INTRODUCTION:HER2 is mutated in 2-4% of non-small cell lung cancers (NSCLC) and is associated with poor prognosis. Tyrosine kinase inhibitors (TKIs) targeting HER2 have historically been hampered by insufficient efficacy against exon 20 insertion mutations and lack of specificity, resulting in off-target adverse events. Zongertinib is an oral, irreversible HER2-selective TKI that spares wild-type EGFR, thereby minimizing associated toxicities. Zongertinib was recently approved in the United States (accelerated), China (conditional), and Japan for patients with previously treated advanced HER2-mutant NSCLC. AREAS COVERED:This article outlines the discovery and clinical development of zongertinib that led to these approvals. We discuss the first-in-human Beamion LUNG-1 trial (NCT04886804), in which zongertinib demonstrated encouraging and durable activity, with a manageable safety profile, in patients with HER2-mutant advanced NSCLC. Finally, we summarize ongoing clinical trials of zongertinib, including its assessment as first-line treatment for advanced HER2-mutant NSCLC. EXPERT OPINION:Zongertinib is the first oral TKI approved for HER2-mutant NSCLC and will provide patients with a convenient, tolerable and effective treatment option in an area of significant unmet need. Next steps include its potential transition to a first-line setting, identification of additional indications, and development of novel combination regimens.

The Gewald reaction is a key method for synthesizing 2‐aminothiophenes, which are valuable scaffolds in drug discovery. Here, we report a practical electrochemical approach to the Gewald reaction using an undivided cell setup in the IKA ElectraSyn 2.0 reactor. Key parameters, including electrode materials, electrolyte type, current, and electron equivalents, were systematically investigated to maximize yields. The optimized protocol demonstrated broad substrate scope, tolerating diverse functional groups and ring systems. Late‐stage functionalization and a one‐pot transformation were achieved on complex molecules, and the method was successfully applied to the synthesis of the non‐steroidal anti‐inflammatory drug tinoridine. This robust, scalable, and sustainable electrochemical strategy offers a convenient and complementary method to traditional Gewald reactions, expanding access to 2‐aminothiophenes for medicinal chemistry.

In vivo tumor responses for zongertinib monotherapy or combinations.

The enabling synthesis of the first route to HER2 inhibitor BI-4142 (1) to deliver a drug substance in kilogram quantity is reported. The synthetic route involves (1) a fit-for-purpose synthesis of pyrimido[5,4-d]pyrimidine 2; (2) a high yielding, scalable synthesis of aniline 3; (3) a safer sodium tungstate-catalyzed sulfide oxidation; (4) SNAr reactions to form C-N bonds; and (5) amidation via Schotten-Baumann conditions. With the speed of delivery prioritized, a purification protocol using silica gel filtration and crystallizations was developed in time to control the quality of API. The overall yield of the delivery route was improved from 22% to 46% over a prior route starting from 2.

Mutations in ERBB2 (encoding HER2) occur in 2% to 4% of non-small cell lung cancer (NSCLC) and confer poor prognosis. ERBB-targeting tyrosine kinase inhibitors, approved for treating other HER2-dependent cancers, are ineffective in HER2-mutant NSCLC due to dose-limiting toxicities or suboptimal potency. We report the discovery of zongertinib (BI 1810631), a covalent HER2 inhibitor. Zongertinib potently and selectively blocks HER2, while sparing EGFR, and inhibits the growth of cells dependent on HER2 oncogenic driver events, including HER2-dependent human cancer cells resistant to trastuzumab deruxtecan. Zongertinib displays potent antitumor activity in HER2-dependent human NSCLC xenograft models and enhances the activities of antibody-drug conjugates and KRASG12C inhibitors without causing obvious toxicities. The preclinical efficacy of zongertinib translates in objective responses in patients with HER2-dependent tumors, including cholangiocarcinoma (SDC4-NRG1 fusion) and breast cancer (V777L HER2 mutation), thus supporting the ongoing clinical development of zongertinib.Significance: HER2-mutant NSCLC poses a challenge in the clinic due to limited options for targeted therapies. Pan-ERBB blockers are limited by wild-type EGFR-mediated toxicity. Zongertinib is a highly potent and wild-type EGFR-sparing HER2 inhibitor that is active in HER2-driven tumors in the preclinical and clinical settings.

