The Scd1 Gene Functions as a Tumor Suppressor In Leukemia Stem Cells

Abstract 201

We have previously shown that the arachidonate 5-lipoxygenase gene (Alox5) functions as a critical regulator of leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML) in mice. The Alox5 pathway appears to represent a major molecular network in LSCs. Taking advantage of our DNA microarray analysis for the identification of critical genes regulated by BCR-ABL in LSCs, we identified a small group of candidate genes that likely play tumor suppressor roles in these stem cells, and among them, a gene called stearoyl-CoA desaturase 1 (Scd1), an endoplasmic reticulum enzyme catalyzing the biosynthesis of monounsaturated fatty acids from saturated fatty acids, was shown to have a strong inhibitory effect on survival of LSCs in CML mice. BCR-ABL transduced bone marrow cells from Scd1^-/- mice induced CML much faster than BCR-ABL transduced wild type bone marrow cells, and overexpression of Scd1 dramatically delayed CML development. Therefore, we further investigated whether Scd1 suppresses LSCs. FACS analysis showed that the percentages and total numbers of LSCs (GFP⁺ Lin⁻c-Kit⁺Sca-1⁺) and long-term (GFP⁺ Lin⁻c-Kit⁺Sca-1⁺ CD34⁻) or short-term (GFP⁺ Lin⁻c-Kit⁺Sca-1⁺ CD34⁺) LSCs in bone marrow of recipients of BCR-ABL transduced Scd1^-/- donor bone marrow cells were significantly higher than those in bone marrow of recipients of BCR-ABL transduced wild type donor bone marrow cells, suggesting that Scd1 suppresses LSCs. Next we did a competitive repopulation assay to examine the function of LSCs. LSCs were sorted by FACS from bone marrow of mice with primary CML induced by transplanting BCR-ABL-transduced Scd1^-/- (CD45.2) or wild type (CD45.1) bone marrow cells. The sorted CD45.2 and CD45.1 LSCs were mixed in a 1:1 ratio, followed by transplantation into lethally irradiated recipient mice to induce secondary CML. At 8 weeks after transplantation, only less than 10% of GFP⁺Gr-1⁺ cells were CD45.1 leukemia cells derived from wild type mice, whereas more than 75%-80% of GFP⁺Gr-1⁺ cells in peripheral blood of the mice were CD45.2 leukemia cells derived from Scd1^-/- mice. To determine how Scd1 deficiency affects the maintenance of LSCs, we examined the cell cycle and apoptosis of LSCs. We found the percentages of apoptotic LSCs (Annexin V⁺ cells) were significantly decreased in bone marrow and spleens from Scd1^-/- CML mice compared to the wild type group; however, we did not observe significant differences in the cell cycle status of LSCs from bone marrow and spleen, indicating that Scd1 regulates apoptosis but not cell cycle of LSCs.

PPARγ agonist rosiglitazone can increase Scd1 expression, and our real time PCR data showed that rosiglitazone significantly induced Scd1 expression in bone marrow cells from CML mice. Therefore, we used PPARγ agonist rosiglitazone to treat these cells, and observed that rosiglitazone treatment dramatically decreased LSCs and that loss of Scd1 partially rescued the effect of PPARγ agonist on LSCs. Further, we investigated the molecular mechanisms that may contribute to the acceleration of CML development resulting from Scd1 deficiency. The decreased apoptosis in LSCs from Scd1^-/- CML mice led us to focus on apoptosis-related genes. Real time PCR analysis showed a significant decrease of p53 in Scd1^-/- immature leukemia cells as compared with that in wild type immature leukemia cells; however, loss of Scd1 resulted in dramatically and modest increased expression of Bcl-2 and Mcl-1 respectively in immature leukemia cells. We also found that the tumor suppressor gene Pten was significantly downregulated in Scd1^-/- LSCs. Together, our results demonstrate a novel tumor suppressor function of Scd1 in LSCs, and provide a rationale for suppressing LSCs by enhancing Scd1 expression.