Recoding the Cancer Epigenome by Intervening in Metabolism and Iron Homeostasis with Mitochondria-Targeted Rhenium(I) Complexes.

Angewandte A Journal of the Gesellschaft Deutscher Chemiker Chemie International Edition www.angewandte.org Accepted Article Title:Recoding Cancer Epigenome by Intervening Metabolism and Iron Homeostasis with Mitochondria-Targeted Re(I) Complexes Authors:Zheng-Yin Pan, Cai-Ping Tan, Lu-Si Rao, Hang Zhang, Yue Zheng, Liang Hao, Liang-Nian Ji, and Zong-Wan Mao This manuscript has been accepted after peer review and appears as an Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article. To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202008624 Link to VoR: https://doi.org/10.1002/anie.202008624 10.1002/anie.202008624 RESEARCH ARTICLE Recoding Cancer Epigenome by Intervening Metabolism and Iron Homeostasis with Mitochondria-Targeted Re(I) Complexes Zheng-Yin Pan, Cai-Ping Tan,* Lu-Si Rao, Hang Zhang, Yue Zheng, Liang Hao, Liang-Nian Ji and Zong-Wan Mao* Abstract: The development and malignancy of cancer cells are required substrates or co-factors of epigenetic enzymes.[9b] As closely related to the changes of epigenome. In this work, a mitochondria play important roles in both material and energy mitochondria-targeted rhenium(I) complex (DFX-Re3) integrating the metabolism, the relationship between mitochondria and clinical iron chelating agent deferasirox (DFX) has been designed. By epigenetic modifications is gradually revealed.[11] For example, α- relocating iron to mitochondria and changing the key metabolic ketoglutarate (α-KG) is an intermediate in tricarboxylic acid cycle species related to epigenetic modifications, DFX-Re3 can elevate the (TCA) cycle, and JmjC domain-containing histone demethylases methylation levels of histone, DNA and RNA. As a consequence, (JHDMs) utilize α-KG, oxygen and Fe(II) as co-factors.[12] DFX-Re3 affects the events related to apoptosis, RNA polymerases Similar mechanism is also adopted by DNA demethylases and T-cell receptor signalling pathways. Finally, we demonstrate that including ten eleven translocation (TET) family enzymes.[13] DFX-Re3 induces immunogenic apoptotic cell death and exhibits Several RNA demethylases, including fat mass and obesity- potent antitumor activity in vivo. Our study provides a new approach associated protein (FTO) and alk B homolog 5 (ALKBH5), also act for the design of novel epigenetic drugs that can recode cancer through similar mechanisms.[14] JHDMs, TETs, FTO and ALKBH5 epigenome by intervening mitochondrial metabolism and iron belong to the family of Fe(II)/2-oxoglutarate-dependent homeostasis. oxygenases, and recent explorations highlight their capability to affect chromatin state and gene transcription by modulating histone, DNA and RNA methylation.[15] Epigenetic regulation is multifaceted with modifications of Introduction histone, DNA and RNA acting coordinately, and modulating them concurrently may achieve a synergistic anticancer effect.[16] Epigenetic modifications mainly include post-translational histone However, co-regulation of these modifications presents a great modifications, DNA methylation, and RNA modifications, which challenge as valid inhibitors/activators are not available for many can influence gene expression by regulating transcription and of the regulatory proteins. On the other hand, manipulating iron chromatin structure.[1] Epigenetic modification machinery includes homeostasis is emerging as an attractive anticancer strategy.[17] writers, readers and erasers that can place, identify and remove It has been reported that breast cancer cells have high these modifications, respectively.[2] Among them, inhibitors for requirement for iron and remodeled iron metabolism pathways histone deacetylases (HDACs) and DNA methyltransferases compared with normal cells.[18] Ferroportin (an exporter of (DNMTs) have been proved for clinical use.