Abstract 12: KMD6A/UTX loss enhances the malignant phenotype of multiple myeloma and sensitizes cells to EZH2 inhibition

Abstract

Abstract In multiple myeloma (MM), inactivating mutations and deletions encompassing the histone demethylase KDM6A locus are found in up to 10% of newly diagnosed patients and associated with poor prognosis. Future sequencing studies may show an increased incidence of mutation with disease relapse. KMD6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) belongs to a family of Jumonji-C (Jmj-C)-containing demethylases working as a scaffold for a multiprotein complex containing H3K4-specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300, and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex appears to remove the gene repression associated methylation of lysine 27 on histone H3 (H3K27me), a mark placed by EZH2. Hence loss-of-function mutations of KDM6A may affect the function of this complex, which is common in B-cell malignancies. We found that KDM6A act as a tumor suppressor in MM in vivo and in vitro, but how this exactly happens and which genes are affected is incompletely understood. We modeled the loss of KDM6A in MM in vitro, using a pair of cell lines, ARP-1 (KDM6A wild type) and ARD (homozygous KDM6A deletion), derived from the same MM patient. As well, we used CRISPR-Cas9 mediated genome editing to disrupt KDM6A gene in cell lines. We examined gene expression profiles in ARP-1 vs. ARD cells, and vs. ARD cells upon reexpression of KDM6A, in a doxycycline-inducible manner, by whole transcriptome (RNA sequencing). To understand how KDM6A demethylase activity contributes to tumor suppressive role of KDM6A, we abolished the demethylase activity using directed mutagenesis targeting the JmjC domain. Disruptive mutations in functional domain of KDM6A provide a growth advantage in MM cell in vitro. Many genes induced upon KDM6A add-back are found upregulated in KDM6A wild-type ARP-1 cells. Mass spectrometry analysis of these cell lines and the add-back system showed no difference in global H3K27me3 levels, suggesting that the tumor suppressive role of KDM6A does not involve alteration of H3K27 methylation or that the changes in this histone mark following KDM6A loss are loci-specific. Reexpression of JmjC-dead KDM6A and wt KDM6A had similar effect on growth and clonogenicity, suggesting that demethylase activity is dispensable. KDM6A demethylase activity is dispensable for the tumor-suppressive effect of KDM6A in MM. Loss of KDM6A alters the transcriptome of MM cells. We therefore hypothesized that the scaffolding properties of KDM6A may be essential for the tumor-suppressive role of KDM6A. We plan to use CRISPR-Cas9 mediated genome editing to create specific mutations and flag-insertion in KDM6A gene, in both cell lines and mouse model, to further investigate the molecular mechanism of KDM6A activity. Citation Format: Daphne Dupere-Richer, Teresa Ezponda, Christine Will, Eliza Caroline Small, Nobish Varghese, Patel Tej, Xiaoxiao Huang, Zheng Yupeng, Giovanni Tonon, Neil Kelleher, Jonathan Keats, Jonathan D. Licht. KMD6A/UTX loss enhances the malignant phenotype of multiple myeloma and sensitizes cells to EZH2 inhibition [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 12.