The YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored. Here, we present IAG933 and its analogs as potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials. Pharmacologic abrogation of the interaction with all four TEAD paralogs resulted in YAP eviction from chromatin and reduced Hippo-mediated transcription and induction of cell death. In vivo, deep tumor regression was observed in Hippo-driven mesothelioma xenografts at tolerated doses in animal models as well as in Hippo-altered cancer models outside mesothelioma. Importantly this also extended to larger tumor indications, such as lung, pancreatic and colorectal cancer, in combination with RTK, KRAS-mutant selective and MAPK inhibitors, leading to more efficacious and durable responses. Clinical evaluation of IAG933 is underway. Chapeau et al. develop a nonallosteric inhibitor of the interaction between YAP and all four TEAD proteins. Treatment with the inhibitor, either as monotherapy or in combination with other treatment modalities, leads to induction of cell death in several in vivo cancer models.

Supplementary Table S2. Summary of variant calls from WES analysis of matched pre-treatment and post-progression biopsies.

PDF file - 104KB, Clinical features of IDH1-mutant intrahepatic cholangiocarcinoma patients in the Screening cohort.

PDF file - 67KB, Characteristics of all intrahepatic cholangiocarcinoma patients evaluated across the two cohorts.

Supplementary Figure S1. KRAS FISH analysis of parental and resistance cell lines.

Supplementary Figure S3. Copy number alterations for Patient #1.

Supplementary Figure 1. Schematic of high-content imaging-based internalization assay. Supplementary Figure 2. OVCAR3 xenografts were grown subcutaneously in NSG mice and treated with a single i.v. dose of 10 mg/kg control IgG1 or CDH6-targeting antibodies conjugated to SMCC-DM1. Supplementary Figure 3. Tumors of the PDX model HOVX2263 were grown subcutaneously in female nude mice randomized into groups of equal mean tumor volume and treated every two weeks with a 5 mg/kg i.v. dose of either IgG1-SPDB-DM4, or CDH6-targeting antibodies conjugated to SPDB-DM4. Supplementary Figure 4. Interaction analysis of anti-CDH6 antibody and CDH6 ECD protein. Supplementary Figure 5. Unenrolled NSG mice bearing OVCAR3 tumors from a separate efficacy study were allowed to grow to ~600 mm3 before being treated with either IgG1-SPDB-DM4 or CDH6-SPDB-DM4 at 8.5 mg/kg i.v. on day 34 post implant and re-dosed as indicated by arrows. Supplementary Figure 6. Representative CDH6 IHC images of the OVCAR3 subcutaneous xenograft grown in NSG mice (A), OVCAR3Luc intraperitoneal xenograft grown in SCID beige mice (B), HOVX2263 ovarian PDX subcutaneous xenograft grown in female nude mice (C), and HOVX4863 ovarian PDX subcutaneous xenograft grown in female nude mice (D). Supplementary Figure 7. (A) Correlation plot of response to HKT288 by best average response vs. CDH6 RNA expression. (B) Waterfall plot of percent best average response to CDH6-sulfoSPDB-DM4 treatment in PCT.

Supplementary Figure S5. Role of ARAF Q489L and MEK F53L in resistance.

Supplementary Figure S4. Wild-type KRAS overexpression drives resistance to RAF inhibitor combinations.

PDF file - 57KB, Levels of serum 2HG relative to tumor burden in IDH1-mutant and IDH2-mutant intrahepatic cholangiocarcinoma patients in the Validation cohort.

This file contains supplementary tables describing parameters for antitumor activity (T/C analyses), PK and protein crystallography.

Table S1. Pharmacokinetic parameters of encorafenib and alpelisib in patients at steady state (cycle 2 day 1). Table S2. Criteria for defining dose-limited toxicities. Supplementary Figure 1. Radiological images of response for a patient treated with the dual-combination therapy of encorafenib and cetuximab. Supplementary Figure 2. Time on study by response for patients treated with the dual-combination therapy of encorafenib and cetuximab and patients treated with the triple-combination therapy of encorafenib, alpelisib, and cetuximab.

