Abstract B32: Loss of the histone demethylase UTX alters the gene expression prolife and contributes to the malignant phenotype of multiple myeloma cells.
Authors: Ezponda Teresa , Popovic Relja , Zheng Yupeng , Nabet Behnam , Will Christine , Small Eliza , Occhionorelli Manuela , Tonon Giovanni , Keats Jonathan , Kelleher Neil , Licht Jonathan D. 
Published: August 2015

Genetic alterations of epigenetic regulators have become a recurrent theme in hematological malignancies. In particular, aberrations that lead to altered levels or distribution of methylation of lysine 27 on histone H3 (H3K27me) have emerged as a common feature of a wide variety of cancers, including multiple myeloma (MM). The histone demethylase UTX/KDM6A activates gene expression by removing the H3K27me3 repressive histone mark, counteracting the activity of EZH2, the enzyme that places this modification. UTX somatic inactivating mutations and deletions are found in up to 10% of multiple myeloma (MM) cases; nevertheless, the epigenetic impact of UTX loss in the MM cell and the mechanisms by which it contributes to this disease remain to be elucidated.

To ascertain the biological impact of UTX loss, we used paired isogenic cell lines isolated from a MM patient before (ARP-1, UTX wild-type) and after (ARD, UTX null) relapse. The UTX-mutant ARD cells were engineered to express UTX in a doxycycline-inducible manner. UTX add-back slowed the proliferation rate of ARD cells, without affecting their viability. Soft agar assays demonstrated that UTX mutant ARD cells have increased clonogenic abilities compared to ARP-1 cells. Re-expression of UTX partially reversed this effect, decreasing the number and size of colonies formed. ARD cells also showed increased adhesion to bone marrow stromal cells Hs-5 and to fibronectin than ARP-1 cells, an ability associated with cell survival and drug resistance. UTX add-back decreased the adhesive properties of ARD mutant cells demonstrating this effect is dependent on UTX loss.

Mass spectrometry analysis of the add-back system and a panel of UTX wild-type and mutant MM cell lines showed that global levels of H3K27me are not altered after UTX loss or upon its add-back, indicating that UTX depletion may lead to alteration of H3K27me only at specific loci, and thus only control the expression of a limited number of genes. To identify the genes and pathways altered upon UTX loss we performed RNA-seq on the paired MM cell lines and the add-back system. This analysis revealed about 5,000 genes differentially expressed between ARP-1 and ARD cells. Re-expression of UTX was able to reverse the expression of about 1,400 genes, most of them being upregulated upon reintroduction of this factor. Gene ontology analysis of the genes responsive to UTX manipulation demonstrated that many of these genes were involved in the regulation of pathways such as the JAK-STAT, the cadherin, the integrin or the Wnt pathway. Many of the pathways were related to cell adhesion properties, correlating with the effects observed in vitro. Single locus chromatin immunoprecipitation (ChIP) demonstrated changes in the levels of H3K27me3 at specific target genes, with the levels of this modification decreasing or increasing in genes activated or repressed upon UTX loss, respectively.

Overall, our data shows that loss of UTX leads to altered H3K27me3 levels at specific loci, resulting in deregulation of gene expression, ultimately promoting the proliferation, clonogenicity and adhesion of MM cells. The characterization of UTX target genes in this disease may help in the identification of targeted therapies for the treatment of this type of MM.

Citation Format: Teresa Ezponda, Relja Popovic, Yupeng Zheng, Behnam Nabet, Christine Will, Eliza Small, Manuela Occhionorelli, Giovanni Tonon, Jonathan Keats, Neil Kelleher, Jonathan D. Licht. Loss of the histone demethylase UTX alters the gene expression prolife and contributes to the malignant phenotype of multiple myeloma cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B32.