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.

Abstract KAT6A and KAT6B encode lysine acetyltransferases that catalyse the transfer of acetyl groups from acetyl-CoA to lysine residues on histone proteins, thereby influencing chromatin structure and gene expression. Amplification of these genes is observed in several cancers, with the 8p11-p12 chromosomal region—containing KAT6A—being increased in copy number in approximately 12-15% of breast cancer cases, leading to higher expression of chromatin-modifying enzymes. In this study, we introduce a novel series of acylsulfonamide-benzofuran compounds that function as selective inhibitors of KAT6A and KAT6B. These inhibitors were first identified via high-throughput screening and subsequently refined through computational modelling and co-crystallization studies. The lead compound from this series, BAY-184, demonstrated efficacy in an in vivo proof-of-concept experiment, confirming its potential as a tool for targeting KAT6A/B activity. Citation Format: Antonius ter Laak, Roman Hillig, Steven Ferrara, Daniel Korr, Peter Staller, Naomi Barak, Philip Lienau, Simon Herbert, Amaury Fernandez, Roland Neuhaus, Matyas Gorjanacz, Vera Puetter, Volker Badock, Wilhelm Bone, Craig Strathdee, Franziska Siegel, Christoph Schatz, Nowak-Reppel Katrin, Olaf Doehr, Stefan Gradl, Ingo Hartung, Matthew Meyerson, Lea Bouche. Development of a new class of KAT6A/B inhibitors with in vivo efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7064.

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

The histone lysine acetyltransferase KAT6A belongs to the MYST family of lysine acetyltransferases which consists of five isoforms. KAT6A (and its paralog KAT6B) have emerged as a promising epigenetic drug targets in cancer, supported by recent clinical data. As support for our high-throughput screening approach aimed at discovering new inhibitors of KAT6A, we developed a crystallization platform system termed KAT6AmutCys in which four surface cysteine residues of KAT6A were mutated to serine. This construct crystallizes readily when in complex with its cofactor AcCoA. The obtained co-crystals were successfully used in a back-soaking approach in which AcCoA was soaked out of the crystals and small-molecule inhibitors were soaked in. In parallel, we explored a previously published surrogate approach termed MYSTcryst in which active-site residues of the related, but easier to crystallize, enzyme KAT8 were mutated to those found in KAT6A. By comparing co-crystal structures of the same ligand bound to KAT6A from our Cys-to-Ser approach and to mutated KAT8 (MYSTcryst) we found that the KAT8 surrogate approach indeed successfully reproduced the binding mode observed in the KAT6A structure, while delivering structures with significantly higher resolution. The MYSTcryst approach was therefore employed to determine the binding modes of two further small-molecule inhibitors, both from our KAT6A inhibitor optimization program and from a competitor lead series. These structures reveal alternative inhibitor conformations at the binding-site entry and the importance of filling a hydrophobic subpocket in the interior of the binding pocket. These insights will aid future efforts towards the development of KAT6A inhibitors as anticancer drugs.

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

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

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.

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

Lung cancer is the most prevalent tumor type worldwide, and despite advancements in providing patients with improved therapies, the 5-year overall survival rate is still only 5.8%. About 2-4% of patients with non-small cell lung cancer (NSCLC) carry either a HER2 exon20 insertion or a HER2 point mutation, which are potent oncogenic drivers. The antibody-drug conjugate fam-trastuzumab deruxtecan-nxki was the first targeted treatment option to be approved for patients with HER2-mutant NSCLC. However, there is still a significant unmet need for more effective and better tolerated therapies with the potential to improve response rates and response durability. Here we present preclinical data for BAY 2927088, a highly potent and selective tyrosine kinase inhibitor targeting HER2. BAY 2927088 shows potent in vitro antiproliferative activity in tumor cell lines carrying HER2 mutations, and strongly blocks HER2 phosphorylation, thereby inhibiting HER2-mediated downstream MAPK signaling. We also confirmed the strong anti-tumor activity of BAY 2927088 in in vivo models with HER2 exon20 insertions. To further explore the therapeutic potential of BAY 2927088, we evaluated combination treatment with current standard-of-care therapies in NSCLC in vitro and in vivo. In addition, we profiled BAY 2927088 vs. other HER2 tyrosine kinase inhibitors by pooled screening of 866 barcoded human cancer cell lines to identify additional oncogenic dependencies and patient populations that might benefit from treatment with BAY 2927088. We found that BAY 2927088 was also active in a subset of HER2-amplified breast and gastroesophageal cancer cell lines. Single-agent BAY 2927088 is currently being evaluated in clinical trials in patients with NSCLC with HER2 mutations. Preliminary results demonstrate efficacy and safety in pretreated patients (NCT05099172, NCT06452277). Franziska Siegel, Kristýna Kotýnková, Gizem Karsli-Uzunbas, Thibaud Jourdan, Melanie Berthold, Stephan Siegel, Daniel Korr, Markus Berger, Haruna Tomono, Sawyer Andersen, Daniel Denney, Jérémie Mortier, Andrew Cherniack, Matthew Meyerson, Heidi Greulich. BAY 2927088: a novel treatment option for patients with NSCLC with HER2 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5610.

