Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, are often hyperactivated in cancer, promoting tumor progression and therapy resistance. Their oncogenic role depends on interaction with TEA domain (TEAD) transcription factors, making the TEAD-YAP/TAZ complex a promising therapeutic target. Using translational mouse models, we show here that sustained systemic depletion of YAP and TAZ (YAP/TAZ) caused severe side effects. These could be avoided through pulsed inhibition, which effectively suppressed tumor growth, even at advanced stages. We identified Tgfb2 as a critical YAP/TAZ target gene for tumor formation and demonstrated that YAP/TAZ drove T cell exclusion via activation of tissue-remodeling genes. Consequently, YAP/TAZ inhibition enhanced immune cell infiltration. However, infiltrating T cells rapidly underwent exhaustion. Combining YAP/TAZ inhibition with immune checkpoint blockade reversed this exhaustion and sensitized resistant tumors to immunotherapy. This combination reshaped the tumor microenvironment to support immune cell infiltration and activation, representing a therapeutic strategy that maximizes antitumor immunity while minimizing toxicity.

YAP and TAZ, key effectors of the Hippo pathway, are frequently hyperactivated in cancer, where they drive tumor progression and resistance to therapy. Their oncogenic activity relies on interaction with TEAD transcription factors, making the TEAD-YAP/TAZ complex an attractive therapeutic target. Using translational mouse models, we demonstrate that sustained systemic YAP/TAZ depletion leads to severe side effects. However, even transient YAP/TAZ inhibition alone is sufficient to suppress tumor growth in advanced stages. Mechanistically, YAP/TAZ activity promotes T cell exclusion from the tumors by inducing target genes involved in tissue remodeling. Consequentially, YAP/TAZ inhibition induces immune infiltration, but the infiltrating T cells rapidly become exhausted. Combining YAP/TAZ inhibition with immune checkpoint blockade (ICB) overcomes this exhaustion and sensitizes previously resistant tumors to immunotherapy. ### Competing Interest Statement The authors declare that they have no competing interests with the exception that T.T. Tang reports employment with Vivace Therapeutics and has equity interest in Vivace Therapeutics. Deutsche Forschungsgemeinschaft, EY 120/4-1, EY 120/9-1, EY 120/10-1, SFB1292 subproject TP21N Wilhelm Sander Stiftung, https://ror.org/02q83sc19, 2022.084.1 Bundesministerium für Bildung und Forschung, 16GW0271K Leibniz Collaborative Excellence, K398/2021 German Cancer Aid, https://ror.org/01wxdd722, 70116078

Constitutive YAP activation resulting from dysregulated Hippo signaling drives tumor progression in mesothelioma and other cancers. VT3989, a first-in-class potent oral TEAD palmitoylation inhibitor, disrupts YAP transcriptional activity. Here we report the first-in-human phase 1/2 trial findings evaluating VT3989 in refractory solid tumors with a focus on mesothelioma. This study is ongoing, and we report results from the dose escalation and non-prespecified interim efficacy results of the expansion cohorts for which recruitment is ongoing. Dose escalation (n = 85) and expansion (n = 87) cohorts included 172 patients (135 mesothelioma). VT3989 exhibited a favorable safety profile with mostly grade 1-2 toxicities, including increased urine albumin:creatinine ratio (UACR), proteinuria, peripheral edema and fatigue. Proteinuria was reversible with dose adjustment and did not result in renal impairment. The overall response rate (ORR) was 26% in 47 patients with mesothelioma treated at clinically optimized doses, whereas the ORR was 32% (disease control rate 86%; median progression-free survival 10 months) in 22 patients with mesothelioma when clinically optimized doses and UACR thresholds were incorporated. These data provide the first early clinical proof of concept for effectively drugging the Hippo-YAP-TEAD pathway. VT3989 was recently awarded orphan drug designation and fast-track designation for the treatment of mesothelioma by the US Food and Drug Administration (FDA). ClinicalTrials.gov Identifier: NCT04665206 .