[3] Demethylases of intracellular iron) is markedly reduced in TNBC cells as compared histone and RNA are also emerging as promising anticancer with normal breast epithelial cells, and two important human iron targets.[4] Interestingly, inhibitors of DNMTs and HDACs show a regulatory protein 1 (IRP1 and IRP2) are overexpressed in synergistic anticancer effect,[5] and histone methylation guides TNBC.[19] As an essential element with critical functions in m6A modification co-transcriptionally,[6] which indicates these intermediate metabolism, energy production and cell proliferation, epigenetic modifications are closely related. Triple-negative the alterations iron homeostasis are closely related to the breast cancer (TNBC), characterized by the absence of estrogen, development, behavior and recurrence of cancer.[20] Ferroptosis, progesterone and HER-2 genes, is a leading cause of breast a form of cell death regulated by iron, plays important roles in the cancer death due to its high mortality and poor prognosis.[7] pathology and treatment of several diseases including cancer.[21] Recent studies show that epigenetic modifications play important By exchanging between its different oxidized forms, iron roles during the progression and treatment of TNBC.[8] imbalance causes free radical formation, lipid peroxidation, DNA The regulation of epigenetics is closely integrated with the and protein damages, which leads to carcinogenesis or cell metabolic states of cancer cells.[9] Firstly, chromatin-modifying death.[22] enzymes are recruited by signaling pathways and transcriptional Based on these considerations and our previous work on factors, and these processes are activated by growth factors, mitochondria-targeted Re(I) complexes,[23] we designed three hormones and cytokines.[10] Secondly, some key metabolites that rhenium(I) complexes incorporating a clinical iron chelating agent can deliver metabolic information to nuclear transcription, are deferasirox (DFX) to break the mitochondrial metabolism and iron Z. Y. Pan, Dr. C. P. Tan, L. S. Rao, H. Zhang, Y. Zheng, L. Hao, Prof. L. N. Ji homeostasis simultaneously (Figure 1A). Among them, DFX-Re3 and Prof. Z. W. Mao can relocate cellular iron to mitochondria and interfere with MOE Key Laboratory of Bioinorganic and Synthetic Chemistry School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, P. R. mitochondrial metabolism including the key metabolites related to China epigenetic modifications (Figure 1B). Moreover, relocating of iron E-mail: tancaip@mail.sysu.edu.cn, cesmzw@mail.sysu.edu.cn causes the down-regulation of the Fe(II)/2-oxoglutarate- Supporting information for this article is given via a link at the end of the dependent demethylases. As a consequence, DFX-Re3 can document.((Please delete this text if not appropriate)) elevate the levels of DNA, RNA and histone methylation simultaneously, which eventually leads to alternations in RNA polymerase II activities and gene expression profiles. 1 tpircsunaM detpeccA Angewandte Chemie International Edition This article is protected by copyright. All rights reserved. 10.1002/anie.202008624 RESEARCH ARTICLE epithelial) cells (Table 1 and Table S2). The cytotoxicity of DFX- Re1–DFX-Re3 and Re1–Re3 is correlated with their lipophilicity and the cellular uptake levels (Table 1, Table S2 and Figure S16). DFX is not active at the concentrations tested. In general, DFX- Re1–DFX-Re3 is more cytotoxic than their corresponding control compound in Re1–Re3. All the Re(I) compounds have a good inhibitory effect on MDA-MB-231 cells, especially DFX-Re3. The cytotoxicity of DFX- Re3 in MDA-MB-231 cells is about 32-fold higher than that in MCF-7 cells. In accordance with the literature reports,[26] cisplatin shows a poor killing effect on MDA-MB-231 cells. It is worth noting that DFX-Re3 shows an 100-fold higher activity on MDA-MB-231 cells than cisplatin. Moreover, DFX-Re3 also shows a high selectivity for MDA-MB-231 cells over MCF-10A cells, and it is 46- fold less cytotoxic on MCF-10A than on MDA-MB-231 cells. Based on the comparison between DFX-Re3 and cisplatin, DFX- Re3 is among the most active rhenium complexes ever reported, especially in TNBC cells.