In vivo tumor responses for zongertinib monotherapy or combinations.

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.

Transcription factors (TFs) are important mediators of aberrant transcriptional programs in cancer cells. In this study, we focus on TF activity (TFa) as a biomarker for cell-line-selective anti-proliferative effects, in that high TFa predicts sensitivity to loss of function of a given gene (i.e., genetic dependencies [GDs]). Our linear-regression-based framework identifies 3,047 pan-cancer and 3,952 cancer-type-specific candidate TFa-GD associations from cell line data, which are then cross-examined for impact on survival in patient cohorts. One of the most prominent biomarkers is TEAD1 activity, whose associations with its predicted GDs are validated through experimental evidence as proof of concept. Overall, these TFa-GD associations represent an attractive resource for identifying innovative, biomarker-driven hypotheses for drug discovery programs in oncology.

During neural tube (NT) development, the notochord induces an organizer, the floorplate, which secretes Sonic Hedgehog (SHH) to pattern neural progenitors. Conversely, NT organoids (NTOs) from embryonic stem cells (ESCs) spontaneously form floorplates without the notochord, demonstrating that stem cells can self-organize without embryonic inducers. Here, we investigated floorplate self-organization in clonal mouse NTOs. Expression of the floorplate marker FOXA2 was initially spatially scattered before resolving into multiple clusters, which underwent competition and sorting, resulting in a stable "winning"floorplate. We identified that BMP signaling governed long-range cluster competition. FOXA2+ + clusters expressed BMP4, suppressing FOXA2 in receiving cells while simultaneously expressing the BMP-inhibitor NOGGIN, promoting cluster persistence. Noggin mutation perturbed floorplate formation in NTOs and in the NT in vivo at mid/hindbrain regions, demonstrating how the floorplate can form autonomously without the notochord. Identifying the pathways governing organizer self-organization is critical for harnessing the developmental plasticity of stem cells in tissue engineering.

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.

Loss-of-function genetic tools are widely applied for validating therapeutic targets, but their utility remains limited by incomplete on- and uncontrolled off-target effects. We describe artificial RNA interference (ARTi) based on synthetic, ultra-potent, off-target-free shRNAs that enable efficient and inducible suppression of any gene upon introduction of a synthetic target sequence into non-coding transcript regions. ARTi establishes a scalable loss-of-function tool with full control over on- and off-target effects.

Genetic loss-of-function methods including short-hairpin RNAs (shRNAs) and CRISPR are key methods for target gene validation across a variety of different disease areas. While these methods have revolutionized target gene discovery and characterization, both methods suffer from limitations in the context of in vivo target validation due to off-target effects and insufficient knock-down for shRNAs and resistant clones for CRISPR. In this study, we describe a method, artificial RNA interference (ARTi) that overcomes these limitations by fundamentally changing the basic experimental strategy of RNAi-based loss-of-function studies. Instead of newly designing gene-specific shRNA for individual target genes, ARTi utilizes ultra-effective and selective artificial miRNA-based shRNAs that do not match any transcribed gene in the target genome. In addition, these sequences are optimized for ultra-efficient miRNA processing and knockdown of synthetic target genes, and are deeply characterized to not trigger major off-target effects, enabling highly stringent target validation in vivo with unprecedented temporal control, selectivity and potency. We validate the approach by studying the in vitro and in vivo phenotypes of EGFR, KRAS and STAG1, genes relevant to cancer biology. This loss-of-function strategy will enable novel experimental strategies in therapeutic target validation and will be instrumental in guiding the lead optimization process by establishing genetic benchmark phenotypes. Citation Format: Ralph Neumüller, Thomas Hoffmann, Alexandra Hörmann, Maja Corcokovic, Johannes Zuber. Precision RNAi using synthetic shRNAmir target sites. [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 3973.

Genetic networks are characterized by extensive buffering. During tumor evolution, disruption of functional redundancies can create de novo vulnerabilities that are specific to cancer cells. Here, we systematically search for cancer-relevant paralog interactions using CRISPR screens and publicly available loss-of-function datasets. Our analysis reveals >2,000 candidate dependencies, several of which we validate experimentally, including CSTF2-CSTF2T, DNAJC15-DNAJC19, FAM50A-FAM50B, and RPP25-RPP25L. We provide evidence that RPP25L can physically and functionally compensate for the absence of RPP25 as a member of the RNase P/MRP complexes in tRNA processing. Our analysis also reveals unexpected redundancies between sex chromosome genes. We show that chrX- and chrY-encoded paralogs, such as ZFX-ZFY, DDX3X-DDX3Y, and EIF1AX-EIF1AY, are functionally linked. Tumor cell lines from male patients with loss of chromosome Y become dependent on the chrX-encoded gene. We propose targeting of chrX-encoded paralogs as a general therapeutic strategy for human tumors that have lost the Y chromosome.