Targeted covalent inhibitors are known to be successful therapeutics used in various indications. Covalent drugs typically target cysteine, as cysteine is well suited due to its high nucleophilicity. However, its low abundance in protein binding sites represents a major limitation. As a result, there is a need to covalently target additional nucleophilic amino acids. Recent literature has reported covalent inhibitors labeling aspartic acid in KRASG12D. However, these compounds also covalently bind to KRASG12C, indicating their cross-reactivity to cysteine along with aspartic acid. We report here carbodiimides as a novel reactive group to selectively target aspartic acid. Covalent inhibitors bearing a carbodiimide moiety are shown to covalently label KRASG12D in biochemical and cellular assays. A high-resolution X-ray crystal structure was obtained, which illustrates the mechanism of the covalent bond formation with KRASG12D. Carbodiimide warheads show selectivity toward KRASG12D over other KRAS alleles and represent a new covalent warhead suitable for covalently binding to aspartic acid in a biochemical and cellular context.

Abstract Alterations of KRAS are observed in approximately one in seven of all human tumors, making KRAS one of the most prevalent oncogenic drivers. Gain-of-function missense mutations in KRAS, leading to its aberrant activation, are found in ~90% of pancreatic cancer, ~40% of colorectal cancer and ~30% of lung adenocarcinoma, with KRASG12V mutations accounting for ~28%, 9% and 6% of the cases, respectively. The poor outcome associated with these tumor types as well as the lack of targeted inhibitors for the KRASG12V-mutant allele calls for the urgent need to identify therapies able to effectively address this allele. BI 3706674 is a novel, potent and orally available small molecule inhibitor of the KRAS oncogene. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including KRASG12V, in the GDP-bound state, and blocks downstream oncogenic signalling. Here, we show that BI 3706674 has a strong anti-proliferative activity in a panel of KRASG12V-mutant cancer cell lines in vitro, along with significant pharmacodynamic (PD) biomarker modulation (including inhibition of ERK1/2 phosphorylation and down-regulation of DUSP6 mRNA). In vivo, a twice daily oral dose of 30 mg/kg was well tolerated, while inducing tumour regression in several mutant KRASG12V patient-derived xenograft (PDX) models across multiple different tumor types. Feedback activation of upstream signalling pathways including receptor tyrosine kinases, such as the EGFR, has been suggested to limit the efficacy of GDP-KRAS-targeting compounds in preclinical studies. Clinical combination trials involving KRASG12C inhibitors such as adagrasib and sotorasib and anti-EGFR modalities are further supporting these findings. Here we show that a combination with anti-EGFR antibodies (e.g., Cetuximab) potently enhances the response observed for BI 3706674 in models of KRASG12V-mutant colorectal cancer. The deeper response observed upon combination of BI 3706674 with Cetuximab across multiple xenograft models provides a strong rationale for the clinical investigation of this combination therapy. Moreover, based on our in vitro mechanistic studies and ex vivo organoid platform we have identified prospective mechanisms of resistance and opportunity for novel drug combinations with BI 3706674 that can potentially translate into clinical trials. Citation Format: David Hwa Peng, Antonio Tedeschi, Lorenz Herdeis, Otmar Schaaf, Fabio Savarese, Francesca Rocchetti, Johannes Popow, Heinrich J. Huber, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Bröeker, Tobias Wunberg, Firoella Schischlik, Jesse Lipp, Mariah Williams, Vaness Chandler, Charles E. Deckard, Vandhana Ramamoorthy, Joseph R. Daniele, Scott Kopetz, Michael Kim, Don L. Gibbons, Christopher P. Vellano, Joseph R. Marszalek, TImothy P. Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. KRASmulti inhibitor BI 3706674, an orally bioavailable, direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in KRASG12V-driven preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3321.

