Abstract
Abstract 449
Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder induced by the BCR-ABL oncogene, and available BCR-ABL kinase inhibitors fail to completely eradicate leukemia stem cells (LSCs) to cure the disease. The challenge lies in the identification of genes that play a critical role in survival regulation of LSCs. Hypoxia-inducible factor-1α (HIF1α), a master transcriptional regulator of the cellular and systemic hypoxia response, is essential for the maintenance of self-renewal capacity of normal hematopoietic stem cells (HSCs). It is still unknown about the role of HIF1α in survival regulation of LSCs in CML. Using a mouse model of CML, here we report that HIF1α plays a crucial role in survival maintenance of LSCs. We conducted a DNA microarray analysis to compare the gene expression profiles between LSCs and normal HSCs in our bone marrow transplantation (BMT) mouse model of CML. We retrovirally transduced bone marrow cells from C57BL/6J (B6) mice with BCR-ABL-GFP or GFP alone (as a normal HSC control) and transplanted the transduced cells into lethally irradiate B6 recipient mice to induce CML. Two weeks after BMT, we sorted GFP+LSK (Lin−Sca-1+c-Kit+) cells from bone marrow of the mice for the Affymetrix microarray analysis. HIF1α gene was up-regulated by BCR-ABL in LSCs. We next examined expression of genes known to be specifically regulated by HIF1α, and found that expression of VEGF, GLUT1 and TGFa, except for PGK1, were significantly higher in LSCs than in HSCs. Real time RT-PCR assay confirmed the up-regulation of HIF1a and other hypoxia-responsive genes by BCR-ABL in LSCs. To determine the role of HIF1α in BCR-ABL leukemiogenesis, we crossed mice carrying a loxP-flanked HIF1a allele with Cre transgenic mice in which expression of Cre is driven by the Vav regulatory element to induce the deletion of the HIF1a gene mainly in the hematopoietic system. We transduced bone marrow cells from 5-FU-treated wild type (WT) or HIF1a−/− mice with BCR-ABL-GFP retrovirus, and then transplanted into lethally irradiated recipient mice to induce primary CML, followed by a secondary transplantation. We found that HIF1α−/− LSCs failed to induce CML in the secondary recipient mice, whereas WT LSCs efficiently induced CML. The defective CML phenotype in the absence of HIF1α was consistent with a gradual decrease of the percentages and total numbers of leukemia cells in peripheral blood and with much less severe splenomegaly. These results indicate that HIF1α is required for CML development, and suggest that HIF1α is required for survival maintenance of LSCs. To understand the underlying mechanisms, we analyzed the effect of HIF1α on cell cycle progression and apoptosis of LSCs, and found that the percentage of HIF1α−/− LSCs in the S-G2/M phase was significantly lower than that of WT LSCs, indicating that the HIF1α deficiency causes a cell cycle arrest of LSCs. Furthermore, we examined whether deletion of HIF1α induces apoptosis of LSCs by staining the cells with annexin V and 7AAD, and found that HIF1α−/− LSCs had a higher apoptotic rate than WT LSCs. We further compared expression levels of three cyclin-dependent kinase inhibitors p16Ink4a, p19Arf, and p57 between HIF1α−/− and WT LSCs, and found that the cell cycle arrest caused by the HIF1α deficiency was associated with significantly higher levels of expression of p16Ink4a, p19Arf and p57 in HIF1α−/− LSCs than in WT LSCs. In addition, we observed an increased expression of the apoptotic gene p53 in HIF1α−/− LSCs, explaining the increased apoptosis of HIF1α−/− LSCs. In summary, our results demonstrate that HIF1α represents a critical pathway in LSCs and inhibition of the HIF1α pathway provides a therapeutic strategy for eradicating LSCs in CML.
Disclosures:
No relevant conflicts of interest to declare.
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