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.

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.

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains. A chemical probe BI-9321 for the PWWP1 domain of NSD3 and its inactive analog were identified. BI-9321 binds to the methyl-lysine binding site, reduces the association of NSD3 with chromatin and inhibits proliferation of acute myeloid leukemia cells.

Targeted cancer therapy is based on exploiting selective dependencies of tumor cells. By leveraging recent functional screening data of cancer cell lines we identify Werner syndrome helicase (WRN) as a novel specific vulnerability of microsatellite instability-high (MSI-H) cancer cells. MSI, caused by defective mismatch repair (MMR), occurs frequently in colorectal, endometrial and gastric cancers. We demonstrate that WRN inactivation selectively impairs the viability of MSI-H but not microsatellite stable (MSS) colorectal and endometrial cancer cell lines. In MSI-H cells, WRN loss results in severe genome integrity defects. ATP-binding deficient variants of WRN fail to rescue the viability phenotype of WRN-depleted MSI-H cancer cells. Reconstitution and depletion studies indicate that WRN dependence is not attributable to acute loss of MMR gene function but might arise during sustained MMR-deficiency. Our study suggests that pharmacological inhibition of WRN helicase function represents an opportunity to develop a novel targeted therapy for MSI-H cancers.

Esophageal squamous cell carcinoma (ESCC) is a highly prevalent tumor type with poor prognosis and limited treatment options. In this study, we aimed to discover potential novel drug targets using a pooled epigenome sgRNA viability screen in a panel of ESCC cell models. We identify the BAF chromatin remodeling helicase subunit SMARCA4/BRG1 as a novel dependency in a subset of ESCC cell lines. In line with the established synthetic lethal interaction of SMARCA4 and SMARCA2/BRM, the two mutual exclusive catalytic subunits of the BAF complex, SMARCA4-dependent cell lines display low or absent expression of SMARCA2. In rescue studies using SMARCA4 variants, we demonstrate that SMARCA4-dependency is linked to its ATPase/helicase activity, but not to bromodomain function. Similarly, ectopic expression of wild-type and bromodomain-mutant SMARCA2, but not a helicase-dead variant, rescues from loss of SMARCA4 in SMARCA2-low ESCC cells. SMARCA2-proficient ESCC cell models are rendered SMARCA4-dependent upon CRISPR/Cas9-mediated knock-out of SMARCA2. We further identify SMARCA4-dependent cell models from additional tumor types (colon, ovarian and pancreas carcinoma) with low or absent SMARCA2 expression. These findings expand the concept of SMARCA2/SMARCA4 paralog dependency and indicate pharmacological inhibition of SMARCA4 as a novel opportunity for targeted therapy of SMARCA2-deficient cancers. Citation Format: Katharina Ehrenhöfer-Wölfer, Teresa Puchner, Silvia Blaha-Ostermann, Alexandra Hörmann, Wolfgang Sommergruber, Norbert Schweifer, Thomas Zichner, Andreas Schlattl, Ralph A. Neumüller, Manfred Koegl, Mark P. Petronczki, Mark Pearson, Simon Wöhrle. SMARCA4 is a selective vulnerability in SMARCA2-deficient esophageal squamous cell carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 397.

SMARCA4/BRG1 and SMARCA2/BRM, the two mutually exclusive catalytic subunits of the BAF complex, display a well-established synthetic lethal relationship in SMARCA4-deficient cancers. Using CRISPR-Cas9 screening, we identify SMARCA4 as a novel dependency in SMARCA2-deficient esophageal squamous cell carcinoma (ESCC) models, reciprocal to the known synthetic lethal interaction. Restoration of SMARCA2 expression alleviates the dependency on SMARCA4, while engineered loss of SMARCA2 renders ESCC models vulnerable to concomitant depletion of SMARCA4. Dependency on SMARCA4 is linked to its ATPase activity, but not to bromodomain function. We highlight the relevance of SMARCA4 as a drug target in esophageal cancer using an engineered ESCC cell model harboring a SMARCA4 allele amenable to targeted proteolysis and identify SMARCA4-dependent cell models with low or absent SMARCA2 expression from additional tumor types. These findings expand the concept of SMARCA2/SMARCA4 paralog dependency and suggest that pharmacological inhibition of SMARCA4 represents a novel therapeutic opportunity for SMARCA2-deficient cancers.

