Targeting NAD+ Metabolism Vulnerability in FH-Deficient Hereditary Leiomyomatosis and Renal Cell Carcinoma with the novel NAMPT Inhibitor OT-82.

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an inherited cancer syndrome caused by germline pathogenic variants in the fumarate hydratase (FH) gene. Affected individuals are at risk for developing cutaneous and uterine leiomyomas and aggressive FH-deficient renal cell carcinoma (RCC) with a papillary histology. Due to a disrupted TCA cycle, FH-deficient kidney cancers rely on aerobic glycolysis for energy production, potentially creating compensatory metabolic vulnerabilities. This study conducted a high-throughput drug screen in HLRCC cell lines, which identified a critical dependency on nicotinamide adenine dinucleotide (NAD), a redox cofactor produced by the biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). Human HLRCC tumors and HLRCC-derived cell lines exhibited elevated NAMPT expression compared to controls. FH-deficient HLRCC cells, but not FH-restored HLRCC or normal kidney cells, were sensitive to NAMPT inhibition. HLRCC cell line viability was significantly decreased in both 2D and 3D in vitro cultures in response to the clinically relevant NAMPT inhibitor OT-82. NAMPT inhibition in vitro significantly decreased the total amount of NAD+, NADH, NADP, NADPH, and PAR levels and the effects of NAMPT inhibition could be rescued by the downstream NAD precursor nicotinamide mononucleotide, confirming the on-target activity of OT-82. Moreover, NAMPT inhibition by OT-82 in two HLRCC xenograft models resulted in severely reduced tumor growth. OT-82 treatment of HLRCC xenograft tumors in vivo inhibited glycolytic flux as demonstrated by reduced lactate/pyruvate ratio in hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging experiments. Overall, our data define NAMPT inhibition as a potential therapeutic approach for FH-deficient HLRCC-associated renal cell carcinoma.

Inhibition of Ribosome Biogenesis in vivo Causes p53-Dependent Death and p53-Independent Dysfunction.

Ribosomes are critical for cell function; their synthesis (known as ribosome biogenesis; "RiBi") is complex and energy-intensive. Surprisingly little is known about RiBi in differentiated cells in vivo in adult tissue. Here, we generated mice with conditional deletion of Nat10 , an essential gene for RiBi and translation, to investigate effects of RiBi blockade in vivo. We focused on RiBi in a long-lived, ribosome-rich cell population, pancreatic acinar cells, during homeostasis and tumorigenesis. We observed a surprising latency of several weeks between Nat10 deletion and onset of structural and functional abnormalities and p53-dependent acinar cell death, which was associated with translocation of ribosomal proteins RPL5 and RPL11 into acinar cell nucleoplasm. Indeed, deletion of Trp53 could rescue acinar cells from apoptotic cell death; however, Nat10 Δ / Δ ; Trp53 Δ / Δ acinar cells remained morphologically and functionally abnormal. Moreover, the deletion of Trp53 did not rescue the lethality of inducible, globally deleted Nat10 in adult mice nor did it rescue embryonic lethality of global Nat10 deletion, emphasizing p53-independent consequences of RiBi inhibition. Deletion of Nat10 in acinar cells blocked Kras -oncogene-driven pancreatic intraepithelial neoplasia and subsequent pancreatic ductal adenocarcinoma, regardless of Trp53 mutation status. Together, our results provide initial insights into how cells respond to defects in RiBi and translation in vivo .

Transfer RNA acetylation regulates in vivo mammalian stress signaling.

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here we investigate the impact of N4-acetylcytidine (ac4C), a highly conserved tRNA modification, using a Thumpd1 knockout mouse model. We find that loss of Thumpd1-dependent tRNA acetylation leads to reduced levels of tRNALeu, increased ribosome stalling, and activation of eIF2α phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Remarkably, concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translation control as well as therapeutic interventions.

Paralogue-Selective Degradation of the Lysine Acetyltransferase EP300.

