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

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous group of hematological tumors composed of distinct subtypes that vary in their genetic abnormalities, gene expression signatures, and prognoses. However, it remains unclear whether T-ALL subtypes differ at the functional level, and, as such, T-ALL treatments are uniformly applied across subtypes, leading to variable responses between patients. Here we reveal the existence of a subtype-specific epigenetic vulnerability in T-ALL by which a particular subgroup of T-ALL characterized by expression of the oncogenic transcription factor TAL1 is uniquely sensitive to variations in the dosage and activity of the histone 3 Lys27 (H3K27) demethylase UTX/KDM6A. Specifically, we identify UTX as a coactivator of TAL1 and show that it acts as a major regulator of the TAL1 leukemic gene expression program. Furthermore, we demonstrate that UTX, previously described as a tumor suppressor in T-ALL, is in fact a pro-oncogenic cofactor essential for leukemia maintenance in TAL1-positive (but not TAL1-negative) T-ALL. Exploiting this subtype-specific epigenetic vulnerability, we propose a novel therapeutic approach based on UTX inhibition through in vivo administration of an H3K27 demethylase inhibitor that efficiently kills TAL1-positive primary human leukemia. These findings provide the first opportunity to develop personalized epigenetic therapy for T-ALL patients.

Keywords: TAL1; UTX; epigenetics; histone demethylase inhibitor; histone demethylation; leukemia.

Figure 1.

Identification of the UTX complex as a TAL1-interacting partner. (A) TAL1-interacting proteins identified by MS after TAL1 immunoprecipitation in Jurkat NEs. (Proba.) Probability of identification determined by ProteinProphet (Nesvizhskii et al. 2003). (B) Western blot analysis of reciprocal immunoprecipitations performed in Jurkat NEs with Abs against endogenous TAL1 and UTX proteins. (SN) Supernatant; (El.) elution. (C) Western blot analysis of TAL1 and UTX pull-downs performed on recombinant purified Flag-UTX and His-TAL1 fusion proteins. (D) Western blot analysis (right panel) of Flag pull-downs performed on recombinant purified Flag-UTX and the indicated GST-TAL1 fusion proteins (left panel). The asterisk indicates the recombinant protein of expected size. (B–D) Molecular masses are indicated (in kilodaltons). Representative examples of three biological replicates are shown.

Figure 2.

TAL1 recruits UTX to aberrantly activate transcription of its target genes through H3K27me3 demethylation. (A) UTX binds to and activates TAL1 target genes through active removal of H3K27me3. UTX knockdown (measured by Western blot) induced by doxycycline (Dox)-mediated expression of an anti-UTX shRNA leads to a decrease in transcription of the indicated TAL1 target genes (measured by quantitative RT–PCR [qRT–PCR]) and an increase in H3K27me3 (measured by ChIP-qPCR). (B) TAL1 recruits UTX to activate transcription of its target genes. TAL1 knockdown (measured by Western blot) induced by Dox-mediated expression of an anti-TAL1 shRNA disrupts UTX binding, which leads to an increase in H3K27me3 (measured by ChIP-qPCR) accompanied by decreased transcription of the indicated genes (measured by qRT–PCR). (C) Venn diagram showing the overlap of TAL1 and UTX binding genome-wide. P-value < 0.001. (D, top panel) ChIP-seq (ChIP combined with deep sequencing) density plots (normalized by reads per million mapped reads) for UTX and TAL1 at the ERG gene locus in TAL1-positive T-ALL cells. The arrowhead indicates the region tested by ChIP-qPCR below. (Bottom panel) Validation of the differential expression, H3K27me3 enrichment, and UTX and TAL1 binding at the ERG gene locus in TAL1-positive versus TAL1-negative T-ALL. (E) Pie chart of the genome-wide distribution of TAL1/UTX-cobound sites based on RefSeq in the TAL1-positive T-ALL. (F) TAL1 recruits an active H3K27me3 demethylase to its binding sites genome-wide in TAL1-positive T-ALL. Histone H3K27me3 ChIP-seq profiles are shown in control (Ctrl) and TAL1 knockdown (KD) Jurkat cells around the 2164 TAL1/UTX-cobound sites. Randomly chosen sites not cobound by TAL1 and UTX are shown for comparison. (TPM) Tags per million. (G) Genomic locations depleted of the H3K27me3 histone mark in the TAL1-positive T-ALL cell line Jurkat (shown in F) are enriched for the H3K27me3 mark in the TAL1-negative T-ALL cell line DND41. Histone H3K27me3 ChIP-seq profiles around the same genomic locations as in F are shown in the TAL1-negative T-ALL cell line DND41. Randomly chosen sites (the same as in F) are shown for comparison. (TPM) Tags per million. (H) UTX positively regulates the expression of TAL1 target genes that are cobound by UTX. Gene set enrichment analysis indicates the degree to which genes that are associated with TAL1/UTX-cobound sites and down-regulated upon TAL1 knockdown are overrepresented at the left (down-regulated upon UTX knockdown) or right (up-regulated upon UTX knockdown) of the entire ranked list (defined by RNA sequencing [RNA-seq] upon UTX knockdown). (NES) Normalized enrichment score. (A,B) Molecular masses (in kilodaltons) are indicated on Western blots. A representative example of three biological replicates is shown. (A,B,D), qRT–PCR results are expressed as mean values relative to the internal control ß2microglobulin (ß2M), with error bars corresponding to standard deviations (SDs). Mean ChIP-qPCR values are presented as a fraction of input, with error bars corresponding to SDs. n = 3. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

Figure 3.

