The interaction of transcriptional enhanced associate domain (TEAD) and transcriptional coactivator yes-associated protein (YAP) mediates oncogenic functions downstream of the Hippo pathway. In this report, we outline our efforts to find a potent inhibitor of this protein-protein interaction with suitable properties for clinical evaluation. We detail the medicinal chemistry program that led to the discovery of IAG933, an inhibitor with a balanced ADME profile, enabling its evaluation as a potential treatment option in clinical settings.

Despite the improvement of therapeutic options, melanoma patients with advanced metastatic disease are still in high need of durable treatments. Analysis of clinical data from patients receiving targeted and/or immunotherapy, along with genetic and functional studies in preclinical melanoma models, demonstrates the key role of the microphthalmia-associated transcription factor (MITF) throughout disease progression, and provides a solid rationale for its therapeutic inhibition. However, direct targeting of MITF or other basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors is unprecedented. Here, we report on the discovery of ligands for the DNA binding domain of MITF, using fragment-based screening (FBS) by nuclear magnetic resonance (NMR). Initial fragments, binding the kink pocket of MITF very weakly, are optimized to sub-micromolar affinities by structure-based design enabled by X-ray crystallography and biophysics. Furthermore, NMR experiments and molecular dynamics simulations reveal a dynamic conformational exchange between helices in the asymmetric homodimer, a phenomenon that is perturbed by ligand binding. This work advances our knowledge on direct targeting of bHLH-LZ DNA binding domains and sets the basis to further explore pharmacological inhibition of MITF.

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients’ tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.

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.

The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens(1-6). Despite advances in treatment with immune checkpoint inhibitors(7-10), there is an unmet need in the treatment of MSI cancers(11-14). Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.

Supplementary Figure 2 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Data from Potent and Selective Inhibition of Polycythemia by the Quinoxaline JAK2 Inhibitor NVP-BSK805

Supplementary Legends for Figures 1-6, Table 1 and Methods and Materials from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary material and methods include the description for the bioanalytical method for HDM201 detection in plasma and tumor, the human and mouse gene expression analysis in vitro and in vivo, the live-cell quantification of cleaved-caspase activation, the western blot analysis, the immunohistochemistry, the splinkerette PCR for the amplification of transposon integration sites and the tumor sequencing, mapping of insertion sequences to the mouse genome and identification of common integration site, additional information on the shRNA screen and the tumor models and supplementary references. Supplementary figures include: • Fig S1: the SJSA-1 inhibition growth curves when treated with HDM201 at different doses and for different times and the data for MOLM-3. • Fig S2: the cumulative percentage of cleaved-caspase-3/7 positive cells over the time, the GI50 of HDM201, CGM097 or nutlin-3a on SJSA-1 cells and the cellular apoptosis, as judged by AUC of cleaved-caspase-3/7 positive cells, induced by these compounds. • Fig S3: the PK profile in plasma and tumor of HDM201 in SJSA-1 tumors-bearing rat after p.o. and i.v. treatment, the Bcl-xl mRNA levels in tumors after HDM201 treatment, representative images of SJSA-1 tumors stained with p53 and cleaved-caspase 3 antibodies after HDM201 treatment and the individual data for the efficacy experiment in SJSA-1 tumor-bearing rats. • Fig S4: the PD of HDM201 in PB tumor bearing nude mice after single dose administration. Supplementary tables include: • Table S1: Biochemical profile of HDM201. • Table S2: List of cell lines tested for their sensitivity to HDM201 (n=291) • Table S3: Contingency table indicating association between sensitivity to HDM201 and TP53 wild-type status. • Table S4: List of cell lines tested for their sensitivity to both MDM2 knock-down by shRNA and HDM201 (n=261) • Table S5: Contingency table indicating association between sensitivity to HDM201 and sensitivity to MDM2 shRNA. • Table S6: List of significant rescuer and sensitizer genes following both HDM201 treatment types • Table S7: Pharmacokinetic parameters for HDM201 after p.o. and i.v. dosing in rat. • Table S8: Summary of primary PK parameters for HDM201 daily regimen after single dose (Day 1) in patients. • Table S9: Summary of primary PK parameters for HDM201 daily regimen on Day 14 in patients. • Table S10: Summary of primary PK parameters for HDM201 q3w regimen after single dose in patients.

Median of the shRNAs and RSA values. File containing median counts and log fold changes of each gene from Supplementary Data File S1. According significance values of each gene as activator or sensitizer compared to DMSO control (see Methods) are included.

Details on materials and methods

Supplementary Figure 6 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

The inhibition of the YAP-TEAD protein-protein interaction constitutes a promising therapeutic approach for the treatment of cancers linked to the dysregulation of the Hippo signaling pathway. The identification of a class of small molecules which potently inhibit the YAP-TEAD interaction by binding tightly to the Ω-loop pocket of TEAD has previously been communicated. This report details the further multi-parameter optimization of this class of compounds resulting in advanced analogs combining nanomolar cellular potency with a balanced ADME and off-target profile, and efficacy of these compounds in tumor bearing mice is demonstrated for the first time.

Supplementary Figure 1 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

List of individual shRNA scores. File containing raw data of individual shRNAs quantified by barcode sequencing of DMSO- and HDM201- treated samples. Log fold changes of individual shRNAs compared to plasmid library and of HDM201-treated samples compared to DMSO-control are also listed.

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 Figure 5 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Insertional results from Arf-/- PB in vivo resistance screens. A and C, Tables listing all sequenced samples with their model number (i.e. X25272), the transplanted fragment number (i.e. P0M1-P1M5-P2M19, P for passage, and M for Mouse), tumor status (DR=drug resistant, or veh=vehicle treated) and treatment schedule (A) QD for daily or (C) 2QW for biweekly. Total number of reads and tumor pathology are labeled for each sample. Insertional landscapes are displayed on the right part of the table where the averaged normalized diversity sequencing counts are indicated for each Common insertion site gene (gCIS) and each tumor. B and D, List of the genes found differentially enriched for PB insertions in HDM201 resistant tumors compared to untreated tumors (B) continuous daily treatment or (D) biweekly intermittent treatment. The data in D were extracted from previous report (28) for intermittent 100 mg/kg biweekly dosing schedule, and reanalyzed with only 6 tumor models used out of 16 in previous report. Fold change, p-value and FDR were calculated for each gene in comparative analyses of untreated tumor samples vs. resistant tumor samples. PercentSample indicated the percentage of resistant tumors with insertion in that gene. The predicted function GOF (gain of function), LOF (loss of function) or uncertain is also indicated.