The transcriptional coactivators EP300 and CREBBP are critical regulators of gene expression that share high sequence identity but exhibit nonredundant functions in basal and pathological contexts. Here, we report the development of a bifunctional small molecule, MC-1, capable of selectively degrading EP300 over CREBBP. Using a potent aminopyridine-based inhibitor of the EP300/CREBBP catalytic domain in combination with a VHL ligand, we demonstrate that MC-1 preferentially degrades EP300 in a proteasome-dependent manner. Mechanistic studies reveal that selective degradation cannot be predicted solely by target engagement or ternary complex formation, suggesting additional factors govern paralogue-specific degradation. MC-1 inhibits cell proliferation in a subset of cancer cell lines and provides a new tool to investigate the noncatalytic functions of EP300 and CREBBP. Our findings expand the repertoire of EP300/CREBBP-targeting chemical probes and offer insights into the determinants of selective degradation of highly homologous proteins.

Paralogue-selective degradation of the lysine acetyltransferase EP300.

The transcriptional coactivators EP300 and CREBBP are critical regulators of gene expression that share high sequence identity but exhibit non-redundant functions in basal and pathological contexts. Here, we report the development of a bifunctional small molecule, MC-1, capable of selectively degrading EP300 over CREBBP. Using a potent aminopyridine-based inhibitor of the EP300/CREBBP catalytic domain in combination with a VHL ligand, we demonstrate that MC-1 preferentially degrades EP300 in a proteasome-dependent manner. Mechanistic studies reveal that selective degradation cannot be predicted solely by target engagement or ternary complex formation, suggesting additional factors govern paralogue-specific degradation. MC-1 inhibits cell proliferation in a subset of cancer cell lines and provides a new tool to investigate the non-catalytic functions of EP300 and CREBBP. Our findings expand the repertoire of EP300/CREBBP-targeting chemical probes and offer insights into the determinants of selective degradation of highly homologous proteins.

Molecular Basis for RNA Cytidine Acetylation by NAT10.

Human NAT10 acetylates the N4 position of cytidine in RNA, predominantly on rRNA and tRNA, to facilitate ribosome biogenesis and protein translation. NAT10 has been proposed as a therapeutic target in cancers as well as aging-associated pathologies such as Hutchinson-Gilford Progeria Syndrome (HGPS). The ∼120 kDa NAT10 protein uses its acetyl-CoA-dependent acetyltransferase, ATP-dependent helicase, and RNA binding domains in concert to mediate RNA-specific N4-cytidine acetylation. While the biochemical activity of NAT10 is well known, the molecular basis for catalysis of eukaryotic RNA acetylation remains relatively undefined. To provide molecular insights into the RNA-specific acetylation by NAT10, we determined the single particle cryo-EM structures of Chaetomium thermophilum NAT10 ( Ct NAT10) bound to a bisubstrate cytidine-CoA probe with and without ADP. The structures reveal that NAT10 forms a symmetrical heart-shaped dimer with conserved functional domains surrounding the acetyltransferase active sites harboring the cytidine-CoA probe. Structure-based mutagenesis with analysis of mutants in vitro supports the catalytic role of two conserved active site residues (His548 and Tyr549 in Ct NAT10), and two basic patches, both proximal and distal to the active site for RNA-specific acetylation. Yeast complementation analyses and senescence assays in human cells also implicates NAT10 catalytic activity in yeast thermoadaptation and cellular senescence. Comparison of the NAT10 structure to protein lysine and N-terminal acetyltransferase enzymes reveals an unusually open active site suggesting that these enzymes have been evolutionarily tailored for RNA recognition and cytidine-specific acetylation.

Loss of NAT10 disrupts enhancer organization via p300 mislocalization and suppresses transcription of genes necessary for metastasis progression.

