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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.

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.

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.