Article Figures & Data
Figures
- Figure 1.
Ectopic expression of ZMIZ1 induces T-ALL in collaboration with NOTCH1. Mice were reconstituted with 5FU-treated donor bone marrow cells after transduction with indicated retrovirus. Representative flow cytometric analysis of peripheral blood of a NGFR/L1601PΔP mouse (A) and a ZMIZ1/L1601PΔP mouse (B). C, Kaplan–Meier curves showing the fraction of mice without T-ALL after bone marrow transplantation, 6 to 8 mice per group. D, representative flow cytometric analysis of CD4/CD8 expression of a ZMIZ1/L1601PΔP mouse with T-ALL. Negative, nontransduced controls are shown (NGFR− GFP− cells).
- Figure 2.
Expression of ZMIZ1 in T-ALL cell lines, primary T-ALL samples, and normal thymopoiesis. A, 14 T-ALL cell lines were screened for ZMIZ1 expression by Western blot. ZMIZ1, positive control; 8946 cells transduced with ZMIZ1 and L1601PΔP retroviruses. B and C, 12 primary pediatric human ETP-ALL samples, 12 primary pediatric human typical T-ALL samples, and 15 primary adult human T-ALL samples were screened for ZMIZ1 expression by qPCR. Expression is shown relative to the CEM cell line. The horizontal line shows median expression. In C, the ETP-ALL samples were divided into samples having more or less MEF2C than CEM cells. D, primary human T-ALL samples were screened for ZMIZ1 and activated NOTCH1 expression by Western blot. Sizes of mutant NOTCH1 proteins vary depending on the sizes of the C-terminal PEST truncations. E, murine thymocytes were sorted and screened for Zmiz1 by qPCR. The expression is shown relative to LSK cells as means ± SEM.
- Figure 3.
ZMIZ1 is a potential therapeutic target. A, CEM cells were transduced with nonsilencing control shRNA or ZMIZ1-silencing shRNA-13. Extrapolated cell counts were determined by flow cytometry. B, T6E cells were transduced with control shRNA or Zmiz1-silencing shRNA-m17 as in A. CEM cells were transduced with ZMIZ1-silencing shRNA-13 (C) or shRNA-15 (D) and then injected into NOD-SCID-γ-chain deficient mice. *, Mice were sacrificed. E–J, transduced CEM cells were pulsed with BrdUrd and analyzed by flow cytometry. Apoptotic cells in the sub-2N gate are indicated in the box in E and graphed in F. Nonapoptotic cells are shown in G and subdivided into various stages of the cell cycle (H) – bottom left box, G1; upper left box, early S; top right box, late S; lower right box, G2–M. NH4+ (I) and lactic acid (J) in the supernatant was determined.
- Figure 4.
Identification of ZMIZ1-regulated genes in CEM cells. A, columns represent biological quadruplicate samples from CEM cells transduced with nonsilencing shRNA control or ZMIZ1-silencing shRNA-13. Human U133 Plus 2.0 GeneChips were used. Probe sets with P < 0.0005 and fold-changes of more than 2-fold are shown (53 up and 35 down probe sets). B, signal intensities of 6 C-MYC-related probe sets were compared. Horizontal lines indicate means. C, expression was validated by qPCR.
- Figure 5.
C-MYC is a target of ZMIZ1. CEM cells were transduced with ZMIZ1-silencing shRNA. C-MYC expression was measured by Western blot (A) and by qPCR (B) relative to the level in nontransduced cells. C, 8946 cells were transduced with the NGFR empty vector (open columns) or ZMIZ1 (black columns) and with the indicated NOTCH1 alleles. After 6 days of doxycycline treatment, the percentage of NGFR+GFP+ cells was measured by flow cytometry and divided by the starting percentage before treatment to derive the fold increase in the percentage of NGFR+GFP+ cells. D–H, sorted 8946 cells were analyzed for c-Myc (D), Cad (E), Dtx1 (F), CD25 (G), and Hes1 (H) by qPCR. Expression is presented as a percentage of the value in ZMIZ1/L1601PΔP-transduced cells. I–L, sorted 8946 cells were treated with doxycycline. Open symbols represent 8946 cells cotransduced with the empty NGFR vector. Closed symbols represent 8946 cells cotransduced with ZMIZ1.
