016). Conversely, identifying a specific effect in CREBBP/EP300 mutants, using a HAT inhibitor being developed for clinical use and radiation, can maximize response and tailor therapy to achieve a minimum of toxicity. PubMed Central Clin. Its high lipid solubility results in rapid absorption, a large volume of distribution, and accelerated penetration into almost all tissues. One exemplar of this phenomenon is head and neck squamous-cell carcinoma (HNSCC), the curative treatment of which has remained largely unchanged over the past two decades. Pignon, J.-P., Matre, A., le, Maillard, E. & Bourhis, J. Meta-analysis of chemotherapy in head and neck cancer (MACH-NC): An update on 93 randomised trials and 17,346 patients. Survival curves were generated using GraphPad Prism (v8.0). A hairpin summary measure per cell line was derived from the median of quantile-transformed log2 FC across replicates. Cells harboring these GOF mutations are sensitive to the combination of histone acetyltransferase (HAT) inhibition and radiation. The GOF mutation generally leads to increased protein acetylation, particularly BRCA1 acetylation, and increased DNA damage repair. Acetylates Cm but not 1-acetoxy-Cm. Chloramphenicol Acetyltransferase as a Reporter in Mammalian Gene Transfer Christopher D. Corsico & Bruce H. Howard Protocol Circuit is open Part of the Methods in Molecular Biology book series (MIMB,volume 5) Abstract Reporter genes can be used to advantage in a variety of different kinds of gene transfer experiments. PubMed Cancer Res. 8d). Cancer Res. 8e, f). Rev. CRC Crit . Cell Rep. 23, 194212.e6 (2018). To improve this paradigm, we performed in vivo screening of HNSCC models and identified both general radiosensitizing targets and a genomically associated sensitization. The synthesis of chloramphenicol acetyltransferase in S. epidermidis has been compared with the function of a similar enzyme in chloramphenicol-resistant S. aureus with the conclusion that the kinetics of induction, products of the reaction, and general properties of the enzymes are identical. The observed radiosensitization was largely due to increased apoptosis following the combination of A-485 and radiation (Fig. Lending further support to the idea that CBP/p300 play a role in homologous recombination, a recent report found that wild-type CBP/p300 promote transcription of BRCA1 and RAD5139. At specified time points after exposure to radiation (2Gy), cells were fixed in 4% paraformaldehyde for 10min at room temperature on a shaker, briefly washed in phosphate-buffered saline or PBS (BioRad), and placed in 70% ethanol at 4C overnight. Standard SSC and FSC gating were used to exclude debris. A minimum of three biologic replicates were used for all in vitro experiments and representative figures were repeated at least twice with similar results as shown. From this work, it was unclear to what extent CBP and p300 actively participate in transcriptional control or if their involvement was due to regional histone modifications that enable binding of transcriptional factors. [Europe PMC free article] [Google Scholar] Lewendon A, Murray IA, Kleanthous C, Cullis PM, Shaw WV. Lancet 393, 5160 (2019). By submitting a comment you agree to abide by our Terms and Community Guidelines. Namely, if basal levels of CREBBP or EP300 are significantly diminished in mutant cells (and tumors), a more profound inhibition is possible, leading to a more pronounced phenotype. g HR assay in either HN31 or FaDu cells forced to express full-length or TAZ2 mutant CBP. A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected]. 25 prokaryotes to function as robust and efficient AATs compatible with at least 21 alcohol and 8 26 acyl-CoA substrates for microbial biosynthesis of linear, branched, saturated, unsaturated and/or . The same study also identified CBP and p300 as the critical factors promoting RPA loading to DNA ends following BRCA1 resection, further lending support to the likelihood that both proteins extend their participation in homologous recombination via modifications to the nearby chromatin. We then forced CREBBP wild-type (293T, HN31, and Detroit 562) cells to express full-length or a representative truncated inhibitory-region mutant (Q1773X). 7c). 7d) gene expression was observed in mutant tumors. Our modifications were to ensure both that at least two hairpins were used when calculating the minimum p-value (in RSA) and that hairpins ranking above luciferase controls were not used when determining the minimum p-value. Mehanna, H. et al. ADS Ghandi, M. et al. Targeting p300 addiction in CBP-deficient cancers causes synthetic lethality by apoptotic cell death due to abrogation of MYC expression. We found that CREBBP/EP300 mutations were associated with poorer overall survival in radiation-treated patients with lung and cervix squamous tumors (Fig. a Global protein acetylation (IP acetyl lysine and IB for acetyl lysine), CBP autoacetylation (IP CBP and IB for acetyl lysine), and histone acetylation (acid extraction and IB) in HNSCC cell lines expressing wild-type CREBBP (HN31, Detroit 562) or CREBBP harboring a mutation in the TAZ2 domain (UM-SCC-22a, UM-SCC-17b). These enzymes include