Acetylation of protein and RNA represent a critical event for development and cancer progression. NAT10 is the only known RNA acetylase that catalyzes the N4-actylcytidine (ac4C) modification of RNAs. Here, we show that the loss of NAT10 significantly decreases lung metastasis in allograft and genetically engineered mouse models of breast cancer. NAT10 interacts with a mechanosensitive, metastasis susceptibility protein complex at the nuclear pore. In addition to its canonical role in RNA acetylation, we find that NAT10 interacts with p300 at gene enhancers. NAT10 loss is associated with p300 mislocalization into heterochromatin regions. NAT10 depletion disrupts enhancer organization, leading to alteration of gene transcription necessary for metastatic progression, including reduced myeloid cell-recruiting chemokines that results in a less metastasis-prone tumor microenvironment. Our study uncovers a distinct role of NAT10 in enhancer organization of metastatic tumor cells and suggests its involvement in the tumor-immune crosstalk dictating metastatic outcomes.

Rewiring of RNA methylation by the oncometabolite fumarate in renal cell carcinoma.

Metabolic reprogramming is a hallmark of cancer that facilitates changes in many adaptive biological processes. Mutations in the tricarboxylic acid cycle enzyme fumarate hydratase (FH) lead to fumarate accumulation and cause hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC is a rare, inherited disease characterized by the development of non-cancerous smooth muscle tumors of the uterus and skin, and an increased risk of an aggressive form of kidney cancer. Fumarate has been shown to inhibit 2-oxoglutarate-dependent dioxygenases (2OGDDs) involved in the hydroxylation of HIF1α, as well as in DNA and histone demethylation. However, the link between fumarate accumulation and changes in RNA post-transcriptional modifications has not been defined. Here, we determine the consequences of fumarate accumulation on the activity of different members of the 2OGDD family targeting RNA modifications. By evaluating multiple RNA modifications in patient-derived HLRCC cell lines, we show that mutation of FH selectively affects the levels of N6-methyladenosine (m6A), while the levels of 5-formylcytosine (f5C) in mitochondrial tRNA are unaffected. This supports the hypothesis of a differential impact of fumarate accumulation on distinct RNA demethylases. The observation that metabolites modulate specific subsets of RNA-modifying enzymes offers new insights into the intersection between metabolism and the epitranscriptome.

Cytidine Acetylation Across the Tree of Life.

Acetylation plays a critical role in regulating eukaryotic transcription via the modification of histones. Beyond this well-documented function, a less explored biological frontier is the potential for acetylation to modify and regulate the function of RNA molecules themselves. N4-Acetylcytdine (ac4C) is a minor RNA nucleobase conserved across all three domains of life (archaea, bacteria, and eukarya), a conservation that suggests a fundamental role in biological processes. Unlike many RNA modifications that are controlled by large enzyme families, almost all organisms catalyze ac4C using a homologue of human Nat10, an essential disease-associated acetyltransferase enzyme.A critical step in defining the fundamental functions of RNA modifications has been the development of methods for their sensitive and specific detection. This Account describes recent progress enabling the use of chemical sequencing reactions to map and quantify ac4C with single-nucleotide resolution in RNA. To orient readers, we first provide historical background of the discovery of ac4C and the enzymes that catalyze its formation. Next, we describe mechanistic experiments that led to the development of first- and second-generation sequencing reactions able to determine ac4C's position in a polynucleotide by exploiting the nucleobase's selective susceptibility to reduction by hydride donors. A notable feature of this chemistry, which may serve as a prototype for nucleotide resolution RNA modification sequencing reactions more broadly, is its ability to drive a penetrant and detectable gain of signal specifically at ac4C sites. Emphasizing practical applications, we present how this optimized chemistry can be integrated into experimental workflows capable of sensitive, transcriptome-wide analysis. Such readouts can be applied to quantitatively define the ac4C landscape across the tree of life. For example, in human cell lines and yeast, this method has uncovered that ac4C is highly selective, predominantly occupying dominant sites within rRNA (rRNA) and tRNA (tRNA). By contrast, when we extend these analyses to thermophilic archaea they identify the potential for much more prevalent patterns of cytidine acetylation, leading to the discovery of a role for this modification in adaptation to environmental stress. Nucleotide resolution analyses of ac4C have also allowed for the determination of structure-activity relationships required for short nucleolar RNA (snoRNA)-catalyzed ac4C deposition and the discovery of organisms with unexpectedly divergent tRNA and rRNA acetylation signatures. Finally, we share how these studies have shaped our approach to evaluating novel ac4C sites reported in the literature and highlight unanswered questions and new directions that set the stage for future research in the field.

