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Metabolic reprogramming is a hallmark of cancer that facilitates changes in many adaptive biological processes. Mutations in the tricarboxylic acid (TCA) 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 a highly metastatic and aggressive form of kidney cancer. Fumarate has been shown to inhibit 2-oxyglutarate-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 alters the activity of demethylases acting upon N6-methyladenosine (m6A), while the demethylase acting upon N1-methyladenosine (m1A) and 5-formylcytosine (f5C) in mitochondrial RNA are unaffected. The observation that metabolites modulate specific subsets of RNA-modifying enzymes offers new insights into the intersection between metabolism and the epitranscriptome.

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FigureS1(A) Cell viability to TAK-243 in our drug screening. (B) SLFN11-KO cell lines in Li-7 by Western blotting. (C) CPT cytotoxicity in Li-7 SLFN11-WT and -KO cell lines. (D) The intensity of colony forming. (E) Cellular apoptosis by flow cytometry (F) Cleaved-PARP and cleaved-Caspase 3 by Western blotting. FigureS2(A, B) EdU incorporation in DU145 treated by TAK-243 for 24h or 48h. (C, D) Fork elongation speed. The length of red or green-colored part. FigureS3 (A) DNA damage response (DDR) proteins after long-term TAK-243 treatment. (B) CHK1 activation by CPT is reduced by the ATR inhibitor. FigureS4(A) UPR Activation by TAK-243 in CCRF-CEM cells. (B, C) UPR Activation by long-term TAK-243 treatment in DU145 and DMS114. (D) Accumulation of short-lived proteins. (E-F) Cell viability with p97 inhibitors in DU145. (G-H) Ubiquitin conjugates in CCRF-CEM with TAK-243 (1 hour). (I-J) Ubiquitin conjugates changes in DU145 cells with TAK-243(24 hours). FigureS5 (A-C) Newly synthesized protein detection by L-HPG in Li-7. Representative image and quantitation (D) Gene ontology (GO) analysis of molecular network associated with SLFN11. (E-F) Validation of SLFN11 interactions with the indicated translation initiation (EIF3B and EIF3L) and protein folding (CCT2) factors. (G) Relative cell viability with gene depletion of the TRiC/CCT chaperonin complex in RNAi screening. (H) Cell viability after transfection with siCCT2.

Butyrophilin 1A1 (BTN1A1) is implicated in the secretion of lipid droplets from mammary epithelial cells as a membrane receptor, which forms a secretion complex with the redox enzyme, xanthine oxidoreductase (XDH). The first evidence that BTN1A1 functions in this process was the generation of Btn1a1−/− mouse lines, in which lipid secretion was disrupted and large unstable droplets were released into alveolar spaces with fragmented surface membranes. We have revisited one of these mutant mouse lines using RNAseq and proteomic analysis to assess the consequences of ablating the Btn1a1 gene on the expression of other genes and proteins. Disruption of intact Btn1a1 protein expression led to a large build‐up of Xdh in the cytoplasm, induction of acute phase response genes and Lif‐activation of Stat3 phosphorylation. At peak lactation, approx. 10% of the cells were dying, as assessed by TUNEL‐analysis of nuclear DNA. Possible cell death pathways included expression of caspase 8 and activated caspase 3, autophagy, Slc5a8‐mediated inactivation of survivin (Birc5), and pStat3‐mediated lysosomal lysis, the latter of which is the principal death route in involuting wild type cells. Milk secretion was prolonged by renewal of the secretory epithelium, as evidenced by the upregulation of Ki67 in approx. 10% of cell nuclei and expression of cyclins and Fos/Jun. These data highlight the plasticity of the mammary epithelium and the importance of functional BTN1A1 expression for maintenance of terminally differentiated secretory cells and optimal milk production throughout lactation.

Background Modulation of metabolic flux through pyruvate dehydrogenase complex (PDC) plays an important role in T cell activation and differentiation. PDC sits at the transition between glycolysis and the tricarboxylic acid cycle and is a major producer of acetyl-CoA, marking it as a potential metabolic and epigenetic node. Methods To understand the role of pyruvate dehydrogenase complex in T cell differentiation, we generated mice deficient in T cell pyruvate dehydrogenase E1A (Pdha) subunit using a CD4-cre recombinase-based strategy. To control for the contribution of exogenous metabolites in vivo, we conducted our T cell functional studies in vitro. T cells were differentiated into memory and effector T cells using standardized protocols. Cells were analyzed using stable isotopic tracing studies, metabolomics, RNAseq, ATACseq, ChIPseq and histone proteomics. Results Herein, we show that genetic ablation of PDC activity in T cells (TPdh-/-) leads to marked perturbations in glycolysis, the tricarboxylic acid cycle, and OXPHOS. Due to depressed OXPHOS, TPdh-/- T cells became dependent upon substrate level phosphorylation via glycolysis. Due to the block of PDC activity, histone acetylation was reduced, as were most other types of post translational modifications. Transcriptional and functional profiling revealed abnormal CD8+ memory T cell differentiation in vitro. Conclusions Collectively, our data indicate that PDC integrates the metabolome and epigenome in memory T cell differentiation. Targeting this metabolic and epigenetic node can have widespread ramifications on cellular function.

