M4N as a potential anti-inflammatory therapy for KDM6A-deficient bladder cancer

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 (M 4 N), 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 M 4 N against KDM6A-deficient BCa using a subcutaneous syngeneic model. Methods: To investigate the anti-proliferative effect of M 4 N 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 M 4 N-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. M 4 N 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 M 4 N suppressed inflammatory pathways such as chemokine signaling and JAK-STAT pathways. In Kdm6a -deficient tumors, M 4 N 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 M 4 N. Conclusions: M 4 N may serve as a novel therapeutic approach for patients with BCa bearing Kdm6a mutations by suppressing inflammation.