P0975 - KDM6B inhibition has anti-tumor activity in anaplastic Wilms tumor cells associated with WT1 and MYCN pathway silencing

Background/Purpose: Histone lysine demethylases (KDMs) regulate embryonal transcription factors in development and pediatric cancers. KDM6B regulates MYCN and WT1 by demethylation of the repressive promoter histone mark H3K37me3. WT1 and NMYC regulate telomerase function by altering the transcription of TERT. Because we observed that KDM6B was highly expressed in Wilms tumor, we hypothesized that KDM6B could regulate levels of WT1 and MYCN in anaplastic WiT49 Wilms tumor cells and that inhibition of KDM6B could have an anti-tumor effect.

Methods: KDM6B expression was compared among Wilms tumor, normal kidney, and fetal kidney samples by RNA-seq. The small molecule GSK-J4 was used to specifically inhibit KDM6B in anaplastic WiT49 cells in vitro. MYCN, WT1, and TERT levels were determined by RNA-seq and western blot or telomerase activity assay. ChIP-PCR was used to determine WT1 promoter-associated H3K27me3. Annexin-V flow assisted cell sorting determined the effect of KDM6B inhibition on apoptosis and cell death. A colony formation assay was used to determine the effect of GSK-J4 in vitro.

Results: RNA-seq KDM6B log2(FPKM) values were higher in Wilms tumor and xenograft samples compared to fetal and normal kidney (p=0.025; Figure 1). GSK-J4 treatment in WiT49 cells resulted in a dose-dependent increase in apoptosis (p<0.001), cell death (p<0.001), and a decrease in colony formation. GSK-J4 treatment reduced WT1 and MYCN expression by RNA-seq and eliminated WT1 and MYCN protein detection. H3K27me3 occupancy at the WT1 promoter was increased by GSK-J4 treatment (p<0.05). GSK-J4 treatment reduced telomerase activity (p<0.05). In vitro results were replicated in transformed human embryonic kidney (HEK293) cells.

Conclusion: KDM6B is highly expressed in Wilms tumor and regulates WT1 and MYCN. Inhibition of KDM6B in anaplastic Wilms tumor cells results in an anti-tumor phenotype and inhibition of telomerase activity. KDM6B may govern a therapeutically relevant regulatory axis involving WT1, NMYC, and TERT.