Exon 20 insertions of HER2, encoded by erb-b2 receptor tyrosine kinase 2 (ERBB2), and other activating HER2 mutations occur in 2% to 4% of lung adenocarcinomas, but there are only limited therapeutic options available for these patients. Sevabertinib (BAY 2927088) is a potent and reversible dual EGFR-HER2 inhibitor that is selective with respect to wild-type EGFR. In this study, we report the preclinical activity of sevabertinib in lung cancer models harboring alterations of HER2, including exon 20 insertions, point mutations, and amplification of wild-type ERBB2. We furthermore demonstrate the activity of sevabertinib in a cancer cell line dependent on a fusion of neuregulin-1, a ligand for the HER2 family member and heterodimerization partner, HER3. Finally, we report patient responses to sevabertinib from a phase 1/2 clinical trial, indicating potential benefit for patients with HER2-mutant lung cancer. SIGNIFICANCE:Additional therapeutic options are needed for patients with lung cancer with HER2 activating mutations, including exon 20 insertions. Sevabertinib shows activity against ERBB2-encoded HER2 exon 20 insertions in preclinical models of lung cancer, corroborated by early data from a phase 1/2 clinical trial.

Supplementary Methods: Crystal Structure Determination of Wild-Type EGFR in Complex with Sevabertinib. Xenograft models

Antiproliferative activity of sevabertinib and EGFR/HER2 tyrosine kinase inhibitors in Ba/F3 cell lines with HER2 point mutations

Antitumor efficacy of sevabertinib in the subcutaneous CTG-2543 xenografts

Cell viability in HER2-amplified cancer cell lines treated with sevarbertinib or approved HER2 inhibitors

The binding mode of sevabertinib within the ATP binding site of wild-type EGFR from two different views

Additional cell viability data of zongertinib and STX-721 in isogenic Ba/F3 cell lines

Antiproliferative activity of sevabertinib and EGFR/HER2 tyrosine kinase inhibitors in Ba/F3 cell lines with HER2 Exon 20 insertion mutations

Abstract Exon 20 insertions of HER2, encoded by erb-b2 receptor tyrosine kinase 2 (ERBB2), and other activating HER2 mutations occur in 2% to 4% of lung adenocarcinomas, but there are only limited therapeutic options available for these patients. Sevabertinib (BAY 2927088) is a potent and reversible dual EGFR–HER2 inhibitor that is selective with respect to wild-type EGFR. In this study, we report the preclinical activity of sevabertinib in lung cancer models harboring alterations of HER2, including exon 20 insertions, point mutations, and amplification of wild-type ERBB2. We furthermore demonstrate the activity of sevabertinib in a cancer cell line dependent on a fusion of neuregulin-1, a ligand for the HER2 family member and heterodimerization partner, HER3. Finally, we report patient responses to sevabertinib from a phase 1/2 clinical trial, indicating potential benefit for patients with HER2-mutant lung cancer. Significance: Additional therapeutic options are needed for patients with lung cancer with HER2 activating mutations, including exon 20 insertions. Sevabertinib shows activity against ERBB2-encoded HER2 exon 20 insertions in preclinical models of lung cancer, corroborated by early data from a phase 1/2 clinical trial.

Crystallographic data collection and refinement statistics for the co-crystal structure of wild-type EGFR with sevabertinib

Kinase inhibition of sevabertinib (1 μM) measured in the presence of 10 μM ATP

Relative body weight change in the CTG-2543, NCI-H1781, and NCI-N87 xenograft models

Sevabertinib response and sensitivity to ERBB family gene CRISPR knockout or RNAi knockdown

This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.