Comparative Analysis of Drug-like EP300/CREBBP Acetyltransferase Inhibitors.

The human acetyltransferase paralogues EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potencies of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize the binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of the relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

Cryptic splice mutation in the fumarate hydratase gene in patients with clinical manifestations of Hereditary Leiomyomatosis and Renal Cell Cancer.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant condition characterized by the development of cutaneous and uterine leiomyomas and risk for development of an aggressive form of papillary renal cell cancer. HLRCC is caused by germline inactivating pathogenic variants in the fumarate hydratase (FH) gene, which encodes the enzyme that catalyzes the interconversion of fumarate and L-malate. We utilized enzyme and protein mobility assays to evaluate the FH enzyme in a cohort of patients who showed clinical manifestations of HLRCC but were negative for known pathogenic FH gene variants. FH enzyme activity and protein levels were decreased by 50% or greater in three family members, despite normal FH mRNA expression levels as measured by quantitative PCR. Direct Nanopore RNA sequencing demonstrated 57 base pairs of retained intron sequence between exons 9 and 10 of polyadenylated FH mRNA in these patients, resulting in a truncated FH protein. Genomic sequencing revealed a heterozygous intronic alteration of the FH gene (chr1: 241498239 T/C) resulting in formation of a splice acceptor site near a polypyrimidine tract, and a uterine fibroid obtained from a patient showed loss of heterozygosity at this site. The same intronic FH variant was identified in an unrelated patient who also showed a clinical phenotype of HLRCC. These data demonstrate that careful clinical assessment as well as biochemical characterization of FH enzyme activity, protein expression, direct RNA sequencing, and genomic DNA sequencing of patient-derived cells can identify pathogenic variants outside of the protein coding regions of the FH gene.

Chemoproteomics Yields a Selective Molecular Host for Acetyl-CoA.

Chemoproteomic profiling is a powerful approach to define the selectivity of small molecules and endogenous metabolites with the human proteome. In addition to mechanistic studies, proteome specificity profiling also has the potential to identify new scaffolds for biomolecular sensing. Here, we report a chemoproteomics-inspired strategy for selective sensing of acetyl-CoA. First, we use chemoproteomic capture experiments to validate the N-terminal acetyltransferase NAA50 as a protein capable of differentiating acetyl-CoA and CoA. A Nanoluc-NAA50 fusion protein retains this specificity and can be used to generate a bioluminescence resonance energy transfer (BRET) signal in the presence of a CoA-linked fluorophore. This enables the development of a ligand displacement assay in which CoA metabolites are detected via their ability to bind the Nanoluc-NAA50 protein "host" and compete binding of the CoA-linked fluorophore "guest". We demonstrate that the specificity of ligand displacement reflects the molecular recognition of the NAA50 host, while the window of dynamic sensing can be controlled by tuning the binding affinity of the CoA-linked fluorophore guest. Finally, we show that the method's specificity for acetyl-CoA can be harnessed for gain-of-signal optical detection of enzyme activity and quantification of acetyl-CoA from cellular samples. Overall, our studies demonstrate the potential of harnessing insights from chemoproteomics for molecular sensing and provide a foundation for future applications in target engagement and selective metabolite detection.

Comparative analysis of drug-like EP300/CREBBP acetyltransferase inhibitors.

The human acetyltransferase paralogs EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potency of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

New insights into the pathophysiology of methylmalonic acidemia.

Methylmalonic acidemia (MMA) is a severe inborn error of metabolism that is characterized by pleiotropic metabolic perturbations and multiorgan pathology. Treatment options are limited and non-curative as the underlying causative molecular mechanisms remain unknown. While earlier studies have focused on the potential direct toxicity of metabolites such as methylmalonic and propionic acid as a mechanism to explain disease pathophysiology, new observations have revealed that aberrant acylation, specifically methylmalonylation, is a characteristic feature of MMA. The mitochondrial sirtuin enzyme SIRT5 is capable of recognizing and removing this PTM, however, reduced protein levels of SIRT5 along with other mitochondrial SIRTs 3 and 4 in MMA and potentially reduced function of all three indicates aberrant acylation may require clinical intervention. Therefore, targeting posttranslational modifications may represent a new therapeutic approach to treat MMA and related organic acidemias.

