Thomas Gerstberger

Structure-Based Design of an in Vivo Active Selective BRD9 Inhibitor

Components of the chromatin remodelling switch/sucrose nonfermentable (SWI/SNF) complex are recurrently mutated in tumors, suggesting that altering the activity of the complex plays a role in oncogenesis. However, the role that the individual subunits play in this process is not clear. We set out to develop an inhibitor compound targeting the bromodomain of BRD9 in order to evaluate its function within the SWI/SNF complex. Here, we present the discovery and development of a potent and selective BRD9 bromodomain inhibitor series based on a new pyridinone-like scaffold. Crystallographic information on the inhibitors bound to BRD9 guided their development with respect to potency for BRD9 and selectivity against BRD4. These compounds modulate BRD9 bromodomain cellular function and display antitumor activity in an AML xenograft model. Two chemical probes, BI-7273 (1) and BI-9564 (2), were identified that should prove to be useful in further exploring BRD9 bromodomain biology in both in vitro and in vivo settings.

Sensitivity and engineered resistance of myeloid leukemia cells to BRD9 inhibition

Here we show that acute myeloid leukemia (AML) cells require the BRD9 subunit of the SWI/SNF chromatin remodeling complex to sustain MYC transcription, rapid cell proliferation, and a block in differentiation. Based on these observations, we derived small-molecule inhibitors of the BRD9 bromodomain, which selectively suppressed the proliferation of mouse and human AML cell lines. To establish these effects as on-target, we engineered a bromodomain-swap allele of BRD9, which retains functionality despite a radically altered bromodomain pocket. Expression of this allele in AML cells conferred resistance to the anti-proliferative effects of our compound series, thus establishing BRD9 as the relevant cellular target. Furthermore, we used an analogous domain-swap strategy to generate an inhibitor-resistant allele of EZH2. Our study provides the first evidence for a role of BRD9 in cancer and reveals a simple genetic strategy for constructing resistance alleles to demonstrate on-target activity of chemical probes in cells.

The novel BET bromodomain inhibitor BI 894999 represses super-enhancer-associated transcription and synergizes with CDK9 inhibition in AML

Bromodomain and extra-terminal (BET) protein inhibitors have been reported as treatment options for acute myeloid leukemia (AML) in preclinical models and are currently being evaluated in clinical trials. This work presents a novel potent and selective BET inhibitor (BI 894999), which has recently entered clinical trials (NCT02516553). In preclinical studies, this compound is highly active in AML cell lines, primary patient samples, and xenografts. HEXIM1 is described as an excellent pharmacodynamic biomarker for target engagement in tumors as well as in blood. Mechanistic studies show that BI 894999 targets super-enhancer-regulated oncogenes and other lineage-specific factors, which are involved in the maintenance of the disease state. BI 894999 is active as monotherapy in AML xenografts, and in addition leads to strongly enhanced antitumor effects in combination with CDK9 inhibitors. This treatment combination results in a marked decrease of global p-Ser2 RNA polymerase II levels and leads to rapid induction of apoptosis in vitro and in vivo. Together, these data provide a strong rationale for the clinical evaluation of BI 894999 in AML.

Abstract LB-206: A bromodomain-swap allele demonstrates that on-target chemical inhibition of BRD9 limits the proliferation of acute myeloid leukemia cells

