In Press
D. Min, J. Byun, E.-J. Lee, A.A. Khan, C. Liu, O. Loudig, W. Hu, Y Zhao, M. Herlyn, B. Tycko, P. A. Cole, and B. Ryu. In Press. “Epigenetic Silencing of BMP6 by Sin3A-HDAC1/2 Repressor Complex Drives Melanoma Metastasis via FAM83G/PAWS1.” Mol. Cancer Res.
Antonieta Salguero, Maggie Chen, Aaron Balana, Nam Chu, Hanjie Jiang, Hwan Bae, Brad Palanski, Katharine Wright, Sara Nathan, Heng Zhu, Sandra Gabelli, Matthew Pratt, and Philip Cole. In Press. “Multifaceted Regulation of Akt by Diverse C-terminal Post-translational Modifications.” ACS Chemical Biology.
S.S Pujari, M. Wu, J. Thomforde, Z. A. Wang, C. Chao, N. Olson, L. Erber, W. Pomerantz, P. A. Cole, and N. Tretyakova. In Press. “Site-specific 5-Formyl Cytosine Mediated DNA-Histone Crosslinks: Synthesis and Polymerase Bypass by Human DA Polymerase h .” Angewandte Chemie.
Hitoshi Shiota, Artyom A Alekseyenko, Zhipeng A Wang, Ivona Filic, Tatiana M Knox, Nhi M Luong, Yeying Huang, David A Scott, Kristen L Jones, Prafulla C Gokhale, Madeleine E Lemieux, Philip A Cole, Mitzi I Kuroda, and Christopher A French. 2021. “Chemical Screen Identifies Diverse and Novel Histone Deacetylase Inhibitors as Repressors of NUT Function: Implications for NUT Carcinoma Pathogenesis and Treatment.” Mol Cancer Res, 19, 11, Pp. 1818-1830.Abstract
NUT carcinoma (NC), characterized most commonly by the BRD4-NUTM1 fusion, is a rare, aggressive variant of squamous carcinoma with no effective treatment. BRD4-NUT drives growth and maintains the poorly differentiated state of NC by activating pro-growth genes such as MYC, through the formation of massive, hyperacetylated, superenhancer-like domains termed megadomains. BRD4-NUT-mediated hyperacetylation of chromatin is facilitated by the chromatin-targeting tandem bromodomains of BRD4, combined with NUT, which recruits the histone acetyltransferase, p300. Here, we developed a high-throughput small-molecule screen to identify inhibitors of transcriptional activation by NUT. In this dCAS9-based GFP-reporter assay, the strongest hits were diverse histone deacetylase (HDAC) inhibitors. Two structurally unrelated HDAC inhibitors, panobinostat and the novel compound, IRBM6, both repressed growth and induced differentiation of NC cells in proportion to their inhibition of NUT transcriptional activity. These two compounds repressed transcription of megadomain-associated oncogenic genes, such as MYC and SOX2, while upregulating pro-differentiation, non-megadomain-associated genes, including JUN, FOS, and key cell-cycle regulators, such as CDKN1A. The transcriptional changes correlate with depletion of BRD4-NUT from megadomains, and redistribution of the p300/CBP-associated chromatin acetylation mark, H3K27ac, away from megadomains toward regular enhancer regions previously populated by H3K27ac. In NC xenograft models, we demonstrated that suppression of tumor growth by panobinostat was comparable with that of bromodomain inhibition, and when combined they improved both survival and growth suppression. IMPLICATIONS: The findings provide mechanistic and preclinical rationale for the use of HDAC inhibitors, alone or combined with other agents, in the treatment of NUT carcinoma.
Zhiqin Ji, Richard F Clark, Vikram Bhat, T Matthew Hansen, Loren M Lasko, Kenneth D Bromberg, Vlasios Manaves, Mikkel Algire, Ruth Martin, Wei Qiu, Maricel Torrent, Clarissa G Jakob, Hong Liu, Philip A Cole, Ronen Marmorstein, Edward A Kesicki, Albert Lai, and Michael R Michaelides. 2021. “Discovery of spirohydantoins as selective, orally bioavailable inhibitors of p300/CBP histone acetyltransferases.” Bioorg Med Chem Lett, 39, Pp. 127854.Abstract
p300 and CREB-binding protein (CBP) are essential for a multitude of cellular processes. Dysregulation of p300/CBP histone acetyltransferase activity is linked to a broad spectrum of human diseases including cancers. A novel drug-like spirohydantoin (21) has been discovered as a selective orally bioavailable inhibitor of p300/CBP histone acetyltransferase. Lead compound 21 is more potent than the first-in-class lead A-485 in both enzymatic and cellular assays and lacks the off-target inhibition of dopamine and serotonin transporters, that was observed with A-485.
