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CORRESPONDENCE Ellen Puré: pure{at}wistar.org
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Leukemia results from genetic defects that lead to enhanced proliferation and survival of bone marrow–derived cells, disrupting homeostasis in the hematopoietic compartment. Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease (MPD) that comprises 15–20% of hematopoietic cancers in humans (1). It is typified by an initial chronic stage, during which patients experience splenomegaly secondary to myeloid cell expansion. This relatively benign phase of the disease often progresses to a fatal blast crisis within 3–5 yr of diagnosis (1). The Philadelphia chromosome, the consequence of a t(9;22) (q34;q11) translocation, is a hallmark of the majority of human CML (2) and results in a fusion between bcr and the tyrosine kinase abl. The product of the Bcr-Abl fusion is altered in its cellular distribution and exhibits constitutive tyrosine kinase activity compared with c-Abl. Consequently, hyperphosphorylation of key Bcr-Abl substrates promotes leukemogenesis in myeloid cells (2).
Exciting advances in molecular medicine have yielded imatinib mesylate/STI571/Gleevec, an inhibitor of Abl activity, which has proved to be effective in many patients with CML (3). Unfortunately, a subset of patients afflicted with myeloid leukemias initially exhibit imatinib resistance, whereas others become refractory to treatment owing to development of drug resistance caused by subsequent mutations in abl (4–7). Moreover, evolution of CML to blast crisis is characterized by the development of a degree of resistance to treatment that is notoriously difficult to overcome (1, 6, 8). The survival of STI571-resistant leukemic cells can be mediated by compensatory activation of the Ras/mitogen-activated protein kinase and phosphatidylinositol 3–kinase (PI3-K)/Akt signaling cascades (9, 10). Thus, targeting these pathways may not only treat leukemic disease but can also circumvent the development of STI571 resistance.
Though several elegant mouse models have been developed to study myeloid leukemias (11–14), many of these develop fulminant blast crisis within weeks of induction, limiting their usefulness for investigating the chronic phase of CML and the critical transition to blast crisis phase (2). One of the few exceptions is the MPD observed in mice deficient in interferon consensus sequence binding protein/interferon regulatory factor 8 (ICSBP/IRF-8), which is characterized by more gradual and less frequent transitions to leukemic blast crisis (15). Importantly, the loss of ICSBP expression in patients with CML correlates with disease progression (16), whereas forced expression induces apoptosis in cell lines established from patients with Bcr-Abl+ CML (17, 18), even in cells which are STI571 resistant (19). However, despite the importance of ICSBP in human CML, none of the regulatory pathways upstream of ICSBP have been defined in the context of leukemia. In this study, we demonstrate that 12/15-lipoxygenase (12/15-LO) regulates ICSBP, and thereby MPD, in a PI3-K–dependent manner.
Lipoxygenases are enzymes that incorporate oxygen into unsaturated lipids and are named according to the position of the carbon double bonds they oxidize (20). These reactions yield short-lived peroxidized products, such as 12(S)-hydroperoxyeicosatetraenoic acid (12(S)-HpETE) and 13(S) -hydroperoxyoctadecadienoic acid, which reduce or are actively converted to several different products, including 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE), lipoxins, and hepoxilins. Importantly, the levels of the lipoxygenase product derivative 12(S)-HETE are reduced in patients with CML compared with normal controls (21), suggesting a role for lipoxygenase activity in the suppression of this disease. Consistent with this hypothesis, expression of platelet 12-lipoxygenase (encoded by Alox12) inversely correlates with the severity of CML disease (22). Similarly, leukocyte 12/15-LO (encoded by Alox15) is located on a region of human chromosome 17 implicated in the suppression of myeloid leukemogenesis (23). Despite these intriguing data, no direct evidence had yet supported a role for lipoxygenases in regulating leukemogenesis. While conducting experiments with 12/15-LO–deficient (Alox15) mice, we discovered that they develop an indolent MPD akin to human CML. We now provide the first description of Alox15 mice as a model for MPD and demonstrate that the pathology in these animals is related to 12/15-LO–mediated PI3-K–dependent regulation of ICSBP and its downstream target genes in vivo.
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RESULTS |
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Overall, the data up to this point indicate that Alox15 mice develop a MPD. Considering the pathology described, including the percentage of myeloblasts in the bone marrow, we classified 10 wk and older asymptomatic Alox15 mice with less severe pathology as being in the chronic phase (
85% of the population) and the moribund mice (15% of the population) as being in crisis phase. Furthermore, the pathology observed in these mice suggests that the most likely cause of death in the moribund mice was the severe anemia in these animals (Fig. S2).
12/15-LO activity suppresses proliferation and survival in mouse and human myeloid cells
We observed significantly increased proliferative capacity in splenic Alox15 myeloid cells based on cell cycle analysis (Fig. 4, a and b).
