Carlo Stresemann

The Human let-7a-3 Locus Contains an Epigenetically Regulated MicroRNA Gene with Oncogenic Function

MicroRNAs (miRNAs) are small noncoding RNAs that repress their target mRNAs by complementary base pairing and induction of the RNA interference pathway. It has been shown that miRNA expression can be regulated by DNA methylation and it has been suggested that altered miRNA gene methylation might contribute to human tumorigenesis. In this study, we show that the human let-7a-3 gene on chromosome 22q13.31 is associated with a CpG island. Let-7a-3 belongs to the archetypal let-7 miRNA gene family and was found to be methylated by the DNA methyltransferases DNMT1 and DNMT3B. The gene was heavily methylated in normal human tissues but hypomethylated in some lung adenocarcinomas. Let-7a-3 hypomethylation facilitated epigenetic reactivation of the gene and elevated expression of let-7a-3 in a human lung cancer cell line resulted in enhanced tumor phenotypes and oncogenic changes in transcription profiles. Our results thus identify let-7a-3 as an epigenetically regulated miRNA gene with oncogenic function and suggest that aberrant miRNA gene methylation might contribute to the human cancer epigenome.

Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine

The cytosine analogues 5‐azacytosine (azacytidine) and 2′‐deoxy‐5‐azacytidine (decitabine) are the currently most advanced drugs for epigenetic cancer therapies. These compounds function as DNA methyltransferase inhibitors and have shown substantial potency in reactivating epigenetically silenced tumor suppressor genes in vitro. However, it has been difficult to define the mode of action of these drugs in patients and it appears that clinical responses are influenced both by epigenetic alterations and by apoptosis induction. To maximize the clinical efficacy of azacytidine and decitabine it will be important to understand the molecular changes induced by these drugs. In this review, we examine the pharmacological properties of azanucleosides and their interactions with various cellular pathways. Because azacytidine and decitabine are prodrugs, an understanding of the cellular mechanisms mediating transmembrane transport and metabolic activation will be critically important for optimizing patient responses. We also discuss the mechanism of DNA methyltransferase inhibition and emphasize the need for the identification of predictive biomarkers for the further advancement of epigenetic therapies.

Methylation of human MicroRNA genes in normal and neoplastic cells

MicroRNAs (miRNAs) are small RNA molecules that control gene expression by inhibition of protein translation or by degradation of cognate target mRNAs. Even though strict developmental and tissue-specific regulation appears to be critical for miRNA function, very little is known about the mechanisms governing miRNA gene expression. Several recent studies have shown that miRNA genes can be regulated by DNA methylation and other epigenetic mechanisms. The observation of altered miRNA gene methylation patterns in human cancers also suggested that miRNA gene methylation is functionally relevant for tumorigenesis. We have now performed a comprehensive analysis of miRNA genes and found that about half of these genes are associated with CpG islands and thus represent candidate targets of the DNA methylation machinery. An expanded analysis of several miRNA-associated CpG islands in five cell lines indicated that miRNA gene methylation is detectable at high frequencies, both in normal and malignant cells. Possible explanations for this phenomenon include the specific structure of miRNA genes and/or their requirement for strict expression regulation.

Human concentrative nucleoside transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation

The DNA methyltransferase inhibitors 5-azacytidine (5-azaCyd) and 5-aza-2′-deoxycytidine have found increasing use for the treatment of myeloid leukemias and solid tumors. Both nucleoside analogues must be transported into cells and phosphorylated before they can be incorporated into DNA and inactivate DNA methyltransferases. The members of the human equilibrative and concentrative nucleoside transporter families mediate transport of natural nucleosides and some nucleoside analogues into cells. However, the molecular identity of the transport proteins responsible for mediating the uptake of 5-azanucleosides has remained unknown. To this end, we have generated a stably transfected Madin-Darby canine kidney strain II cell line expressing recombinant hCNT1. An antiserum directed against hCNT1 specifically detected the protein in the apical membrane of hCNT1-expressing Madin-Darby canine kidney cells. Using [14C]5-azaCyd, we show here that hCNT1 mediated the Na+-dependent uptake of this drug with a Km value of 63 μmol/L. Na+-dependent transport of radiolabeled cytidine, uridine, and 5-fluoro-5′-deoxyuridine further showed the functionality of the transporter. hCNT1-expressing cells were significantly more sensitive to 5-azaCyd, and drug-dependent covalent trapping of DNA methyltransferase 1 was substantially more pronounced. Importantly, these results correlated with a significant sensitization of hCNT1-expressing cells toward the demethylating effects of 5-azaCyd and 5-aza-2′-deoxycytidine. In conclusion, our study identifies 5-azaCyd as a novel substrate for hCNT1 and provides direct evidence that hCNT1 is involved in the DNA-demethylating effects of this drug. [Mol Cancer Ther 2009;8(1):225–31]

Abstract 5239: Probing the cancer epigenome: empowering target validation by open innovation