BI 3706674 is a novel small molecule inhibitor targeting KRAS in its inactive (GDP-bound) state and is currently undergoing IND enabling studies. Here, we assess the sensitivity of BI 3706674 in a large panel of cancer cell lines with the aim to identify biomarkers predictive of patient response. We employ two complementary, high-throughput cell viability screening setups: the pooled PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) platform (868 cancer cell lines representing 80 tumor types) and a well-based custom screen performed in collaboration with Horizon Discovery (292 cell lines representing 32 tumor types). Based on correlation with publicly available data of over 1464 drug sensitivity profiles, the top 10 correlated drugs (ranked by Pearson correlation) are exclusively MEK inhibitors. This analysis shows that compounds with high similarity in their sensitivity profile to our KRAS inhibitor target proteins in the MAPK pathway. Correlation of drug sensitivity data with CRISPR gene dependency data for KRAS showed that drug selectivity of BI 3706674 is specific for KRAS (Pearson R = -0.49) compared to other members of the RAS family, such as HRAS or NRAS (R=0.03 & R=0.16). BI 3706674 shows sensitivity across a wide range of KRAS alleles (KRAS wild-type (wt) copy number amplification, G12V, G12C, G13D, G12D, G12A, Q61H) with the highest sensitivity for cell lines with KRAS wt amplifications (relative copy number of > 10) followed by cell lines with a KRAS G12V and G12C mutant alleles. Efficacy was observed in 8/9 cell lines with KRAS wt relative copy number of > 10 (sensitivity threshold of 1- AUC = 0.25) in the PRISM screen, emphasizing the utility of KRAS copy number as a predictive biomarker for drug response. Furthermore, KRAS copy number amplifications and KRAS expression are highly correlated features across cell lines (Pearson R = 0.72, P=2.2e-16), indicating that both KRAS copy number amplification and KRAS expression could serve as sensitivity biomarkers for BI 3706674. In this study, we show that BI 3706674 is a potent and selective KRAS inhibitor and that KRAS copy number alterations represent a highly predictive biomarker. Conclusively, high-throughput drug screens are powerful tools to define and further refine biomarkers and study drug mechanism of actions. Citation Format: Fiorella Schischlik, Antonio Tedeschi, Dorothea Rudolph, Daniel Gerlach, Birgit Wilding, Matthias Treu, Julian Fuchs, Lorenz Herdeis, Joachim Broeker, Tobias Wunberg, Andrew S. Boghossian, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Darryl McConnell, Mark Pearson, Norbert Kraut, Christian Haslinger, Jesse Lipp. Determinants of sensitivity to BI KRASmulti inhibitor using high-throughput in-vitro drug screens [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A092.

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.