In addition, the percentage of apoptotic cells was decreased in Alox15 compared with wild-type cells, indicating enhanced survival of Alox15 cells (Fig. 4 c). We then investigated the relevance of 12/15-LO to human MPD. Consistent with the potential role of 12/15-LO in suppressing human disease, we found that human Bcr-Abl+ leukemia K562 cells (28), despite being derived from the myeloid lineage (which generally expresses 12/15-LO), do not express detectable levels of 12/15-LO protein (Fig. 4 d). However, forced expression resulted in levels of 12/15-LO similar to those found in mouse macrophages (Fig. 4 d) and led to a decrease in cell survival and proliferation that could be overcome by treatment with the 12/15-LO inhibitor PD146176 (Fig. 4, e and f). Thus, 12/15-LO can suppress human as well as mouse MPD.
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To ascertain whether the regulation of ICSBP nuclear accumulation by 12/15-LO corresponds with changes in the expression of ICSBP target genes, we measured levels of Bcl-2, an oncoprotein known to be suppressed by ICSBP (19, 35), in myeloid cells. We observed a dramatic up-regulation of Bcl-2 in the Alox15 myeloid population (Fig. 8 d) that correlated inversely with the nuclear accumulation of ICSBP.
PI3-K regulates Alox15 myeloproliferative cell survival and ICSBP phosphorylation and nuclear accumulation
Given the impact of tyrosine phosphorylation events on the ability of ICSBP to serve as a repressor of gene expression (36), we postulated that 12/15-LO regulates the nuclear accumulation and function of this transcription factor by influencing its phosphorylation status. Indeed, immunoprecipitation studies revealed an increase in tyrosine phosphorylation of ICSBP in Alox15 spleen (Fig. 9 a), indicating an important role for 12/15-LO in suppressing ICSBP phosphorylation.
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We found that Alox15 mice older than 10 wk develop MPD with 100% penetrance. In considering a novel mouse model for leukemic disease, it is important to evaluate the preleukemic state, pathology in knockouts as well as controls, and to assess the transferability of the leukemic syndrome (29), all of which we have done. However, one should, if possible, also investigate whether the disease is clonal in nature using antigen receptor rearrangement or retroviral insertion sites. Unfortunately, given the strictly myeloid nature of the syndrome in our mice and the fact that these animals are free of retroviral insertions, none of the conventional methods were available to us to establish clonality (29). Therefore, though the persistence of transplanted Alox15 cells 10 wk after transfer to wild-type recipients strongly suggests the clonal derivation of these cells, we have opted to make the more conservative conclusion that Alox15 mice develop a MPD that may become leukemic. Further studies, perhaps using a nontransforming retrovirus, will be required to completely elucidate this issue.
Given the indolent nature of the MPD that occurs in Alox15 mice, these animals provide a relatively unique opportunity to study the mechanisms underlying the chronic stage of MPD and its transition to blast crisis. In fact, as with humans with CML, we have found in this study that ICSBP is regulated differently in chronic and crisis stage cells, which appears to affect downstream targets of this transcription factor. In the future, it will be important to distinguish whether the switch to crisis is a result of transcriptional regulation of ICSBP by 12/15-LO or is caused by mutations in this gene. Indeed, it is possible that the occurrence of genetic aberrancies in ICSBP are a prerequisite of blastic transformation given the near uniform reduction in ICSBP transcripts in patients with crisis stage CML (16). Thus, further studies to define mechanisms of blastic transformation in indolent models of CML such as Alox15 mice are critical to developing new therapies. Moreover, as we have shown that 12/15-LO is expressed selectively in the myeloid lineage among hematopoietic cells, this pathway may present a selective target for treating MPD.
Curiously, though Alox15 mice were generated nearly a decade ago, the MPD we describe in this manuscript was not previously reported. There are several possible explanations for this. One possibility is that the syndrome in Alox15 may have become more pronounced on the pure C57BL/6 genetic background described in this study, as opposed to the F = 7 animals previously studied. Additionally, the majority of Alox15 mice manifests a degree of splenomegaly that might not be obvious to investigators handling the mice for other purposes and, therefore, who are not focused on the integrity of lymphoid organs. This is less likely in the case of the more dramatic splenomegaly observed in mice in blast crisis, but the incidence of these mice is relatively low (15%). Importantly, scientists at the Jackson Laboratory, the vendor of Alox15 mice (see Materials and methods), have recently examined Alox15 mice versus wild-type controls and have observed a similar phenotype of splenomegaly and flow cytometric abnormalities, confirming our findings in an independent facility (Nicholson, A., personal communication). The history of Alox15 mice mirrors the plight of human CML patients who, because of their initially mild symptoms, are often considered healthy for some time before being diagnosed.