The Hippo pathway is a highly conserved signalling network that controls tissue growth and cell fate, responding to physical properties of the tissue microenvironment and cell biological features such as adhesion and polarity. Hippo signalling perturbation is associated with several human diseases, particularly various solid cancers. Hippo pathway-targeted therapies are beginning to emerge for the treatment of cancer, most of which are focused on disrupting the ability of the YAP and TAZ transcription co-activator proteins to promote transcription of genes with their cognate TEAD1-4 DNA binding proteins. Recently, TEAD inhibitors have shown promise in a phase I clinical trial in cancers that are enriched for Hippo pathway mutations, such as mesothelioma. Moreover, Hippo pathway-targeted therapies have great potential to be combined with RAS-MAPK pathway inhibitors, given the close functional relationship that these signalling pathways share in development and disease.

The Hippo tumor suppressor pathway controls transcription by regulating nuclear abundance of YAP and TAZ, which activate transcription with the TEAD1-TEAD4 DNA-binding proteins. Recently, several small-molecule inhibitors of YAP and TEADs have been reported, with some entering clinical trials for different cancers with Hippo pathway deregulation, most notably, mesothelioma. Using genome-wide CRISPR/Cas9 screens we reveal that mutations in genes from the Hippo, MAPK, and JAK-STAT signaling pathways all modulate the response of mesothelioma cell lines to TEAD palmitoylation inhibitors. By exploring gene expression programs of mutant cells, we find that MAPK pathway hyperactivation confers resistance to TEAD inhibition by reinstating expression of a subset of YAP/TAZ target genes. Consistent with this, combined inhibition of TEAD and the MAPK kinase MEK, synergistically blocks proliferation of multiple mesothelioma and lung cancer cell lines and more potently reduces the growth of patient-derived lung cancer xenografts in vivo. Collectively, we reveal mechanisms by which cells can overcome small-molecule inhibition of TEAD palmitoylation and potential strategies to enhance the anti-tumor activity of emerging Hippo pathway targeted therapies.

Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant tumor cells which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

Abstract Introduction: Diffuse gastric cancer (DGC) accounts for one third of gastric cancers and is associated with poor differentiation, discohesive growth, chemoresistance, and worse survival compared to intestinal-type tumors. DGC is typically genomically stable, and there are no approved DGC-targeted therapies. Recent studies have identified a potential tumor-promoting role for Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif (YAP/TAZ), which interact with TEAD transcription factors to regulate gene expression. Therefore, we evaluated the activity and mechanism of TEAD inhibitors in DGC preclinical models. Methods: Gastric cancer patient-derived organoids (PDOs) and cell lines were treated with TEAD inhibitors (K-975, VT103, VT104, VT107) and/or 5-fluorouracil (5-FU) for in vitro assays evaluating cell proliferation or invasion. Additional experiments were conducted to test the in vivo activity of TEAD inhibitors or shRNA-mediated knockdown of YAP in subcutaneous xenograft, orthotopic xenograft, and lung metastasis models using a genetically engineered mouse model of gastric cancer driven by oncogenic KrasG12D and loss of Trp53/Cdh1. Results: TEAD inhibitors K-975, VT103, VT104, and VT107 reduced proliferation, colony formation, and invasion in DGC cell lines and PDOs. We observed no effect of TEAD inhibitors on the viability of normal gastric organoids. Combining the TEAD1-selective inhibitor VT103 with 5-FU chemotherapy resulted in further reduction of DGC PDO viability relative to monotherapy, including in a PDO derived from a patient with locally advanced DGC that demonstrated intrinsic resistance to neoadjuvant chemotherapy. In flank tumors and orthotopic models, inhibition of the YAP/TAZ-TEAD pathway using YAP shRNA and VT103, either alone or in combination with 5-FU, abrogated primary tumor formation, with the strongest inhibition observed with combination treatment. Loss of YAP or VT103 treatment also significantly reduced lung metastasis after tail vein injection of mouse gastric cancer cells. We hypothesized that TEAD inhibition blocks an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Consistent with this hypothesis, TEAD1 inhibition with VT103 or YAP knockdown reduced expression of the CSC marker CD44 in vitro and decreased expression of CD44 and the EMT marker Slug in tumor xenografts. Conclusions: The YAP/TAZ-TEAD pathway is an important driver of gastric cancer growth and metastasis, particularly in DGC where it may promote EMT and cancer stemness. TEAD inhibition reduces gastric cancer growth and invasion in vitro and in vivo in preclinical models and demonstrates synergistic activity with 5-FU chemotherapy. These results suggest a potential role for evaluating the efficacy of TEAD inhibitors in patients with DGC. Citation Format: Anastasiia Bulakhova, Jin Sun Cho, Jack T. Rifkin, Jaewon Kim, Thomas J. Ryan, Tracy T. Tang, Ryan H. Moy. Targeting the YAP/TAZ-TEAD pathway with TEAD inhibitors synergizes with chemotherapy and blocks diffuse gastric cancer progression [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 7281.

Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly understood how phosphatidylinositol, which is synthesized in the ER, reaches those membranes where PPIns are formed. Here, we used VT01454, a recently identified inhibitor of class I PI transfer proteins (PITPs), to unravel their roles in lipid metabolism, and solved the structure of inhibitor-bound PITPNA to gain insight into the mode of inhibition. We found that class I PITPs not only distribute PI for PPIns production in various organelles such as the plasma membrane (PM) and late endosomes/lysosomes, but that their inhibition also significantly reduced the levels of phosphatidylserine, di- and triacylglycerols, and other lipids, and caused prominent increases in phosphatidic acid. While VT01454 did not inhibit Golgi PI4P formation nor reduce resting PM PI(4,5)P2 levels, the recovery of the PM pool of PI(4,5)P2 after receptor-mediated hydrolysis required both class I and class II PITPs. Overall, these studies show that class I PITPs differentially regulate phosphoinositide pools and affect the overall cellular lipid landscape.

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Abstract Genetic alterations of the Hippo signaling pathway components resulting in YAP/TAZ activation have been reported in a variety of human malignancies. YAP/TAZ activation and functional requirement have also been linked to resistance to targeted therapies by providing the essential survival signal in drug-tolerant persister/dormant cells. As the main drivers for YAP/TAZ recruitment to chromatin, TEAD transcription factors are the major effectors of the Hippo-YAP/TAZ pathway involved in the regulation of cell proliferation, survival, and cell migration. There are four members in the TEAD family: TEAD1, TEAD2, TEAD3, and TEAD4. All four members have a conserved cysteine residue that gets auto-palmitoylated and a highly conserved central pocket in which the palmitate is buried. TEAD auto-palmitoylation is required for TEAD interaction with coactivator YAP/TAZ and transcriptional activity. We have discovered and developed highly potent and selective TEAD auto-palmitoylation inhibitors that interact directly with TEAD by occupying the central palmitate pocket, disrupt YAP/TAZ-TEAD protein interaction, suppress TEAD transcriptional activity, and selectively block NF2-deficient mesothelioma proliferation in vitro and inhibit NF2 mutant tumor growth in vivo. One of these compounds, VT3989, is being evaluated in an ongoing phase 1 clinical trial, where partial responses in mesothelioma patients have been demonstrated, showing for the first time that the Hippo pathway is druggable and that the Hippo pathway is now a validated target for cancer therapy. It remained a question, however, which TEAD members are more important and whether it would be safer and as efficacious to inhibit one TEAD member than multiple TEAD members. Thus, we utilized our TEAD inhibitors with differential TEAD selectivity to determine the importance of TEAD1 selectivity in anti-tumor efficacy and in renal safety. We evaluated the anti-tumor efficacy of several potent TEAD1-selective and pan-TEAD inhibitors in in vivo combination studies using EGFR mutant/KRAS mutant xenograft models. We found that TEAD1-selective TEAD palmitoylation inhibitors are less efficacious than pan-TEAD/multiple-TEAD inhibitors in combination studies with targeted therapies (as such EGFRi and KRASi). In 14-day/28-day rat studies, TEAD1-selective TEAD inhibitors also exhibited proteinuric nephropathy similar to that observed with pan-/multiple-TEAD inhibitors. Therefore, based on our findings, we can conclude that TEAD1-selective TEAD palmitoylation inhibitors can have similar on-target effect on kidneys as TEAD inhibitors with broader TEAD selectivity while having reduced anti-tumor efficacy and durability of response in combination with targeted therapies. Citation Format: Tracy T. Tang, Leonard Post. Comparing TEAD palmitoylation inhibitors with differential TEAD selectivity in combination efficacy with targeted therapies and in renal safety [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 7282.