Here we report pre-clinical characterization of the clinical compound BI 1810631, a HER2 specific, EGFR wild-type-sparing tyrosine kinase inhibitor (TKI). BI 1810631 has shown early signs of clinical activity in patients carrying tumors with HER2 aberrations [NCT04886804], mainly comprising HER2 mutations (Heymach et al. 2023). In non-small cell lung cancer, activating mutations in HER2 are found in 2-4% of patients and predominantly cluster in exon 20 within the tyrosine kinase domain (TKD), of which the most frequent variant is A775_G776insYVMA. Studies across tumor cell lines and xenograft mouse models show that BI 1810631 is a potent and selective inhibitor of HER2-driven oncogenic signaling. Compound-mediated reduction of cell growth and survival was observed in mutant HER2 driven Ba/F3 cell systems, in human tumor cell lines in vitro and translated into tumor regressions in a genome-engineered HER2-YVMA mutant xenograft model. The in vivo efficacy of BI 1810631 was further confirmed in HER2 exon 20 mutant patient-derived tumor models. Corresponding biomarker studies in vitro and in vivo showed modulation of pharmacodynamic markers, corroborating the on-target mechanism of action of the compound. These findings demonstrate that HER2 mutations can be effectively addressed by BI 1810631 and support the ongoing and future clinical trials. Citation Format: Ralph A. Neumüller, Birgit Wilding, Dirk Scharn, Anke Baum, Martin Augsten, Irene Waizenegger, Shinji Kohsaka, Valeria Santoro, Paolo Chetta, Lydia Woelflingseder, Johannes Popow, Daniel Gerlach, Peter Ettmayer, Thomas Gerstberger, Julian Fuchs, Matthias Treu, Stephan Zahn, Mark Pearson, Mark Petronczki, Darryl B. McConnell, Norbert Kraut, Flavio Solca. BI 1810631 is a novel EGFR wild-type sparing, HER2-selective small molecule inhibitor that efficiently blocks HER2 mutant-driven lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C140.

Abstract Alterations of KRAS are found in approximately one in seven of all human tumors, making KRAS one of the most prevalent oncogenic drivers. Gain-of-function missense mutations in KRAS, leading to its aberrant activation, are found in ~90% of pancreatic cancer, ~40% of colorectal cancer and ~30% of lung adenocarcinoma, with KRASG12V mutations accounting for ~28%, 9% and 6% of the cases, respectively. The poor outcome associated with these tumor types, particularly pancreatic cancer, as well as the lack of targeted inhibitors for KRASG12V calls for the urgent need to identify therapies able to effectively address the KRASG12V mutant allele. BI 3706674 is a novel, potent and orally available small molecule inhibitor of the KRAS oncogene. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including KRASG12V, in the GDP-bound state, and blocks downstream oncogenic signalling. Here, we show that BI 3706674 has a strong anti-proliferative activity in a panel of KRASG12V mutant cancer cell lines in vitro, along with significant pharmacodynamic (PD) biomarker modulation (including inhibition of ERK1/2 phosphorylation and down-regulation of DUSP6 mRNA).In vivo, a twice daily oral dose of 30 mg/kg was well-tolerated, while inducing tumour regression in several mutant KRASG12V cell line-derived and patient-derived xenograft (CDX and PDX) models across multiple different human tumor types. We have selected different non-small cell lung cancer xenograft models for extensive PK/PD/efficacy analysis in vivo. Results of the ongoing analysis will be shared. Feedback activation of upstream signalling pathways including receptor tyrosine kinase, such as the EGFR, has been suggested to be able to limit the efficacy of GDP-KRAS targeting compounds in preclinical studies. These findings have been supported by clinical combination trials involving KRASG12C inhibitors such as Adagrasib and Sotorasib and anti-EGFR modalities. We demonstrate for the first time that combination with anti-EGFR antibodies (e.g., Cetuximab) potently enhances the response observed with BI 3706674 in settings of KRASG12V mutant pancreatic, colorectal, and lung cancer. The deeper response observed upon combination of BI 3706674 with Cetuximab across multiple xenograft models provides a strong rationale for the clinical investigation of this combination therapy. Moreover, we utilize an ex vivo organoid platform to rapidly identify novel drug combinations with BI 3706674 that can potentially translate to clinical trials. The KRASmulti inhibitor BI 3706674 is currently undergoing IND enabling studies. Single agent dose escalation will include patients with cancers harbouring KRASG12V mutations. Citation Format: David H Peng, Antonio Tedeschi, Lorenz Herdeis, Fabio Savarese, Francesca Rocchetti, Popow Johannes, Heinrich J Huber, Nicola Melillo, Jake Dickinson, Hitesh B Mistry, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Broker, Tobias Wunberg, Fiorella Schischlik, Jesse Lipp, Vandhana Ramamoorthy, Joseph R Daniele, Scott Kopetz, Michael Kim, Don L Gibbons, Christopher P Vellano, Joseph R Marszalek, Timothy P Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. BI KRASmulti, a first-in-class, orally bioavailable and direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in KRAS G12V-driven preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A087.