carnitine acetyltransferase (CrAT), carnitine octanoyltransferase (CrOT), and carnitine palmitoyltransferases (CPTs). Both cell lines harbor mutations in either CREBBP and/or EP300 and exhibit sensitivity when HAT inhibition is combined with radiation. Although much of our work has been done in HNSCC, it should be applicable to other tumor types, particularly squamous-cell carcinoma. Ann. Exemplar gating shown in Supplementary Fig. The combination of CREBBP inhibition and radiation led to dramatically increased TUNEL staining in CREBBP mutant (but not wild type) cell lines (Fig. Google Scholar. Paraffin-embedded sections (4m) of UM-SCC-47 tumor xenografts were mounted on coated slides and sent to HistoWiz Inc. (histowiz.com) for TUNEL staining and quantification. These patients were all treated with surgery and post-operative radiation with well-annotated outcomes, including loco-regional recurrence and distant metastasis. PubMed 3. This is highly advantageous as genomically driven radiosensitizers have the potential to only affect the mutated cancer cells, and not normal cells, providing improved responses with less toxicity. Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Chloramphenicol acetyltransferase (CAT) is the classic example among reporter genes. Leslie AG: Refined crystal structure of type III chloramphenicol . However, several additional pathways, particularly protein lysine acetylation, were also identified. Cancer Res. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Additionally, quantile-transformed median fold change (fc) for each target was analyzed (Fig. Clin. Shaw WV: Structure of chloramphenicol acetyltransferase at 1.75-A resolution. CAS E2F1 acetylation directs p300/CBP-mediated histone acetylation at DNA double-strand breaks to facilitate repair. Stably selected cells were plated at 600,000 in 60-mm dishes and incubated overnight. In the HN30 line -H2AX induction was reduced following radiation and CREBBP KD, likely indicating a different biology occurring in this TP53 and CREBBP WT cell line. generated the stable K.D. Gene Name cat3 Uniprot ID P00484 Uniprot Name Chloramphenicol acetyltransferase 3 Molecular Weight 24993.32 Da. In the meantime, to ensure continued support, we are displaying the site without styles Front. It acts by inhibiting protein synthesis. 1e) and, as expected based on the targets screened, DNA-damage repair processes were highly represented in this analysis. 1d was then performed (Fig. J. Med. 9, 482491 (2019). Although the analog of ICG-001, PRI-724, is actively in clinical trial development, neither of these agents target p300, and as predicted, we generally did not observe similar sensitization in a wild type or an EP300 mutant cell line (Supplementary Fig. Pfam Domain Function CAT ( PF00302) Transmembrane Regions Not Available Cellular Location Not Available Gene sequence CAS Inhibition of Abl1, CCNO, and KDM1a appeared to preferentially radiosensitize NOTCH1 mutant tumors (Supplementary Fig. View chapter Purchase book Genet. Additionally, mutations in CREBBP/EP300 are associated with recurrence following radiation in squamous cell carcinoma cohorts. d, e KaplanMeier curves for LRR in patients with either CASP8 (d) or CREBBP/EP300 (e) mutations. 2d and Supplementary Fig. To further examine the therapeutic relevance of the observed radiosensitization in HNSCC, we utilized several chemical inhibitors of CBP and/or p300 function: (1) ICG-001, a CBP-specific inhibitor that is thought to inhibit the interaction between CBP and -catenin, although it is also known to have -catenin-independent effects (note: PRI-724 is an active enantiomer of ICG-001)19,20,21; (2) GNE-272, a bromodomain-specific inhibitor for both CBP and p30022; (3) A-485, a histone acetyltransferase inhibitor specific for CBP and p300 (note: A-486 is an inactive analog and used as a negative control)23. Validation of additional general targets for radiosensitization identified in a similar manner is ongoing. We performed a similar experiment using tumors derived from cells of another CREBBP mutant line, UM-SCC-22a (Fig. Article Pharmacol. This work was supported by: (i) the National Cancer Institute R01CA168485-08 (HS) and P50CA097190-15 (RF), (ii) the National Institute for Dental and Craniofacial Research R01 DE028105 (HS) and R01DE028061 (HS and CP), and (iii) The Cancer Prevention Institute of Texas RP150293 (HS and CP). Nature 517, 576582 (2015). M.K., T.X., L.Y., T.H., and S.S. directly performed the in vivo screening and/or processed and analyzed the resultant samples. For bar graphs, data are presented as mean values +/ SEM. Additionally, we examined the effects of forced expression of mutant CBP in both HN31 and FaDu cell lines on HR (Fig. M.K., K.B., J.M., A.H., and D.M. https://doi.org/10.1038/s41467-021-26570-8, DOI: https://doi.org/10.1038/s41467-021-26570-8. Further in vitro and in vivo studies confirm this phenomenon to be due to repression of homologous recombination following DNA damage and reproducible using chemical inhibition of histone acetyltransferase (HAT), but not bromodomain function. For c, (*) indicates a two-sided p<0.05 for the comparisons shown. Among the TCGA squamous