Reference compounds for characterizing cellular injury in high-content cellular morphology assays.

Robust, generalizable approaches to identify compounds efficiently with undesirable mechanisms of action in complex cellular assays remain elusive. Such a process would be useful for hit triage during high-throughput screening and, ultimately, predictive toxicology during drug development. Here we generate cell painting and cellular health profiles for 218 prototypical cytotoxic and nuisance compounds in U-2 OS cells in a concentration-response format. A diversity of compounds that cause cellular damage produces bioactive cell painting morphologies, including cytoskeletal poisons, genotoxins, nonspecific electrophiles, and redox-active compounds. Further, we show that lower quality lysine acetyltransferase inhibitors and nonspecific electrophiles can be distinguished from more selective counterparts. We propose that the purposeful inclusion of cytotoxic and nuisance reference compounds such as those profiled in this resource will help with assay optimization and compound prioritization in complex cellular assays like cell painting.

Conditional Covalent Lethality Driven by Oncometabolite Accumulation.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a cancer predisposition syndrome driven by mutation of the tumor suppressor fumarate hydratase (FH). Inactivation of FH causes accumulation of the electrophilic oncometabolite fumarate. In the absence of methods for reactivation, tumor suppressors can be targeted via identification of synthetic lethal interactions using genetic screens. Inspired by recent advances in chemoproteomic target identification, here, we test the hypothesis that the electrophilicity of the HLRCC metabolome may produce unique susceptibilities to covalent small molecules, a phenomenon we term conditional covalent lethality. Screening a panel of chemically diverse electrophiles, we identified a covalent ligand, MP-1, that exhibits FH-dependent cytotoxicity. Synthesis and structure-activity profiling identified key molecular determinants underlying the molecule's effects. Chemoproteomic profiling of cysteine reactivity together with clickable probes validated the ability of MP-1 to engage an array of functional cysteines, including one lying in the Zn-finger domain of the tRNA methyltransferase enzyme TRMT1. TRMT1 overexpression rescues tRNA methylation from inhibition by MP-1 and partially attenuates the covalent ligand's cytotoxicity. Our studies highlight the potential for covalent metabolites and small molecules to synergistically produce novel synthetic lethal interactions and raise the possibility of applying phenotypic screening with chemoproteomic target identification to identify new functional oncometabolite targets.

Antisense pairing and SNORD13 structure guide RNA cytidine acetylation.

N4-acetylcytidine (ac4C) is an RNA nucleobase found in all domains of life. The establishment of ac4C in helix 45 (h45) of human 18S ribosomal RNA (rRNA) requires the combined activity of the acetyltransferase NAT10 and the box C/D snoRNA SNORD13. However, the molecular mechanisms governing RNA-guided nucleobase acetylation in humans remain unexplored. After applying comparative sequence analysis and site-directed mutagenesis to provide evidence that SNORD13 folds into three main RNA helices, we report two assays that enable the study of SNORD13-dependent RNA acetylation in human cells. First, we demonstrate that ectopic expression of SNORD13 rescues h45 in a SNORD13 knockout cell line. Next, we show that mutant snoRNAs can be used in combination with nucleotide resolution ac4C sequencing to define structure and sequence elements critical for SNORD13 function. Finally, we develop a second method that reports on the substrate specificity of endogenous NAT10-SNORD13 via mutational analysis of an ectopically expressed pre-rRNA substrate. By combining mutational analysis of these reconstituted systems with nucleotide resolution ac4C sequencing, our studies reveal plasticity in the molecular determinants underlying RNA-guided cytidine acetylation that is distinct from deposition of other well-studied rRNA modifications (e.g., pseudouridine). Overall, our studies provide a new approach to reconstitute RNA-guided cytidine acetylation in human cells as well as nucleotide resolution insights into the mechanisms governing this process.