Targeting dual oncogenic machineries driven by TAL1 and PI3K-AKT pathways in T-cell acute lymphoblastic leukemia

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of thymic T-cell precursors. Overexpression of oncogenic transcription factor TAL1 is observed in 40-60% of human T-ALL cases, frequently together with activation of the NOTCH1 and PI3K-AKT pathways. In this study, we performed chemical screening to identify small molecules that can inhibit the enhancer activity driven by TAL1 using the GIMAP enhancer reporter system. Among approximately 3,000 compounds, PIK- 75, a known inhibitor of PI3K and CDK, was found to strongly inhibit the enhancer activity. Mechanistic analysis demonstrated that PIK-75 blocks transcriptional activity, which primarily affects TAL1 target genes as well as AKT activity. TAL1-positive, AKT-activated T-ALL cells were very sensitive to PIK-75, as evidenced by growth inhibition and apoptosis induction, while T-ALL cells that exhibited activation of the JAK-STAT pathway were insensitive to this drug. Together, our study demonstrates a strategy targeting two types of core machineries mediated by oncogenic transcription factors and signaling pathways in T-ALL.

Figure 1.

Establishment of the GIMAP enhancer reporter system. (A) Overview of the strategy used to screen for small-molecule compounds. The GIMAP enhancer luciferase reporter construct (eGIMAP-pGL4.26) was cloned and transfected into Jurkat cells. Stable clones were selected and subjected to drug screening or genetic knockdown. (B) Jurkat cells stably expressing the GIMAP reporter construct were subjected to short hairpin RNA (shRNA) knockdown using lentivirus infection. Cell viability and luciferase activity were measured 3 days post infection. Relative luminescence was determined by normalizing luciferase activity to cell viability and is presented as the fold change compared to the control scrambled RNA (shScrambled RNA). The values are shown as individual dots and the mean ± standard deviation of technical triplicates. (C) Jurkat cells stably expressing the GIMAP reporter construct were treated with DBZ or THZ1. Cell viability and luciferase activity were measured after 5 hours. Relative luminescence was determined by normalizing luciferase activity to cell viability and is presented as the fold change compared to untreated cells (0 nM). The values are shown as individual dots and the mean ± standrard devaition of technical triplicates. Representative results from multiple independent experiments were shown (B and C).

Figure 2.

Chemical screening using the GIMAP enhancer reporter system. (A) Overview of the chemical screening strategy. Using a liquid handling workstation, 2,961 compounds from 3 chemical libraries, a negative control (dimethyl sulfoxide [DMSO]) and a positive control (THZ1), were added to Jurkat cells that stably expressed the GIMAP enhancer reporter construct. Cell viability and luciferase activity were measured after 5 hours using a microplate reader. Images were created by BioRender. (B) Scatterplot showing luminescence (representing the GIMAP enhancer activity) and fluorescence (representing cell viability) of the cells treated with each of the compounds from the anticancer library. The values shown are the means of technical triplicates, presented as fold change compared to THZ1. (C) Jurkat cells stably expressing the GIMAP enhancer construct were treated with PIK-75 and WP1130 at various concentrations. Cell viability and luciferase activity were measured after 5 hours. Relative luminescence was determined by normalizing luciferase activity to cell viability and is presented as the fold change compared to untreated cells. The values are shown as individual dots and the mean ± standard deviation of technical triplicates. Representative results from multiple independent experiments were shown (C).

Figure 3.

Growth inhibitory effect of PIK-75 on T-cell acute lymphoblastic leukemia cell lines. (A) Western blot showing the expression of phosphorylated AKT and TAL1 in T-cell acute lymphoblastic leukemia (T-ALL) cell lines. (B and C) T-ALL cell lines were treated with PIK-75. Cell viability was measured after 24 hours by CellTiter-Glo. The values shown are the mean ± standard deviation of technical triplicates and are normalized to untreated cells. Cell lines with viability greater than 20% after treatment with 1 mM PIK-75 were considered to be insensitive. Not able to be determined (N.D.), half maximal inhibitory concentration (IC₅₀) values could not be determined by the dose response inhibition function using variable slope by GraphPad Prism. Cell lines grouped based on sensitivity to PIK-75 (B) or TAL1 and AKT status (C). (D) Cell lines ranked according to IC₅₀ value. TAL1 and AKT status are annotated by color. (E) Jurkat cells were seeded at 10,000 cells per well, treated with dimethyl sulfoxide (DMSO) or PIK-75 (120 nM) for 4 hours and stained with Annexin V-APC and PI. The values shown are the proportions of the total population ± standard deviation of technical triplicates. Representative results from multiple independent experiments were shown (B to E).

Figure 4.

