Cell Cycle 8:21, 3488-3492; November 1, 2009; © 2009 Landes Bioscience

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3488 Cell Cycle volume 8 issue 21

Key words: Alox5, BCR-ABL, Ph+ leuke-mia, leukemic stem cells, zileuton

Submitted: 06/11/09

Accepted: 08/19/09

Previously published online: www.landesbioscience.com/journals/cc/article/9852

*Correspondence to:Shaoguang Li; Email: shaoguang.li@umassmed.edu

Cancer stem cells (CSCs) are believed to be the initiating cells for many

types of blood cancer and some solid tumors, and curative therapies of these cancers require eradicating CSCs. Specific targeting of CSCs but not nor-mal stem cell counterparts is a correct strategy for developing new anti-can-cer therapies, and the success of this approach relies on identification of spe-cific target genes in CSCs. Using BCR-ABL-induced chronic myeloid leukemia (CML) as a cancer model, we recently identified arachidonate 5-lipoxygenase (5-LO) gene (Alox5) as a critical regu-lator for leukemia stem cells (LSCs) in CML. Without Alox5, BCR-ABL fails to induce CML in mice due to the impair-ments of the functions of LSCs. The lack of Alox5 does not significantly affect the functions of normal hematopoietic stem cells. In addition, Zileuton, a specific 5-LO inhibitor, also causes the impair-ments of the functions of LSCs in a similar manner. Our results prove the principle that CSC-specific genes that play key roles in cancer development can be identified and inhibition of these genes can lead to eradication of these cells for cure. Here, we further discuss the mechanisms of Alox5 in CML, and the use of Zileuton as a potential and promising drug in eradicating LSCs in CML and other myeloproliferative diseases. We believe that our discovery of the role of Alox5 in regulating the function of LSCs in CML reminds us of viewing CSCs at a different angel. We predict that CSCs in other types of cancer also utilize specific regulatory

pathways to control their survival and self-renewal, and inhibition of these pathways profoundly suppresses CSCs but not their normal stem cell counter-parts. Specific targeting of CSCs with-out causing significant harm to normal stem cells should be a correct direction to go in developing novel therapeutic strategies in the future.

Introduction

Human Philadelphia chromosome arises from a chromosomal translocation between chromosome 9 and 22[t(9;22)(q34;q11)], resulting in the formation of a chimeric gene called BCR-ABL.1 Philadelphia chromosome-positive (Ph+) leukemias induced by the BCR-ABL oncogene include chronic myeloid leuke-mia (CML) and B cell acute lymphoblas-tic leukemia (B-ALL). CML is a clonal expansion of BCR-ABL-expressing hematopoietic stem cells, and often ini-tiates in a chronic phase and eventually progresses to a terminal blastic phase.

The BCR-ABL tyrosine kinase inhibi-tor imatinib mesylate is the standard of care for Ph+ leukemia, and induces a complete hematologic and cytoge-netic response in most CML patients.2 However, imatinib is ineffective in sup-pressing BCR-ABL mutants that have mutations in the kinase domains of BCR-ABL, and among these, the BCR-ABL-T315I mutant, which is present in 15–20% of imatinib-resistant patients, is not inhibited by either imatinib or dasa-tinib and other clinically available BCR-ABL kinase inhibitors.3 Imatinib will

The Alox5 gene is a novel therapeutic target in cancer stem cells of chronic myeloid leukemia

Yaoyu Chen,1 Dongguang Li2 and Shaoguang Li1,*1Division of Hematology/Oncology; Department of Medicine; University of Massachusetts Medical School; Worcester, MA USA; 2School of Computer and

Security Science; Edith Cowan University; Mount Lawley, WA Australia

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Alox5, it was striking that recipients of BCR-ABL transduced bone marrow cells from Alox5-/- donor mice failed to develop CML, albeit leukemia cells transiently existed. Specifically, myeloid leukemia cells grew initially, reached a peak after 2 weeks, then started to decline, and even-tually disappeared after 7 weeks. In con-trast, recipients of BCR-ABL transduced bone marrow cells from wild type donor mice developed and died of CML within 4 weeks. In the same CML mice receiv-ing BCR-ABL transduced Alox5-/- bone marrow cells, non-BCR-ABL-expressing myeloid cells were found to continue to grow while BCR-ABL-expressing myeloid cells gradually declined and disappeared in the absence of Alox5. Because CML develops from LSCs, together these results suggest that loss of Alox5 leads to a func-tional defect in LSCs rather than in nor-mal HSCs.

