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Maturation Stage of T-cell Acute Lymphoblastic Leukemia Determines BCL-2 versus BCL-XL Dependence and Sensitivity to ABT-199

Triona Ni Chonghaile, Justine E. Roderick, Cian Glenfield, Jeremy Ryan, Stephen E. Sallan, Lewis B. Silverman, Mignon L. Loh, Stephen P. Hunger, Brent Wood, Daniel J. DeAngelo, Richard Stone, Marian Harris, Alejandro Gutierrez, Michelle A. Kelliher and Anthony Letai
Triona Ni Chonghaile
1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
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Justine E. Roderick
2Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts.
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Cian Glenfield
3Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland.
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Jeremy Ryan
1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
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Stephen E. Sallan
4Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.
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Lewis B. Silverman
4Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.
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Mignon L. Loh
5Department of Pediatrics, Benioff Children's Hospital, University of California, San Francisco, San Francisco, California.
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Stephen P. Hunger
6Pediatric Hematology/Oncology/BMT, University of Colorado School of Medicine and Children's Hospital Colorado, Aurora, Colorado.
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Brent Wood
7Department of Medicine, University of Washington, Seattle, Washington.
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Daniel J. DeAngelo
1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
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Richard Stone
1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
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Marian Harris
8Department of Pathology, Boston Children's Hospital, Boston, Massachusetts.
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Alejandro Gutierrez
4Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.
9Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
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Michelle A. Kelliher
2Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts.
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  • For correspondence: anthony_letai@dfci.harvard.edu michelle.kelliher@umassmed.edu
Anthony Letai
1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
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  • For correspondence: anthony_letai@dfci.harvard.edu michelle.kelliher@umassmed.edu
DOI: 10.1158/2159-8290.CD-14-0353 Published September 2014
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    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.

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    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.

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    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.

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    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.

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    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.

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    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.

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    • 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).
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Cancer Discovery: 4 (9)
September 2014
Volume 4, Issue 9
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Maturation Stage of T-cell Acute Lymphoblastic Leukemia Determines BCL-2 versus BCL-XL Dependence and Sensitivity to ABT-199
Triona Ni Chonghaile, Justine E. Roderick, Cian Glenfield, Jeremy Ryan, Stephen E. Sallan, Lewis B. Silverman, Mignon L. Loh, Stephen P. Hunger, Brent Wood, Daniel J. DeAngelo, Richard Stone, Marian Harris, Alejandro Gutierrez, Michelle A. Kelliher and Anthony Letai
Cancer Discov September 1 2014 (4) (9) 1074-1087; DOI: 10.1158/2159-8290.CD-14-0353

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Maturation Stage of T-cell Acute Lymphoblastic Leukemia Determines BCL-2 versus BCL-XL Dependence and Sensitivity to ABT-199
Triona Ni Chonghaile, Justine E. Roderick, Cian Glenfield, Jeremy Ryan, Stephen E. Sallan, Lewis B. Silverman, Mignon L. Loh, Stephen P. Hunger, Brent Wood, Daniel J. DeAngelo, Richard Stone, Marian Harris, Alejandro Gutierrez, Michelle A. Kelliher and Anthony Letai
Cancer Discov September 1 2014 (4) (9) 1074-1087; DOI: 10.1158/2159-8290.CD-14-0353
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