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We have shown that Alox5is a critical regulator of leukemia stem cells (LSCs) in a BCR-ABL–induced chronic myeloid leukemia (CML) mouse model, and we hypothesize that the Alox5pathway represents a major molecular network that regulates LSC function. Therefore, we sought to dissect this pathway by comparing the gene expression profiles of wild type and Alox5−/−LSCs. DNA microarray analysis revealed a small group of candidate genes that exhibited changes in the levels of transcription in the absence of Alox5expression. In particular, we noted that the expression of the Msr1gene was upregulated in Alox5−/−LSCs, suggesting that Msr1suppresses the proliferation of LSCs. Using CML mouse model, we show that Msr1is downregulated by BCR-ABLand this down-regulation is partially restored by Alox5deletion, and that Msr1deletion causes acceleration of CML development. Moreover, Msr1deletion markedly increases LSC function through its effects on cell cycle progression and apoptosis. We also show that Msr1affects CML development by regulating the PI3K-AKT pathway and β-Catenin. Together, these results demonstrate that Msr1suppresses LSCs and CML development. The enhancement of the tumor suppressor function of Msr1may be of significance in the development of novel therapeutic strategies for CML.

The myeloproliferative neoplasms (MPNs) are hematologic malignancies with a chronic clinical course and a risk of developing thrombosis and acute leukemia. JAK2 is a member of the Janus family of non-receptor tyrosine kinases, and the somatic activating point mutation of JAK2(JAK2V617F) has been found in Philadelphia chromosome-negative MPNs in approximately 95% of polycythemia (PV), 60% of essential thrombocytosis (ET), and 50% of JAK2 V617F-positive MPD (PMF) patients. Although several JAK2 inhibitors are in the early stage of clinical trials and have been shown to help to improve symptoms and quality of life in patients, their long-term effectiveness in patients remains to be determined. New therapeutic strategies need to be developed for treating PV more effectively. Because hematopoietic stem cells (HSCs) harbor JAK2V617Fin PV patients, PV should be compared to Philadelphia chromosome-positive chronic myeloid leukemia (CML) that is also derived from HSCs and has a myeloproliferative phenotype similar to PV in mice. Therefore, we reasoned that a gene essential for CML development might also play a critical role in PV development. We have shown that the survival and self-renewal of CML-initiating cells require the arachidonate 5-lipoxygenase gene (Alox5) and that Alox5is essential for CML development. Therefore, in this study we investigated the role of Alox5in pathogenesis of PV in mice. We showed that JAK2V617Fupregulates Alox5 expression both in vitro(Ba/F3 cells) and in vivo(the spleen cells in PV mice). To examine whether Alox5 is required for induction of PV by JAK2V617F, we transduced bone marrow (BM) cells from wild type (WT) or Alox5homozygous knockout (Alox5-/-) mice with JAK2V617F-GFP, followed by transplantation into lethally-irradiated recipient mice. We found that the average percentage of JAK2V617F-expressing white blood cell counts (GFP+WBCs) in recipients of JAK2V617F-transduced Alox5-/-donor BM cells was significantly lower than that in recipients of JAK2V617F-transduced WT donor BM cells (P< 0.001). Total numbers of WBCs, red blood cells (RBCs), hemoglobin (HGB) and hematocrit (HCT) were also significantly lower in the absence of Alox5. With time (at 9 months after induction of PV), the effect of Alox5deficiency on PV development became more significant. Because JAK2V617F-transformed HSCs is shown to be a PV-initiating cell population, we further tested whether loss of Alox5affects this cell population in PV mice. We found that the percentage and total number of HSCs (Lin-Sca-1+c-Kit+) in mice receiving JAK2V617F-transduced Alox5-/-BM cells was significantly lower than that in mice receiving JAK2V617F-transduced WT BM cells. These findings suggest that Alox5 could be an effective target gene for PV. We tested this idea by treating PV mice with an Alox5 inhibitor zileuton (300 mg/kg, once a day) or a placebo. Western blot analysis of protein lysates from the spleens of PV mice showed that 5-lipoxygines (5-LO, a protein product of the Alox5gene) is upregulated by JAK2V617F, and inhibition of Alox5 function by zileuton restored the 5-LO level back to the control level. In addition, the number of WBCs in placebo-treated mice rose significantly with time, whereas the number of WBCs in zileuton-treated mice did not. The inhibition of PV development by zileuton correlated with much smaller infiltrated spleen in zileuton-treated PV mice than in placebo-treated PV mice. Furthermore, we compared the levels of JAK2V617F-expressing cells (GFP+) in PV mice. FACS analysis showed that the percentage of Gr-1+or Mac+PV cells in peripheral blood was significantly lower in zileuton-treated PV mice than in placebo-treated PV mice. Consistent with the inhibition of PV cells by zileuton in mice, survival of zileuton-treated PV mice was significantly improved compared to placebo-treated PV mice (Fig. 3I). At 5 months after induction of PV, about 40% placebo-treated PV mice died, but all zileuton-treated mice survived. To understand the underlying mechanisms, we treated JAK2V617F-expressing BaF/3 cells with zileuton, and found that zileuton inhibited activation of AKT and β-catenin by JAK2V617F, and did not affect activation of PI3K, MAPK and JAK2. Together, these results demonstrate that Alox5 represents a critical pathway in JAK2V617F-induced PV and that targeting of Alox5 provides a new strategy for treating PV.

Abstract 1666