To show the effect of Alox5 on LSCs, FACS-sorted wild type and Alox5-/- LSCs were mixed in a 1:1 ratio and then trans-planted into the same recipient mice. At day 25 after transplantation, more than 90% of myeloid leukemia cells in periph-eral blood of the mice were wild-type (CD45.1+) in origin, and all these mice developed CML and died. These results show that the ability of Alox5-/- LSCs to induce CML is significantly lower than that of wild type LSCs, demonstrating that the Alox5 deficiency severely impairs the function of LSCs. This idea was more firmly demonstrated by a failure of Alox5-/- LSCs to induce CML when sorted Alox5-/- LSCs were transplanted into lethally irradiated recipient mice. In these mice, myeloid leukemia cells could be detected initially in peripheral blood of recipi-ent mice receiving BCR-ABL transduced Alox5-/- bone marrow cells; however, these leukemia cells soon disappeared, and the mice became leukemia-free and survived. Importantly, the Alox5 deficiency did not cause a functional defect in normal HSCs, because wild-type or Alox5-/- bone mar-row cells re-populated lethally irradiated recipient mice in a similar degree. These results indicate that loss of Alox5 specifi-cally causes the impairment in LSCs but not normal HSCs.

Because LSCs (GFP+Lin-c-Kit+Sca-1+) contain BCR-ABL-expressing long-term

Our Strategy for Targeting LSCs

A straightforward strategy for targeting LSCs is through inhibiting development-related genes that play roles in regulation of both normal HSCs and LSCs, as many of these genes have been identified and initially found to play critical roles in the development of normal HSCs. For exam-ple, Wnt/β-catenin, hedgehog and Bim-1 have been known to play important roles in normal embryonic or HSCs, and later reported to functionally regulate CML stem cells.14-17 As expected, these genes essential for normal development of HSCs should serve as effective targets for inhib-iting LSCs.17,18 Specifically, cyclopamine, a hedgehog inhibitor, reduces CML stem cell population and prolongs survive of CML mice.18 However, a potential prob-lem of this strategy is that normal stem cells could be simultaneously inhibited during a long-term cancer treatment, lead-ing to severe side effects potentially. One strategy for inhibiting LSCs is to target genes that play crucial roles in functional regulation of LSCs but not normal HSCs, and obviously, the challenge for taking this approach is to first identify these key stem cell-specific genes, as expression of many genes can be altered by an oncogene including BCR-ABL in CML and not all these genes are essential for survival and self-renewal of LSCs. The Alox5 gene is probably the first LSC-specific gene iden-tified and shown to play a critical role in LSCs but not normal HSCs,13 and our identification of this gene in LSCs shows a good example for how we design a strategy for targeting LSCs. In our study, the Alox5 gene was picked for further functional study based on our comparison of gene expression profiles between normal HSCs and LSCs by DNA microarray analysis, in which Alox5 is shown to be upregulated by BCR-ABL, which was confirmed by real-time PCR analysis. Alox5 has been shown to be associated with many important sig-naling pathways including p53 and PI3K, and is thought to be involved in many dif-ferent types of diseases.19,20 For the above-mentioned reasons, we decided to test the role of Alox5 in the development of CML induced by BCR-ABL in mice. Although we anticipated to observe an attenuation of CML development in the absence of

unlikely provide a cure, as CML stem cells exist.4 CD34+Lin- cells from bone marrow of CML patients contain CML stem cells and are thought to be respon-sible for disease progression and resistance to imatinib.4 These CML stem cells are insensitive to imatinib both in vitro and in vivo.5,6 Specifically, CML stem cells were found in the undivided CD34+ cell population that was insensitive to ima-tinib inhibition after being cultured with imatinib, although imatinib killed almost all dividing cells.5 Furthermore, the BCR-ABL transcript could still be detected in CD34+ cells in the bone marrow of CML patients after a long-term treatment with imatinib.6

