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Blood, Vol. 113, Issue 11, 2375-2385, March 12, 2009
HOXA9 is required for survival in human MLL-rearranged acute leukemias
Blood Faber et al.
113: 2375
Supplemental materials for: Faber et al
Files in this Data Supplement:
- Figure S6. Time course analysis of expression of the myeloid differentiation marker CD14 by FACS (JPG, 74.2 KB)
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A progressive increase in CD14 expression in cells transduced with HOXA9shRNA constructs is demonstrated. This results were confirmed three times in independent experiments.
- Figure S7. Comparative marker analysis in t(9;11) MOLM-14 AML cells 44h after HOXA9 suppression (3 1F3-HOXA9shRNA; 3 2A5-HOXA9shRNA; 6 GFP control shRNA) (JPG, 301 KB)
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(A) Gene set enrichment demonstrated a highly similar top 500 set of probes with decreased expression after HOXA9 suppression using either the 1F3-HOXA9 or 2A5-HOXA9shRNA construct (Enrichment Score (ES)=0.82; p<0.001). Therefore both suppression gene sets were combined for further analysis. (B) The top 500 genes demonstrating decreased expression in HOXA9 depleted cells 44h post transduction are shown (3 1F3-HOXA9shRNA; 3 2A5-HOXA9shRNA; 6 GFP-controlshRNA). The group of genes included genes previously identified as important in leukemogenesis and self-renewal of leukemia stem cells such as the HOXA9 cofactor MEIS1, MEF2C, and BMI1.
- Figure S8. Induction of Apoptosis by HOXA9 suppression is correlated with the presence of the MLL-fusion oncogene (JPG, 56.6 KB)
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A time course analysis of cell viability for all four HOXA9-directed shRNA’s without puromycin selection in MLL-wildtype Kasumi (AML) and Jurkat (T-ALL) is shown.
- Figure S9. Analysis of effects after HOXA9 suppression in AML/ALL cell lines (7 MLL-rearranged, 10 non-rearranged) utilizing the 1F3-HOXA9 shRNA (JPG, 101 KB)
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(A) Determination of baseline HOXA9 mRNA expression before shRNA transduction as measured by quantitative real-time–PCR. AML as well as ALL cell lines carrying either the MLL-AF9 or MLL-AF4 translocation (red bars) or no MLL-translocation (blue bars) were assessed. The right panel shows a box plot with mean HOXA9 expression in the MLL-rearranged or MLL-germline samples. The MLL-rearranged samples were found to have a significantly higher HOXA9 expression level when compared to MLL-wildtype controls (p=0.014). (B) Analysis of cell number 48 hours after shRNA mediated HOXA9 suppression (MTT assay) for MLL-rearranged samples (red bars) and MLL-wildtype samples (blue bars) is shown on the left. The right panel demonstrates a significant difference in the mean cell number for MLL-rearranged as compared to MLL-wildtype cell lines (p=0.007).
- Figure S10 (JPG, 56.2 KB)
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Quantitative real-time PCR analysis of HOXA9 expression 48 hours after transduction of primary human AML cells with the 1F3-HOXA9shRNA construct demonstrated efficient HOXA9 suppression as compared to GFP-shRNA transduced controls.
- Figure S11. Assessment of the effects of HOXA9 suppression with the 2A5-HOXA9shRNA construct in vivo using bioluminescent imaging in an independent experiment (JPG, 116 KB)
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(A) Prior to transplantation, SEMK2-M1 cells were transduced with either 2A5-HOXA9shRNA or GFP-controlshRNA and injected intravenously 20 hours after transduction. Mice were imaged 2, 24 and 72 hours after injection and then weekly. (B) There was no significant difference in total body luminescence two hours after injection confirming that an equal number of treated cells was injected (p=0.25). Repetitive longitudinal in vivo bioluminescent imaging revealed a progressive and highly significant difference in total body luminescence at later time points. (C) The degree of leukemic organ infiltration in both groups was assessed. The total number of human SEMK2 cells in spleens from SCID-beige mice that received either 2A5-HOXA9shRNA transduced or GFP-control-shRNA–transduced cells was evaluated. There is a highly significant difference in the total number of SEMK2-M1 between groups (p=0.00042). (D) Human cells were isolated from mice that received either GFP-controlshRNA (“control group”) or 2A5-HOXA9shRNA (“2A5-HOXA9shRNA group”) transduced SEMK2-M1 cells 14 days after injection, and the HOXA9 expression levels were compared to SEMK2-M1 cells growing in culture. The HOXA9 expression level at this time point was similar in the sorted SEMK2-M1 cells from the 2A5-HOXA9shRNA group as in those sorted from the control group (p=0.195). (E) Isolated human cells from the 2A5-HOXA9shRNA group were subsequently transduced with either the 1F3-HOXA9shRNA or GFP-controlshRNA construct and the total number of viable cells was assessed 7 days after transduction by Annexin V/PI staining. A highly significant reduced number of viable cells was observed in the cells transduced with the 1F3-HOXA9shRNA construct as compared to GFP-shRNA transduced controls (p=0.0000057).
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