Consequences of UTX Dysfunction in Myelodysplastic Syndrome

Abstract 2427

While various mechanisms of chromosomal instability in myeloid malignancies and myelodysplastic syndrome (MDS) have been proposed based on inherited and acquired genetic mutations, the molecular basis of the epigenetic instability has been essentially unexplored. However, the newly discovered mutations in various genes involved in epigenetic regulation may represent a link between genomic and epigenetic dysfunction of DNA.

The histone methyltransferase EZH2 and UTX (KDM6A) histone demethylase are among these genes, and potentially inactivating mutations that result in their defective function can lead to alteration of histone H3 lysine 27 trimethylation (H3K27me3), thus likely contributing to transcriptional repression or activation of distinct target gene groups. When 102 patients with myeloid disorders were screened, homozygous (LOH) and heterozygous UTX mutations were found in 9% of cases, including frame shift mutations, stop codons, and missense variants. Among mutant cases, chronic myelomonocytic leukemia (CMML) and secondary acute myeloid leukemia (AML) were most prevalent. In addition, deletions in the UTX region of the X chromosome were identified (N=4), thus increasing the overall number of affected cases. Homozygous inactivating mutations in UTX were also found in AML THP-1 cells, which showed no detectable protein, and in MOLM-13 in which UTX mRNA was absent. In 15/17 patients tested, UTX was expressed and transcripts were easily quantifiable in different hematopoietic cell fractions, including CD34+ cells. Of note is that in addition to mutations, the function of 2-ketoglutarate-dependent UTX is likely affected by the presence of 2-hydroxyglutarate generated by IDH/2 mutations in an additional 2% of cases with CMML and AML.

To further investigate role of UTX and the consequences of its dysfunction in CMML and AML, we transduced THP-1 and MOLM-13 (both UTX null cell lines) with a lentiviral vector containing UTX cDNA (UTX+) or an empty vector. Multiple clones were generated and mRNA and protein levels were determined. Following clone selection, the resultant UTX+ cell lines showed 1000-fold and 500-fold UTX mRNA increase and distinct protein over expression by western blot (WB), respectively, as compared to the baseline UTX null cells. However, despite successful UTX reintroduction, ectopic expression did not result in a significant reduction of baseline H3K27me3 levels, as demonstrated by WB and immunofluoresence. However, when cells were stimulated with PMA, a significant reduction of H3K27me3 levels (∼50%) was observed in UTX+ cells as compared to UTX-null cells. In THP-1 cells, overexpression of UTX resulted in increased cell proliferation by MTT assay, whereas in MOLM-13 growth rate was unchanged. Reintroduction of UTX did not lead to greater apoptotic susceptibility in modified cell lines challenged with PMA as measured by APO-BRDU intake. However, PMA treatment significantly enhanced the adherence of the UTX-reintroduced cells relative to unmodified cells and resulted in monocytic differentiation as judged by increased surface CD13 or CD14 expression. This effect was paralleled by increased C/EBP-ε, a transcription activator associated with differentiation and decreased pERK in UTX+ cells. In unstimulated UTX+ cells, up-regulation of surface markers linked to differentiation was present but was less pronounced. These findings were supported by morphologic changes in both cell lines showing more mature morphology (e.g., less condensed chromatin and vacuolization) of transduced cells. We have also applied expression arrays to UTX+ and UTX- control cell lines, and found that a majority of the top 10 overexpressed genes in UTX+ transfectants were involved in differentiation. The biological network of these genes showed involved in NfkB and MAPK pathways. Using pyrosequencing and methylation arrays, there were 68 differentially demethylated and 85 hypermethylated sites in UTX-overexpressing cells, suggesting site-specific UTX epigenetic regulation.

In summary, our results suggest that UTX may be involved in epigenetic regulation of promoters through site-specific histone demethylation function. UTX mutations may compromise this function, thereby promoting repression of tumor suppressor genes associated with differentiation arrest.