[23, 24b, 25, 27] Table 1. IC50 values of Re complexes towards different cell linesa Figure 1 (A) Chemical structures of DFX-Re1–DFX-Re3. (B) Schematic Compound log Po/w IC50 (μM) illustration of the anticancer mechanism of DFX-Re3. DFX-Re3 can relocate iron to mitochondria and affect mitochondrial metabolism, especially key MDA-MB-231 MCF-7 MCF-10A components of TCA cycle and one-carbon metabolism, including α-KG, fumarate (FU), succinate (SU) and the ratio of S-Adenosylmethionine (SAM) to S-Adenosylhomocysteine (SAH). Moreover, DFX-Re3 can downregulate the DFX-Re1 1.86 25.3±2.1 15.6±1.6 28.9±1.2 demethylases. DFX-Re2 2.01 0.7±0.8 15.2±0.5 21.2 ±1.5 Finally, we demonstrate that DFX-Re3 can induce immunogenic DFX-Re3 2.51 0.4±0.1 13.3±0.4 18.9±2.1 apoptosis and exhibits prominent antitumor activity in vivo. In all, our research provides a novel strategy to regulate cancer DFX u.d. >100 >100 >100 epigenome by intervening mitochondrial metabolism and iron homeostasis. Cisplatin u.d. 40.5±2.2 6.0±1.1 20.7±2.3 Results and Discussion a IC50 values are drug concentrations necessary for 50% inhibition of cell viability. Data are presented as means ± standard deviations (SD) and cell viability is assessed after 72 h of incubation. log Po/w value represents oil-water Synthesis and characterization partition coefficient. The ligand L1 was obtained by the condensation reaction of 3-(4- DFX-Re3 affects mitochondrial metabolism and key pyridyl) propylamine with DFX. The control compounds Re1–Re3 epigenetic metabolites (Scheme S1) were synthesized by literature methods.[24] DFX- We then use DFX-Re3, the most active compound, as a model Re1–DFX-Re3 were synthesized similarly by refluxing the compound to study the anticancer mechanisms. Confocal corresponding precursors with ligand L1 in tetrahydrofuran under microscopy shows that DFX-Re3 can effectively penetrated into an inert atmosphere (Scheme S2). The ligand L1 and complexes MDA-MB-231 cells after 2 h incubation. A high colocalization DFX-Re1–DFX-Re3 were characterized by ESI-MS, 1H NMR coefficient (about 94%) is obtained for DFX-Re3 and MitoTracker spectroscopy, 13C NMR spectroscopy and HPLC analysis (Figure Deep Red (MTDR; Figure 2A). Inductively coupled plasma-mass S1−S13). UV-Vis absorption spectra of DFX-Re1–DFX-Re3 show spectrometry (ICP-MS; Figure 2B) shows that DFX-Re3 gradually stronger bands at about 250–350 nm assigned to ligand-centered enriches in mitochondria. transition, and weaker broad bands centered at 350–450 nm The impact of DFX-Re3 on cancer cell metabolism was attributed to a metal-to-ligand charge transfer (1MLCT; Figure studied by metabolimic analysis using gas chromatography-time- S14).[23b] DFX-Re1–DFX-Re3 exhibit emission maxima in the of-flight mass spectrometry (GC-TOF-MS). The data were yellow region (ca. 560–590 nm) that is ascribed to the triplet analyzed by principal component analysis to find outliers (Figure MLCT excited state of diimine Re(I) tricarbonyl complexes (Figure S17). The orthogonal projections to latent structures discriminant S15).[25] The quantum yields of DFX-Re1–DFX-Re3 fall in the analysis score plots show a distinct metabolic profile for DFX- range of 0.044–0.325 with lifetimes between 320.1–1715.6 ns Re3-treated samples compared with the control (Figure S18). (Table S1). Based on a criterion of variable importance projection > 1 and p < 0.05, 679 (up-regulation:159; down-regulation: 520) and 605 (up- DFX-Re3 shows selective cytotoxicity in TNBC cells regulation: 192; down-regulation: 413) differential metabolites are The cytotoxicity of DFX-Re1–DFX-Re3 and Re1–Re3 were detected for the positive and negative ion modes in DFX-Re3- evaluated on human breast cell lines including MDA-MB-231 treated samples, respectively (Figure S19 and Table S3 and S4). (TNBC), MCF-7 (breast cancer) and MCF-10A (normal mammary These biomarkers are selected and further analyzed with 2 tpircsunaM detpeccA Angewandte Chemie International Edition This article is protected by copyright. All rights reserved. 