Abstract Genomic instability is a hallmark of cancer and can result in the deletion of genes with no apparent or as yet unknown relevance to tumor survival and proliferation. Although the deletion of such genes may be tolerated due to functional compensation it, however, entails a cancer cell specific vulnerability as the tumor cell relies on the function of compensating redundant genes. Recurrent deletions of cleavage stimulation factor subunit 2 Tau (CSTF2T) have been observed in several tumor samples (TCGA). During meiosis CSTF2, a paralogue of CSTF2T, which resides on the X-chromosome, is transcriptionally silenced and the zygote depends on the activity of CSTF2T. As a result, CSTF2T knockout mice exhibit male sterility which corroborates that CSTF2 and CSTF2T form a functionally redundant pair of genes with an essential function (Dass et al. 2007). With the aim to apply these findings to tumor biology, we therefore tested whether tumor cells with homozygous CSTF2T deletions depend on CSTF2 using CRISPR-based growth competition assays and confirmed this concept in lung adenocarcinoma and melanoma cell line models. Our data establish synthetic lethality between CSTF2 and CSTF2T and suggest inhibition of CSTF2 in CSTF2T deficient tumors as a new therapeutic concept. Citation Format: Johannes Popow, Corinna Wieshofer, Andreas Schlattl, Simon Woehrle, Ralph Neumüller, Mark P. Petronczki, Jark Boettcher, Manfred Koegl, Mark Pearson. Synthetic lethality between CSTF2 and CSTF2T in lung adenocarcinoma and melanoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4449.

Genotype specific vulnerabilities of cancer cells constitute a promising strategy for the development of new therapeutics. Deletions of non-essential genes in tumors can generate unique vulnerabilities which could be exploited therapeutically. The MTAP gene is recurrently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. Recent studies have uncovered an increased dependency of MTAP-deleted cancer cells on the function of a PRMT5 containing complex, including WDR77, PRMT5 and the kinase RIOK1. As RIOK1 kinase activity constitutes a potential therapeutic target, we wanted to test if MTAP deletion confers increased sensitivity to RIOK1 inhibition. Using CRISPR/Cas9-mediated genome engineering we generated analog sensitive alleles of RIOK1 in isogenic cell lines differing only by MTAP status. While we were able to independently confirm an increased dependency of MTAP-deleted cells on PRMT5, we did not detect a differential requirement for RIOK1 kinase activity between MTAP-proficient and deficient cells. Our results reveal that the kinase activity of RIOK1 is required for the survival of cancer cell lines irrespective of their MTAP status and cast doubt on the therapeutic exploitability of RIOK1 in the context of MTAP-deleted cancers.

Drosophila melanogaster neural stem cells (neuroblasts [NBs]) divide asymmetrically by differentially segregating protein determinants into their daughter cells. Although the machinery for asymmetric protein segregation is well understood, the events that reprogram one of the two daughter cells toward terminal differentiation are less clear. In this study, we use time-resolved transcriptional profiling to identify the earliest transcriptional differences between the daughter cells on their way toward distinct fates. By screening for coregulated protein complexes, we identify vacuolar-type H+-ATPase (v-ATPase) among the first and most significantly down-regulated complexes in differentiating daughter cells. We show that v-ATPase is essential for NB growth and persistent activity of the Notch signaling pathway. Our data suggest that v-ATPase and Notch form a regulatory loop that acts in multiple stem cell lineages both during nervous system development and in the adult gut. We provide a unique resource for investigating neural stem cell biology and demonstrate that cell fate changes can be induced by transcriptional regulation of basic, cell-essential pathways.

Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi) or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila. miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a, and the previously uncharacterized gene MESR4, in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola) gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.

The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs) in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ISC self-renewal, ISC proliferation, ISC maintenance of diploid status, ISC survival, ISC-to-enterocyte (EC) lineage differentiation, and ISC-to-enteroendocrine (EE) lineage differentiation. By comparing regulators among ISCs, female germline stem cells, and neural stem cells, we found that factors related to basic stem cell cellular processes are commonly required in all stem cells, and stem-cell-specific, niche-related signals are required only in the unique stem cell type. Our findings provide valuable insights into stem cell maintenance and lineage-specific differentiation.