Low reproducibility of published target validation studies as well as the frequent failure of genetic knock-down effects to phenocopy those of small molecule inhibitors have been recognized as road blocks for cancer drug discovery. Academic and industrial institutions have started to address these issues by providing access to high quality small molecular probes for novel targets of interest. Here we discuss probe discovery challenges and quality criteria based on the generation of three novel inhibitors for epigenetic targets. ATAD2 (ATPase family AAA-domain containing protein 2) is an epigenetic regulator that binds to chromatin through its bromodomain (BD). ATAD2 has been proposed to act as a co-factor for oncogenic transcription factors such as ERα and Myc. A more thorough validation of ATAD2 as a therapeutic target has been hampered by the lack of appropriate ATAD2 inhibitors. Here we disclose a structurally unprecedented series of ATAD2 BD inhibitors identified from a DNA-encoded library screen. Optimization delivered BAY-850, a highly potent and exceptionally selective ATAD2 BD inhibitor, which fully recapitulates effects seen by genetic mutagenesis studies in a cellular assay. The three BD and PHD-finger (BRPF) family members are found in histone acetyltransferase complexes. Whereas bromodomain inhibitors with dual activity against BRPF1 and 2 have been described before, we now disclose BAY-299, the first nanomolar inhibitor of the BRPF2 BD with high selectivity against its paralogs. Isoform selectivity was confirmed in cellular protein-protein interaction assays and rationalized based on X-Ray structures. BAY-598, a highly selective, cellularly active and orally bioavailable inhibitor of the protein lysine methyl transferase SMYD2, had been disclosed previously (Stresemann et al., AACR 2015). Development of BAY-598 allowed the identification of new methylation targets of SMYD2 as well as a proposed role of SMYD2 in pancreatic cancer. These results support further development of small molecule inhibitors as research tools to probe the functional role of novel epigenetic targets and underscore the power of open innovation for advancing our understanding of cancer target biology. Citation Format: Ingo V. Hartung, Cheryl Arrowsmith, Volker Badock, Naomi Barak, Markus Berger, Peter J. Brown, Clara D. Christ, Erik Eggert, Ursula Egner, Oleg Fedorov, Amaury E. Fernandez-Montalvan, Matyas Gorjanacz, Andrea Haegebarth, Bernard Haendler, Roman C. Hillig, Simon H. Holton, Kilian V. Huber, Seong J. Koo, Antonius ter Laak, Susanne Mueller, Anke Mueller-Fahrnow, Cora Scholten, Stephan Siegel, Timo Stellfeld, Detlef Stoeckigt, Carlo Stresemann, Masoud Vedadi, Joerg Weiske, Hilmar Weinmann. Probing the cancer epigenome: empowering target validation by open innovation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5239. doi:10.1158/1538-7445.AM2017-5239

Abstract 2829: Discovery and in vitro and in vivo characterization of aminopyrazoline-based SMYD2 inhibitors

SMYD2 (SET and MYND domain-containing protein 2) is a protein lysine methyltransferase (PKMT) which was initially described as a histone H3K36 and H3K4 methyltransferase involved in transcriptional regulation. SMYD2 has recently been reported to methylate and regulate several non-histone cancer relevant proteins such as p53, retinoblastoma protein (Rb) and the estrogen receptor alpha. Given the reports that overexpression of SMYD2 is linked to poor prognosis in certain cancers, SMYD2 is proposed to be an oncogene and an attractive cancer drug target. Here we report the discovery of a novel potent and selective SMYD2 inhibitor, SMYD2-BAY-01, by high throughput screening and extensive biophysical validation. The co-crystal structure revealed that SMYD2-BAY-01 binds to the substrate binding site and occupies the hydrophobic pocket for lysine binding using an unprecedented hydrogen bond pattern. The competitive behavior of the inhibitor in biochemical assays is consistent with the binding mode observed in the crystal structure. Further optimization generated SMYD2-BAY-02, which shows improved low nanomolar potency and is selective against kinases and other PKMTs. Furthermore, SMYD2-BAY-02 specifically inhibits SMYD2 methylation activity in a cellular assay with similar potency and reduces methylation of the tumor suppressor protein p53. Based on promising in vitro and in vivo DMPK data, SMYD2-BAY-02 was further characterized in vivo for SMYD2-specific methylation inhibition. In vivo activity could be shown upon in vivo administration at doses as low as 30 mg/kg p.o. in a SMYD2 overexpressing esophageal squamous cell carcinoma model. In summary, SMYD2-BAY-02 is a promising selective and potent SMYD2 inhibitor in vitro and in vivo and may represent a new treatment option for cancers overexpressing SMYD2. Citation Format: Carlo Stresemann, Ingo Hartung, Timo Stellfeld, Naomi Barak, Jeffrey Mowat, Clara Christ, Antonius ter Laak, Silke Koehr, Jorg Weiske, Roman Hillig, Volker Badock, Detlef Stoeckigt, Karl Ziegelbauer, Hilmar Weinmann, Volker Gekeler. Discovery and in vitro and in vivo characterization of aminopyrazoline-based SMYD2 inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2829. doi:10.1158/1538-7445.AM2015-2829