The Nguyen

All Publications

• Activating p53Y220C with a mutant-specific small molecule. Nature communications Zhu, X., Byun, W. S., Pieńkowska, D. E., Nguyen, K. T., Wang, M., Nettles, S. A., Gourisankar, S., Phillips, N. A., Gerhartz, J., Geng, Q., Qiu, T., Zhong, J., Jiang, Z., Sarott, R. C., Hinshaw, S. M., Zhang, T., Attardi, L. D., Nowak, R. P., Gray, N. S. 2026

  Abstract

  TP53 is the most commonly mutated gene in cancer, but it remains recalcitrant to clinically meaningful therapeutic reactivation. We present here the discovery and characterization of a small molecule chemical inducer of proximity that activates mutant p53. We named this compound TRanscriptional Activator of p53 (TRAP-1) due to its ability to engage p53Y220C and BRD4 in a ternary complex, which potently activates mutant p53 and triggers robust p53 target gene transcription. Treatment of p53Y220C-expressing cell lines with TRAP-1 results in rapid upregulation of CDKN1A and other p53 target genes and induces cellular senescence and apoptosis. Negative control compounds that are unable to form a ternary complex lack these activities, demonstrating the necessity of chemically induced proximity for the observed pharmacology. This approach to activating mutant p53 highlights how chemically induced proximity can be used to restore the functions of tumor suppressor proteins that have been inactivated by mutation in cancer.

  View details for DOI 10.1038/s41467-026-72165-6

  View details for PubMedID 42067562

• p53 drives lung cancer regression through a TSC2/TFEB-dependent senescence program. Cancer discovery Wang, M., Bieging-Rolett, K. T., Kaiser, A. M., Brady, C. A., Lockhart, J. H., Ferreira, S., Nguyen, K. T., Rajeevan, A., Evans, S. A., Zhao, T., Raj, N., Elkrief, A., Tischfield, S. E., Ladanyi, M., Ozawa, M. G., Bui, N. Q., Chen, C. T., Flores, E. R., Attardi, L. D. 2025

  Abstract

  Pharmacological restoration of p53 tumor suppressor function is a conceptually appealing therapeutic strategy for the many deadly cancers with compromised p53 activity, including lung adenocarcinoma (LUAD). However, the p53 pathway has remained undruggable, partly because of insufficient understanding of how to drive effective therapeutic responses without toxicity. Here, we use mouse and human models to deconstruct the transcriptional programs and sequelae underlying robust therapeutic responses in LUAD. We show that p53 drives potent tumor regression by direct Tsc2 transactivation, leading to mTORC1 inhibition and TFEB nuclear accumulation, which in turn triggers lysosomal gene expression programs, autophagy, and cellular senescence. Senescent LUAD cells secrete factors to recruit macrophages, precipitating cancer cell phagocytosis and tumor regression. Collectively, our analyses reveal a surprisingly complex cascade of events underlying a p53 therapeutic response in LUAD and illuminate targetable nodes for p53 combination therapies, thus establishing a critical framework for optimizing p53-based therapeutics.

  View details for DOI 10.1158/2159-8290.CD-25-0525

  View details for PubMedID 41115251

• Epigenetic priming targets tumor heterogeneity to shift transcriptomic phenotype of pancreatic ductal adenocarcinoma towards a Vitamin D susceptible state. Cell death & disease He, B., Stoffel, L., He, C. J., Cho, K., Li, A. M., Jiang, H., Flowers, B. M., Nguyen, K. T., Wang, K. W., Zhao, A. Y., Zhou, M. N., Ferreira, S., Attardi, L. D., Ye, J. 2024; 15 (1): 89

