Article Figures & Data
Figures
- Figure 1.
BH3 profiling and in vitro testing of ABT-263 and ABT-199 reveals BCL-XL dependencies in T-ALL. A, the binding affinities of BH3 peptides BAD and HRK for the antiapoptotic BCL-2 family. Red, high-affinity binding; green, nondetectable binding measured by a fluorescence polarization assay (14). B, the BAD and HRK responses from the BH3 profiles of T-ALL cell lines are plotted. The means ± SDs of three independent experiments are graphed. C, the cell lines were treated with a six-point dose range from 1 nmol/L to 10 μmol/L of ABT-263 and ABT-199 for 48 hours, and apoptosis was measured by Annexin V and propidium iodide (PI) staining. The average of three independent experiments was used to generate dose–response curves in GraphPad Prism. The IC50 in μmol/L is graphed for each cell line. D, there is a statistical difference between the IC50 for ABT-263 and ABT-199 in the T-ALL lines. The ETP cell line LOUCY is shown in red. E, Western blot analysis shows expression of BCL-2 and BCL-XL in the T-ALL cell lines. F, the mean ratio ± SEM of BCL-2 expression divided by BCL-XL expression measured by densitometry of three independent plots is graphed.
- Figure 2.
BH3 profiling reveals BCL-2 dependencies in primary ETP COG T-ALL samples. A, BH3 profiling of pediatric COG T-ALL primary samples before initiating treatment. The mean of the BAD and HRK responses is graphed ± SD from three replicate wells. B, a dot plot of the BAD peptide response versus the HRK peptide response is graphed. The ETP cases, as defined by immunophenotypic analysis, are marked in red. Yellow indicates probable BCL-2 dependence, whereas blue indicates probable BCL-XL dependence. The BAD peptide response (C), the HRK peptide response (D), and BAD minus HRK peptide response were graphed for ETP versus typical T-ALL samples (E). Statistical significance was calculated using the nonparametric Mann–Whitney test.
- Figure 3.
BCL-2 and BCL-XL expression alters with maturation stage of the T cell. BCL-2 and BCL-XL protein expression was measured by FACS analysis, and the gating strategy for distinguishing the different stages of T-cell differentiation is shown in Supplementary Fig. S3. A, the expression of BCL-2 and BCL-XL is normalized to the double-positive stage and expressed in mean fluorescent units (MFU); the experiment was repeated three times and the mean ± SD is graphed. B, the mRNA expression of BCL-2 is shown for primary human cells at the listed stages of differentiation. These data are modified from the online database (37). C, the mRNA expression of BCL-2 and BCL-XL in both ETP-ALL and typical T-ALL is graphically depicted in a heat map, with red indicating high expression and blue indicating low expression. The data are modified from the published online database (38). D, the BCL-2/BCL-XL and MCL-1 protein levels are shown for the typical human T-ALL cell lines and samples (TALL-x-4, -9, -1, and -2) as well as the ETP-ALL cell line LOUCY and the relapsed ETP-ALL sample (TALL-x-11). E, ratio of BCL-2:BCL-XL protein is shown for the cell lines and primary samples examined in D.
- Figure 4.
BH3 profiling reveals BCL-2 dependence in ETP-ALL in a separate cohort of DFCI patient samples. A, BH3 profiling of pediatric and adult DFCI T-ALL primary samples before initiating treatment. A dot plot of BAD peptide versus HRK peptide is graphed; ETP-ALL are marked in red, and green indicates that ETP status was not determined. Yellow indicates putative BCL-2 dependence, whereas blue indicates putative BCL-XL dependence. The BAD peptide response (B) and the HRK peptide response (C) are plotted for ETP-ALL versus typical T-ALL samples. Statistical significance was calculated using the nonparametric Mann–Whitney test. The primary DFCI T-ALL samples were treated with a six-point dose range from 1 nmol/L to 10 μmol/L of ABT-263 and ABT-199 for 6 hours, and apoptosis was measured by Annexin V and PI staining. The IC50 of ABT-263 and ABT-199 are graphed (D). The ABT-263 (E) and ABT-199 (F) response was compared between the typical T-ALL and ETP-ALL samples. The IC50 for ABT-199 is correlated with the BAD minus the HRK peptide percentage mitochondrial depolarization (G). Correlation was calculated using the nonparametric Spearman r test. Red marks the ETP-ALL cases, green marks cases with undetermined ETP status, and black marks typical T-ALL.
- Figure 5.