Abstract Schwannoma tumours typically arise on the eighth cranial nerve and are mostly caused by loss of the tumour suppressor Merlin (NF2). There are no approved chemotherapies for these tumours and the surgical removal of the tumour carries a high risk of damage to the eighth or other close cranial nerve tissue. New treatments for schwannoma and other NF2-null tumours such as meningioma are urgently required. Using a combination of human primary tumour cells and mouse models of schwannoma, we have examined the role of the Hippo signalling pathway in driving tumour cell growth. Using both genetic ablation of the Hippo effectors YAP and TAZ as well as novel TEAD palmitoylation inhibitors, we show that Hippo signalling may be successfully targeted in vitro and in vivo to both block and, remarkably, regress schwannoma tumour growth. In particular, successful use of TEAD palmitoylation inhibitors in a preclinical mouse model of schwannoma points to their potential future clinical use. We also identify the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) as a Hippo signalling target, driven by the TAZ protein in human and mouse NF2-null schwannoma cells, as well as in NF2-null meningioma cells, and examine the potential future role of this new target in halting schwannoma and meningioma tumour growth.

Supplementary Table S10 Differential gene expression analysis of scRNA-seq data from tumor cluster in patient #1778 (AMG-510-resistant tumor) vs patient #1566 (control, KRASG12V tumor)

Supplementary Table S11 Differential gene expression analysis of scRNA-seq data from tumor cluster in patient #2349 (MRTX-849/TNO155-resistant) vs patient #1566 (control, KRASG12V tumor)

Abstract Primary/intrinsic and treatment-induced acquired resistance limit the initial response rate to and long-term efficacy of direct inhibitors of the KRASG12C mutant in cancer. To identify potential mechanisms of resistance, we applied a CRISPR/Cas9 loss-of-function screen and observed loss of multiple components of the Hippo tumor suppressor pathway, which acts to suppress YAP1/TAZ-regulated gene transcription. YAP1/TAZ activation impaired the antiproliferative and proapoptotic effects of KRASG12C inhibitor (G12Ci) treatment in KRASG12C-mutant cancer cell lines. Conversely, genetic suppression of YAP1/WWTR1 (TAZ) enhanced G12Ci sensitivity. YAP1/TAZ activity overcame KRAS dependency through two distinct TEAD transcription factor–dependent mechanisms, which phenocopy KRAS effector signaling. First, TEAD stimulated ERK-independent transcription of genes normally regulated by ERK (BIRC5, CDC20, ECT2, FOSL1, and MYC) to promote progression through the cell cycle. Second, TEAD caused activation of PI3K–AKT–mTOR signaling to overcome apoptosis. G12Ci treatment-induced acquired resistance was also caused by YAP1/TAZ-TEAD activation. Accordingly, concurrent treatment with pharmacologic inhibitors of TEAD synergistically enhanced KRASG12C inhibitor antitumor activity in vitro and prolonged tumor suppression in vivo. In summary, these observations reveal YAP1/TAZ-TEAD signaling as a crucial driver of primary and acquired resistance to KRAS inhibition and support the use of TEAD inhibitors to enhance the antitumor efficacy of KRAS-targeted therapies. Significance: YAP1/TAZ-TEAD activation compensates for loss of KRAS effector signaling, establishing a mechanistic basis for concurrent inhibition of TEAD to enhance the efficacy of KRASG12C-selective inhibitor treatment of KRASG12C-mutant cancers. See related commentary by Johnson and Haigis, p. 4005

Supplementary Table S8 RNA-seq analysis of MTRX-849/SHP099-resistant tumor nodules from KCL mice