- Figure 6.
ZMIZ1 may regulate c-Myc through a novel MIZ-independent mechanism. A, schematic diagram of the mutants of ZMIZ1 used in this experiment. B, 8946 cells were transduced with the indicated ZMIZ1 constructs in the NGFR vector with the activated NOTCH1 allele L1601PΔP. After 6 days of doxycycline treatment, the absolute number of NGFR+GFP+ cells was counted and divided by the starting number to derive the absolute cell number fold increase. C, sorted NGFR+GFP+ 8946 cells were analyzed for c-Myc by qPCR relative to the NGFR vector. Control, 8946 cells transduced with MigR1 and NGFR vectors. D, disordered profile plot of ZMIZ1 generated by DISOPRED2 (http://bioinf.cs.ucl.ac.uk/disopred/).
- Figure 7.
ZMIZ1 inhibition sensitizes leukemic cells to NOTCH inhibitors and glucocorticoids. A, CEM cells were transduced with ZMIZ1-silencing shRNA and treated with DMSO or GSI. P < 0.0001 for interaction tests using an ANOVA model of log-transformed data on day 9 counts (B–C). Transduced CEM cells (80,000 cells/mL) were treated with dexamethasone and cultured (B). Relative cell number is shown in C. P values are derived from an ANOVA model with 12 means. D–E, CEM cells were treated as in B–C in the presence of 1 mmol/L mifepristone. F–G, a similar experiment was conducted as in B–C, but using DND-41 cells. B–G, dexamethasone concentration is plotted on the x-axis.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods - PDF file - 90K, Supplementary Methods. Viral constructs, viral production, transduction, bone marrow transplantation, flow cytometry, cell culture, Western blotting, immunoprecipitation, metabolic assays, xenotransplantation, dexamethasone treatment of T-ALL cell lines, GSI treatment of T-ALL cell lines, and supplementary references.
- Supplementary Figure 9 - PDF file - 5445K, Figure S9. Enforced expression of C-MYC increased apoptosis.
- Supplementary Figure 10 - PDF file - 5445K, Figure S10. ZMIZ1 inhibition sensitizes leukemic cells to NOTCH inhibitors.
- Supplementary Table 1 - PDF file - 5445K, Table S1. Q-PCR primers used in this study.
- Supplementary Table 2 - PDF file - 5445K, Table S2. Enrichment analysis of differentially regulated genes (fold change>1.3, p<0.01) after ZMIZ1 knockdown using C-MYC-related gene lists in the MSigDB Version 3.0 database.
- Supplementary Figure 1 - PDF file - 5445K, Figure S1. Retroviral transduction efficiency.
- Supplementary Figure 2 - PDF file - 651K, Figure S2. Gene expression profiling showing expression of ZMIZ1 in human T-ALL and normal thymopoiesis.
- Supplementary Figure 3 - PDF file - 5445K, Figure S3. ZMIZ1 RNA and protein levels were reduced by ZMIZ1-targeted shRNA.
- Supplementary Figure 4 - PDF file - 5445K, Figure S4. Cellular consequences of ZMIZ1 knockdown.
- Supplementary Figure 5 - PDF file - 5445K, Figure S5. The C-MYC pathway is downregulated after ZMIZ1 knockdown.
- Supplementary Figure 6 - PDF file - 5445K, Figure S6. ZMIZ1 does not regulate C-MYC post-transcriptionally or bind NOTCH1.
- Supplementary Figure 7 - PDF file - 5445K, Figure S7. ZMIZ1 appears to regulate C-MYC expression indirectly.
- Supplementary Figure 8 - PDF file - 5445K, Figure S8. C-MYC cannot substitute for ZMIZ1, but is a required effector of ZMIZ1 function.
Volume 73, Issue 2