The YAP-TEAD protein-protein interaction (PPI) is a critical event known to mediate YAP oncogenic functions downstream of the Hippo pathway. Current advanced pharmacological agents which aim at inhibiting YAP-TEAD oncogenic function do so by engaging into the lipid pocket of TEAD. Thereby the consequences of a direct pharmacological disruption of the interface of YAP and TEADs remain unexplored. Here we report the identification of IAG933, the first molecule able to potently and directly disrupt the YAP/TAZ-TEADs PPI with suitable properties to enter in clinical trial. The path to drug discovery was established by structure-based optimization of a truncated natural YAP peptide allowing the pharmacophore mapping of TEAD coil binding site. Based on in silico screening, validated hit was optimized using structure- and property-based lead optimization yielding IAG933, whose chemical structure will be for the first time disclosed here. Biochemical and cellular assays demonstrate that IAG933 specifically abrogates the interaction between YAP/TAZ coactivators and all four TEAD isoforms, thus selectively inhibiting TEAD-driven transcriptional activity and inducing anti-cancer effects. At the epigenome level, YAP eviction from chromatin was observed upon treatment with IAG933, while leaving TEADs genomic occupancy unaffected. Concomitantly, engagement of co-repressor VGLL4 translated to a decrease in enhancer activity with rapid and progressive changes in transcription of Hippo target genes. In preclinical experiments, IAG933 linear pharmacokinetics was consistent with dose proportional TEAD transcriptional inhibition and anti-tumor efficacy in xenograft and primary-tumor derived malignant pleural mesothelioma models. Daily treatment with IAG933 elicited complete tumor regression in the MSTO-211H xenograft model at well-tolerated doses. In line with the current clinical strategy for IAG933, robust anti-tumor efficacy in cancer models bearing NF2 loss of function or expressing TAZ-fusions was observed. Moreover, we provide evidence for combination benefits of IAG933 with several MAPK/KRAS inhibitors, both in vitro and in vivo, in non-Hippo altered models including lung, pancreatic and colorectal cancer. Overall, our results provide a rationale of progressing IAG933 as a monotherapy in patients with Hippo-mutated cancers, and as a combination partner in MAPK-dependent cancers, with the potential to treat patient populations of high unmet medical need. Citation Format: Tobias Schmelzle, Emilie Chapeau, Daniel Bauer, Patrick Chene, Jason Faris, Cesar Fernandez, Pascal Furet, Giorgio Galli, Jiachang Gong, Stephanie Harlfinger, Francesco Hofmann, Eloisa Jimenez Nunez, Joerg Kallen, Thanos Mourikis, Laurent Sansregret, Paulo Santos, Clemens Scheufler, Holger Sellner, Markus Voegtle, Markus Wartmann, Peter Wessels, Frederic Zecri, Nicolas Soldermann. IAG933, a selective and orally efficacious YAP1/WWTR1(TAZ)-panTEAD protein-protein interaction inhibitor with pre-clinical activity in monotherapy and combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB319.

Supplementary Figures, Tables, Methods

This file describes supplementary material and methods used.

Supplementary Table S1. Patient characteristics and MAPK pathway reactivating resistance mechanisms of BRAF-mutant CRC patients with clinical acquired resistance to RAF/EGFR or RAF/MEK inhibitor combinations.

This file contains the figure legends for supplementary figures S1-S7.

PURPOSE:This first-in-human study (NCT02947152) evaluated the safety, tolerability, pharmacokinetics, and preliminary efficacy of HKT288, a first-in-class CDH6-targeting antibody-drug conjugate (ADC).EXPERIMENTAL DESIGN:HKT288 was administered intravenously (IV) every 3 weeks until patients experienced unacceptable toxicity or progressive disease (PD). The starting dose of 0.3 mg/kg was determined based on the highest nonseverely toxic dose in monkeys, which was 2 mg/kg IV weekly. Based on preclinical toxicology, skin, eyes, bone marrow, and liver were expected targets of toxicity.RESULTS:Nine patients were enrolled: 5 with renal cell carcinoma and 4 with epithelial ovarian cancer. The best overall response on the 0.3 mg/kg cohort in patients with measurable disease was RECIST v1.1 stable disease in 3 patients and PD in 2 patients. The most frequent adverse events (AEs) regardless of causality were pyrexia (44.4%), constipation (44.4%), fatigue (33.3%), and vomiting (33.3%). Three suspected-related neurologic AEs (Grade 2) were reported on the 0.75 mg/kg cohort: seizure in 1 patient and another patient with aphasia and encephalopathy. Further studies were unable to identify the underlying mechanism of the neurologic AEs, and the study was terminated early.CONCLUSIONS:Preclinical toxicology did not predict the neurotoxicity observed with HKT288, and a comprehensive assessment performed post hoc did not identify the mechanism of toxicity. The development of further CDH6-targeting ADCs should be pursued with caution.