Histone deacetylase inhibitors (HDACis) are part of a growing class of epigenetic therapies used for the treatment of cancer. While elevated levels of the efflux pump P-gp are associated within vitroresistance to romidepsin, this mechanism does not translate to the clinic. We developed a romidepsin-resistant cell line with a resistance mechanism independent of P-gp function that acts upstream of the deacetylation process. We found that expression of the methyltransferase METTL7A is necessary for resistance, and that expression of METTL7A in naïve cells can drive resistance to thiol-containing HDACis. We demonstrate that METTL7A can methylate romidesinin vitroand that the ability of METTL7A to drive resistance to thiol-containing HDACis can be blocked by the methyltransferase inhibitor DCMB. Our data supports a model whereby exposure of cells to romidepsin selects for upregulation of the methyltransferase METTL7A, which in turn modifies the zinc-binding thiol, inactivating the drug.

Upon exit from the totipotent 2-cell (2C) embryo stage, the 2C-associated transcriptional program needs to be efficiently silenced. However, the molecular mechanisms involved in this process remain mostly unknown. Here, we demonstrate that the 2C-specific transcription factor DUX directly induces the expression of DUXBL to promote this silencing. Indeed, DUX expression in Duxbl-knockout ESC causes increased induction of the 2C-transcriptional program, whereas DUXBL overexpression impairs 2C-associated transcription. CUT&RUN analyses show that DUXBL gains accessibility to DUX-bound regions in DUX-induced ESC while it is unable to bind those regions in uninduced cells. Mechanistically, we determined that DUXBL interacts with TRIM24 and TRIM33, two members of the tripartite motif superfamily involved in gene silencing and co-localizes with them in nuclear foci upon DUX expression. Furthermore, DUXBL downregulation in mouse zygotes leads to a penetrant 2C-stage arrest. Our data reveals an unexpected role for DUXBL in controlling the exit from totipotency.

Although BTB–zinc finger (BTB-ZF) transcription factors control the differentiation of multiple hematopoietic and immune lineages, how they function is poorly understood. The BTB-ZF factor Thpok controls intrathymic CD4 + T cell development and the expression of most CD4 + and CD8 + lineage genes. Here, we identify the nucleosome remodeling and deacetylase (NuRD) complex as a critical Thpok cofactor. Using mass spectrometry and coimmunoprecipitation in primary T cells, we show that Thpok binds NuRD components independently of DNA association. We locate three amino acid residues within the Thpok BTB domain that are required for both NuRD binding and Thpok functions. Conversely, a chimeric protein merging the NuRD component Mta2 to a BTB-less version of Thpok supports CD4 + T cell development, indicating that NuRD recruitment recapitulates the functions of the Thpok BTB domain. We found that NuRD mediates Thpok repression of CD8 + lineage genes, including the transcription factor Runx3 , but is dispensable for Cd4 expression. We show that these functions cannot be performed by the BTB domain of the Thpok-related factor Bcl6, which fails to bind NuRD. Thus, cofactor binding critically contributes to the functional specificity of BTB-ZF factors, which control the differentiation of most hematopoietic subsets.

During routine genome duplication, many potential replication origins remain inactive or ‘dormant’. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that are distinguished from baseline (i.e. ordinarily active) origins by their preferential association with two phospho-isoforms of the helicase component MCM2. During normal unperturbed cell growth, baseline origins, but not dormant origins, associate with a form of MCM2 that is phosphorylated by DBF4-dependent kinase (DDK) on serine 139 (pS139-MCM2). This association facilitates the initiation of DNA replication from baseline origins. Concomitantly, SIRT1 inhibits Ataxia Telangiectasia and Rad3-related (ATR)-kinase-mediated phosphorylation of MCM2 on serine 108 (pS108-MCM2) by deacetylating the ATR-interacting protein DNA topoisomerase II binding protein 1 (TOPBP1), thereby preventing ATR recruitment to chromatin. In cells devoid of SIRT1 activity, or challenged by replication stress, this inhibition is circumvented, enabling ATR-mediated S108-MCM2 phosphorylation. In turn, pS108-MCM2 enables DDK-mediated phosphorylation on S139-MCM2 and facilitates replication initiation at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational MCM modifications that reflect a balance between SIRT1 activity and ATR signaling.

Ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase orchestrates DNA damage response and repair pathways stimulated by replicative stresses. Recent studies have established that pharmacological inhibition of ATR is clinically promising. As M1774 is an oral ATR inhibitor in clinical development, we explored the molecular basis by which M1774 induces cancer cell death. As a single agent, we found that M1774 suppresses cancer cell viability at nanomolar concentrations with a potency higher than ceralasertib and berzosertib, but lower than gartisertib (M4344) and elimusertib in the small cell lung cancer (SCLC) cell lines H146, H82, and DMS114. We found that M1774 efficiently suppresses the ATR/CHK1 checkpoints. While M1774 alone induced apoptosis and G2/M cell cycle arrest at micromolar concentrations, at a non-toxic low dose, M1774 enhanced TOP1 inhibitor-mediated cancer cell death by preventing replication arrest and inducing DNA damage detected by EdU and γH2AX staining. Tandem mass tagging (TMT) coupled with mass spectrometry revealed that M1774 combined with SN-38 increases the expression of replication-related proteins (TIPIN, CDC45, TIMELESS, and RPA1) and G2/M-related proteins (PLK1 and CCNB1). To establish the synergistic combinations of M1774 with clinical anticancer DNA damaging agents in preclinical models, we performed experiments in cancer cell lines, patient-derived organoids, and xenograft models. Low doses of M1774 significantly synergized with the clinical TOP1 inhibitor SN-38, the TOP2 inhibitor etoposide, cisplatin, and the PARP inhibitor talazoparib in SCLC cell lines. We also found that M1774 significantly reversed chemoresistance to DNA-damaging agents in cancer cells lacking SLFN11 expression, suggesting that SLFN11 expression can be utilized for combination therapy with M1774 as a biomarker. The synergistic efficacy between M1774 and DNA-damaging agents was confirmed in SCLC patient-derived organoids, colon cancer patient-derived organoids, and H82 SCLC xenografts. Together, these results provide insights into the molecular mechanism and potential combination strategies for M1774 in cancer therapy. Citation Format: Ukhyun Jo, Yasuhiro Arakawa, Astrid Zimmermann, Daiki Taniyama, Makito Mizunuma, Lisa M Jenkins, Suresh Kumar, Frank T Zenke, Yves Pommier. Molecular pharmacology and broad synergy of the novel ATR inhibitor M1774 with DNA damaging anticancer agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB301.

Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, translocation of phospholipase-Cγ1 (PLC-γ1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC-γ1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC-γ1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC-γ1 activation are regulated both independently and additively by recruitment of PLC-γ1 to phosphorylated LAT, by formation of the LAT-Gads-SLP-76-PLC-γ1 tetramer, and by tyrosine phosphorylation of PLC-γ1. The recently solved structure of PLC-γ1 indicates that, in the resting state, several PLC-γ1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC-γ1, enabling contact with its membrane substrate, disinhibition of PLC-γ1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation.

CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of T cell EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47

The present study demonstrates how TOP3B is involved in resolving R-loops. We observed elevated R-loops in TOP3B knockout cells (TOP3BKO), which are suppressed by TOP3B transfection. R-loop-inducing agents, the topoisomerase I inhibitor camptothecin, and the splicing inhibitor pladienolide-B also induce higher R-loops in TOP3BKO cells. Camptothecin- and pladienolide-B-induced R-loops are concurrent with the induction of TOP3B cleavage complexes (TOP3Bccs). RNA/DNA hybrid IP-western blotting show that TOP3B is physically associated with R-loops. Biochemical assays using recombinant TOP3B and oligonucleotides mimicking R-loops show that TOP3B cleaves the single-stranded DNA displaced by the R-loop RNA-DNA duplex. IP-mass spectrometry and IP-western experiments reveal that TOP3B interacts with the R-loop helicase DDX5 independently of TDRD3. Finally, we demonstrate that DDX5 and TOP3B are epistatic in resolving R-loops in a pathway parallel with senataxin. We propose a decatenation model for R-loop resolution by TOP3B-DDX5 protecting cells from R-loop-induced damage.

Cellular quiescence and cell cycle reentry regulate vital biological processes such as cellular development and tissue homeostasis and are controlled by precise regulation of gene expression. The roles of long noncoding RNAs (lncRNAs) during these processes remain to be elucidated. By performing genome-wide transcriptome analyses, we identify differential expression of several hundreds of lncRNAs, including a significant number of the less-characterized class of microRNA-host-gene (MIRHG) lncRNAs or lnc-MIRHGs, during cellular quiescence and cell cycle reentry in human diploid fibroblasts. We observe that MIR222HG lncRNA displays serum-stimulated RNA processing due to enhanced splicing of the host nascent pri-MIR222HG transcript. The pre-mRNA splicing factor SRSF1 negatively regulates the microprocessor-catalyzed cleavage of pri-miR-222, thereby increasing the cellular pool of the mature MIR222HG. Association of SRSF1 to pri-MIR222HG, including to a mini-exon, which partially overlaps with the primary miR-222 precursor, promotes serum-stimulated splicing over microRNA processing of MIR222HG. Further, we observe that the increased levels of spliced MIR222HG in serum-stimulated cells promote the cell cycle reentry post quiescence in a microRNA-independent manner. MIR222HG interacts with DNM3OS, another lncRNA whose expression is elevated upon serum-stimulation, and promotes cell cycle reentry. The double-stranded RNA binding protein ILF3/2 complex facilitates MIR222HG:DNM3OS RNP complex assembly, thereby promoting DNM3OS RNA stability. Our study identifies a novel mechanism whereby competition between the splicing and microprocessor machinery modulates the serum-induced RNA processing of MIR222HG, which dictates cell cycle reentry.