UTX is involved in leukemia maintenance of TAL1-positive but not TAL1-negative T-ALL cells. (A) UTX is expressed in TAL1-positive and TAL1-negative T-ALL subtypes. Western blot analysis of UTX and TAL1 expression in NEs prepared from the indicated cell lines and patients’ primary blasts. Representative examples of three biological replicates are shown. (B) Knockdown of UTX by lentiviral delivery of anti-UTX shRNA in T-ALL cell lines and primary blasts from patients. qRT–PCR results are presented as mean values relative to the internal control ß2M ±SD. n = 3. (***) P < 0.001. (C) UTX knockdown decreases the growth of primary blasts from TAL1-positive but not TAL1-negative T-ALL patients. Viable cells after UTX knockdown (shUTX) relative to viable cells without knockdown (shCtrl) are reported as mean ± standard error of the mean (SEM). n = 4. (**) P < 0.01; (ns) not significant. (D) UTX knockdown triggers apoptosis in primary blasts from TAL1-positive but not TAL1-negative T-ALL patients. Apoptosis is reported as the percentage of Annexin V-positive cells. Data are shown as mean ± SEM. n = 4. (***) P < 0.001; (ns) not significant. Representative FACS plots are shown at the right. (E, top panel) Scatter plot summarizing genome-wide changes in the expression of genes (adjusted P-value ≤ 0.05) that belong to the indicated GO categories upon UTX knockdown (KD). RNA-seq was performed in duplicate in control and UTX knockdown Jurkat cells. (Bottom panels) Validation of RNA-seq results by qRT–PCR. Fold changes in expression (knockdown/control) are presented as mean values relative to the internal control ß2M ±SD. n = 3. (F,G) Overexpression (OE) of UTX wild-type but not its enzymatically dead mutant (mut) counterpart increases the growth of TAL1-positive Jurkat T-ALL cells (F) but decreases the growth of TAL1-negative DND41 T-ALL cells (G). (Top panels) qRT–PCR results are presented as mean values relative to the internal control ß2M ±SD. n = 3. (Middle panels) Western blots are shown as representative examples of three biological replicates. (Bottom panels) Concentrations of viable cells are presented as mean ± SEM. n = 4. (Red) TAL1-positive T-ALL; (blue) TAL1-negative T-ALL.

Figure 4.

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 (−log₁₀) versus fold change (log₂). 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 ß2M ±SD. n = 3. (**) P < 0.01.

Figure 5.

The H3K27 demethylase inhibitor GSK-J4 is efficient in vivo against patient-derived xenotransplant models of TAL1-positive T-ALL. (A) Experimental strategy. NSG mice were transplanted by intrafemoral (IF) injection of primary human T-ALL cells. When the engraftment level reached 5%–10% of human leukemic blasts in the BM, mice were injected intraperitoneally with GSK-J4 or a vehicle control. (B–D) In vivo administration of GSK-J4 strongly reduces the percentage of TAL1-positive human leukemic blasts in the BM (B) and spleen (C,D) but has no visible effect on TAL1-negative T-ALL transplanted mice. (B,C) The percentages of human CD45⁺ CD7⁺ leukemic blasts are presented as mean values ± SEM. (ns) Not significant (P ≥ 0.5); four mice per group. Representative FACS plots are shown at the right. (D) Immunohistochemistry using a human CD45 Ab on spleen histological sections. The results are representative of three biological replicates. (E) In vivo administration of GSK-J4 significantly reduces leukemia-induced splenomegaly in TAL1-positive but not TAL1-negative T-ALL transplanted mice. Spleen weights are presented as mean values ± SEM. (ns) Not significant (P ≥ 0.5); four mice per group.

Figure 6.

Model of UTX inhibition as a selective therapy in T-ALL. In TAL1-positive T-ALL, leukemia is maintained through a failure to down-regulate the oncogenic transcription factor TAL1 that in turn tethers the H3K27me3 demethylase UTX to genomic sites that should normally be silenced. Aberrant maintenance of an open chromatin configuration at these sites through active removal of the repressive histone mark H3K27me3 permits the establishment of a TAL1-mediated leukemic gene expression program. As such, in the TAL1-positive molecular subtype of T-ALL, UTX is a major pro-oncogenic cofactor, and a therapy based on UTX inhibition is efficient at eliminating leukemic blasts through down-regulation of the TAL1 leukemic gene expression program. On the other hand, in TAL1-negative T-ALL, leukemia is maintained independently of UTX, which explains that, even though UTX is expressed in these cells, inhibition of its enzymatic activity is not efficient as a therapy to eliminate TAL1-negative leukemic blasts. Thus, UTX inhibition may provide a useful therapeutic strategy for TAL1-positive (but not TAL1-negative) T-ALL patients.