Abstract 962: Activation of natural killer cells by small molecule inhibits growth of BCG-resistant bladder cancer cells

Abstract Bacillus Calmette-Guérin (BCG) is the standard treatment for non-muscle invasive bladder cancer (NMIBC) patients, but resistance declines its efficacy, emphasizing the need for alternative therapies. BCG therapy triggers a specific cytotoxic immune response by activating and expanding CD56/natural killer (NK) cells and T cells, which effectively target and eliminate bladder cancer cells. This priming of NK and T cells by BCG leads to the release of cytokines such as interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα), which stimulate adaptive immunity and enhance tumor cell destruction. Evidence suggests that impaired T-cell and cytotoxic responses are key factors in immunotherapy failure. We designed the study to identify compounds which have the ability to activate the NK cells and generate a robust immune response within the bladder microenvironment. Our preliminary studies exhibited a notable elevation in HLA-B expression and an increased NK cell presence in recurrent NMIBC patients. Yet, despite the increased NK cell population, our findings suggest that NK cell activation may be insufficient, possibly due to inhibitory signals that suppress NK cell cytotoxicity. Bioinformatics analyses revealed a strong association between HLA-B and the NK cell inhibitory receptor KIR3DL3, prompting us to explore potential inhibitors of this interaction. In-silico screening identified 4′, 5, 7-trihydroxyflavone, a natural flavone as a small-molecule blocker of the KIR3DL3-HLA-B interaction. We hypothesized that inhibiting this pathway, 4′, 5, 7-trihydroxyflavone could potentially reduce inhibitory signaling, thus enhancing NK cell-mediated cytotoxic activity against bladder cancer cells. Treatment of BCG-resistant human bladder cancer T24 cells with BCG or 4′, 5, 7-trihydroxyflavone alone for 8 hours no notable effect was noted on cell viability. Incubation of NK cells with 4′, 5, 7-trihydroxyflavone, without BCG, resulted in a significant increase in cancer cell death, indicating that the compound alone could induce a potent cytotoxic response. Furthermore, qRT-PCR analysis demonstrated elevated expression of CD244, a type-I transmembrane protein involved in immune system regulation, and CD160, a glycoprotein closely linked to NK cell activity, specifically following treatment with 4′, 5, 7-trihydroxyflavone but not with BCG. Taken together, our data provide evidence that 4′, 5, 7-trihydroxyflavone may act as a reactivator of NK and T cell-mediated responses in BCG-resistant bladder cancer cells. By effectively blocking the inhibitory KIR3DL3-HLA-B interaction, this compound enhances NK cell cytotoxicity, resulting in a higher percentage and more sustained immune-mediated cancer cell death. This suggests a promising therapeutic approach for patients who are unresponsive to BCG, potentially leading to improved outcomes in resistant NMIBC. Citation Format: Anusha Gupta, Shiv Verma, Sanjay Gupta. Activation of natural killer cells by small molecule inhibits growth of BCG-resistant bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 962.