Recent studies have revealed vital roles of SWI/SNF complexes in leukemia and a variety of other cancers, making this chromatin remodeler a candidate drug target in human malignancy. Chemical modulation of SWI/SNF activity, however, remains to be achieved. Given the success of pharmacological bromodomain inhibition, we evaluated the role of bromodomain-carrying SWI/SNF subunits and identified Bromodomain-containing protein 9 (BRD9) as critical for the growth of Acute Myeloid Leukemia (AML). In AML cells, BRD9 binds the enhancer of the MYC proto-oncogene and sustains MYC transcription, rapid cell proliferation, as well as a block in differentiation. Based on these observations, we derived a small-molecule inhibitor of the BRD9 bromodomain, which partially displaces BRD9 from MYC enhancer elements and selectively suppresses the proliferation of mouse and human AML cell lines. Given the known role of other bromodomains, namely those of BRD4, in leukemia growth, ruling out potential off-target activity of our BRD9 inhibitor was critical. Traditionally, bromodomain inhibitor selectivity is tested using in vitro binding assays that examine a subset of other bromodomains. To sample the entire space of potential off-target proteins, we sought an in-cell selectivity assay. To this end, we engineered a bromodomain-swap allele of BRD9, which retains functionality despite a radically altered bromodomain pocket. Expression of this allele in AML cells confers resistance to the anti-proliferative effects of our BRD9 inhibitor, thus establishing BRD9 as the relevant cellular target. Furthermore, we used an analogous domain-swap strategy to generate an inhibitor-resistant allele of EZH2. Our study provides the first evidence for a role of BRD9 in cancer and further highlights a simple genetic strategy for constructing resistance alleles to demonstrate on-target activity of chemical probes in cells. Citation Format: Anja F. Hohmann, Laetitia J. Martin, Jessica Minder, Jae-Seok Roe, Junwei Shi, Steffen Steurer, Gerd Bader, Darryl McConnell, Mark Pearson, Thomas Gerstberger, Teresa Gottschamel, Diane Thompson, Yutaka Suzuki, Manfred Koegl, Christopher Vakoc. A bromodomain-swap allele demonstrates that on-target chemical inhibition of BRD9 limits the proliferation of acute myeloid leukemia cells. [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 LB-206.

Abstract B79: BI 894999, a novel BET inhibitor: Treatment of hematological malignancies by repression of super-enhancer driven oncogenes

Background: Bromodomain and extra-terminal (BET) protein inhibitors comprising the family members BRD2, 3, 4 and T are being extensively studied as treatment options in human haematological malignancies as well as in solid cancers. BRD4 is a key epigenetic regulator playing an important role in activating p-TEFb and governing expression of various oncogenes including MYC by contributing to multi-protein complexes forming so-called super-enhancers. BI 894999 is a novel, potent and selective orally bioavailable inhibitor of the BET family which has recently entered clinical trials. Results: Analysis of BI 8949999s activity in cell proliferation assays has revealed that it is a highly potent drug, particularly in hematological cell lines (n>40) such as MM, AML and lymphoma. Compound profiling using the BROMOscanTM assay revealed high selectivity for BRD2/3/4 and BRDT. On the cellular level, BI 894999 treatment leads to G1 arrest and subsequent apoptosis. Importantly, the compound also displayed excellent activity (low NM range) on 18/20 primary patient derived AML samples tested ex vivo. Potent tumor growth inhibition of this compound has been demonstrated in disseminated xenograft models of AML (MV-4-11, THP-1) and MM (MOLP-8), and in a transgenic model (Vk*MYC MM) as single agent or in combination with established and investigational therapeutic agents. When comparing the epigenetic regulator BI 894999 to other drugs, active in AML and also targeting chromatin pathways such as histone acetylation or DNA methylation, BI 894999 is found to regulate a very distinct set of genes. As already known, MYC expression and MYC target genes are affected by BET inhibitor treatment, as well as multiple other cancer relevant transcriptional signatures. HEXIM1, a negative regulator of p-TEFb was shown at RNA and protein level to be highly induced by BI 894999 in all cell lines tested so far and was verified as an excellent PD biomarker in extended PK/PD analyses. Detailed molecular analyses employing BRD4 ChIP-seq in MV-4-11 AML cells confirmed the published data that the strong repressive effect of BET inhibition on MYC expression is at least partly mediated by the displacement of BRD4 from a distant 39 MYC super-enhancer region. In addition, we have identified multiple other oncogene driving super-enhancers in AML cells. Conclusion: BI 894999 is a potent BET inhibitor which is currently being evaluated in clinical trials. This compound is highly active in MM and AML cell lines and patient samples, as well as in in vivotumor models. HEXIM1 was identified as a robust PD biomarker. Finally, we have demonstrated that BI 894999 is active by antagonizing super-enhancer driven oncogene expression such as MYC. Citation Format: Ulrike Tontsch-Grunt, Fabio Savarese, Daniel Gerlach, Davide Gianni, Anke Baum, Dirk Scharn, Harald Engelhardt, Onur Kaya, Norbert Schweifer, Thomas Gerstberger, Norbert Kraut. BI 894999, a novel BET inhibitor: Treatment of hematological malignancies by repression of super-enhancer driven oncogenes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B79.