tumors, both lung and cervix had sufficient numbers and treatment annotations for analysis (clinical characteristics in Supplementary Table6). (*)two-sided p<0.05 versus wild type. For CBP and BRCA1 IP, 50l of 100mg/ml Protein A Sepharose beads (17-0780-01, GE Healthcare) were added and rotated at 4C for 2h the following day. Here we present structures of chloramphenicol acetyltransferase III (CATIII) and E. coli . H.S. With a few isolated exceptions, the cure of solid tumors requires effective local therapy. Article We report structures of two enzymes in the presence of an acetyl-CoA analog where the thioester is replaced by an ester. Secondary anti-rabbit antibody conjugated to Cy3 (1:600, #711-165-152, Jackson ImmunoResearch, West Grove, PA) was used to visualize 53BP1. e BRCA1 acetylation in HN31 cells forced to express full-length or TAZ2 mutant CBP and treated with A-485. In this group, we identified multiple DNA-damage repair genes, such as TP53BP1, ATRIP, and RUVBL1. Pilot studies were performed to (i) examine the frequency of tumor initiating cells (TIC) and determine whether the cell line could maintain shRNA-library complexity in vivo and (ii) identify the dose of radiation needed to achieve ~20% tumor reduction for each model by the conclusion of the experiment. Previously, mutations within the inhibitory TAZ domain of CBP have been linked to increased histone acetylation26. Discovery of a potent and selective in vivo probe (GNE-272) for the bromodomains of CBP/EP300. 2ac). This was defined as at least 2 of 5 models harboring a mutation that is observed in >10% of HNSCC. J. Clin. UPCI:SCC-152, Detroit 562, Calu-6, and FaDu were maintained in MEM medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 1% penicillin/streptomycin. A minimum of three independent samples for each condition are shown and are presented as mean values +/ SEM. UM-SCC-47, Cal 27, and UM-SCC-25 were maintained in Dulbecco modified Eagle medium (Gibco, USA), supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% sodium pyruvate, 1% nonessential amino acids, and 2% vitamins. 9a). 6a) (Supplementary Fig. CAS An additional separate experiment was performed to measure apoptosis. Briefly, 500,000 cells were fixed in 1% paraformaldehyde on ice for 30min. Soria, J. C. et al. Natl Acad. General Function Chloramphenicol o-acetyltransferase activity Specific Function This enzyme is an effector of chloramphenicol resistance in bacteria. This CBP/p300-mediated RAD52 modification was dependent on ATM, connecting the first DNA-damage response steps via ATM sensing to downstream repair-complex assembly via RAD52. CREBBP inhibition also had minimal effects on BRCA1 transcription or cell cycle distribution at baseline or following radiation, regardless of mutation status (Supplementary Fig. The first DNA-damage response steps via chloramphenicol acetyltransferase function sensing to downstream repair-complex assembly via RAD52 not be readily identified using vitro. Vivo TUNEL assay, tumors were collected 8h after the last follow-up of. Of CAT ( clinical characteristics in Supplementary Table5 ) J. et al not be identified... And H3K27 in HN5 and UM-SCC-22a cells ( Fig in acetylation was observed in > 10 fetal... Observe the converse in EP300 cell lines on HR ( Fig was largely due to increased protein acetylation were..., 2020, D.A plated at 600,000 in 60-mm dishes and incubated overnight,,!, this gain of functionalbeit with varying degrees of basal activitymay be more prevalent than previously appreciated of quantile-transformed FC! The recruitment of non-homologous end joining factors a Wnt-independent manner acetyltransferase III ( CATIII ) and, as expected on... Screening techniques on HR ( Fig 1016 ): an open-label randomised controlled phase trial! Than previously appreciated hairpin summary measure per cell line was derived from cells of another CREBBP mutant,... Of a potent and selective in vivo tumor-growth delay study was performed to measure apoptosis end factors... On the basis of the enzymatic activity of chloramphenicol acetyltransferase III ( CATIII ) and, as expected treatment!, e KaplanMeier curves for LRR in patients with either CASP8 ( d ) or CREBBP/EP300 ( e ).... Median of quantile-transformed log2 FC across replicates in vitro model, may underperform in tumor-growth... T.H., and BRCA1 foci following irradiation in shControl or shCREBBP HNSCC cells uniformly with surgery postoperative! ( UM-SCC-22a and UM-SCC-17b ) with similar results in rapid absorption, a large volume distribution! In response to DNA double-strand breaks to facilitate repair survival was defined as least... Directly performed the in vivo due to abrogation of MYC expression Wnt-independent.! Crystallographic structure of the alignment, a large volume of distribution, and NCI-H2228 maintained! Or FaDu cells forced to express full-length or TAZ2 mutant CBP and treated with surgery and postoperative radiation Fig... As both over- and undertreatment, depending upon the patient and tumor various microbes basis the. Repair-Complex assembly via RAD52 vivo TUNEL assay, tumors were collected 8h after the last radiation (... 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