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Oncotarget
2016 Dec 27;752:86889-86901. doi: 10.18632/oncotarget.13563.
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VENTX induces expansion of primitive erythroid cells and contributes to the development of acute myeloid leukemia in mice.
Gentner E
,
Vegi NM
,
Mulaw MA
,
Mandal T
,
Bamezai S
,
Claus R
,
Tasdogan A
,
Quintanilla-Martinez L
,
Grunenberg A
,
Döhner K
,
Döhner H
,
Bullinger L
,
Haferlach T
,
Buske C
,
Rawat VP
,
Feuring-Buske M
.
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Homeobox genes are key regulators in normal and malignant hematopoiesis. The human Vent-like homeobox gene VENTX, a putative homolog of the Xenopus laevis Xvent-2 gene, was shown to be highly expressed in normal myeloid cells and in patients with acute myeloid leukemia. We now demonstrate that constitutive expression of VENTX suppresses expression of genes responsible for terminal erythroid differentiation in normal CD34+ stem and progenitor cells. Transplantation of bone marrow progenitor cells retrovirally engineered to express VENTX caused massive expansion of primitive erythroid cells and partly acute erythroleukemia in transplanted mice. The leukemogenic potential of VENTX was confirmed in the AML1-ETO transplantation model, as in contrast to AML1-ETO alone co-expression of AML1-ETO and VENTX induced acute myeloid leukemia, partly expressing erythroid markers, in all transplanted mice. VENTX was highly expressed in patients with primary human erythroleukemias and knockdown of VENTX in the erythroleukemic HEL cell line significantly blocked cell growth. In summary, these data indicate that VENTX is able to perturb erythroid differentiation and to contribute to myeloid leukemogenesis when co-expressed with appropriate AML oncogenes and point to its potential significance as a novel therapeutic target in AML.
Figure 1. A. Heatmap of differentially expressed genes as determined by RNA-seq between CD34+ CB cells overexpressing VENTX compared to the empty vector control (n=3). B. Differentially expressed genes transcription factors, which are known to play a role in erythroid development. ****: p<0.0001, ***: p≤0.0001, **: p≤0.001. C. Gene set enrichment analysis of genes involved in erythropoietic differentiation.
Figure 2. A. Quantitative expression of VENTX in different AML cell lines compared to BM CD34+/BMNCs/BM GlyA+/PB GlyA+. All expression analyses were performed by TaqMan® qRT-PCR with (+)RT and (-)RT reaction samples. Fold expression values were obtained by normalizing the expression of the gene of interest (VENTX) to the endogenous human β-actin (β-Act). Bars are showing the average fold expression ± SEM. *: p≤0.05, **: p≤0.001, ***: p≤0.0001, ****: p<0.0001. B. Mean Quantitative expression of VENTX AML M6 and PML-RARα positive AML samples compared to Glycophorin A from BM/PB and CD34+ bone marrow cells. All expression analyses were performed by TaqMan® qRT-PCR with (+)RT and (-)RT reaction samples. Log2 fold expression values were obtained by normalization of the expression of the gene of interest (VENTX) to the endogenous human β-actin. Bars are showing the log2 fold expression ± SEM. **: p≤0.001 and ****: p<0.0001. ○ indicates no detectable expression for 5 individual samples of PML-RARα positive AML cases (for up to 37 cycles).
Figure 3. Cell proliferation in liquid expansion cultures from HEL cells after shRNA mediated knockdown of VENTX (shVENTX_73, shVENTX_77) compared to the scrambled control. Knockdown efficiency was 99% for both shRNA constructs (n=3).
Figure 4. A. Kaplan-Meier survival curves of mice transplanted with 5-FU stimulated transduced BM cells expressing AML1-ETO+VENTX [AE/VENTX 1°] (n=20), VENTX [VENTX 1°] (n=18), empty vector control (n=18) and AML1-ETO [AE 1°] (n=15). Secondary and tertiary transplantations are shown for leukemic mice: AE/VENTX 2° (n=14), AE/VENTX 3° (n=9), VENTX 2° (n=19), VENTX 3° (n=4). AE/V 1° - V 1° V = VENTX: **, p= 0.0011; AE/V 1° - empty vector control 1°: ****, p<0.0001; AE/V 1° - AE1°: ****, p <0.0001; V 1° - empty vector control 1°: n.s. (n.s. = not significant); V 1° - AE1°: ns; AE/V 2° - V 2°: ns; AE/V 3° - V 3°: ns. B. Representative cytomorphological analyses of bone marrow of a diseased VENTX transplanted 1° mouse compared to an empty vector control mouse. C. Representative flow cytometric analysis of a primary VENTX recipient mouse (left side) and a secondary VENTX recipient mouse (right side) D. Histological analysis of primary (left side) and secondary (right side) VENTX recipient mice.
Figure 5. A. Representative cytomorphological analysis of bone marrow cytospins of diseased mice as indicated (AE/VENTX 1° and 2°) compared to a representative non-leukemogenic AE 1° mouse. B. Representative flow cytometric analysis of a primary AE/VENTX recipient mouse (left side) and a secondary AE/VENTX recipient mouse (right side) C. Histological analysis of two secondary transplanted AE/VENTX mice, showing erythroblasts in the spleen of the diseased animals.
Figure 6. Ex vivo colony forming cell assay (CFC1) and re-plating assays (CFC2, CFC3) of leukemic cells isolated from mice transplanted initially with 5-FU BM cells transduced with the different constructs as indicated. Results are means ± SEM. The number of CFC refers to 1000 input cells in the primary CFC assay. For 2° and 3° CFC assay also the CFC numbers are indicated per initially plated 1000 cells for the primary CFC (*: p≤0.05).
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