10.1002/anie.202008624 RESEARCH ARTICLE Figure 2 (A) Colocalization of DFX-Re3 with MTDR in MDA-MB-231 cells. Cells were labeled with MTDR (150 nM, 15 min) and incubated with DFX-Re3 (5 μM, 2 h). DFX-Re3: λex = 405 nm; λem = 560 ± 20 nm. MTDR: λex = 633 nm; λem = 650 ± 20 nm. (B) Distribution of DFX-Re3 (5 μM; 2 h or 6 h) in cellular compartments of MDA-MB-231 cells measured by ICP-MS. (C) The impact of DFX-Re3 (5 μM, 6 h) on key metabolites related to epigenetics analyzed by GC-TOF-MS. (D) Quantitative analysis of the impact of DFX-Re3 (5 μM, 6 h) on ɑ-KG levels using UHPLC-MS. (E) The change in SAM/SAH ratio after treatment with DFX-Re3 (1 μM or 5 μM, 6 h) was quantitatively detected with ELISA. (F) Respiratory curves of MDA-MB-231 cells treated with DFX-Re3 for 4 h. The OCR was measured under basal conditions, and after the sequential addition of oligomycin (1 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.8 μM), and a mixture of rotenone (0.5 μM) and antimycin A (0.5 μM). (G) Glycolysis profiles of MDA-MB-231 cells treated with DFX-Re3 for 4 h. The ECAR was measured under basal conditions, and after the sequential addition of glucose (25 mM), oligomycin (1 μM), and 2-Deoxyglucose (2-DG, 100 mM). Data are mean ± SD. *p<0.05, **p<0.01. MetaboAnalyst to show the potential metabolic pathways (Figure changes of these metabolites are all conducive to the increase of S20 and S21). DFX-Re3 treatment influences several metabolic methylation level.[28] pathway including alanine, aspartate and glutamate metabolism, The impact of DFX-Re3 on mitochondrial respiration and pyrimidine metabolism, purine metabolism and pentose glycolysis was determined using a Seahorse XF24 Extracellular phosphate pathway. Flux Analyzer (Figure 2F). DFX-Re3 induces a dose-dependent Then we analyzed the key metabolites involved in epigenetic decrease in ATP production, maximum respiration and non- regulation.[28] Changes in the contents of one-carbon metabolism mitochondrial respiration (Figure S23). DFX-Re3 causes and TCA cycle are detected (Figure S22).[10] DNA/RNA significantly dose-dependent inhibition of glycolytic capacity and demethylase and most histone demethylation reactions utilize α- glycolytic reserve (Figure 2G and Figure S24). Moreover, DFX- kG as the cofactor, while succinate (SU) and fumarate (FU) act as Re3 induces the loss of mitochondrial membrane potential (MMP), the inhibitors of these reactions.[29] SAM is the methyl group donor and the proportion of cells with depolarized mitochondria reaches for histone/DNA/RNA methyltransferases, while SAH is the 86.4% after treatment for 6 h at 4 μM (Figure S25). reaction product and a competitive inhibitor of them.[30] In cells treated with DFX-Re3, α-kG (ca. 1.36-fold) and SAH (ca. 21.2- DFX-Re3 relocates cellular iron to mitochondria and induces fold) are down-regulated, while SU (ca. 1.57-fold) and FU (ca. 4.3- mitochondrial reactive oxygen species (ROS) fold) are up-regulated (Figure 2C). Ultra-high-performance liquid After incubation of the cells with DFX-Re3, ICP-MS shows that chromatography-mass spectrometry (UHPLC-MS) shows that the the content of Fe in mitochondria increases, while that in cellular α-KG level in DFX-Re3-treated group is decreased by cytoplasm decreases (Figure 3A). No obvious change in the 0.87-fold as compared with that in the control group (Figure 2D). content of Fe is detected in whole cells as well as in nuclei. Re3 An enzyme linked immunosorbent assay (ELISA) measurement without the DFX group shows no obvious effect on the subcellular shows that the ratio of SAM/SAH is increased by 25.7-fold in cells distribution of iron. treated with DFX-Re3 (5 µM, 6 h; Figure 2E). Interestingly, the 3 tpircsunaM detpeccA Angewandte Chemie International Edition This article is protected by copyright. All rights reserved. 