Supplementary figure 1. CDK6 overexpression promotes FAK inhibitor resistance in SNU668. Supplementary figure 2. CDK4/6 inhibitor enhanced the efficacy of FAK inhibitors in Cdh1-/-RHOAY42C/+ organoids. Supplementary figure 3. Combination of defactinib and palbociclib in patient-derived organoids (PDOs). Supplementary figure 4. CDK4/6 inhibitor enhanced the efficacy of FAK inhibitors in DGC cell lines. Supplementary figure 5. In vivo experiment of VS-4718 and Palbociclib combination. Supplementary figure 6. FAK inhibitor VS-4718 induces MAPK activation in DGC organoids and cell lines. Supplementary figure 7. Phospho-RTK and RAS-GTP detection in Cdh1-/-RHOAY42C/+ organoids treated with FAK inhibitors. Supplementary figure 8. MAPK inhibitor and FAK inhibitor have synergistic effect in Cdh1-/-RHOAY42C/+ organoids. Supplementary figure 9. MAPK inhibitor and FAK inhibitor have synergistic effect in PDOs. Supplementary figure 10. MAPK inhibitor and FAK inhibitor have synergistic effect in DGC cell lines. Supplementary figure 11. In vivo experiment of VS-4718 with VS-6766 or palbociclib combination.

Abstract Purpose: Diffuse gastric cancer (DGC) is an aggressive and frequently lethal subtype of gastric cancer. Because DGC often lacks genomic aberrations that indicate clear candidate therapeutic targets, it has been challenging to develop targeted therapies for this gastric cancer subtype. Our previous study highlighted the contribution of focal adhesion kinase (FAK) in the tumorigenesis of DGC and the potential efficacy of small-molecule FAK inhibitors. However, drug resistance to monotherapy often hinders the efficacy of treatment. Experimental Design: We generated a genome-scale library of open reading frames (ORF) in the DGC model of Cdh1−/−RHOAY42C/+ organoids to identify candidate mechanisms of resistance to FAK inhibition. Compensatory activated pathways were also detected following treatment with FAK inhibitors. Candidates were investigated by cotargeting in vitro and in vivo experiments using DGC. Results: We found that cyclin-dependent kinase 6 (CDK6) promoted FAK inhibitor resistance in ORF screen. In addition, FAK inhibitor treatment in DGC models led to compensatory MAPK pathway activation. Small-molecule CDK4/6 inhibitors or MAPK inhibitors effectively enhanced FAK inhibitor efficacy in vitro and in vivo. Conclusions: Our data suggest that FAK inhibitors combined with MAPK inhibitors or CDK4/6 inhibitors warrant further testing in clinical trials for DGC.

WEE1 kinase is renowned as an S-G2 checkpoint inhibitor activated by ATR-CHK1 in response to replication stress. WEE1 inhibition enhances replication stress and effectively circumvents checkpoints into mitosis, which triggers significant genetic impairs and culminates in cell death. This approach has been validated clinically for its promising anti-tumor efficacy across various cancer types, notably in cases of ovarian cancers. Nonetheless, the initial stage of clinical trials has shown that the first-in-human WEE1 inhibitor adavosertib is limited by dose-limiting adverse events. As a result, recent efforts have been made to explore predictive biomarkers and smart combination schedules to alleviate adverse effects. In this review, we focused on the exploration of therapeutic biomarkers, as well as schedules of combination utilizing WEE1 inhibitors and canonical anticancer drugs, according to the latest preclinical and clinical studies, indicating that the optimal application of WEE1 inhibitors will likely be as part of dose-reducing combination and be tailored to specific patient populations.