KAT6A and KAT6B genes are two closely related lysine acetyltransferases that transfer an acetyl group from acetyl coenzyme A (AcCoA) to lysine residues of target histone substrates, hence playing a key role in chromatin regulation. KAT6A and KAT6B genes are frequently amplified in various cancer types. In breast cancer, the 8p11-p12 amplicon occurs in 12-15% of cases, resulting in elevated copy numbers and expression levels of chromatin modifiers like KAT6A. Here, we report the discovery of a new acylsulfonamide-benzofuran series as a novel structural class for KAT6A/B inhibition. These compounds were identified through high-throughput screening and subsequently optimized using molecular modeling and cocrystal structure determination. The final tool compound, BAY-184 (29), was successfully validated in an in vivo proof-of-concept study.

HER2 mutations are potent oncogenic drivers in various cancer indications. In NSCLC, about 2% of patients carry either a HER2 exon20 insertion mutation or a HER2 point mutation. With the recent approval of fam-trastuzumab deruxtecan-nxki, the first targeted treatment option became available for HER2 mutant NSCLC patients. However, there remains a high unmet need for more effective and better tolerated therapies with the potential to improve response rates and response durability. BAY 2927088 is a reversible small molecule inhibitor that is currently being evaluated in a first-in-human, phase I clinical trial in patients with EGFR mutant NSCLC (NCT05099172). Here, we present the preclinical activity profile of BAY 2927088 in HER2 mutant NSCLC. BAY 2927088 shows strong antiproliferative activity in an isogenic Ba/F3 cell line panel of HER2 exon20 insertion mutations as well as HER2 point mutations. BAY 2927088 is highly potent on the most frequent HER2 exon20 insertion mutations A775insYVMA and G776del insVC, as well as in the HER2 point mutations S310F, S335C, and L755S, among others. In addition, the compound was active in a subset of endogenously HER2 mutant cancer cell lines. The in vitro activity of BAY 2927088 was validated in vivo in a patient-derived xenograft model carrying the HER2 exon20 insertion mutation A775insYVMA. The strong preclinical activity of BAY 2927088 in HER2 mutant NSCLC supports clinical evaluation in this indication and might offer a novel targeted therapy option for NSCLC patients that carry HER2 mutations. Citation Format: Franziska Siegel, Gizem Karsli-Uzunbas, Kristyna Kotynkova, Quinn McVeigh, Stephan Siegel, Daniel Korr, Volker Schulze, Markus Berger, Georg Beckmann, Andrew Cherniack, Matthew Meyerson, Heidi Greulich. Preclinical activity of BAY 2927088 in HER2 mutant non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4035.

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.

Abstract Several targeted therapeutic options exist for non-small cell lung cancer (NSCLC) patients whose tumors harbor mutations of EGFR, including L858R, exon 19 deletions, and the acquired resistance mutation, T790M. However, there is no approved targeted therapy for patients with EGFR exon 20 insertions, highlighting an ongoing unmet medical need. Here, we describe a potent and selective inhibitor of EGFR exon 20 insertion mutants with decreased activity on the wild-type EGFR for an improved therapeutic window. Our compound, BAY-568, exhibits greater than 20-fold selectivity for EGFR exon 20 insertions compared to wild-type EGFR in isogenic Ba/F3 models and in cancer cell lines endogenously expressing EGFR exon 20 insertions. This activity is also observed in xenograft models in vivo, correlating with reductions in phospho-EGFR and phospho-Erk in tumors but not skin samples from treated mice. BAY-568 furthermore has even greater activity towards the “classical” erlotinib-sensitive mutations, L858R and exon 19 deletions. Importantly, our compound is reversible, differentiating it from other investigational compounds currently in clinical trials. Consistent with this, the presence of a C797S mutation, typically found in patients with acquired resistance to osimertinib, has no effect on the activity of BAY-568. Taken together, these results demonstrate the ability of BAY-568 to kill cancer cells harboring exon 20 insertions and other EGFR mutations with decreased activity on wild-type EGFR, irrespective of C797S mutation status. Citation Format: Franziska Siegel, Stephan Siegel, Keith Graham, Bethany Kaplan, Kirstin Petersen, Ulf Boemer, Uwe Eberspaecher, Daniel Korr, Ursula Moenning, Detlev Suelzle, Jens Schroeder, Florian Prinz, Sabine Zitzmann-Kolbe, Gizem Karsli-Uzunbas, Timothy Lewis, Mario Hermsen, Andrew Cherniack, Franz von Nussbaum, Knut Eis, Matthew Meyerson, Heidi Greulich. Preclinical activity of the first reversible, potent and selective inhibitor of EGFR exon 20 insertions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1470.