The effect of PIK-75 on target proteins and pathways. (A) Western blot showing the expression of phosphorylated AKT and RNA polymerase II in 4 T-cell acute lymphoblastic leukemia (T-ALL) cell lines (Jurkat, CCRF-CEM, DND-41, and LOUCY). All cell lines were treated with dimethyl sulfoxide (DMSO) or PIK-75 (1 mM). (B) Western blot analysis showing the expression of γH2AX in Jurkat, CCRF-CEM, DND-41, and LOUCY cells treated with either DMSO or PIK-75 (1 mM).

Figure 5.

Gene expression changes afer PIK-75 treatment in T-cell acute lymphoblastic leukemia cells. (A and B) mRNA expression levels of GIMAP cluster genes (A) and other TAL1 targets (B) in Jurkat cells 4 hours after PIK-75 treatment (120 nM). Expression values were normalized to spike-in RNA and shown as individual dots and mean ± standard deviation of biological duplicates and technical duplicates. Representative results from multiple independent experiments were shown. (C) MA plot showing the average of normalized counts vs. log₂-normalized fold change (FC) between samples treated with dimethyl sulfoxide (DMSO) and PIK-75, estimated using DESeq2 with or without spike-in normalization. Each individual gene and spike-in control are represented by a single dot. RefSeq genes that showed statistically significant differences (adjusted P value <0.01) are colored blue. Similarly, spike-in controls are shown in purple (adjusted P value <0.01) or orange. N.S.: not significant.

Figure 6.

The effect of PIK-75 on TAL1 targets and super-enhancer-associated genes. (A) Gene set enrichment analysis (GSEA) to determine overall correlation between the change in gene expression after PIK-75 treatment in Jurkat cells and specific set of genes. The list of high-confidence TAL1 target genes (bound by TAL1 and downregulated after TA L 1 knockdown in Jurkat cells) and of super-enhancer associated genes were used as gene sets. (B) Heatmap showing the expression of TAL1 targets after PIK-75 treatment. (C) Gene ontology analysis was performed using genes that were significantly downregulated (base mean >10, log₂ (fold change) × -log₁₀ (P value) <-1, adjusted P value <0.01) in Jurkat cells 4 hours after PIK-75 treatment. The top 10 terms were selected according to the adjusted P value and are shown according to the combined score. Adjusted P value was calculated using Fisher’s exact test.

Figure 7.

Potential involvement of JAK-STAT pathway in drug sensitivity to PIK-75. (A) mRNA expression levels of PIM1 and CISH in Jurkat and DND-41 cells 4 hours after PIK-75 treatment (120 nM). Expression values were normalized to spike-in RNA and shown as individual dots and mean ± standard deviation of biological duplicates and technical duplicates. Representative results from multiple independent experiments were shown. (B) Western blot analysis showing the expression of phosphorylated STAT5 proteins in a panel of T-cell acute lymphoblastic leukemia (T-ALL) cell lines. (C) Western blot analysis showing the expression of multiple JAK and STAT proteins and their phosphorylated forms in Jurkat and DND-41 cell lines after treatment with dimethyl sulfoxide (DMSO) (control) or PIK-75 (120 nM) for 4 hours. (D) Western blot analysis showing the expression of phosphorylated JAK and STAT proteins as well as BCL2 protein with or without IL-7 induction and PIK-75 treatment (HPB-ALL half maximal inhibitory concentration [IC₅₀] =640 nM). (E) HPB-ALL was induced with IL-7 (50 ng/mL) for 24 hours before treatment with PIK-75. Cell viability was measured after 24 hours by CellTiter-Glo. The values shown are the mean ± standard deviation of technical triplicates and are normalized to untreated cells. (F) mRNA expression level of BCL2 in HPB-ALL cells after 24 hours of IL-7 treatment and/or 4 hours of PIK-75 treatment (640 nM) was measured by qunatitative reverse transcription polymerase chain reaction (qRT-PCR). Expression values were normalized to spike-in RNA and shown as individual dots and mean of technical duplicates. Representative results from multiple independent experiments were shown.

Figure 8.

Growth inhibitory effect of PIK-75 on primary T-cell acute lymphoblastic leukemia cells. (A) Two patient-derived mouse xenograft samples (DFCI-9 and DFCI-15) were treated with PIK-75. Cell viability was measured after 24 hours by CellTiter-Glo. The values shown are the mean ± standard deviation of technical triplicates and are normalized to untreated cells. (B) Western blot analysis showing the expression of RNA polymerase II, AKT, and their phosphorylated forms in DFCI-15 cells after treatment with dimethyl sulfoxide (control) or PIK-75 (DFCI-15 half maximal inhibitory concentration [IC₅₀] =210 nM) for 4 hours.