Abstract Alterations of KRAS are found in approximately one in seven of all human cancers, making KRAS one of the main oncogenic drivers in cancer. Gain-of-function missense mutations in KRAS are found in ~90% of pancreatic cancer, ~40% of colorectal cancer and ~30% of lung adenocarcinoma. In addition, focal high-level amplification of the KRAS wild-type (wt) allele, an alternative means of activating this oncoprotein, is observed in ~7% of cancers and lacks effective targeted therapies. Thus, there is an urgent need to identify effective therapies to address KRAS-driven tumors. Recently, we have reported the identification of compounds active against a broad range of oncogenic KRAS variants (KRASmulti) that selectively inactivate downstream signaling and tumor growth (Kim et al., Nature 2023). Herein we describe BI 3706674, a novel, potent and orally available small molecule inhibitor of the KRAS oncogene. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including the KRAS wt allele, in the GDP-bound state and thereby disrupts oncogenic signaling. Using several in vitro assays, we show that BI 3706674 is a) highly selective for KRAS vs HRAS or NRAS; b) blocks the interaction between KRAS mutant/wt and its guanidine exchange factor (GEF) SOS1 and c) shows potent antiproliferative activity in isogeneic cell lines that are dependent on various KRAS mutant alleles. In two large cancer cell line panels, BI 3706674 potently and selectively inhibits proliferation of KRAS mutant cancer cell lines as well as cancer cell lines with an amplification of the KRAS wt allele. Significant pharmacodynamic (PD) biomarker modulation (including inhibition of ERK1/2 phosphorylation and DUSP6 mRNA down-regulation) was observed in cell lines with a KRAS wt relative copy number (CN) > 10, treated with BI 3706674. Amplification of the KRAS wt allele is most frequently observed in gastric (~5%), esophageal (~13%) and gastroesophageal junction cancers (~12%). BI 3706674 was well-tolerated and showed dose-dependent efficacy in cell line-derived and patient-derived xenograft models (CDX and PDX) of human gastric cancer with a KRAS wt CN > 10. A twice daily oral dose of 30 mg/kg was sufficient to induce significant tumor regression. BI 3706674 treatment induced PD biomarker modulation (including inhibition of ERK1/2 phosphorylation, DUSP6 down-regulation, repression of the MAPK Pathway Activity Score (MPAS) signature and Ki67 reduction) in two KRAS wt amplified gastric cancer xenograft models. The depth and duration of PD biomarker responses following treatment correlated well with the doses used in the respective efficacy studies. Finally, these results support the clinical use of biomarkers such as the MPAS signature score and Ki67 to investigate treatment effects elicited by BI 3706674 in tumor biopsies. The KRASmulti inhibitor BI 3706674 is undergoing IND enabling studies and is intended to target tumors driven by KRAS wt amplifications as well as oncogenic KRAS missense mutations. Citation Format: Antonio Tedeschi, Lorenz Herdeis, Valeria Santoro, Fabio Savarese, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Bröker, Tobias Wunberg, Michael Gmachl, Rumpel Klaus, Fiorella Schischlik, Jesse Lipp, David H. Peng, Mariah Williams, Charles Deckard, Vandhana Ramamoorthy, Joseph R. Daniele, Jaffer A. Ajani, Funda Meric-Bernstam, Christopher P. Vellano, Joseph R. Marszalek, Timothy P. Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. BI KRASmulti, a first-in-class, orally bioavailable and direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in preclinical models and validates wild-type amplified KRAS as a therapeutic target [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A085.