10.1002/anie.202008624 RESEARCH ARTICLE Figure 3 (A) Distribution of Iron in cellular compartments of MDA-MB-231 cells measured by ICP-MS. The cells were incubated with Re(I) (5 μM, 4 h). (B) Generation of mitochondrial ROS caused by DFX-Re3 treatment. MDA-MB-231 cells were treated with DFX-Re3 (1 μM, 2 h). DCF: λex = 488 nm, λem = 530 ± 20 nm; MTDR: λex = 633 nm, λem = 683 ± 20 nm. Absorption (C) and Emission (D) spectra of DFX-Re3 (20 μM) upon the addition of Fe3+ measured in H2O/DMSO (v/v = 1/1) at 298 K. (E) The assignment of the main peak in the ESI-MS spectrum of DFX-Re3 with FeCl3 measured in H2O/MeCN (v/v = 1/1) at 298 K. As expected, the accumulation of Fe by DFX-Re3 in mitochondria produces massive ROS (Figure S26A). DFX-Re3 DFX-Re3 increases the methylation levels of DNA, RNA and treatment causes a dose-dependent increase in the fluorescence histone of DCF (2',7'-dichlorofluorescein, a ROS probe; Figure S26). The As DFX-Re3 can affect the cellular distribution of iron and key fluorescence of DCF overlaps well with that of MTDR (Figure 3B), epigenetic metabolites, which are closely related to the activity of indicating mitochondria are the major ROS-generating sites. Pre- Fe(II)/2-oxoglutarate-dependent demethylases,[33] we then incubation of cells with N-acetyl-cysteine (NAC, a ROS inhibitor) investigated the impact of DFX-Re3 on methylation levels. After partially rescues cells from death (Figure S26B). treatment with DFX-Re3, the methylation levels of H3 at the five In the presence of Fe3+ (Figure 3C) and Fe2+ (Figure S27A), most common sites are all increased (Figure 3A). DFX-Re3 the absorption peak of DFX-Re3 at about 295 nm decreases, treatment causes an increase in 5mC (5-methylcytosine, Figure accompanied by an increase of the absorption at 330 nm. An iso- 3B), which may be caused by the increased SAM/SAH ratio.[30] A absorption point is formed at 305 nm, indicating the formation of genome-wide hypomethylation is observed for TNBC, however, a single species. When the molar ratio of DFX-Re3/Fe is 1/3, the the function of altered DNA methylation status on the fluorescence of DFX-Re3 decreases by about 13- and 4.5-fold for development of TNBC needs further investigation.[34] 5hmC (5- Fe3+ (Figure 3D) and Fe2+ (Figure S27B), respectively. The hydroxymethylcytosine) is markedly decreased in the presence of phenomena can be attributed to the enhanced intramolecular DFX-Re3 (Figure 3C), which is consistent with literature reports photo-induced electron transfer from the Re chromophore to the showing that the inhibition of TET will block the formation of electron deficient iron-bound DFX moiety.[31] The cellular emission 5hmC.[35] Consistently, an increase in N6-methyladenosine (m6A, of DFX-Re3 is gradually decreased with the increase of incubation the most prevalent form of methylation on mRNA) is detected in time, which further confirms that Fe is relocated to mitochondria cells treated with DFX-Re3 (Figure 3D), which may be attributed (Figure S28). No significant change in the fluorescence intensity to the decreased activity of FTO.[36] of DFX-Re3 is observed in the presence of other typical biological In contrast, Re3 partially reduces histone methylation, metal ions (Figure S29). Although in most cases cellular iron is especially H3K4Me3 and H3K9Me3. Re3 shows a dose- transported and stored in the ferrous form, iron shuttles between dependent effects on the content of 5hmC. Re3 also increases the ferrous and ferric forms.[32] Both absorption and emission 5mC and m6A, which is less obvious than that observed for DFX- spectra show that DFX-Re3 has stronger binding ability towards Re3. Moreover, the expression of JMJD2A, TET2 and FTO are Fe3+, and ESI-MS confirms that a new complex assigned as decreased in a concentration-dependent manner in cells treated [Fe(DFX-Re3−PF −) −3H+]2+ is formed by mixing DFX-Re3 with with DFX-Re3 (Figure 3E), which may be ascribed to decrease of 6 2 Fe3+ (Figure 3E and S30). 4 tpircsunaM detpeccA Angewandte Chemie International Edition This article is protected by copyright. All rights reserved. 