David D Aufhauser, Paul Hernandez, Seth J Concors, Ciaran O'Brien, Zhonglin Wang, Douglas R Murken, Arabinda Samanta, Ulf H Beier, Lauren Krumeich, Tricia R Bhatti, Yanfeng Wang, Guanghui Ge, Liqing Wang, Shayan Cheraghlou, Florence F Wagner, Edward B Holson, Jay H Kalin, Philip A Cole, Wayne W Hancock, and Matthew H Levine. 2021. “HDAC2 targeting stabilizes the CoREST complex in renal tubular cells and protects against renal ischemia/reperfusion injury.” Sci Rep, 11, 1, Pp. 9018.Abstract
Histone/protein deacetylases (HDAC) 1 and 2 are typically viewed as structurally and functionally similar enzymes present within various co-regulatory complexes. We tested differential effects of these isoforms in renal ischemia reperfusion injury (IRI) using inducible knockout mice and found no significant change in ischemic tolerance with HDAC1 deletion, but mitigation of ischemic injury with HDAC2 deletion. Restriction of HDAC2 deletion to the kidney via transplantation or PAX8-controlled proximal renal tubule-specific Cre resulted in renal IRI protection. Pharmacologic inhibition of HDAC2 increased histone acetylation in the kidney but did not extend renal protection. Protein analysis demonstrated increased HDAC1-associated CoREST protein in HDAC2-/- versus WT cells, suggesting that in the absence of HDAC2, increased CoREST complex occupancy of HDAC1 can stabilize this complex. In vivo administration of a CoREST inhibitor exacerbated renal injury in WT mice and eliminated the benefit of HDAC2 deletion. Gene expression analysis of endothelin showed decreased endothelin levels in HDAC2 deletion. These data demonstrate that contrasting effects of HDAC1 and 2 on CoREST complex stability within renal tubules can affect outcomes of renal IRI and implicate endothelin as a potential downstream mediator.
He Huang, Di Zhang, Yejing Weng, Kyle Delaney, Zhanyun Tang, Cong Yan, Shankang Qi, Chao Peng, Philip A Cole, Robert G Roeder, and Yingming Zhao. 2021. “The regulatory enzymes and protein substrates for the lysine β-hydroxybutyrylation pathway.” Sci Adv, 7, 9.Abstract
Metabolism-mediated epigenetic changes represent an adapted mechanism for cellular signaling, in which lysine acetylation and methylation have been the historical focus of interest. We recently discovered a β-hydroxybutyrate-mediated epigenetic pathway that couples metabolism to gene expression. However, its regulatory enzymes and substrate proteins remain unknown, hindering its functional study. Here, we report that the acyltransferase p300 can catalyze the enzymatic addition of β-hydroxybutyrate to lysine (Kbhb), while histone deacetylase 1 (HDAC1) and HDAC2 enzymatically remove Kbhb. We demonstrate that p300-dependent histone Kbhb can directly mediate in vitro transcription. Moreover, a comprehensive analysis of Kbhb substrates in mammalian cells has identified 3248 Kbhb sites on 1397 substrate proteins. The dependence of histone Kbhb on p300 argues that enzyme-catalyzed acylation is the major mechanism for nuclear Kbhb. Our study thus reveals key regulatory elements for the Kbhb pathway, laying a foundation for studying its roles in diverse cellular processes.
Daniel R Dempsey, Thibault Viennet, Reina Iwase, Eun Young Park, Stephanie Henriquez, Zan Chen, Jeliazko R Jeliazkov, Brad A Palanski, Kim L Phan, Paul Coote, Jeffrey J Gray, Michael J Eck, Sandra B Gabelli, Haribabu Arthanari, and Philip A Cole. 2021. “The structural basis of PTEN regulation by multi-site phosphorylation.” Nat Struct Mol Biol, 28, 10, Pp. 858-868.Abstract
Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN's catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN's cellular functions.
