Repression of BIM mediates survival signaling by MYC and AKT in high-risk T-cell acute lymphoblastic leukemia

Abstract

Treatment resistance in T-cell acute lymphoblastic leukemia (T-ALL) is associated with phosphatase and tensin homolog (PTEN) deletions and resultant phosphatidylinositol 3'-kinase (PI3K)-AKT pathway activation, as well as MYC overexpression, and these pathways repress mitochondrial apoptosis in established T-lymphoblasts through poorly defined mechanisms. Normal T-cell progenitors are hypersensitive to mitochondrial apoptosis, a phenotype that is dependent on the expression of proapoptotic BIM. In a conditional zebrafish model, MYC downregulation induced BIM expression in T-lymphoblasts, an effect that was blunted by expression of constitutively active AKT. In human T-ALL cell lines and treatment-resistant patient samples, treatment with MYC or PI3K-AKT pathway inhibitors each induced BIM upregulation and apoptosis, indicating that BIM is repressed downstream of MYC and PI3K-AKT in high-risk T-ALL. Restoring BIM function in human T-ALL cells using a stapled peptide mimetic of the BIM BH3 domain had therapeutic activity, indicating that BIM repression is required for T-ALL viability. In the zebrafish model, where MYC downregulation induces T-ALL regression via mitochondrial apoptosis, T-ALL persisted despite MYC downregulation in 10% of bim wild-type zebrafish, 18% of bim heterozygotes and in 33% of bim homozygous mutants (P=0.017). We conclude that downregulation of BIM represents a key survival signal downstream of oncogenic MYC and PI3K-AKT signaling in treatment-resistant T-ALL.

Figure 1

BIM is downregulated downstream of AKT and MYC in a conditional zebrafish model of MYC-induced T-ALL. (a and c) One representative rag2:MYC-ER; rag2:EGFP; rag2:mCherry triple-transgenic zebrafish shown at the time of T-ALL onset (a) and 4 weeks after removal from 4-hydroxytamoxifen (4HT). (b and d) Representative rag2:MYC-ER; rag2:EGFP; rag2:myr-Akt2 zebrafish, shown at the time of T-ALL onset and 4 weeks after 4-hydroxytamoxifen removal (-4HT). (e) Quantitation of T-ALL phenotypes after MYC-ER downregulation (by removal from 4-hydroxytamoxifen), comparing animals expressing a rag2:myr-Akt2 transgene (encoding constitutively active Akt) to rag2:mCherry controls. P value calculated using Fisher’s exact test. (f) Q-RT-PCR analysis of bim mRNA expression, performed using RNA from T-ALL cells isolated from rag2:MYC-ER; rag2-EGFP-bcl2 zebrafish that also expressed either rag2:myr-Akt2 or rag2:mCherry control. T-ALL cells were sorted from animals in 4-hydroxytamoxifen (“MYC On”), or 4 days after tamoxifen removal (“MYC Off”). Bcl2-transgenic T-ALL cells were used in all conditions to avoid comparing live versus dying cells. bactin2 was the control for Q-RT-PCR analysis. Error bars represent mean +/− standard error of the mean for experiments performed in triplicate. Significance was assessed using the Kruskal-Wallis test, a statistical method designed to assess whether at least one of the conditions is significantly different from the others.

Figure 2

BIM is repressed downstream of AKT and MYC in human T-ALL cell lines. (a and b) Q-RT-PCR analysis of BIM mRNA expression in four human T-ALL cell lines treated with 500 nM BEZ235, a dual ATP-competitive PI3K and mTOR inhibitor versus DMSO for 24 hours (a), or 1 μM JQ1 (a bromodomain inhibitor that downregulates MYC activity) versus DMSO for 24 hours (b). P values calculated using the Welch t test. (c to f) Western blot analysis of human T-ALL cell lines treated for 24 hours using DMSO (vehicle control), 1 μM JQ1, 500 nM BEZ235, or both drugs in combination, using the indicated antibodies.

Figure 3

BIM is repressed by MYC and AKT in human treatment-resistant T-ALL. (a) Correlation of BIM and MYC expression levels, as assessed using microarray gene expression profiling of primary childhood T-ALL patient samples. Data are from the following probe sets: BIM, 1561844_at. MYC, 202431_s_at. Line shows results of a linear regression analysis, and P value was calculated by Pearson correlation analysis. (b and c) Q-RT-PCR analysis of BIM mRNA expression in four treatment-refractory primary pediatric T-ALL samples, expanded in immunocompromised mice and treated in short-term culture assays (24 hrs) with 500 nM BEZ235 (c) or 1 μM JQ1 (d), versus DMSO vehicle control. Error bars represent mean +/− standard error of the mean for experiments performed in triplicate. P values calculated using a Welch t test.

Figure 4

Restoration of BIM function has therapeutic activity in human T-ALL. Human T-ALL cell lines (a) ALL-SIL, (b) CCRF-CEM, (c) MOLT4 or (d) PF382 were treated with a stapled peptide mimetic of the BIM BH3 domain, BIM SAHB_A (amino acids 146-166), or with the analogous peptide harboring an inactivating R153D reverse polarity mutation, for 24 hours at the indicated doses. Cell viability was assessed by CellTiter Glo, and is shown relative to DMSO control. Error bars represent mean +/− standard error of the mean for experiments performed in triplicate.

Figure 5

Therapeutic apoptosis induction by inhibition of MYC and PI3K-AKT pathways in treatment-resistant human T-ALL. (a to d) Effect of BEZ235 (500 nM), JQ1 (1 μM), or both drugs in combination on apoptosis induction in primary human treatment-resistant T-ALL samples treated in short-term culture assays (48 hrs), as assessed using FACS analysis for Annexin V and 7AAD staining. Data for early apoptotic (annexin V-positive, 7AAD-negative) and late apoptotic (annexin V-positive, 7AAD-positive) cells are shown separately, and error bars indicate mean +/− standard error of the mean for each apoptotic subset. P values were calculated using 2-way ANOVA analysis on the combined data for all apoptotic cells (early + late). In all four patient samples, the effects of the drugs were at best additive; no interaction terms were statistically significant at the 0.10 level.

Figure 6

Mutation of bim does not accelerate onset of MYC-induced T-ALL in the zebrafish. (a) Experimental design to test the effect of bim mutation on onset of MYC-induced T-ALL, and on tumor regression after MYC-ER downregulation following removal from 4-hydroxytamoxifen (4HT). (b) Analysis of T-ALL onset in zebrafish from the experiment described in a. Tumor-free survival was calculated using the method of Kaplan-Meier. and P value was calculated using the log rank test.

Figure 7

BIM mediates T-ALL regression following MYC inactivation in vivo. (a and c) One representative rag2:MYC-ER;rag2:EGFP double-transgenic, bim wild-type zebrafish, shown at the time of T-ALL onset (a) and 8 weeks after removal from 4HT (c). (b and d) One rag2:MYC-ER; rag2:EGFP double-transgenic zebrafish that harbored homozygous mutations of bim, shown at time of T-ALL onset (b) and 8 weeks after removal from 4-hydroxytamoxifen (d). (e) Quantitation of T-ALL persistence after downregulation of MYC by removal from 4-hydroxytamoxifen. P value was calculated using a Kruskal-Wallis test. (f) Proposed model to explain our findings.