PPAR gamma (PPARG) is a ligand activated transcription factor that regulates genes involved in inflammation, bone biology, lipid homeostasis, as well as a master regulator of adipogenesis and a potential lineage driver of luminal bladder cancer. While PPARG agonists lead to transcriptional activation of canonical target genes, inverse agonists have the opposite effect through inducing a transcriptionally repressive complex leading to repression of canonical target gene expression. While many agonists have been described and tested clinically, inverse agonists offer an underexplored avenue to modulate PPARG biology in vivo. Current inverse agonists lack favorable in vivo properties; herein we describe the discovery and characterization of a series of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists, BAY-5516, BAY-5094, and BAY-9683. Structural studies of this series revealed distinct pre- and post-covalent binding positions, which led to the hypothesis that interactions in the pre-covalent conformation are primarily responsible for driving affinity, while interactions in the post-covalent conformation are more responsible for cellular functional effects by enhancing PPARG interactions with its corepressors. The need to simultaneously optimize for two distinct states may partially explain the steep SAR observed. Exquisite selectivity was achieved over related nuclear receptors in the subfamily due in part to a covalent warhead with low reactivity through an SNAr mechanism in addition to the specificity gained through covalent binding to a reactive cysteine uniquely positioned within the PPARG LBD. BAY-5516, BAY-5094, and BAY-9683 lead to pharmacodynamic regulation of PPARG target gene expression in vivo comparable to known inverse agonist SR10221 and represent new tools for future in vivo studies to explore their potential utility for treatment of disorders of hyperactivated PPARG including luminal bladder cancer and other disorders.

Velcrin compounds are a class of small molecules that induce complex formation between PDE3A and SLFN12, killing cancer cells that express elevated levels of these two proteins by a mechanism independent of PDE3A enzymatic inhibition. Instead, PDE3A binding stimulates the RNase activity of SLFN12, resulting in cleavage of the specific SLFN12 substrate, tRNA-Leu-TAA. Cleavage of tRNA-Leu-TAA in turn causes ribosomal pausing, inhibition of protein synthesis, and cancer cell death. Unlike traditional targeted therapies that leverage dependencies created in cancer cells by genomic alterations, velcrins instead kill cancer cells by a gain-of-function mechanism dependent on the RNase activity of SLFN12. In a collaboration between the Broad Institute and Bayer Pharmaceuticals, we developed the first velcrin, BAY 2666605, to enter Phase I clinical trials. BAY 2666605 is active in cell line and patient-derived xenografts of several tumor types, specifically where elevated levels of the two biomarkers, PDE3A and SLFN12, are expressed. Biomarker-positive tumors are especially enriched among melanomas, and we have consistently observed tumor regression in biomarker-positive melanoma tumor models in vivo. BAY 2666605 furthermore shows drug-like properties, excellent brain penetration, increased stimulation of SLFN12 RNase activity, and reduced inhibition of PDE3A enzymatic activity compared with most other velcrins and approved PDE3A inhibitors. BAY 2666605 has recently entered a First-in-Human study (NCT04809805) in patients with advanced solid tumors that co-express PDE3A and SLFN12, including melanoma, ovarian cancer, and sarcoma. Citation Format: Stefan Gradl, Sooncheol Lee, Martin Lange, Xiaoyun Wu, Silvia Goldoni, Timothy Lewis, Charlotte Kopitz, Colin Garvie, Philip Lienau, Stephanie Hoyt, Henrik Seidel, Stephan Kaulfuss, Manuel Ellermann, Luc de Waal, Adrian Tersteegen, Sven Golfier, Detlev Suelzle, Christa Hegele-Hartung, James Carr, Frederick Brookfield, Michael Bruening, Melanie Berthold, Thibaud Jourdan, Monica Schenone, Galen Gao, Joseph McGaunn, Antje Wengner, Elisa Aquilanti, Franziska Siegel, Marine Garrido, Annette Walter, Isabelle Genvresse, Andrew Cherniack, Stuart Schreiber, Knut Eis, Ashley Eheim, Matthew Meyerson, Heidi Greulich. BAY 2666605: The first PDE3A-SLFN12 complex inducer for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND04.