  Abstract

  As a highly heterogeneous tumor, pancreatic ductal adenocarcinoma (PDAC) exhibits non-uniform responses to therapies across subtypes. Overcoming therapeutic resistance stemming from this heterogeneity remains a significant challenge. Here, we report that Vitamin D-resistant PDAC cells hijacked Vitamin D signaling to promote tumor progression, whereas epigenetic priming with glyceryl triacetate (GTA) and 5-Aza-2'-deoxycytidine (5-Aza) overcame Vitamin D resistance and shifted the transcriptomic phenotype of PDAC toward a Vitamin D-susceptible state. Increasing overall H3K27 acetylation with GTA and reducing overall DNA methylation with 5-Aza not only elevated the Vitamin D receptor (VDR) expression but also reprogrammed the Vitamin D-responsive genes. Consequently, Vitamin D inhibited cell viability and migration in the epigenetically primed PDAC cells by activating genes involved in apoptosis as well as genes involved in negative regulation of cell proliferation and migration, while the opposite effect of Vitamin D was observed in unprimed cells. Studies in genetically engineered mouse PDAC cells further validated the effects of epigenetic priming for enhancing the anti-tumor activity of Vitamin D. Using gain- and loss-of-function experiments, we further demonstrated that VDR expression was necessary but not sufficient for activating the favorable transcriptomic phenotype in respond to Vitamin D treatment in PDAC, highlighting that both the VDR and Vitamin D-responsive genes were prerequisites for Vitamin D response. These data reveal a previously undefined mechanism in which epigenetic state orchestrates the expression of both VDR and Vitamin D-responsive genes and determines the therapeutic response to Vitamin D in PDAC.

  View details for DOI 10.1038/s41419-024-06460-9

  View details for PubMedID 38272889

  View details for PubMedCentralID 5858034

• A non-proteolytic release mechanism for HMCES-DNA-protein crosslinks EMBO JOURNAL Donsbach, M., Duerauer, S., Gruenert, F., Nguyen, K. T., Nigam, R., Yaneva, D., Weickert, P., Bezalel-Buch, R., Semlow, D. R., Stingele, J. 2023: e113360

  Abstract

  The conserved protein HMCES crosslinks to abasic (AP) sites in ssDNA to prevent strand scission and the formation of toxic dsDNA breaks during replication. Here, we report a non-proteolytic release mechanism for HMCES-DNA-protein crosslinks (DPCs), which is regulated by DNA context. In ssDNA and at ssDNA-dsDNA junctions, HMCES-DPCs are stable, which efficiently protects AP sites against spontaneous incisions or cleavage by APE1 endonuclease. In contrast, HMCES-DPCs are released in dsDNA, allowing APE1 to initiate downstream repair. Mechanistically, we show that release is governed by two components. First, a conserved glutamate residue, within HMCES' active site, catalyses reversal of the crosslink. Second, affinity to the underlying DNA structure determines whether HMCES re-crosslinks or dissociates. Our study reveals that the protective role of HMCES-DPCs involves their controlled release upon bypass by replication forks, which restricts DPC formation to a necessary minimum.

  View details for DOI 10.15252/embj.2022113360

  View details for Web of Science ID 001039430500001

  View details for PubMedID 37519246

• N-Terminally arginylated ubiquitin is attached to histone H2A by RING1B E3 ligase in human cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Seo, D., Kim, D., Nguyen, K., Oh, J., Lee, J., Hwang, C. 2023; 666: 186-194

  Abstract

  Ubiquitin (Ub) is highly conserved in all eukaryotic organisms and begins at the N-terminus with Met and Gln. Our recent research demonstrates that N-terminally (Nt-) arginylated Ub can be produced in the yeast Saccharomyces cerevisiae. However, the existence of Nt-arginylated Ub in multicellular organisms remains unknown. Here we explore the mechanism for creating Nt-arginylated Ub using human embryonic kidney HEK293 cells that express various Nt-modified Ubs. We found that Gln-starting Q-Ub was converted into Glu-starting E-Ub by NTAQ1 Nt-deamidase and subsequently Nt-arginylated by ATE1 arginyltransferase in HEK293 cells. We also found that the resulting Arg-Glu-starting RE-Ub was mainly deposited on the Lys119 residue of histone H2A. Furthermore, RING1B E3 Ub ligase mediated the attachment of RE-Ub to H2A. These findings reveal a previously unknown type of histone ubiquitylation which greatly increases the combinatorial complexity of histone and ubiquitin codes.

  View details for DOI 10.1016/j.bbrc.2023.02.022

  View details for Web of Science ID 001012085000001

  View details for PubMedID 36932026

• Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, B., Kim, M., Oh, S., Nguyen, K., Kim, J., Varshavsky, A., Hwang, C., Song, H. 2022; 119 (31): e2209597119

  Abstract

  N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNAArg as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis. The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNAArg, and a 3'-proximal segment of the tRNAArg moiety. The enzyme's active site is located, at least in part, between the two domains. In vitro and in vivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe3+-heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.