10.1002/anie.202008624 RESEARCH ARTICLE Figure 4 (A) Dose-dependent effects of DFX-Re3 and Re3 on the expression of methylated histone H3 after 24 h treatment. (B–D) Determination of the content of 5mC/5hmC in DNA and m6A in RNA in MDA-MB-231 cells treated with DFX-Re3/Re3 for 24 h via dot blot assay. Methylene blue (MB) represents loading control of DNA/RNA samples. (E) Dose-dependent effects of DFX-Re3 on the expression of JMJD2A, TET2, FTO after 24 h treatment. free iron content in the cytoplasm. These results suggest that RNA polymerase II are attenuated (Figure 5B; Table S5), which relocation of iron to mitochondria in cells can concurrently is consistent with previous reports indicating that changes in increase the DNA, RNA and histone methylation levels. epigenetic states can affect RNA polymerase II activity.[39] The expression of the top five positively/negatively regulate genes is DFX-Re3 alters transcriptome especially RNA polymerase II verified by real-time quantitative PCR (RT-qPCR; Figure 5C). By activity referring to the reported functions of these genes (Table S6), we Histone/DNA/ RNA methylation can affect chromatin states and find that DFX-Re3 causes up-regulation of tumor suppressor gene transcription, we then use RNA-seq to study the impact of genes (e.g., EGR1) and down-regulation of oncogenes (e.g., DFX-Re3 on transcriptome. The correlation coefficient between WNT7B). Gene set enrichment analysis (GSEA) shows that the every four individual samples is above 0.83, which indicates that change of genes is positively related to the regulation of experiment is reproducible (Figure S31). transcription from RNA polymerase II promoter (Figure 5D) and The overall Q30 percentage is above 93.9%, more than 97.5% o apoptosis (Figure 5E), and negatively related to T cell receptor f readings are mapped to reference genes in all samples, signaling pathway (Figure 5F). and 85.4% of readings are located in exons (Figure S32). The expression levels of 754 genes are found to be significantly DFX-Re3 exhibits potent anticancer activities in vitro and in changed, among which 470 and 284 genes are up-regulated and vivo down- regulated, respectively (Figure 5A). The heat-map of RNA- Next, we evaluated the anticancer mechanisms of DFX-Re3 both seq shows that expression patterns are very similar across in vitro and in vivo. Transmission electron microscopy (TEM) samples in each group (Figure S33). Kyoto Encyclopedia of observation shows that mitochondria swell obviously and Genes and Genomes (KEGG) enrichment analysis shows that the ridge disappears after DFX-Re3 (2 µM, 24 h) treatment DFX-Re3 mainly influences signaling pathway closely associated (Figure 6A), which indicates the loss of MMP. At a higher with mitochondrial[37] and epigenetic modulation[38], which include concentration, condensation of chromatin appears on the edge tumor necrosis factor (TNF), forkhead box O (FoxO), apoptosis, and center of the nucleus, and fragmented nucleus indicating late p53, phosphatidylinositol-3-kinases/Ser-ine-threonine protein apoptosis can also be observed. Annexin V/propidium iodide (PI) kinase (PIK3/Akt) and mitogen-activated protein kinase (MAPK) double staining assay shows that the proportion of cells in early signaling pathways (Figure S34). and late apoptosis phase increases dote-dependently in DFX- Gene ontology analysis shows that gene categories include Re3-treated samples (Figure S36). DFX-Re3 activates caspase positive transcription from RNA polymerase II promoter, 3/7 and pretreatment of Z-VAD-FMK (a pan-caspase inhibitor) chromatin, growth factor activity, intracellular receptor signaling inhibits cell death rates (Figure S37). Consistent with the previous pathway are significantly changed upon DFX-Re3 treatment RNA-seq results showing that DFX-Re3 can induce changes in (Figure S35). Interestingly, the expression of 61 genes related to the expression of many immune-related genes, DFX-Re3 induces 5 tpircsunaM detpeccA Angewandte Chemie International Edition This article is protected by copyright. All rights reserved. 