Steffen Steurer

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.

A Novel RAF Kinase Inhibitor with DFG-Out–Binding Mode: High Efficacy in BRAF-Mutant Tumor Xenograft Models in the Absence of Normal Tissue Hyperproliferation

BI 882370 is a highly potent and selective RAF inhibitor that binds to the DFG-out (inactive) conformation of the BRAF kinase. The compound inhibited proliferation of human BRAF–mutant melanoma cells with 100× higher potency (1–10 nmol/L) than vemurafenib, whereas wild-type cells were not affected at 1,000 nmol/L. BI 882370 administered orally was efficacious in multiple mouse models of BRAF-mutant melanomas and colorectal carcinomas, and at 25 mg/kg twice daily showed superior efficacy compared with vemurafenib, dabrafenib, or trametinib (dosed to provide exposures reached in patients). To model drug resistance, A375 melanoma–bearing mice were initially treated with vemurafenib; all tumors responded with regression, but the majority subsequently resumed growth. Trametinib did not show any efficacy in this progressing population. BI 882370 induced tumor regression; however, resistance developed within 3 weeks. BI 882370 in combination with trametinib resulted in more pronounced regressions, and resistance was not observed during 5 weeks of second-line therapy. Importantly, mice treated with BI 882370 did not show any body weight loss or clinical signs of intolerability, and no pathologic changes were observed in several major organs investigated, including skin. Furthermore, a pilot study in rats (up to 60 mg/kg daily for 2 weeks) indicated lack of toxicity in terms of clinical chemistry, hematology, pathology, and toxicogenomics. Our results indicate the feasibility of developing novel compounds that provide an improved therapeutic window compared with first-generation BRAF inhibitors, resulting in more pronounced and long-lasting pathway suppression and thus improved efficacy. Mol Cancer Ther; 15(3); 354–65. ©2016 AACR.

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.

PO-449 Optimisation of an AlphaLISA assay for the characterisation of PROTAC-induced ternary complexes within cell lysates

Introduction Proteolysis Targeting Chimaeras (PROTACs) offer a powerful strategy to degrade cancer-associated proteins. PROTACs recruit an E3 ubiquitin ligase, such as von Hippel-Lindau (VHL), to a protein of interest (POI) to form a ternary complex. Measuring the formation of this ternary species is important for understanding PROTAC mode of action. Proximity AlphaLISA assays can be used for this purpose but have, to date, focused on using purified recombinant protein in vitro. Here, we report the development and optimisation of an AlphaLISA assay for monitoring ternary complex formation using full-length POI within cell lysates. Material and methods AlphaLISA assays were used to profile PROTAC-induced complex formation of FLAG-tagged full-length POI in cell lysate to purified biotinylated VCB. Signal was measured from close proximity of FLAG-tagged acceptor beads to streptavidin donor beads and benchmarked against a well-characterised PROTAC, MZ1, purified His-tagged Brd4 bromodomain 2 (BD2) and biotinylated VCB. A FLAG-tagged full-length POI containing an insert of Brd4 BD2 (POI-fusion) was utilised, such that MZ1 could be employed as a tool compound for assay development. Results and discussions Signal intensity was dependent on total lysate protein and VCB concentration, allowing for optimisation of component concentrations for maximal signal. A mild, detergent-free hypotonic cell lysis buffer gave enhanced signal over harsher buffers containing detergents. Additionally, the cell lysate cytosolic fraction provided superior signal compared to nuclear fractions and whole cell lysates, further highlighting the reliance of transient ternary complex formation on assay conditions. By utilising these optimal assay conditions determined with the POI-fusion, it is now possible to detect ternary complex formation between POI-targeting PROTACs and full-length POI. Conclusion To our knowledge, this optimised cell lysate AlphaLISA assay provides the first report and proof of concept for the interrogation of PROTAC-induced complex formation of full-length proteins with E3 ligases, which will be a valuable tool to the burgeoning PROTAC field. Consideration of assay parameters that permit optimal conditions for ternary complex formation in a POI-dependent manner allows the application of the AlphaLISA method as a screening platform. This application could profile PROTAC efficiency for a wide range of full-length, cell-derived proteins that are more relevant to the endogenous context encountered by the PROTAC intracellularly.

Abstract 2344: Modeling 1st- and 2nd-line therapy of BRAF mutant melanoma using a novel BRAF inhibitor with DFG-out binding mode.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The BRAF inhibitor vemurafenib (VEM) was approved for treatment of patients with BRAFV600E melanoma based on an ORR of 48% and a PFS of 5.3 months. Phase III data reported for the BRAF inhibitor dabrafenib (DAB) as well as the MEK inhibitor trametinib (TRA) in BRAFmut melanoma indicate similar outcomes. Phase I/II data published recently for a combination of DAB and TRA suggest improved efficacy compared with single-agent therapy; nevertheless, the short duration of responses even for the combination points to an urgent need for further improvement. BI 882370, a potent and selective B/CRAF inhibitor, binds to the DFG-out (inactive) conformation of BRAF, whereas VEM as well as DAB occupy the DFG-in (active) conformation. BI 882370 inhibited proliferation of BRAFmut melanoma cell lines with about 100x higher potency (EC50 1 - 10 nM) than VEM; BRAFwt cell lines were not affected at 1 μM. For studies in nude mouse xenograft models, VEM and DAB were dosed to provide exposures achieved in patients; TRA was used at the MTD for mice, resulting in exposures at or above the exposures tolerated by humans. In the G361 melanoma model (BRAFV600E), 1st line treatment with 25 mg/kg BI 882370 administered twice daily (bid) resulted in partial regression of all tumors, superior to results achieved by VEM, DAB as well as their combination. To model clinical resistance, a large cohort of A375 melanoma (BRAFV600E) bearing mice was treated with VEM; without exception, all tumors responded with partial regression and their median volume reached a nadir on day 11. Thereafter, the majority of tumors resumed growth in spite of continued treatment, whereas tumors in a subset of animals regressed further. Progressing tumors were randomized to 2nd line therapy on day 36 (median volume 280 mm3). TRA did not show any efficacy in this resistant population. BI 882370 induced tumor regression, however, resistance developed within 3 weeks. BI 882370 in combination with TRA resulted in more pronounced regressions and resistance was not observed until the end of the experiment, 6 weeks after initiation of 2nd line therapy. Importantly, in multiple in vivo studies performed to date, mice treated with BI 882370 at doses of 25 mg/kg bid for several weeks did not show any body weight loss or clinical signs of intolerability. Mice treated for 2 weeks were euthanized and major organs subjected to histological analysis. No changes were observed in any of the animals; in particular, there was no evidence for epidermal hyperplasia. Further, a pilot study in male rats dosed at 25 mg/kg bid for 2 weeks did not result in any toxicologically relevant findings in terms of clinical chemistry, hematology, pathology and toxicogenomics of liver and skin. Our results suggest that compared to 1st generation BRAF inhibitors, BI 882370 may provide an improved therapeutic window, enabling more pronounced and longer-lasting pathway suppression and thus resulting in improved efficacy. Citation Format: Irene C. Waizenegger, Anke Baum, Heinz Stadtmuller, Steffen Steurer, Gerd Bader, Pilar Garin-Chesa, Norbert Schweifer, Andreas Bernthaler, Christian Haslinger, Otmar Schaaf, Sien Mousa, Florian Colbatzky, Gunther R. Adolf. Modeling 1st- and 2nd-line therapy of BRAF mutant melanoma using a novel BRAF inhibitor with DFG-out binding mode. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2344. doi:10.1158/1538-7445.AM2013-2344