  View details for DOI 10.1073/pnas.2209597119

  View details for Web of Science ID 000907752700017

  View details for PubMedID 35878037

  View details for PubMedCentralID PMC9351520

• The MARCHF6 E3 ubiquitin ligase acts as an NADPH sensor for the regulation of ferroptosis. Nature cell biology Nguyen, K. T., Mun, S. H., Yang, J., Lee, J., Seok, O. H., Kim, E., Kim, D., An, S. Y., Seo, D. Y., Suh, J. Y., Lee, Y., Hwang, C. S. 2022; 24 (8): 1239-1251

  Abstract

  Ferroptosis is a unique form of cell death caused by excessive iron-dependent lipid peroxidation. The level of the anabolic reductant NADPH is a biomarker of ferroptosis sensitivity. However, specific regulators that detect cellular NADPH levels, thereby modulating downstream ferroptosis cascades, are largely unknown. We show here that the transmembrane endoplasmic reticulum MARCHF6 E3 ubiquitin ligase recognizes NADPH through its C-terminal regulatory region. This interaction upregulates the E3 ligase activity of MARCHF6, thus downregulating ferroptosis. We also found that MARCHF6 mediates the degradation of the key ferroptosis effectors ACSL4 and p53. Furthermore, inhibiting ferroptosis rescued the growth of MARCHF6-deficient tumours and peri-natal lethality of Marchf6-/- mice. Together, these findings identify MARCHF6 as a previously unknown NADPH sensor in the ubiquitin system and a crucial regulator of ferroptosis.

  View details for DOI 10.1038/s41556-022-00973-1

  View details for PubMedID 35941365

  View details for PubMedCentralID 8142022

• N-terminal acetylation and the N-end rule pathway control degradation of the lipid droplet protein PLIN2. The Journal of biological chemistry Nguyen, K. T., Lee, C. S., Mun, S. H., Truong, N. T., Park, S. K., Hwang, C. S. 2019; 294 (1): 379-388

  Abstract

  Perilipin 2 (PLIN2) is a major lipid droplet (LD)-associated protein that regulates intracellular lipid homeostasis and LD formation. Under lipid-deprived conditions, the LD-unbound (free) form of PLIN2 is eliminated in the cytosol by an as yet unknown ubiquitin (Ub)-proteasome pathway that is associated with the N-terminal or near N-terminal residues of the protein. Here, using HeLa, HEK293T, and HepG2 human cell lines, cycloheximide chase, in vivo ubiquitylation, split-Ub yeast two-hybrid, and chemical cross-linking-based reciprocal co-immunoprecipitation assays, we found that TEB4 (MARCH6), an E3 Ub ligase and recognition component of the Ac/N-end rule pathway, directly targets the N-terminal acetyl moiety of Nα-terminally acetylated PLIN2 for its polyubiquitylation and degradation by the 26S proteasome. We also show that the TEB4-mediated Ac/N-end rule pathway reduces intracellular LD accumulation by degrading PLIN2. Collectively, these findings identify PLIN2 as a substrate of the Ac/N-end rule pathway and indicate a previously unappreciated role of the Ac/N-end rule pathway in LD metabolism.

  View details for DOI 10.1074/jbc.RA118.005556

  View details for PubMedID 30425097

  View details for PubMedCentralID PMC6322874

• Control of protein degradation by N-terminal acetylation and the N-end rule pathway. Experimental & molecular medicine Nguyen, K. T., Mun, S. H., Lee, C. S., Hwang, C. S. 2018; 50 (7): 1-8

  Abstract

  Nα-terminal acetylation (Nt-acetylation) occurs very frequently and is found in most proteins in eukaryotes. Despite the pervasiveness and universality of Nt-acetylation, its general functions in terms of physiological outcomes remain largely elusive. However, several recent studies have revealed that Nt-acetylation has a significant impact on protein stability, activity, folding patterns, cellular localization, etc. In addition, Nt-acetylation marks specific proteins for degradation by a branch of the N-end rule pathway, a subset of the ubiquitin-mediated proteolytic system. The N-end rule associates a protein's in vivo half-life with its N-terminal residue or modifications on its N-terminus. This review provides a current understanding of intracellular proteolysis control by Nt-acetylation and the N-end rule pathway.

  View details for DOI 10.1038/s12276-018-0097-y

  View details for PubMedID 30054456

  View details for PubMedCentralID PMC6063864