UTX Mutations and Epigenetic Changes In MDS/MPN and Related Myeloid Malignancies

Abstract 121

Balanced and unbalanced chromosomal lesions and genetic mutations are hallmarks of myeloid malignancies. In addition, aberrant methylation of CpG islands, leading to epigenetic silencing appears to play a significant role in tumor suppressor gene inactivation and malignant progression. While various mechanisms of chromosomal instability have been identified, the pathogenesis of epigenetic instability remains unexplored. ASXL1, EZH2 and TET2 mutations, found in myeloid disorders provide a potential link between genetic and epigenetic events. UTX is a histone H3K27 demethylase belonging to the polycomb group of proteins. Methylation at lysine 27 correlates with gene silencing and repression. A recent mutational screen of cancer cell lines has identified mutations in UTX in AML THP-1 cells.

We screened a series of myeloid disorders for UTX gene mutations and identified an index case in a CMML patient with a somatic UTX missense mutation. This sample demonstrated × chromosome copy neutral loss of heterozygosity (CN-LOH) that included UTX on a whole genome SNP-A array. UTX was then sequenced in 49 additional patients with MDS/MPN and 24 with secondary AML. Two of the MDS/MPN (2/49, 4%) and 3 of the sAML patients (3/24; 12%) showed UTX mutation. Three previously unreported missense polymorphisms were noted (present in paired CD3+ cells and detected with a frequency of <1/400 of controls). No mutations were found in MDS (N=15). The identified lesions were not associated with loss of sex chromosomes. Both homozygous (UPD) and heterozygous UTX mutations were identified and included frame shift or stop codons, as well as missense mutations. Inactivating or hypomorphic mutations may result in similar functional consequences with decreased expression of UTX. We quantitated UTX mRNA abundance in 15 hematopoietic cell lines using TaqMan PCR, and THP1 cells (UTX null) showed the lowest UTX mRNA expression. In primary patient samples, decreased UTX mRNA levels were found in the bone marrow of MDS/MPN patients (N=21) as compared to controls. To examine UTX expression in normal hematopoietic differentiation, CD34+ cells, along with myeloid CD33+ or monocytic CD14+ cell fractions (N=5) were tested and expressed easily quantifiable UTX transcripts, while the highest UTX expression was found in Gly-A+ erythroid precursors.

UTX mutation and dysfunction might be predicted to lead to altered histone repression marks and alterations in epigenetic regulation. Consistent with this hypothesis, we tested for H3K27-Me3 methylation by ELISA and observed a 45% methylation increase in a UTX mutant patient sample.

To further investigate the function of UTX, consequences and treatment implications of the corresponding mutations we transduced THP-1 cells with a lentiviral vector containing the UTX cDNA (UTX+) or an empty vector. Following clone selection, the resultant UTX+ cell line showed 1000-fold UTX mRNA increase and distinct protein overexpression by western blot (WB), as compared to the baseline UTX null cells. Proliferation kinetics up to 120h indicated that overexpression of UTX results in increased cell proliferation by 20% compared to controls. Treatment with decitabine (120h; 0.5μ M) resulted in a differential effect on UTX+ cells: proliferation increased 4.6-fold vs. 1.3-fold in UTX null cells.

Based on the hypothesis that mutation in UTX may lead to epigenetic instability and accumulation of aberrant methylated CpG sites, we further investigated epigenetic changes in UTX+ and UTX null cells. WB showed comparable levels of global H3K27-Me3 and H3 acetylation levels in UTX+ and UTX null cells, and only modest decreases (9%) of trimethylation in a more precise ELISA assay. Similarly, when methylation array (14K genes; 27K CpG sites) was used to assess the effects of UTX overexpression, global methylation levels expressed by averaged β-values did not significantly differ. However, 153 specific CpG sites were found to be differentially methylated on the array: with 68 genes hypo- and 85 hypermethylated. In particular SSRP1, NCOR2 and DIRAS3 genes showed hypomethylated promoters in the UTX+ cells, suggesting that UTX is involved in gene specific demethylation.

In sum, our results suggest that UTX gene may be involved in epigenetic regulation of promoters through site-specific histone demethylation function. UTX mutation may compromise this function, thereby promoting repression of tumor suppressor genes.