Abstract Introduction and Objective: Histone modifications are important for tissue homeostasis, and their mutations are involved in carcinogenesis. Mutations in an X-linked histone demethylase KDM6A are most frequently observed in bladder cancer (BCa) among human malignancies. We previously demonstrated that loss-of-function of KDM6A contributes to BCa development by promoting inflammation through activating the JAK-STAT pathway. Tetra-O-methyl-nordihydroguaiaretic acid (M4N), a methylated derivative of nordihydroguaiaretic acid, inhibits transcription factor Hypoxia-Inducible Factor (HIF) and Specificity protein 1 (Sp1) and exerts anti-inflammatory activities through inhibiting the JAK-STAT pathway. Here, we investigated the therapeutic efficacy of M4N against KDM6A-deficient BCa using a subcutaneous syngeneic model. Methods: To investigate the anti-proliferative effect of M4N on KDM6A-deficient BCa, we used CRISPR-Cas9 genome editing and established Kdm6a-deficient sublines from mice BCa cell lines, MB49 and MBT2. Kdm6a-deficient and control parental cells were subcutaneously implanted into syngeneic mice and tumor growth on the recipients fed either a normal diet and an M4N-supplemented diet was continuously monitored. Resected tumors were subjected to pathological, molecular, and cellular analyses using real-time PCR and immunohistochemical staining. Results: In subcutaneous syngeneic models, Kdm6a-deficient cells formed significantly larger tumors compared to control cells. M4N efficiently suppressed tumor growth of Kdm6a-deficient cells but showed no effect on tumors of control cells. By performing RNA sequencing and pathway analyses using resected tumors, we found that M4N suppressed inflammatory pathways such as chemokine signaling and JAK-STAT pathways. In Kdm6a-deficient tumors, M4N significantly suppressed the expression of pro-inflammatory cytokines and chemokines, such as Il-6 and Ccl2, and also reduced the expression of Vegfa and Nampt, the downstream targets of Sp1 and HIF, involved in inflammatory pathways. Moreover, immunostaining of the tumors with F4/80, a macrophage marker, and with p-STAT, a JAK-STAT pathway marker, revealed significant macrophage accumulation and activation of the JAK-STAT pathway, specifically in tumors lacking Kdm6a, which was efficiently suppressed by M4N. Conclusions: M4N may serve as a novel therapeutic approach for patients with BCa bearing Kdm6a mutations by suppressing inflammation. Citation Format: Nobuhito Muramoto, Masayuki Iwasaki, Yasuyuki Sera, Kohei Kobatake, Kyosuke Iwane, Tsuyoshi Fukushima, Kotohiko Kimura, Ru Chih C. Huang, Hiroaki Honda. M4N as a potential anti-inflammatory therapy for KDM6A-deficient bladder cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7474.
Muramoto et al. (Fri,) studied this question.