Journal: Genes & Development

Article Title: UTX inhibition as selective epigenetic therapy against TAL1-driven T-cell acute lymphoblastic leukemia

doi: 10.1101/gad.276790.115

Figure Lengend Snippet: The H3K27 demethylase inhibitor GSK-J4 represses TAL1/UTX target genes and selectively kills TAL1-positive T-ALL cells through UTX inhibition. ( A , B ) TAL1-positive T-ALL cells are more sensitive to GSK-J4 treatment. ( A ) Dose-dependent effect of GSK-J4 on the viability of primary blasts from T-ALL patients. Data are presented as mean values of the percentage of CD7-positive Annexin V-negative cells ±SEM. n = 3. The reduction of apoptosis in TAL1-positive T-ALL is statistically significant compared with TAL1-negative T-ALL (e.g., at 5 µM GSK-J4, P -value = 0.016; at 10 µM GSK-J4, P -value = 8.8 × 10 −3 by t -test [unpaired, two-tail, nonequal variance]). ( B ) GSK-J4 (5 µM) decreases growth ( top panel) and increases apoptosis ( bottom panel) of primary blasts from TAL1-positive T-ALL patients. ( Top panel) Viable cell numbers after GSK-J4 treatment relative to viable cell numbers after vehicle control treatment are reported as mean values ± SEM. n = 4. ( Bottom panel) Apoptosis is reported as the percentage of Annexin V-positive cells. Data are shown as mean ± SEM. n = 4. (*) P < 0.05; (***) P < 0.001; (ns) not significant. Representative FACS plots are shown at the right . ( C , D ) In TAL1-positive T-ALL, GSK-J4 acts predominantly through UTX inhibition. ( C ) Correlation of changes in gene expression between UTX knockdown (shUTX) and GSK-J4 treatment. RNA-seq was performed with pairs of duplicates for (1) control and UTX knockdown Jurkat cells and (2) vehicle-treated and GSK-J4-treated Jurkat cells. ( D ) Overexpression (OE) of UTX, but not JMJD3, rescues GSK-J4-mediated apoptosis in TAL1-positive T-ALL. Overexpression of UTX (wild type or enzymatically dead mutant) or JMJD3 (wild type or enzymatically dead mutant) was induced in TAL1-positive Jurkat cells ( top ) or TAL1-negative DND41 cells ( bottom ) followed by treatment with GSK-J4 or a vehicle control. ( Left panels) Western blot analyses of UTX and JMJD3 overexpression. Representative examples of three biological replicates are shown. ( Right panels) The percentage of apoptotic cells (Annexin V-positive) after GSK-J4 treatment is reported relative to the percentage of apoptotic cells after treatment with vehicle control. Data are shown as mean ± SEM. n = 4. ( E ) GSK-J4 treatment increases the repressive histone mark H3K27me3 globally (as measured by Western blot; representative example of three biological replicates) ( left panel) and on specific TAL1/UTX target genes (as measured by ChIP-qPCR) ( right panel). Mean values are presented as a fraction of input ± SD. n = 3. (**) P < 0.01; (***) P < 0.001). ( F ) GSK-J4 treatment leads to down-regulation of the TAL1–UTX transcriptional regulatory network. ( Left panel) Volcano plot showing genome-wide changes in gene expression upon GSK-J4 treatment in Jurkat cells as measured by RNA-seq. Adjusted P -value (−log 10 ) versus fold change (log 2 ). The entire set of TAL1/UTX target genes (defined as TAL1/UTX-cobound genes that are significantly down-regulated upon TAL1 knockdown and UTX knockdown) are highlighted in red, with gene names shown for representative examples. ( Right panel) Validation of RNA-seq by qRT–PCR for a subset of genes. Results are reported as mean values relative to the internal control ß 2M ±SD. n = 3. (**) P < 0.01.

Article Snippet: Retroviral particles expressing JMJD3 (Addgene plasmid no. 21212) or an enzymatically dead JMJD3 mutant (H1390A) (Addgene plasmid no. 21214) ( ) were generated in 293-GP cells using the Retro-X universal packaging system with the Ampho envelope protein vector (Clontech).

Techniques: Inhibition, Control, Gene Expression, Knockdown, RNA Sequencing, Over Expression, Mutagenesis, Western Blot, ChIP-qPCR, Genome Wide, Biomarker Discovery, Quantitative RT-PCR