We report a new chemoenzymatic cascade starting with aldehyde synthesis by carboxylic acid reductase (CAR) followed by chemical in situ oxime formation. The final step to the nitrile is catalyzed by aldoxime dehydratase (Oxd). Full conversions of phenylacetic acid and hexanoic acid were achieved in a two-phase mode.

Oncogenic alterations in human epidermal growth factor receptor 2 (HER2) occur in approximately 2% of patients with non-small cell lung cancer and predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. Most clinical-grade tyrosine kinase inhibitors are limited by either insufficient selectivity against wild-type (WT) epidermal growth factor receptor (EGFR), which is a major cause of dose-limiting toxicity or by potency against HER2 exon 20 mutant variants. Here we report the discovery of covalent tyrosine kinase inhibitors that potently inhibit HER2 exon 20 mutants while sparing WT EGFR, which reduce tumor cell survival and proliferation in vitro and result in regressions in preclinical xenograft models of HER2 exon 20 mutant non-small cell lung cancer, concomitant with inhibition of downstream HER2 signaling. Our results suggest that HER2 exon 20 insertion-driven tumors can be effectively treated by a potent and highly selective HER2 inhibitor while sparing WT EGFR, paving the way for clinical translation.

ERAP1 is a zinc-dependent M1-aminopeptidase that trims lipophilic amino acids from the N-terminus of peptides. Owing to its importance in the processing of antigens and regulation of the adaptive immune response, dysregulation of the highly polymorphic ERAP1 has been implicated in autoimmune disease and cancer. To test this hypothesis and establish the role of ERAP1 in these disease areas, high affinity, cell permeable and selective chemical probes are essential. DG013A 1, is a phosphinic acid tripeptide mimetic inhibitor with reported low nanomolar affinity for ERAP1. However, this chemotype is a privileged structure for binding to various metal-dependent peptidases and contains a highly charged phosphinic acid moiety, so it was unclear whether it would display the high selectivity and passive permeability required for a chemical probe. Therefore, we designed a new stereoselective route to synthesize a library of DG013A 1 analogues to determine the suitability of this compound as a cellular chemical probe to validate ERAP1 as a drug discovery target.

Abstract Activating mutations in ERBB2 receptors are tractable oncogenes in 2-3% of NSCLC patients for whom no approved targeted therapies are available. In this indication, oncogenic mutations in HER2 predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. We initiated a drug discovery program aiming at discovering novel HER2 selective inhibitors sparing EGFR WT activity. Focus was set on the most frequent HER2 mutation (ERBB2 A775 insYVMA), which is least sensitive to current compounds tested in clinical trials. Here, we report the identification and pharmacological characterization of novel selective HER2 exon 20 mutation TKIs that differ from currently tested TKIs such as poziotinib, TAK-788 or BDTX-189. We could demonstrate that selective inhibition of oncogenic HER2 signaling abrogates oncogenic signaling in in vitro models. Cell survival and proliferation was reduced, which translated into tumor regressions in preclinical CRISPR engineered xenotransplantation models of HER2 exon 20 mutants. The in vivo efficacy was confirmed in patient-derived tumor models. Our results suggest that HER2 exon 20 insertions can be effectively treated by a potent and highly selective HER2 inhibitor that spares EGFR wild type. These findings warrant the upcoming clinical testing in HER2 mutant NSCLC patients in order to effectively treat this aggressive type of cancer. Citation Format: Ralph A. Neumüller, Birgit Wilding, Dirk Scharn, Dietrich Böse, Valeria Santoro, Daniel Gerlach, Peter Ettmayer, Thomas Gerstberger, Julian Fuchs, Matthias Treu, Stephan Zahn, Anke Baum, Paolo Chetta, Mark Pearson, Darryl B. McConnell, Norbert Kraut, Flavio Solca. Novel EGFR WT sparing, HER2 selective inhibitors for the treatment of HER2 exon 20 insertion driven tumors address a clear unmet medical need [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 1472.