Combined inhibition of Bcl-2 family members and YAP induces synthetic lethality in metastatic gastric cancer with RASA1 and NF2 deficiency

(2023) 22:156 Kwon et al. Molecular Cancer https://doi.org/10.1186/s12943-023-01857-0 Molecular Cancer Open Access RESEARCH Combined inhibition of Bcl‑2 family members and YAP induces synthetic lethality in metastatic gastric cancer with RASA1 and NF2 deficiency Jong‑Wan Kwon1†, Jeong‑Seop Oh2,3†, Sang Hyeok Seok1, Hyeok‑Won An1, Yu Jin Lee1, Na Yun Lee1, Taehun Ha1, Hyeon Ah Kim1, Gyeong Min Yoon1, Sung Eun Kim1, Pu‑Reum Oh1, Su‑Hyung Lee4, Dominic C. Voon5,6, Dae‑Yong Kim2,3* and Jun Won Park1* Abstract Background Targetable molecular drivers of gastric cancer (GC) metastasis remain largely unidentified, leading to limited targeted therapy options for advanced GC. We aimed to identify molecular drivers for metastasis and devise corresponding therapeutic strategies. Methods We performed an unbiased in vivo genome-wide CRISPR/Cas9 knockout (KO) screening in peritoneal dissemination using genetically engineered GC mouse models. Candidate genes were validated through in vivo transplantation assays using KO cells. We analyzed target expression patterns in GC clinical samples using immuno‑ histochemistry. The functional contributions of target genes were studied through knockdown, KO, and overexpres‑ sion approaches in tumorsphere and organoid assays. Small chemical inhibitors against Bcl-2 members and YAP were tested in vitro and in vivo. Results We identified Nf2 and Rasa1 as metastasis-suppressing genes through the screening. Clinically, RASA1 mutations along with low NF2 expression define a distinct molecular subtype of metastatic GC exhibiting aggressive traits. NF2 and RASA1 deficiency increased in vivo metastasis and in vitro tumorsphere formation by synergistically amplifying Wnt and YAP signaling in cancer stem cells (CSCs). NF2 deficiency enhanced Bcl-2-mediated Wnt signal‑ ing, conferring resistance to YAP inhibition in CSCs. This resistance was counteracted via synthetic lethality achieved by simultaneous inhibition of YAP and Bcl-2. RASA1 deficiency amplified the Wnt pathway via Bcl-xL, contributing to cancer stemness. RASA1 mutation created vulnerability to Bcl-xL inhibition, but the additional NF2 deletion con‑ ferred resistance to Bcl-xL inhibition due to YAP activation. The combined inhibition of Bcl-xL and YAP synergistically suppressed cancer stemness and in vivo metastasis in RASA1 and NF2 co-deficiency. Conclusion Our research unveils the intricate interplay between YAP and Bcl-2 family members, which can lead to synthetic lethality, offering a potential strategy to overcome drug resistance. Importantly, our findings support † Jong-Wan Kwon and Jeong-Seop Oh equally contributed to this work. *Correspondence: Dae‑Yong Kim Jun Won Park Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑ mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kwon et al. Molecular Cancer (2023) 22:156 Page 2 of 24 a personalized medicine approach where combined therapy targeting YAP and Bcl-2, tailored to NF2 and RASA1 status, could effectively manage metastatic GC. Keywords Cancer stem cells, CRISPR/Cas9, Wnt pathway, YAP signaling Background Gastric cancer (GC) is a highly aggressive malignancy and it is the fourth leading cause of cancer-related death worldwide [1, 2]. Despite advances in chemotherapy, patients with metastatic GC still have poor outcomes, with a 3-year overall survival of 5% [3, 4]. Targeted approaches based on genetic and molecular features of metastatic GC are urgently required to pave the way for effective therapy. However, options for targeted therapy in metastatic GC are limited. Identification of targetable molecular drivers of metastasis and the development of targeted therapies for metastatic GC are challenging on multiple fronts. Foremost, the molecular and genetic profiles of GC metastasis are not well characterized owing to mutational heterogeneity [5, 6]. Additionally, most somatic mutations in advanced GC arise in tumor suppressor genes [5], complicating efficient targeting and treatment. Furthermore, preclinical models that are genetically well defined and closely mimic human GC malignancy are scarce. Only few genetically well-defined immunocompetent mouse models demonstrating metastasis, which are essential for validating candidate genes associated with GC metastasis, have been reported [7–10]. Notably, advanced, metastatic mouse GC models have been developed through conditional mutations targeting the Wnt, Ras, and p53 pathways in gastric epithelia, expanding our understanding of GC metastasis [7]. Additionally, the Cdh1F/+;Trp53F/F;Smad4F/F (ChetPS) GC mouse model represents a unique preclinical model that recapitulates the malignant progression of human GC [8, 11, 12]. The ChetPS GC mouse model has provided insights into GC metastasis and therapeutic vulnerability based on genetic features [9, 13]. Further comprehensive gene screening approaches to identify functional drivers of metastasis using such preclinical models may offer valuable clues for rationalized and personalized treatment options for metastatic GC. Previous studies have identified the B-cell lymphoma 2 (Bcl-2) family of proteins, particularly Bcl-2 and Bcl-xL, as promising targets for suppressing gastrointestinal cancer stemness and metastasis [9, 14]. These are anti-apoptotic proteins that are frequently overexpressed in cancer cells and contribute to tumorigenesis, drug resistance, and metastasis [15]. Beyond a classical role in inhibiting apoptosis, these proteins also play non-canonical roles in promoting carcinogenesis by regulating other cellular processes, including proliferation and differentiation [15, 16]. Although inhibitors of Bcl-2 and Bcl-xL are effective in treating hematopoietic malignancies [17], efficacy is limited in primary solid cancers including GC partly because of a complex microenvironment that promotes cancer cell survival and resistance to treatment. Clinical trials of inhibitors of the Bcl-2 famil (...truncated)