Hanjie Jiang, Daniel R Dempsey, and Philip A Cole. 2021. “Ubiquitin Ligase Activities of WWP1 Germline Variants K740N and N745S.” Biochemistry.Abstract
WWP1 is an E3 ubiquitin ligase that has been reported to target the tumor suppressor lipid phosphatase PTEN. K740N and N745S are recently identified germline variants of WWP1 that have been linked to PTEN-associated cancers [Lee, Y. R., et al. (2020) ]. These WWP1 variants have been suggested to release WWP1 from its native autoinhibited state, thereby promoting enhanced PTEN ubiquitination as a mechanism for driving cancer. Using purified proteins and enzymatic assays, we investigate the possibility that K740N and N745S WWP1 possess enhanced ubiquitin ligase activity and demonstrate that these variants are similar to the wild type (WT) in both autoubiquitination and PTEN ubiquitination. Furthermore, K740N and N745S WWP1 show dependencies similar to those of WT in terms of allosteric activation by an engineered ubiquitin variant, upstream E2 concentration, and substrate ubiquitin concentration. Transfected WWP1 WT and mutants demonstrate comparable effects on cellular PTEN levels. These findings challenge the idea that K740N and N745S WWP1 variants promote cancer by enhanced PTEN ubiquitination.
Hanjie Jiang and Philip A Cole. 2021. “N-Terminal Protein Labeling with N-Hydroxysuccinimide Esters and Microscale Thermophoresis Measurements of Protein-Protein Interactions Using Labeled Protein.” Curr Protoc Mol Bio, Pp. e14.Abstract
Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein-protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy-to-apply protocol for high-stoichiometric, site-specific labeling of a protein at its N-terminus with N-hydroxysuccinimide (NHS) esters as a means to measure protein-protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent-labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2-E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Labeling a protein of interest at its N-terminus with NHS esters through stepwise reaction Alternate Protocol: Labeling a protein of interest at its N-terminus with NHS esters through a one-pot reaction Basic Protocol 2: Purifying the N-terminal fluorescent-labeled protein and determining its concentration and labeling efficiency Basic Protocol 3: Using MST to determine the binding affinity of an N-terminal fluorescent-labeled protein to a binding partner. Basic Protocol 4: NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay.
M. Na, G.M. Martin, D.L. Karl, J. Sun, M. Torres-Martin, H. Itonaga, C. Martinez, S. Chen, Y. Xu, S. Duffort, P.-J. Hamard, C. Chen, L. Cimmino, F.-C. Yang, B. Zucconi, P. A. Cole, M. Xu, M.E. Figueroa, and S.D. Nimer. 2021. “P300 Suppresses the Transition of Myelodysplastic Syndrome to Acute Myeloid Leukemia.” JCI Insights, 6, Pp. e138478.
Z. A. Wang and P. A. Cole. 2020. “The Chemical Biology of Reversible Lysine Post-translational Modifications.” Cell Chem Biol.Abstract
Lysine (Lys) residues in proteins undergo a wide range of reversible post-translational modifications (PTMs), which can regulate enzyme activities, chromatin structure, protein-protein interactions, protein stability, and cellular localization. Here we discuss the "writers," "erasers," and "readers" of some of the common protein Lys PTMs and summarize examples of their major biological impacts. We also review chemical biology approaches, from small-molecule probes to protein chemistry technologies, that have helped to delineate Lys PTM functions and show promise for a diverse set of biomedical applications.
C. D. Morrison-Smith, T. M. Knox, I. Filic, K. M. Soroko, B. K. Eschle, M. K. Wilkens, P. C. Gokhale, F. Giles, A. Griffin, B. Brown, G. I. Shapiro, B. E. Zucconi, P. A. Cole, M. E. Lemieux, and C. A. French. 2020. “Combined Targeting of the BRD4-NUT-p300 Axis in NUT Midline Carcinoma by Dual Selective Bromodomain Inhibitor, NEO2734.” Mol. Cancer Ther., 19, 7, Pp. 1406–1414.Abstract
NUT midline carcinoma (NMC) is a rare, aggressive subtype of squamous carcinoma that is driven by the BRD4-NUT fusion oncoprotein. BRD4, a BET protein, binds to chromatin through its two bromodomains, and NUT recruits the p300 histone acetyltransferse (HAT) to activate transcription of oncogenic target genes. BET-selective bromodomain inhibitors have demonstrated on-target activity in patients with NMC, but with limited efficacy. P300, like BRD4, contains a bromodomain. We show that combining selective p300/CBP and BET bromodomain inhibitors, GNE-781 and OTX015, respectively, induces cooperative depletion of MYC and synergistic inhibition of NMC growth. Treatment of NMC cells with the novel dual p300/CBP and BET bromodomain-selective inhibitor, NEO2734, potently inhibits growth and induces differentiation of NMC cells in vitro; findings that correspond with potentiated transcriptional effects from combined BET and p300 bromodomain inhibition. In three disseminated NMC xenograft models, NEO2734 provided greater growth inhibition, with tumor regression and significant survival benefit seen in two of three models, compared with a lead clinical BET inhibitor or "standard" chemotherapy. Our findings provide a strong rationale for clinical study of NEO2734 in patients with NMC. Moreover, the synergistic inhibition of NMC growth by CBP/p300 and BET bromodomain inhibition lays the groundwork for greater mechanistic understanding of the interplay between p300 and BRD4-NUT that drives this cancer.