PDX of ETP-ALL is very sensitive to in vivo treatment with ABT-199, whereas typical T-ALL is relatively resistant. Primagrafts were generated from two primary T-ALL samples; one sample was identified as ETP-ALL by immunophenotypic analysis (TALL-x-11), whereas the other was identified as typical T-ALL (TALL-x-2). The mean BAD and HRK peptide response of triplicate wells are plotted ± SD for the ETP-ALL sample (A). The in vitro response to ABT-199 and ABT-263 was measured using Annexin V and PI following 6 hours of treatment for the ETP-ALL sample. The percentage of survival is graphed on the dose–response curve, and the subsequent IC50 values are listed (B). Similar results are shown for the typical T-ALL sample, and the BAD and HRK response from the BH3 profile are graphed (C) and percentage survival following ABT-199 and ABT-263 treatment (D). Cells (1 × 106) of either the ETP-ALL sample or the typical T-ALL sample were injected in the tail vein of NOD scid gamma (NSG) mice until an engraftment of 65% human CD45+ cells (E). The animals were then randomized into vehicle, ABT-199, or ABT-263 treatment (100 mg/kg by oral gavage daily). The in vivo response to ABT-199 and ABT-263 was measured by counting the total human CD45+ leukocytes in the blood (F) and in the bone marrow (G) at the end of the 2 weeks of treatment in the ETP-ALL sample. Similar in vivo experiments were carried out with the typical T-ALL sample with total human CD45+ in the blood (H) and in the bone marrow (I) measurements shown following 2 weeks of treatment. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005.
- Figure 6.
Schematic of T-cell differentiation and BCL-2/BCL-XL dependence in ETP-ALL and typical T-ALL. During T-cell maturation, the most immature T cells express CD34, but do not express CD4 or CD8; hence, they are referred to as double negative (DN). As the cells mature, they express both CD4 and CD8 to become double-positive thymocytes. Thymocytes that survive both positive and negative selection become mature CD4 or CD8 single-positive T-cells. There is a reciprocal dependence on BCL-2 during the immature double-negative stage, which changes to BCL-XL dependence during the immature single-positive (Isp) and double-positive (CD4+ and CD8+) stage of differentiation. Malignancy arising in an immature T-cell ETP-ALL is dependent on BCL-2 and sensitive to both the BCL-2–selective BH3 mimetic ABT-199 and ABT-263 (binds BCL-2, BCL-XL, and BCL-W). In contrast, malignancy arising from the more mature double-positive T cells (typical T-ALL) is dependent on BCL-XL and selectively sensitive to the ABT-263 BH3 mimetic.
Additional Files
Supplementary Figures S1-S6
Files in this Data Supplement:
- Data Supplement - Supplementary Figure S1. T-cell ALL cell lines were treated for 48hr with five doses of ABT-199 and ABT-263. Apoptosis was assessed by Annexin V and Propidium Iodide (PI) staining. The mean % viability of three independent experiments is graphed +/- standard error. The calculated IC50 in μM for each of the BH3 mimetics is listed.
- Data Supplement - Supplementary Figure S2. The percentage of cells expressing CD8 is shown for both ETP-ALL and for Typical T-ALL primary human samples. The data is modified from the published online dataset (2). The majority of typical T-ALL samples are positive for CD8 expression by flow cytometry analysis.
- Data Supplement - Supplementary Figure S3. Flow cytometry gating strategy to identify the early progenitor cells using CD44 and CD25 antibodies on the double negative CD4-/CD8- gate. BCL-2 expression and BCL-XL expression was measured on each of the gated regions listed.
- Data Supplement - Supplementary Figure S4. The mean of the BAD and the HRK peptide responses are plotted for triplicate wells +/- SD of the pediatric and adult Dana-Farber patient samples (A). The BAD peptide minus the HRK peptide was used as a variable to measure BCL-2 dependence in both the typical T-ALL samples and the ETP-ALL samples (B).
- Data Supplement - Supplementary Figure S5. Primary T-ALL samples were treated for six hours in short term culture with both ABT-199 and ABT-263. Apoptosis was assesed by Annexin V and Propidium Iodide staining. The calculated IC50 in μM is listed beside each of the BH3 mimetics.
- Data Supplement - Supplementary Figure S6. NSG mice were injected in the tail with either ETP-ALL (TALL-x-11) or typical T-ALL (TALL-x-2) 1X106 cells and following engraftment to 65% the mice were randomized to receive vehicle, ABT-199 or ABT-263 treatment for two-weeks. The spleen weights (A) and absolute human CD45+ counts (B) along with % human CD45+ cell in the blood (C) were measured for the ETP-ALL primagraft following treatment. The leg bones were harvested and representative images are shown for the different treatments (D). Similar experiments were performed for the typical T-ALL primagraft spleen weights (E), absolute human CD45+ count in the spleen (F) with % human CD45+ cells in the blood were measured (G). Similarly the legs bones were harvested and representative images are shown (H).
Volume 4, Issue 9