Leukemia Stem Cells in CML

Leukemia stem cells (LSCs) in many types of hematologic malignancies are believed to be a cell population required for initiating and sustaining growth of leukemia cells.7-11 In our study of LSCs in CML induced by BCR-ABL in mice, we show that LSCs are similar pheno-typically to normal hematopoietic stem cells (HSCs).12 In these CML mice, BCR-ABL-expressing HSCs (GFP+Lin-c-Kit+Sca-1+) are able to induce secondary CML in recipient mice,12 indicating that this cell population functions as LSCs. Mechanistically, it is reasonable to think that BCR-ABL causes aberrant expres-sion of genes responsible for consequently turning a normal stem cell into a cancer stem cell. To support this idea, we show that the Alox5 gene is overexpressed in LSCs in CML mice, and these BCR-ABL-expressing cells also express the same cell surface markers that are expressed on normal HSCs.13 Although the underly-ing molecular mechanisms for the dif-ferent properties between normal HSCs and LSCs are still unclear, our identifi-cation of Alox5 as a critical regulator of LSCs provides us with the first clue for further investigation. As described above, bone marrow CD34+Lin- CML stem cells in human patients are insensitive to the BCR-ABL kinase inhibitor imatinib, and are difficult to be eradicated.5,6 This cell population isolated from CML patients will provide a more physiological system for studying CML stem cells.

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myeloproliferative disease (MPD). We ask this question because we postulate that some MPDs may have a common cellular origin same as LSCs in CML, and if so, the development of these MPDs should be affected by Alox5. To test this idea, we chose to determine whether Alox5 plays a role in MPD induced by the TEL-PDGFRβ oncogene.24,25 In mice, recipients of TEL-PDGFRβ trans-duced bone marrow cells rapidly develops MPD that recapitulates some aspects of human CMML, including leukocyto-sis, splenomegaly, and extramedullary hematopoiesis.24,25 To examine the role of Alox5 in myeloid leukemia induced by TEL-PDGFRβ, we transduced wild type or Alox5-/- donor bone marrow cells in B6 background with TEL-PDGFRβ to induce leukemia. We found that recipi-ents of TEL-PDGFRβ transduced bone marrow cells from 5-FU-treated wild-type donor mice developed and died of myeloid leukemia within 40 days, whereas recipi-ents of TEL-PDGFRβ transduced bone marrow cells from Alox5-/- donor mice were largely resistant to the induction of myeloid leukemia (Fig. 1A). This defec-tive disease phenotype correlated with less spleen weight (Fig. 1B). In addition, FACS analysis of myeloid leukemia cells in peripheral blood showed that Gr-1+ myeloid leukemia cells grew initially, reached a peak level quickly, and then decline to a low level at 60 days after the induction of leukemia (Fig. 1C). These results indicate that myeloid leukemia induced by TEL-PDGFRβ cannot fully develop in the absence of Alox5. It will be interesting to further study what cell lin-eage is affected by the Alox5 deficiency in TEL-PDGFRβ induced leukemia. It is possible that the development of other types of MPDs also requires Alox5, and that the Alox5 pathway may represent a major regulatory network in MPDs. On the other hand, Alox5 is dispensable in regulation of self-renewal and differen-tiation of normal HSCs. This different requirement of Alox5 in LSCs and nor-mal HSCs provides a unique opportu-nity to develop specific anti-LSC therapy without causing harm to normal HSCs. Importantly, Alox5 may serve as an effec-tive target in preventing the development of CML and perhaps other MPDs.

