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

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 immunohistochemistry. The functional contributions of target genes were studied through knockdown, KO, and overexpression 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 signaling, 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 conferred 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 a personalized medicine approach where combined therapy targeting YAP and Bcl-2, tailored to NF2 and RASA1 status, could effectively manage metastatic GC.

A synergistic partnership between IL-33/ST2 and Wnt pathway through Bcl-xL drives gastric cancer stemness and metastasis.

ST2 functions as a receptor for the cytokine IL-33. It has been implicated in carcinogenesis. In this study, we sought to mechanistically determine how ST2 and IL-33 function to support cancer stem cell (CSC) activity and drive gastric cancer (GC) pathogenesis. ST2+ subpopulation spontaneously arose during gastric tumorigenesis. A thorough evaluation of ST2 and IL-33 expression in gastric tumors revealed that they show an overlapping expression pattern, notably in poor differentiated GC and metastasis foci. Moreover, their expression levels are clinically correlated to cancer progression. Using a genetic model of CSC-driven gastric carcinogenesis, ST2+ subpopulation displays increased tumorigenicity, chemoresistance and metastatic potentials through increased survival fitness endowed by an elevated MAPK-regulated Bcl-xL. The IL-33/ST2 axis enhances the self-renewal and survival of GC stem cells and organoids. Importantly, we observed a synergistic cooperation between IL-33/ST2 and the canonical Wnt pathway in transactivating Wnt-dependent transcription and supporting CSC activity, a partnership that was abrogated by inhibiting Bcl-xL. Concordant with this, ST2+ subpopulation was targeted by MEK1/2 and Bcl-xL-specific inhibitors. These findings establish ST2 as a functional CSC marker that fortifies the Wnt signal while availing a novel therapeutic strategy to suppress GC progression by targeting the IL-33/ST2/Bcl-xL signaling axis.

The loss of epithelial Smad4 drives immune evasion via CXCL1 while displaying vulnerability to combinatorial immunotherapy in gastric cancer.

SMAD4 is frequently mutated and inactivated in human gastric cancer (GC). Although the epithelial cell-autonomous functions of Smad4 have been extensively studied, its contribution to tumor immunity is largely undetermined. Here, we report that the loss of Smad4 expression in GC cells endows them with the ability to evade tumor immunity. Unlike their Smad4-proficient counterparts, Smad4-deficient stomach organoids can evade host immunity to form tumors in immunocompetent mice. Smad4-deficient GC cells show expanded CD133+ cancer stem-like cells while suppressing dendritic cell (DC) differentiation and cytotoxic T cells with granulocytic myeloid-derived suppressor cell (G-MDSC) accumulation through a secretome containing CXCL1. Moreover, Smad4 deficiency increases programmed cell death ligand-1 (PD-L1) and decreases 4-1BBL expressions, indicating a change in immunogenicity. Combinatorial immune checkpoint blockade (ICB) of anti-PD-L1 and anti-CTLA-4 or agonistic anti-4-1BB antibodies effectively treats ICB monotherapy-resistant Smad4-deficient allografts, exposing a specific vulnerability. Collectively, these data provide a rational basis for ICB strategies in treating advanced GC with Smad4 deficiency.

In vivo CRISPR-Cas9 knockout screening using quantitative PCR identifies thymosin beta-4 X-linked that promotes diffuse-type gastric cancer metastasis.

Gastric cancer (GC) is histologically classified into intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC), and the latter is poorly differentiated and highly metastatic. In this study, using quantitative real-time polymerase chain reaction, we described a complete protocol for in vivo CRISPR-Cas9-based knockout screening of essential genes for DGC metastasis. We functionally screened 30 candidate genes using our mouse DGC models lacking Smad4, p53, and E-cadherin. Pooled knockout mouse DGC cells were transplanted into a spleen of syngeneic immunocompetent mice to study clonal advantages in context of a complex process of liver metastasis. Tmsb4x (thymosin beta-4 X-linked), Hmox1, Ifitm3, Ldhb, and Itgb7 were identified as strong candidate genes that promote metastasis. In particular, Tmsb4x enhanced DGC metastasis and stomach organoid-generated tumor growth in in vivo transplantation models. Tmsb4x promoted tumor clonogenicity and anoikis resistance. In situ hybridization analysis showed that Tmsb4x is highly expressed in E-cadherin-negative mouse DGC models compared with mouse IGC and intestinal cancer models. E-cadherin deficiency also increased Tmsb4x expression in stomach organoids via Wnt signaling activation. Collectively, these results demonstrate that Tmsb4x promotes DGC metastasis. In addition, this experimental system will aid in the identification of novel target genes responsible for DGC metastasis.