Cells respond to ionizing radiation (IR) through the activities of constitutively expressed proteins and through changes in gene expression. Constitutively expressed proteins play well-defined roles in major components of the cellular radioresponse, such as DNA repair and cell cycle. However, the contribution of changes in gene expression to the radioresponse is less understood. We have previously demonstrated through polysome profiling and microarray analysis that translational control is a key component of radiation-induced changes in gene expression. In response to IR, specific transcripts are recruited to or away from polysomes without affecting the global polysome profile of the cells. One mechanistic explanation for this observation is that IR influences the activity of RNA binding proteins (RBPs), which regulate the inclusion or exclusion of transcripts from polysomes. Here, we utilized RNA interactome capture (RIC) to identify RBPs bound to transcripts in control and irradiated human cancer cells. Cells were either mock irradiated or treated with 2Gy. One hour post-IR, RNA and protein complexes were crosslinked with 254 nm UV light. mRNA was isolated from cell lysates with oligo(dT) magnetic beads followed by several rounds of highly stringent washes to remove non-specific interactors. RNA was removed from crosslinked proteins by RNase digestion. The proteins were separated on SDS-PAGE gels and subjected to in-gel trypsin digestion, and the subsequent peptides were analyzed by label-free quantification mass spectrometry using an Orbitrap Fusion mass spectrometer. Proteome Discoverer 2.2 was used to search the data against human proteins from the UniProt database using SequestHT with a 1% false discovery rate. We found that we were able to efficiently pull down RNA:protein complexes with a crosslinking dose of 150 mJ/cm2. As determined by silver staining, the protein pattern of crosslinked samples differed greatly from protein isolated from whole cell lysates. By western blot, this method highly enriched for known RBPs, such as PTBP1 and CUGBP1, while contamination from non-RBPs, such as actin and tubulin, was not detected. By mass spectrometry, we identified hundreds of RBPs in each replicate above the no crosslinking background control for both untreated and irradiated cells. However, more RBPs were consistently detected in the control cells. Examples of RBPs preferentially binding in control cells include SSB, a protein involved in a variety of RNA metabolic processes, and U2AF2, a protein involved in RNA splicing. Overall, these data demonstrate that RIC is a sensitive and rigorous method to identify RBPs and that RBPs may play a role in radiation-induced post-transcriptional gene regulation. Preferential binders identified by this method may provide further insight into pathways regulating tumor cell radiosensitivity. Citation Format: Stacey L. Lehman, Theresa Wechsler, Gaelyn C. Lyons, Lisa M. Jenkins, Kevin Camphausen, Philip J. Tofilon. Identification of RNA binding proteins influenced by ionizing radiation through RNA interactome capture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3741.

The N-end rule is a conserved mechanism found in Gram-negative bacteria and eukaryotes for marking proteins to be degraded by ATP-dependent proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target a protein to the degradation machinery. In Escherichia coli, the adaptor ClpS binds an N-degron and delivers the protein to ClpAP for degradation. As ClpS recognizes N-terminal Phe, Trp, Tyr, and Leu, which are not found at the N terminus of proteins translated and processed by the canonical pathway, proteins must be post-translationally modified to expose an N-degron. One modification is catalyzed by Aat, an enzyme that adds leucine or phenylalanine to proteins with N-terminal lysine or arginine; however, such proteins are also not generated by the canonical protein synthesis pathway. Thus, the mechanisms producing N-degrons in proteins and the frequency of their occurrence largely remain a mystery. To address these issues, we used a ClpS affinity column to isolate interacting proteins from E. coli cell lysates under non-denaturing conditions. We identified more than 100 proteins that differentially bound to a column charged with wild-type ClpS and eluted with a peptide bearing an N-degron. Thirty-two of 37 determined N-terminal peptides had N-degrons. Most of the proteins were N-terminally truncated by endoproteases or exopeptidases, and many were further modified by Aat. The identities of the proteins point to possible physiological roles for the N-end rule in cell division, translation, transcription, and DNA replication and reveal widespread proteolytic processing of cellular proteins to generate N-end rule substrates.