10.1002/anie.202008624 RESEARCH ARTICLE Figure 6 (A) TEM pictures of MDA-MB-231 cells treated with DFX-Re3 at the indicated doses for 24 h. The red box indicates the enlarged area. (B) Calreticulin immunofluorescence of MDA-MB231 cells treated with DFX-Re3 for 24 h. Calreticulin: λex = 633 nm; λem = 660 ± 20 nm; DAPI: λex = 405 nm; λem = 450 ± 20 nm. (C) Tumor volumes of mice after treatment with phosphate buffered saline (PBS), DFX-Re3 and cisplatin. Intratumoral injections were performed as indicated by the red arrows. *, p < 0.05; **, p < 0.01. (D) Tumors separated from nude mice at day 14. Conclusions In this work, we designed a Re(I) complexes that can selectively kill TNBC cells by attenuating mitochondrial metabolism and iron Figure 5 (A) Volcano plots showing the differentially expressed genes in MDA- MB-231 cells treated with DFX-Re3 (5 μM, 24 h). Standard: P-value < 0.05, hemostasis simultaneously. DFX-Re3 can change the key variable importance projection (VIP) > 1. FC: fold change; FDR: False positive metabolites involved in epigenetic regulation, accumulate iron to rate. (B) Overlapping of differentially expressed genes and genes in the RNA mitochondria and down-regulate the expression of Fe(II)/2- polymerase II pathway. (C) Validation of the top 5 up/down-regulated genes oxoglutarate-dependent demethylases, so as to concurrently related to RNA polymerase II using RT-qPCR. Relative fold changes in gene expression were normalized according to the average expression of the increase the methylation levels of DNA, RNA and histone. The housekeeping gene β-actin. The full names of these genes are listed in Table recoding of TNBC epigenome alters the RNA polymerase activity, S7. (D–F) GSEA reveals negative and positive enrichment of DFX-Re3-altered reshapes the transcriptome and induces immunogenic apoptosis. genes in three different cellular pathways. NES: normalized enrichment score. Finally, DFX-Re3 shows potent anticancer activity and low systemic toxicity in vivo. In all, we propose a new strategy to recode the cancer epigenome and develop treatment for tumors up-regulation of calreticulin (Figure 6B), an important molecular resistant to traditional chemotherapy. marker of immunogenic death.[40] These results show that DFX- Re3 can induce caspase-dependent immunogenic apoptosis. For in vivo antitumor evaluation, nude mice bearing Acknowledgements mouse breast cancer 4T1 tumors with initial volumes of ca. 100 mm3 were randomly divided into three groups (n = 4). After two consecutive intratumoral injections at the indicated dosages, This study was supported by the National Natural Science DFX-Re3 can inhibit the tumor growth effectively. The inhibitory Foundation of China (nos. 21778078, 91953117 and 21837006), activity of DFX-Re3 is better than that of cisplatin (Figure 6C and the innovative team of Ministry of Education (no. IRT_17R111), Figure S38A). After 14 days of treatment, the inhibition rates of the Guangdong Natural Science Foundation (2015A030306023), DFX-Re3 and cisplatin are 89% and 63%, respectively (Figure and the Fundamental Research Funds for the Central Universities. 6D). Additionally, no mouse death or substantial body weight loss is found for DFX-Re3 during the treatment (Figure S38B), and no Keywords: Epigenetic Modification • Iron Homeostasis • obvious pathological change in the organs is detected for DFX- Mitochondrial Metabolism • Rhenium Complex • Anticancer Re3 at the end of the treatment (Figure S38C). These data indicate that DFX-Re3 possesses high anticancer effect and low [1] H. P. Mohammad, O. Barbash, C. L. Creasy, Nat. Med. 2019, 25, 403-418. systemic toxicity in vivo. [2] a) Y. Bergman, H. Cedar, Nat. Struct. Mol. Biol. 2013, 20, 274-281; b) H. Shi, J. Wei, C. He, Mol. Cell 2019, 74, 640-650. [3] Y. Cheng, C. He, M. Wang, X. Ma, F. Mo, S. Yang, J. Han, X. Wei, Signal. 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