Simon Holton

ATAD2 is an epigenetic reader of newly synthesized histone marks during DNA replication.

ATAD2 (ATPase family AAA domain-containing protein 2) is a chromatin regulator harboring an AAA+ ATPase domain and a bromodomain, previously proposed to function as an oncogenic transcription co-factor. Here we suggest that ATAD2 is also required for DNA replication. ATAD2 is co-expressed with genes involved in DNA replication in various cancer types and predominantly expressed in S phase cells where it localized on nascent chromatin (replication sites). Our extensive biochemical and cellular analyses revealed that ATAD2 is recruited to replication sites through a direct interaction with di-acetylated histone H4 at K5 and K12, indicative of newly synthesized histones during replication-coupled chromatin reassembly. Similar to ATAD2-depletion, ectopic expression of ATAD2 mutants that are deficient in binding to these di-acetylation marks resulted in reduced DNA replication and impaired loading of PCNA onto chromatin, suggesting relevance of ATAD2 in DNA replication. Taken together, our data show a novel function of ATAD2 in cancer and for the first time identify a reader of newly synthesized histone di-acetylation-marks during replication.

Isoform-Selective ATAD2 Chemical Probe with Novel Chemical Structure and Unusual Mode of Action

ATAD2 (ANCCA) is an epigenetic regulator and transcriptional cofactor, whose overexpression has been linked to the progress of various cancer types. Here, we report a DNA-encoded library screen leading to the discovery of BAY-850, a potent and isoform selective inhibitor that specifically induces ATAD2 bromodomain dimerization and prevents interactions with acetylated histones in vitro, as well as with chromatin in cells. These features qualify BAY-850 as a chemical probe to explore ATAD2 biology.

Benzoisoquinolinediones as Potent and Selective Inhibitors of BRPF2 and TAF1/TAF1L Bromodomains

Bromodomains (BD) are readers of lysine acetylation marks present in numerous proteins associated with chromatin. Here we describe a dual inhibitor of the bromodomain and PHD finger (BRPF) family member BRPF2 and the TATA box binding protein-associated factors TAF1 and TAF1L. These proteins are found in large chromatin complexes and play important roles in transcription regulation. The substituted benzoisoquinolinedione series was identified by high-throughput screening, and subsequent structure–activity relationship optimization allowed generation of low nanomolar BRPF2 BD inhibitors with strong selectivity against BRPF1 and BRPF3 BDs. In addition, a strong inhibition of TAF1/TAF1L BD2 was measured for most derivatives. The best compound of the series was BAY-299, which is a very potent, dual inhibitor with an IC50 of 67 nM for BRPF2 BD, 8 nM for TAF1 BD2, and 106 nM for TAF1L BD2. Importantly, no activity was measured for BRD4 BDs. Furthermore, cellular activity was evidenced using a BRPF2– or TAF1–histone H3.3 or H4 interaction assay.

Abstract 287: BAY 1816032, a novel BUB1 kinase inhibitor with potent antitumor activity

The spindle assembly checkpoint represents a highly conserved surveillance mechanism which safeguards correct chromosome segregation by delaying anaphase onset until all chromosomes are properly bi-oriented on the spindle apparatus. Non-catalytic functions of the mitotic kinase BUB1 (budding uninhibited by benzimidazoles 1) were reported to be essential for spindle assembly checkpoint activation. In contrast, the catalytic function of BUB1 plays a minor role in spindle assembly checkpoint activation but is required for chromosome arm resolution and positioning of the chromosomal passenger complex for resolution of spindle attachment errors. Here, we disclose for the first time the structure and functional characterization of a novel, first-in-class Bub1 kinase inhibitor. Medicinal chemistry efforts resulted in BAY 1816032 featuring high potency, long target residence time and good oral bioavailablity. It inhibits BUB1 enzymatic activity with an IC50 of 7 nanomol/L, shows slow dissociation kinetics resulting in a long target residence time of 87 min, and an excellent selectivity on a panel of 395 kinases. Mechanistically BAY 1816032 abrogated nocodazole-induced Thr-120 phosphorylation of the major BUB1 target protein histone H2A in HeLa cells with an IC50 of 29 nanomol/L, induced lagging chromosomes and mitotic delay. Persistent lagging chromosomes and missegregation were observed upon combination with low concentrations of paclitaxel. Single agent BAY 1816032 inhibited proliferation of various tumor cell lines with a median IC50 of 1.4 micromol/L and demonstrated synergy or additivity with paclitaxel or docetaxel in almost all cell lines evaluated (minimal combination index 0.3). In tumor xenograft studies BAY 1816032 only marginally inhibited tumor growth as single agent upon oral administration, however, upon combination with paclitaxel or docetaxel a strong and statistically significant reduction of tumor size as compared to the respective monotherapy was observed. Intratumoral levels of phospho-Thr120 H2A were found to be strongly reduced, and no hints on drug-drug interactions were found. In line with the good tolerability in xenograft studies, no relevant findings from non-GLP 2 weeks toxicological studies in rat and dog were reported. Our findings validate the innovative concept of interference with mitotic checkpoints and justify clinical proof of concept studies evaluating BUB1 inhibitor BAY 1816032 in combination with taxanes in order to enhance their efficacy and potentially overcome resistance. Citation Format: Gerhard Siemeister, Anne Mengel, Wilhelm Bone, Jens Schroder, Sabine Zitzmann-Kolbe, Hans Briem, Amaury E. Fernandez-Montalvan, Simon Holton, Ursula Monning, Oliver von Ahsen, Sandra Johanssen, Arwed Cleve, Marion Hitchcock, Kirstin Meyer, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. BAY 1816032, a novel BUB1 kinase inhibitor with potent antitumor activity [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 287. doi:10.1158/1538-7445.AM2017-287