We demonstrated that 12/15-LO–mediated suppression of leukemic cell growth is dependent on its enzymatic activity, and our ex vivo data suggest that 12/15-LO is responsible for producing several lipid mediators in vivo, including 12(S)-HETE. Interestingly, others have shown decreased levels of 12(S)-HETE in the bone marrow of humans with CML (21). This effect may be caused in part by reduced levels of platelet 12-lipoxygenase RNA levels in CML patients (22). The common products between leukocyte and platelet 12-lipoxygenases likely explain the fact that these enzymes are often jointly implicated in disease pathology, as exemplified in the case of osteoporosis (38, 39). Another reason for the overlapping functions between these two enzymes may be transcellular oxidation products formed between platelets and leukocytes, such as lipoxins and their intermediates (40–42). Alternatively, other consequences of lipoxygenase activity, such as direct membrane oxidation and reactive oxygen species, may be involved (20).
Consistent with the mechanistic links between them, the ICSBP and 12/15-LO–deficient mouse models for human MPD share several characteristics. Indeed, the decreased survival in ICSBP heterozygotes (9%) (15) is similar to what we observed in 12/15-LO–null mice, which would be expected in the context of the reduced (but not completely ablated) nuclear levels of ICSBP observed in 12/15-LO–deficient animals. Both models also display an initial MPD that transitions to a transplantable leukemia in a subset of animals over time. Additionally, it is important to note that both ICSBP and 12/15-LO are involved in inflammation (43–46), which may mediate in part their effects on leukemic disease, as many contemporary studies indicate that there may be a stronger connection between inflammation and carcinogenesis than previously recognized (47). Finally, mouse ICSBP and 12/15-LO share a high degree of homology with their human counterparts (48, 49), each are down-regulated in cells from CML patients (16, 21), and both can suppress human CML growth; therefore, both of these molecules are mechanistically relevant to human pathology. The lack of requirement for Abl activation in the 12/15-LO and ICSBP-deficient models is of particular interest considering the resistance to Abl inhibitors in a substantial proportion of myeloid leukemia patients and the role of the PI3-K pathway in many of these cases (9).
PI3-K is activated by cellular stress and various cytokine pathways, promoting cell survival, metabolism, and growth (50). Mice deficient in PTEN, a suppressor of PI3-K–mediated Akt activation, succumb to myeloid leukemias (51). Moreover, PI3-K inhibitors have been shown to kill imatinib-resistant leukemic cells, underscoring the importance of the PI3-K pathway in CML. However, because activation of PI3-K can be both Abl-dependent and -independent (9, 52), the PI3-K–dependent nature of the Alox15 syndrome does not readily elucidate whether 12/15-LO may be acting downstream of or in parallel to the Abl signaling cascade.
The role of 12/15-LO in regulating PI3-K signaling, especially in myeloid cells, was previously unknown. However, others have shown that the 12/15-LO products 13(S)-HODE and 15(S)-HETE up- and down-regulate, respectively, Akt phosphorylation in prostate cells (37), whereas 12/15-LO products uniformly up-regulate Akt phosphorylation in vascular smooth muscle cells (53). Thus, a cell-type selective effect of 12/15-LO on PI3-K signaling is apparent and will be of great interest to study in myeloid cells.
We have demonstrated decreased nuclear accumulation of ICSBP in Alox15 myeloid cells, which may be the result of PI3-K–mediated aberrant phosphorylation of ICSBP. In agreement with our study, it has been reported that tyrosine phosphorylation of ICSBP can decrease its direct binding to DNA, thereby limiting its ability to repress gene transcription (36). However, this phosphorylation also increases its binding to other transcription factors (36), enabling ICSBP to induce gene expression. Indeed, others have shown that phosphorylation of ICSBP on tyrosines 92 and 95 promotes its positive regulation of neurofibromin 1 expression without affecting ICSBP suppressor function, thereby promoting myeloid cell differentiation in vitro (54, 55). On the other hand, our data suggest that increased tyrosine phosphorylation of ICSBP can also promote MPD, likely through acting on distinct tyrosines. Thus, it will be important in future studies to characterize which tyrosine residues are phosphorylated as a result of 12/15-LO–mediated PI3-K pathway activity. In either case, the increased phosphorylation of ICSBP in our model may either subject it to a more rapid nuclear turnover secondary to decreased DNA binding or directly suppress its nuclear translocation. Our working model of the 12/15-LO–dependent regulation of ICSBP is presented in Fig. 10.
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Further dissecting the mechanism by which 12/15-LO suppresses MPD will uncover novel regulatory pathways in hematopoiesis and may pave the way for the development of additional targeted therapies for the treatment of myeloid leukemias and other cancers.
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MATERIALS AND METHODS |
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Lipid quantification.