Abstract CSNK1A1 is a serine/threonine kinase involved in multiple cellular processes, including cell division, beta catenin signaling, and TP53 activation. Inhibition of CSNK1A1 has previously been validated as a therapeutic strategy in hematologic malignancy, and degradation of CSNK1A1 protein is the downstream mechanism of action for lenalidomide in 5q- myelodysplasia (Krönke, et al. Nature. 2015.). However, lenalidomide is inactive in most solid tumor models, thus limiting the study of CSNK1A1 inhibition in other contexts. Analysis of genetic loss-of-function data from the Cancer Dependency Map reveals multiple sensitive models, including lineage-specific enrichment in colorectal and gastric cancer. In an academic-industry collaboration, we a) developed first-in-class potent and selective ATP-competitive CSNK1A1 small molecule inhibitors with preclinical anti-cancer efficacy in vivo, and b) identified FAM83 expression as a key determinant of inhibitor sensitivity. We identified a tetrahydro-pyrrolopyridinone scaffold that was subsequently optimized to yield BAY-888 (CSNK1A1 IC50 4 nM @ 10 μM ATP; 63 nM @ 1 mM ATP) and BAY-204 (CSNK1A1 IC50 2 nM @ 10 μM ATP; 12 nM @ 1 mM ATP). The crystal structure of CSNK1A1 in complex with BAY-888 confirmed compound binding in the ATP binding pocket. Across the PRISM barcoded cell line panel of more than 500 solid tumor cell lines, inhibitors phenocopy the CSNK1A1 shRNA knockdown profile. To determine downstream mediators of CSNK1A1 inhibitor sensitivity, we performed co-IP mass spectrometry following CSNK1A1 pulldown and global phosphoproteomic assays following inhibitor treatment. We identified multiple interacting proteins that are also phosphorylation targets, including FAM83 family members. FAM83 was recently reported to mediate the subcellular localization of CSNK1A1 (Fulcher, et al. Sci Signal. 2018.). Excitingly, the baseline expression of FAM83B and FAM83H correlates with inhibitor and shRNA cell line sensitivity. Modulation of FAM83H expression altered CSNK1A1 localization and sensitivity to CSNK1A1 inhibition. BAY-888 and BAY-204 are orally bioavailable and were evaluated in multiple murine cell line xenograft models. We observed promising efficacy in DLBCL (TMD8) in vivo as well as in multiple FAM83-high solid tumor models, including colorectal (HCT116 and HT29), gastric (IM95), and urothelial cancer (KU19-19). We identified RPS6 phosphorylation as one of the PD biomarkers correlating with efficacy in vivo. In summary, CSNK1A1 is a promising target with anti-tumor efficacy and achievable therapeutic index in preclinical models of FAM83-high solid tumors. Citation Format: Steven M. Corsello, Huajia Zhang, Rajesha Rupaimoole, Volker K. Schulze, Clara Lemos, Kasia B. Handing, Douglas L. Orsi, Mrinal Shekhar, Ulrike Sack, Sven Christian, Wilhelm Bone, Ranad Humeidi, William Colgan, Stephanie Hoyt, Andrew Cherniack, Jens Schroder, Stefan Kaulfuss, Krzysztof Brzezinka, Oliver von Ahsen, Anne Mengel, Roman C. Hillig, Detlev Suelzle, Jeremie Mortier, Caitlin Harrington, Rohith Nagari, Justyna Wierzbinska, Derek Chiang, Georg Beckmann, Meagan Olive, Namrata Udeshi, Annie Apffel, Steven Carr, Philip Lienau, Christian Lechner, Ulf Boemer, Alisha Caliman, David McKinney, Florence Wagner, Dominik Mumberg, Marcus Bauser, Andrea Haegebarth, Knut Eis, Ashley Eheim, Todd R. Golub. Discovery of potent and selective CSNK1A1 inhibitors for solid tumor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3588.