Abstract 5084: Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action

ATAD2 (ATPase family AAA-domain containing protein 2, also called ANCCA) is an epigenetic regulator that binds to chromatin through its bromodomain (BD), a motif specialized for acetyl-lysine recognition. ATAD2 directly associates with multiple transcription factors such as ERα, AR, E2F, and Myc; hence, ATAD2 has been proposed to act as a co-factor for oncogenic transcription factors. Furthermore, we have recently reported a novel role for ATAD2 during DNA replication, uncovering interactions between ATAD2 and histone acetylation marks on newly synthesized histone H4. High expression of ATAD2 strongly correlates with poor patient prognosis in multiple tumor types, including gastric, endometrial, hepatocellular, ovarian, breast and lung cancers. However, the exact function of ATAD2 in these tumor types remains unclear. A more thorough validation of ATAD2 as a therapeutic target is hampered by the lack of isoform-selective, potent and cellularly active ATAD2 inhibitors. A systematic assessment of crystal structures of BD-containing protein family predicted that development of selective inhibitors of ATAD2 would be challenging. In line with this prediction, only limited progress in developing lead compounds targeting ATAD2 has been reported so far. A few notable exceptions relied on fragments as starting points, however, their weak potency, insufficient selectivity against other BDs, permeability limitations or modest cellular activity have curbed their further development towards drug candidates. Here we embarked on a novel strategy to identify ATAD2 inhibitors: 11 different DNA-encoded libraries adding up to 67 billion unique encoded compounds were combined and incubated with ATAD2 BD followed by two rounds of affinity-mediated selection. This approach provided with several series of binders, for which specific target engagement of their SMOL moiety upon off-DNA synthesis was confirmed in biochemical and biophysical assays. Several rounds of potency optimization led to the identification of BAY-850, a highly potent and ATAD2 (isoform A) mono-selective inhibitor, which holds an amine substituted 3-(2-furyl)benzamide core. This compound shows - as revealed by size exclusion chromatography and native mass spectrometry - a novel mode of action for a BD inhibitor based on specific target dimerization. In a cellular fluorescence recovery after photobleaching (FRAP) assay BAY-850 displaced wild-type ATAD2 from the chromatin to the same extent as the genetic mutagenesis of ATAD2 BD. In contrast, chemically very similar inactive control compounds showed no major effects on ATAD2 association with the chromatin. These results qualify BAY-850 as the first biologically active ATAD2 isoform A-specific chemical probe, which will enable further elucidation of the cancer biology of this intriguing protein. Citation Format: Amaury E. Fernandez-Montalvan, Markus Berger, Benno Kuropka, Seong Joo Koo, Volker Badock, Joerg Weiske, Simon J. Holton, Apirat Chaikuad, Laura Diaz-Saez, James Bennett, Oleg Federov, Kilian Huber, Paolo Centrella, Matthew A. Clark, Christoph E. Dumelin, Eric A. Sigel, Holly S. Soutter, Dawn M. Troast, Ying Zhang, John W. Cuozzo, Anthony D. Keefe, Didier Roche, Vincent Rodeschini, Jan Hubner, Hilmar Weinmann, Ingo V. Hartung, Matyas Gorjanacz. Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action [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 5084. doi:10.1158/1538-7445.AM2017-5084

Functional and Structural Characterization of Bub3·BubR1 Interactions Required for Spindle Assembly Checkpoint Signaling in Human Cells