then analyzed LSCs in bone marrow of the treated mice by FACS at days 20 and 90 after the induction of CML by BCR-ABL. We find that the ratio between the percentage of LT-LSCs and that of ST-LSCs/MPP cells is similarly increased as shown in recipients of BCR-ABL trans-duced Alox5-/- bone marrow cells, suggest-ing that inhibition of 5-LO by Zileuton also causes the blockade of differentiation of LT-LSCs. These results indicate that targeting of LSCs with Zileuton could be beneficial to improving treatment of CML, and we tested this idea in mice. We treated CML mice with a placebo, Zileuton or imatinib alone, or two agents in combination. As expected, all placebo-treated mice developed and died of CML within 4 weeks after the induction of CML by BCR-ABL, and Zileuton alone was even more effective than imatinib in prolonging survival of CML mice. The therapeutic effect of Zilueton is likely through inhibiting LSCs, because at the early stage of the disease Zileuton treat-ment caused a less marked reduction of white blood cell counts than did imatinib treatment, presumably because Zileuton targeted LSCs and imatinib inhibited more differentiated leukemia cells. About 7 weeks after the treatment with Zileuton, GFP+Gr-1+ leukemia cells in peripheral blood of the mice gradually declined and dropped from over 50% to less than 2%, indicating that myeloid leukemia is even-tually eliminated. Treatment of CML mice with both Zileuton and imatinib had a better therapeutic effect than with either Zileuton or imatinib alone in prolonging survival of the mice. These findings are consistent with those from our genetic studies using Alox5-/- mice, demonstrat-ing that targeting of the Alox5 pathway is potentially curative for CML.

Alox5 is Also Required for Myeloproliferative Disease Induced by TEL-PDGFRβ

The specific role of the Alox5 gene in reg-ulating the function of LSCs but not nor-mal HSCs suggests different mechanisms of self-renewal and differentiate between LSCs in CML and normal HSCs. We wonder whether Alox5 is also required for the development of other types of

(LT), short-term (ST) HSCs and multipo-tent progenitor (MPP) cells, in our study we further investigated which of these cell populations is affected by the Alox5 deficiency. At an early time point after the induction of CML by BCR-ABL, the numbers of LSCs were similar between recipients of BCR-ABL transduced wild-type and Alox5-/- bone marrow cells. Among these LSCs, the percentage or total number of LT-LSCs was about half of those of ST-LSCs/MPP cells. After 90 days, few LSCs could be found in bone marrow of mice receiving BCR-ABL trans-duced Alox5-/- bone marrow cells, and the percentage or total number of LT-LSCs was about eight-fold higher than those of ST-LSCs/MPP cells. This reversal of the LT-LSCs to ST-LSCs/MPPs ratio suggests that the Alox5 deficiency causes a blockade of differentiation of LT-LSCs. Although more experiments are needed to confirm this “check point” between LT-LSCs and ST-LSCs, if true, this could be the first evidence showing how differentiation of LT-LSCs is mechanistically regulated. Expression of Alox5 is also differentially expressed in human CML.21,22 We believe that the Alox5 pathway represents a major molecular network that regulates the func-tion of LSCs, and much more work needs to be done to fully dissect this pathway.

Zileuton is a Promising Drug for Eradicating LSCs in Human CML

Several drugs, such as cyclopamine (inhibiting the function of hedgehog) and rapamycin (restoring the function of Pten), have been reported to have an inhibitory effect on leukemia stem cells,18,23 and their effectiveness in treat-ing human leukemias remains to be seen. Our identification of the role of the Alox5 gene in regulating LSCs in CML pro-vides a unique opportunity to develop a novel anti-stem cell therapy for CML, as Zileuton, a FDA-approved drug for treat-ing human asthma,22 specifically inhibits the enzymatic activity of arachidonate 5-lipoxygenase (5-LO),23 the product of the Alox5 gene. In our Alox5 study, we further tested whether Alox5 is a poten-tial therapeutic target in LSCs.13 With Zileuton, we treated recipients of BCR-ABL transduced bone marrow cells, and

