Anti-NSCLC efficacy of a novel ROR1/PI3Kα/BRD4 multi-target inhibitor

Receptor tyrosine kinase-like orphan receptor 1 (ROR1), phosphatidylinositol 3-kinase alpha (PI3Kα) and bromodomain-containing protein 4 (BRD4) are key therapeutic targets in non-small cell lung cancer (NSCLC), playing critical roles in tumourigenesis and disease progression. Despite this, no multi-targeted inhibitors that simultaneously target these three targets have been reported to date. Gefitinib improves survival in many NSCLC patients. However, its clinical utility is limited by drug resistance and adverse effects. We further discovered compound BIQO-9, which exhibits superior antitumor activity, based on a structure-activity relationship study of compound 9a. MTT assays demonstrated that BIQO-9 exhibited potent activity against A549 and H1975 cells, with IC₅₀ values of 0.49 μM and 0.22 μM, respectively-significantly lower than those of compound 9a (0.83 μM and 1.02 μM). Kinase activity assays further revealed enhanced inhibition of PI3Kα by BIQO-9 (IC₅₀ = 4.59 nM) compared to compound 9a (IC₅₀ = 13.12 nM). Additionally, Kinase profiling, biotin pull-down and cellular thermal shift assay (CETSA) identified BRD4 and ROR1 as additional direct targets of BIQO-9. Flow Cytometry analysis showed that BIQO-9 induces G₂ phase cell cycle arrest and promotes apoptosis in A549 cells. Western Blot analysis confirmed that its anti-NSCLC effect was achieved by simultaneously inhibiting the downstream signaling pathways of ROR1, PI3K and BRD4. Notably, BIQO-9 could significantly enhance the efficacy of Gefitinib in inducing cell apoptosis and G₂ phase arrest. This synergistic effect has also been verified in mouse xenograft model. To our knowledge, BIQO-9 represents the first ROR1/PI3Kα/BRD4 multi-target inhibitor with therapeutic potential in NSCLC. Moreover, the combination of BIQO-9 and Gefitinib offers a promising novel strategy for NSCLC treatment.