The spindle assembly checkpoint (SAC) is an essential safeguarding mechanism devised to ensure equal chromosome distribution in daughter cells upon mitosis. The proteins Bub3 and BubR1 are key components of the mitotic checkpoint complex, an essential part of the molecular machinery on which the SAC relies. In the present work we have performed a detailed functional and biochemical characterization of the interaction between human Bub3 and BubR1 in cells and in vitro. Our results demonstrate that genetic knockdown of Bub3 abrogates the SAC, promotes apoptosis, and inhibits the proliferation of human cancer cells. We also show that the integrity of the human mitotic checkpoint complex depends on the specific recognition between BubR1 and Bub3, for which the BubR1 Gle2 binding sequence motif is essential. This 1:1 binding event is high affinity, enthalpy-driven and with slow dissociation kinetics. The affinity, kinetics, and thermodynamic parameters of the interaction are differentially modulated by small regions in the N and C termini of the Gle2 binding domain sequence, suggesting the existence of “hotspots” for this protein-protein interaction. Furthermore, we show that specific disruption of endogenous BubR1·Bub3 complexes in human cancer cells phenocopies the effects observed in gene targeting experiments. Our work enhances the current understanding of key members of the SAC and paves the road for the pursuit of novel targeted cancer therapies based on SAC inhibition.

Abstract 2718: Synthesis and characterization of novel benzylpyrazole-based BUB1 kinase inhibitors with anti-tumor activity

BUB1 (budding uninhibited by benzimidazoles 1) is a serine/threonine protein kinase. The protein is bound to kinetochores and plays a key role in the establishment of the mitotic spindle checkpoint and chromosome congression prior to anaphase. Inhibition of BUB1 kinase represents a novel approach for cancer treatment: whereas cell cycle arrest is the predominant mode of action of a number of antimitotic cancer drugs (e.g. taxanes and vinca alkaloids), BUB1 inhibition results in aneuploidy and cell death by driving cells through mitosis irrespective of DNA damage and misattached chromosomes. Here, we report the characterization of a novel benzylpyrazole lead-structure series inhibiting BUB1 exemplified by BAY-320, a novel, first-in-class small molecule inhibitor of BUB1 kinase. This structure class was initially discovered as a single hit in a high-throughput screen, and resulted in a lead compound by chemical optimization. Benzylpyrazole BAY-320 is highly selective for BUB1 with single digit nanomolar biochemical potency and single-digit micromolar cellular potency (HeLa proliferation assay). Synergistic effects can be observed when BUB1 inhibitor BAY-320 is combined with low doses of paclitaxel affecting chromosome segregation and cell proliferation. X-ray data of benzylpyrazoles allowed for a better understanding the binding mode for rational property design. Further data on structure-activity relationship including pharmacokinetic, drug metabolism and the synthesis of BAY-320 and analogues will be presented. These results validate the benzylpyrazoles as novel selective BUB1 inhibitors and BUB1 as a promising approach for cancer treatment. Citation Format: Marion Hitchcock, Gerhard Siemeister, Hans Briem, Amaury Ernest Fernandez-Montalvan, Simon Holton, Anne Mengel, Ursula Monning, Michael Brands, Karl Ziegelbauer, Dominik Mumberg, Franz von Nussbaum. Synthesis and characterization of novel benzylpyrazole-based BUB1 kinase inhibitors with anti-tumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2718.

Abstract 4703: The BET inhibitor BAY 1238097 shows efficacy in BRAF wild-type and mutant melanoma models