Z. A. Wang, C. J. Millard, C. L. Lin, J. E. Gurnett, M. Wu, K. Lee, L. Fairall, J. W. Schwabe, and P. A. Cole. 2020. “Diverse nucleosome Site-Selectivity among histone deacetylase complexes.” Elife, 9.Abstract
Histone acetylation regulates chromatin structure and gene expression and is removed by histone deacetylases (HDACs). HDACs are commonly found in various protein complexes to confer distinct cellular functions, but how the multi-subunit complexes influence deacetylase activities and site-selectivities in chromatin is poorly understood. Previously we reported the results of studies on the HDAC1 containing CoREST complex and acetylated nucleosome substrates which revealed a notable preference for deacetylation of histone H3 acetyl-Lys9 vs. acetyl-Lys14 (Wu et al, 2018). Here we analyze the enzymatic properties of five class I HDAC complexes: CoREST, NuRD, Sin3B, MiDAC and SMRT with site-specific acetylated nucleosome substrates. Our results demonstrate that these HDAC complexes show a wide variety of deacetylase rates in a site-selective manner. A Gly13 in the histone H3 tail is responsible for a sharp reduction in deacetylase activity of the CoREST complex for H3K14ac. These studies provide a framework for connecting enzymatic and biological functions of specific HDAC complexes.
Wang L. Han R. Belfer U. Kalin J. Cole P. A. Hancock W. Xiong, Y. 2020. “Essential Role of the CoREST Complex in Foxp3+ T-Regulatory Cell Functions.” J. Clin. Invest., 130, Pp. 1830-1842.
Dagil L. Fairall L. Robertson N. Wu M. Ragan T. J. Savva G. C. Morone N. Kunze M. B. A. Jamieson A. G Song, Y. 2020. “Functional and Structural Coupling Between LSD1 and HDAC1 in the CoREST Corepressor Complex.” Cell Reports, 30, Pp. 2699-2711.
S. A. Miller, R. A. Policastro, S. S. Savant, S. Sriramkumar, N. Ding, X. Lu, H. P. Mohammad, S. Cao, J. H. Kalin, P. A. Cole, G. E. Zentner, and H. M. O'Hagan. 2020. “Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer.” Mol. Cancer Res., 18, 2, Pp. 264–277.Abstract
Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine-specific demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer and enhances cell migration. In this study, we determine that LSD1 expression is significantly elevated in patients with colorectal cancer with mutation of the catalytic subunit of PI3K, PIK3CA, compared with patients with colorectal cancer with WT PIK3CA. LSD1 enhances activation of the AKT kinase in colorectal cancer cells through a noncatalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. In addition, growth of PIK3CA-mutant colorectal cancer cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall, we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA-mutant cells. IMPLICATIONS: Our data support the hypothesis that inhibitors targeting the CoREST complex may be clinically effective in patients with colorectal cancer harboring PIK3CA mutations.
Z. A. Wang and P. A. Cole. 2020. “Methods and Applications of Expressed Protein Ligation.” Methods Mol. Biol., 2133, Pp. 1–13.Abstract
Expressed protein ligation is a method of protein semisynthesis and typically involves the reaction of recombinant protein C-terminal thioesters with N-cysteine containing synthetic peptides in a chemoselective ligation. The recombinant protein C-terminal thioesters are produced by exploiting the action of nature's inteins which are protein modules that catalyze protein splicing. This chapter discusses the basic principles of expressed protein ligation and recent advances and applications in this protein semisynthesis field. Comparative strengths and weaknesses of the method and future challenges are highlighted.
P. A. Cole and T. Shapiro. 2020. “Paul Talalay 1923-2019 A Biographical Memoir.” National Academy of Sciences . Publisher's Version
J. W. Hsu, M. Bai, K. Li, J. S. Yang, N. Chu, P. A. Cole, M. J. Eck, J. Li, and V. W. Hsu. 2020. “The protein kinase Akt acts as a coat adaptor in endocytic recycling.” Nat. Cell Biol., 22, 8, Pp. 927–933.Abstract
Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins1-3. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling4-6. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event.