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ABL kinase activity alone is insufficient to completely shut down BCR-ABL, and that other strategies need to be developed. Our demonstration of the role of Alox5 in regulating the function of LSCs but not normal HSCs identifies the first LSC-specific gene in CML, and provides us with an exciting opportunity to explore the regulatory molecular pathways in LSCs. In our Alox5 study, we have pri-marily linked the Alox5 pathway to acti-vation of β-catenin, as we showed that the loss of Alox5 caused downregulation of β-catenin expression in LSCs, but not in normal HSCs. Several important ques-tions remain to be answered: (1) How does BCR-ABL activate Alox5 expression in LSCs? (2) How do Alox5 and β-catenin interact to regulate the function of LSCs? (3) What are other signaling molecules in the Alox5 pathway? (4) Is the 5-LO inhibitor Zileution effective in eradicating human cancer stem cells in CML patients? (5) Does LTB4 produced by 5-LO play a

in LSCs, and the key is to select candidate genes for further functional tests. Prior to the functional tests, we always confirm expression change of a gene of interest by real-time PCR. The function of a candi-date gene can be primarily determined based on the literature or database search. The last step is to functionally test a can-didate gene using knockout or transgenic strains.

Future Directions

Imatinib effectively inhibits BCR-ABL kinase activity, but does not remove BCR-ABL protein, which may partially explain why imatinib does not eradicate LSCs in CML. In fact, we have shown that total number and percentage of LSCs in bone marrow of CML mice gradually increase with time during imatinib treatment, although the BCR-ABL kinase activity is greatly inhibited by imatinib.26 These results indicate that inhibition of BCR-

Our Strategies for Identifying New Therapeutic Targets in Cancer

Stem Cells

Targeting of cancer stem cells (CSCs) has become an important issue in develop-ing new therapies of cancer. As described above, we believe that the key to eradicat-ing CSCs is to identify critical pathways responsible for regulating self-renewal and differentiation of CSCs but not normal stem cells. Our Alox5 study has shown that this strategy is feasible.13 Using CML induced by BCR-ABL as a model disease, here we propose a strategy for identifying new therapeutic targets in CSCs (Fig. 2). Isolation of LSCs and normal HSCs is a key initial step to obtaining high-quality RNA for microarray analysis. In our study, BCR-ABL-expressing HSCs (GFP+Lin-c-Kit+Sca-1+) represent LSCs in CML mice. It is important to realize that the microar-ray analysis will show many genes that were up and downregulated by BCR-ABL

Figure 1. Alox5 is essential for the induction of MPD by teL-PDGFrβ. Kaplan-Meier survival curves for recipients of teL-PDGFrβ-transduced bone marrow cells from wild type or Alox5-/- donor mice. all recipients of BCR-ABL-transduced bone marrow cells from wild type donor mice developed MPD and died within 40 days after bone marrow transplantation (days post BMt), whereas the majority of recipients of teL-PDGFrβ-transduced bone marrow cells from Alox5-/- donor mice survived.

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21. Graham SM, Vass JK, Holyoake TL, Graham GJ. Transcriptional analysis of quiescent and proliferat-ing CD34+ human hemopoietic cells from normal and chronic myeloid leukemia sources. Stem Cells 2007; 25:3111-20.

22. Radich JP, Dai H, Mao M, Oehler V, Schelter J, Druker B, et al. Gene expression changes associated with progression and response in chronic myeloid leu-kemia. Proc Nat Acad Sci USA 2006; 103:2794-9.

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26. Hu Y, Chen Y, Douglas L, Li S. beta-Catenin is essential for survival of leukemic stem cells insensitive to kinase inhibition in mice with BCR-ABL-induced chronic myeloid leukemia. Leukemia 2009; 23:109-16.

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12. Hu Y, Swerdlow S, Duffy TM, Weinmann R, Lee FY, Li S. Targeting multiple kinase pathways in leukemic progenitors and stem cells is essential for improved treatment of Ph+ leukemia in mice. Proc Nat Acad Sci USA 2006; 103:16870-5.

13. Chen Y, Hu Y, Zhang H, Peng C, Li S. Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia. Nat Genet 2009; 41:783-92.

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role in regulating the function of LSCs? We believe that our Alox5 story has shown a new strategy for how to effectively target cancer stem cells for developing curative therapies.

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Figure 2. Our strategy for identification of genes that play key roles in regulating the functions of LSCs. One of the most critical steps in this ap-proach is to analyze DNa microarray data to provide a short list of candidate genes.