BAY 2666605, co-developed by the Broad Institute and Bayer Pharmaceuticals, is a selective and potent molecular glue, part of a family of small molecules recently baptized as ‘velcrins’, that induces complex formation between phosphodiesterase 3A (PDE3A) and SLFN12. BAY 2666605 has recently entered a First-in-Human study (NCT04809805) in patients with advanced solid tumors and here we describe its pre-clinical pharmacology profile. DNMDP, the precursor to BAY 2666605, was discovered in a phenotypic screen of genomically annotated cancer cell lines and sensitivity to treatment correlated to high expression of PDE3A (1). Upon treatment, SLFN12 is recruited into a stable complex with PDE3A where its RNase activity is enhanced and required for response (2). BAY 2666605 is a potent complex inducer (EC50 = 7 nM) and cytotoxic in vitro with nanomolar potency (IC50 = 1nM, in the most sensitive cell lines). Cancer cells with high expression of PDE3A and co-expression of SLFN12 are killed by a mechanism independent of PDE3A enzymatic inhibition. PDE3A-SLFN12 binding is required for cytotoxicity. Biomarker-positive lines are enriched in the melanoma lineage and show dose-dependent sensitivity to BAY 2666605 both in vitro and in vivo. Notably, we have consistently observed tumor regression in biomarker-positive melanoma models, including in PDX models (10mg/kg po BID). Based on target expression data from TCGA and tumor arrays, various other tumor types also co-express PDE3A and SLFN12, such as sarcomas and ovarian cancer. To this end, we show that BAY 2666605 inhibits tumor growth of PDX models of sarcoma and ovarian cancer in vivo. BAY 2666605 has excellent brain penetration, making glioblastoma a promising indication. Biomarker-positive GBM models are sensitive to BAY 2666605 both in vitro and in vivo. In a subset of orthotopic GBM models BAY 2666605 treatment has significant impact on survival. In BAY 2666605 treated models we have observed MCL1 downregulation and this biomarker will be evaluated in clinical settings. Our pre-clinical data indicate that BAY 2666605 is a potent anti-tumor agent with first-in-class potential and broad indication space. 1. de Waal et al. Identification of cancer-cytotoxic modulators of PDE3A by predictive chemogenomics, Nat. Chem. Biol. 12, 102-108 (2016) 2. Garvie et al. Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase, Nat. Commun. 12, 4375 (2021) Citation Format: Silvia Goldoni, Martin Lange, Charlotte Kopitz, Stefan Kaulfuss, Sven Golfier, Adrian Tersteegen, Stefanie Bunse, Melanie Berthold, Thibaud Jordan, Philip Lienau, Franziska Siegel, Annette Walter, Henrik Seidel, Elisa Aquilanti, Andrew Baker, Xiaoyun Wu, Sooncheol Lee, Stefan Gradl, Emmanuelle di Tomaso, Matthew Meyerson, Knut Eis, Ashley Eheim, Heidi Greulich. Preclinical profiling of BAY 2666605: The first PDE3A-SLFN12 complex inducer for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2663.

The ligand-activated nuclear receptor peroxisome-proliferator-activated receptor-γ (PPARG or PPARγ) represents a potential target for a new generation of cancer therapeutics, especially in muscle-invasive luminal bladder cancer where PPARγ is a critical lineage driver. Here we disclose the discovery of a series of chloro-nitro-arene covalent inverse-agonists of PPARγ that exploit a benzoxazole core to improve interactions with corepressors NCOR1 and NCOR2. In vitro treatment of sensitive cell lines with these compounds results in the robust regulation of PPARγ target genes and antiproliferative effects. Despite their imperfect physicochemical properties, the compounds showed modest pharmacodynamic target regulation in vivo. Improvements to the in vitro potency and efficacy of BAY-4931 and BAY-0069 compared to those of previously described PPARγ inverse-agonists show that these compounds are novel tools for probing the in vitro biology of PPARγ inverse-agonism.