BET proteins recognize histone acetylation marks and play an essential role in transcription elongation. BRD4, the best studied family member, binds to the regulatory regions of oncogenes such as MYC, thereby controlling its expression and that of the downstream transcriptome. Following the identification of the first selective inhibitors, BET proteins have been shown to play essential roles in hematological and, more recently, in solid tumors. Here we studied the efficacy of the novel BET inhibitor BAY 1238097 in melanoma models. BAY 1238097 was a potent BET inhibitor with IC50 values of 47 and 295 nM for BRD4 BD1 and BD2, respectively. In NanoBRET assays, the interaction between BRD4, BRD3 or BRD2 with histone H4 was blocked in intact cells with IC50 values of 65 nM, 294 nM and 642 nM, respectively. In addition to its strong anti-proliferative activity in several hematological tumor models, BAY 1238097 was also effective in several melanoma cell lines with GI50 values below 500 nM in BRAF wild-type (CHL-1, COLO-792, B16F10, IPC-298, MeWo) as well as BRAF mutant (A375, G-361, SK-MEL-30, LOX-IMVI, SK-MEL-5, MEL-HO) models. Resistant cell lines with GI50 values above 10 μM were identified in both the BRAF wild-type and mutant groups. ChIP analysis performed in BRAF wild-type CHL-1 cells showed that BAY 1238097 displaced BRD4 from regulatory regions of oncogenes such as MYC, leading to loss of expression. The oxygen consumption rates of the melanoma cell lines were measured and we found that cell lines sensitive to BAY 1238097 depended on oxidative phosphorylation rather than glycolysis for energy production. In vivo efficacy was determined for BAY 1238097 in three patient-derived melanoma models harboring the wild-type BRAF gene. Daily, oral treatment with 7.5 mg/kg BAY 1238097 led to significantly reduced tumor growth (39% T/C) in one model. Interestingly, this model was resistant to dacarbazine given i.p. at 100 mg/kg daily. No biologically significant anti-tumor activity was observed for the two other models treated with the same conditions (62% and 70% T/C, respectively). Altogether the results show BAY 1238097 to be a potent inhibitor of BRD4 binding to histones. The compound has strong anti-proliferative activity in different melanoma models, regardless of the BRAF mutation status but related to the metabolic activity. One out of three patient-derived melanoma models with wild-type BRAF responded to BAY 1238097 treatment in vivo. Future studies will help to better characterize the impact of BET inhibition on melanoma. Citation Format: Bernard Haendler, Kathy A. Gelato, Laura Schockel, Tatsuo Sugawara, Pascale Lejeune, Heidrun Ellinger-Ziegelbauer, Amaury E. Fernandez-Montalvan, Simon Holton, Stephan Siegel, Melanie Heroult, Annette Walter, Stuart Ince, Matthias Ocker. The BET inhibitor BAY 1238097 shows efficacy in BRAF wild-type and mutant melanoma models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4703.

Abstract 4539: ATAD2 mediates DNA replication in cancer

ATAD2 (ATPase family AAA domain-containing protein 2) is an epigenetic regulator which associates with chromatin through its Bromodomain specialized in Acetyl-Lys binding of histones. ATAD2 was also shown to directly associate with multiple transcription factors such as ERα, AR, E2F and MYC, and is believed to function as an oncogenic transcription factor in breast cancer. Here, we propose that ATAD2 facilitates DNA replication. ATAD2 is specifically expressed in S and G2 phase during which it co-localizes with newly synthesized DNA. We found ATAD2 on nascent chromatin together with newly synthesized histone H4 acetylated on K12 and Proliferating Cell Nuclear Antigen (PCNA), a central protein coupling replication with chromatin restoration, but not on post-replicative chromatin. In line with these observations depletion of ATAD2 by siRNA led to reduced DNA replication, perturbed loading of PCNA onto chromatin and inhibition of cell proliferation. Interestingly, a brief cycloheximide treatment of the cells to prevent the deposition of newly synthesized histones (e.g. H4K5,12diac) abrogated the recruitment of ATAD2 to nascent chromatin suggesting that ATAD2 might recognize and interact with these histone marks. Indeed, extensive biochemical and biophysical analyses involving TR-FRET, MST (MicroScale Thermophresis), Biocore, and NMR revealed that the bromodomain of ATAD2 preferentially interacts with these marks characteristic of newly synthesized histones. Consequently, overexpression of ATAD2 mutants unable to interact with these marks impaired DNA replication and recruitment of PCNA onto chromatin. Taken together, our data suggest that ATAD2 is essential for DNA replication and thus predicts that it is expressed in cells undergoing S phase. To further strengthen this hypothesis we compared the expression of ATAD2 with the proliferation marker Ki67, and the late S and G2/M marker TOP2A, in various cancer types such as colorectal, gastric, lung, prostate and breast cancers by immunohistochemistry. Indeed ATAD2 expression was restricted to Ki67 and TOP2A expressing areas of tumors, independent of cancer type. Moreover, aggressive tumors, such as triple negative breast cancer and metastatic castration-resistant prostate cancer, showed more intense and abundant expression of ATAD2 whereas slow-growing tumors showed low expression of ATAD2. This research identifies a role for ATAD2 in replication, providing mechanistic and translational support for therapeutic development in cancer. Citation Format: Seong Joo Koo, Amaury Ernesto Fernandez-Montalvan, Simon Holton, Oliver von Ahsen, Volker Badock, Sarah Vittori, Christopher J. Ott, James E. Bradner, Matyas Gorjanacz. ATAD2 mediates DNA replication in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4539.