Unperfused fresh spleens were cut into sections in duplicate and stimulated for 20 min with 1 µM ionomycin and 200 nM PMA in serum-free media. Some of the supernatant was used for lipoxin A4 analysis by ELISA (Oxford Biomedical Research, Inc.). The rest of the supernatant was extracted for analysis by stable isotope dilution normal phase chiral liquid chromatography (LC) coupled with electron capture atmospheric pressure chemical ionization (APCI)/mass spectrometry (MS) as described previously (49). In brief, samples were spiked with deuterium-labeled internal standards, adjusted to pH 3 with 2.5 N hydrochloric acid, and extracted with 2 x 4 ml diethyl ether. The organic layer was evaporated to dryness under nitrogen, derivatized with 2,3,4,5,6-pentafluorobenzyl bromide in the presence of diisopropylethylamine, and evaporated to dryness under a stream of nitrogen. Derivatized samples were reconstituted in 100 µl of hexane/ethanol (97:3, vol/vol) and 20 µl was analyzed by LC/electron capture APCI/MS system for analysis. Quantitation was performed by comparison of peak area ratios of the analytes to their relevant stable isotope internal standard and interpolation of area ratios from a standard curve (49).
Transfer experiments.
8–10-wk-old wild-type mice were injected via the tail vein with 0.5 x 106 (Alox15 crisis) or 107 (Alox15 chronic and C57BL/6) splenocytes or 2 x 106 bone marrow cells, monitored every other day, and killed at 6 or 10 wk after transfer. Tumor transfer was assessed by blinded morphological characterization of the spleens by hematoxylin and eosin (H&E). The presence of Alox15 donor cells in the transplanted mice was confirmed by PCR of the spleen DNA with primers specific for the neomycin cassette introduced by the Alox15 targeting construct (see Primer sequences).
Histology.
Blood samples were collected from the inferior vena cava, anticoagulated with EDTA, and kept on ice before slide preparation. Bone marrow cells were collected by flushing the femurs of wild-type and Alox15 mice. Cytospin slides were stained using Kwik-Diff reagents for Wright stains (Thermo Electron). Tissue samples were frozen in Tissue-Tek OCT and stored at –80°C. Morphology was analyzed in 8-µm sections stained with Gill's H&E. Tdt-mediated dUTP-biotin nick-end labeling staining for apoptotic cells was performed on three sections of spleen per mouse using a staining kit (Roche Applied Science) according to the manufacturer's instructions. The number of positive cells was quantified using Image-Pro software (Media Cybernetics).
Flow cytometry.
Single cell suspensions of splenocytes, bone marrow cells, and Histopaque 1083–purified (Sigma-Aldrich) peripheral mononuclear cells were prepared and depleted of red blood cells by lysis in ammonium chloride buffer, and cells were stained using 2 µg/ml directly labeled specific antibody or isotype-matched antibodies as controls. Compensation was performed using anti-CD4 or Mac-1 (FITC) and anti-CD8 or Mac-1 (PE) staining. All flow cytometric antibodies were purchased from BD Biosciences. Cells were washed and analyzed using a BD FACSCalibur machine (BD Biosciences). For sorting, cells were stained as described earlier in this paragraph and sorted for the populations indicated in the figures at the Wistar Institute Flow Cytometry Core facility.
Propidium iodide staining.
Single cell suspensions were fixed in ice-cold 70% ethanol at 4C° for 1 h, washed with PBS, and resuspended in 50 µg/ml propidium iodide containing 1.5 mM sodium citrate and 5 µg/ml RNase A in PBS. Samples were analyzed by flow cytometry.
Lysates, immunoprecipitation, and Western blots.
RBC-depleted splenocytes were prepared as in Flow cytometry, and nuclear lysates were prepared using the NucBuster kit (EMD Biosciences) according to the manufacturer's instructions. For cytoplasmic lysates, extracts were prepared from isolated cells using 1% NP-40 lysis buffer with phosphatase and protease inhibitors, whereas total extracts were prepared using M-PER (Bio-Rad Laboratories) according to manufacturer's instructions. For immunoprecipitation, extracts were normalized to total protein concentration using a Bradford assay (Bio-Rad Laboratories), according to manufacturer's instructions, and immunoprecipitated overnight with 1 µg of antibody, captured with protein G beads, and washed five times with 0.1% SDS buffer before electrophoresis. For Western analysis of unprecipitated extracts, samples were normalized to total protein using Bradford assay according to manufacturer's instructions, resolved by 10% SDS page gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoblotted with specific antibodies, as described in Results. All immunoblotting antibodies were obtained from Santa Cruz Biotechnology, Inc., except the anti-Crk antibody, which was purchased from BD Biosciences, and anti-phosphoAkt (Ser473), which was purchased from Cell Signaling.
Reverse transcription reaction/quantitative real-time PCR.
Total RNA was extracted from splenocytes using TRIZOL (Invitrogen) according to manufacturer's instructions. RNA was treated with Turbo DNase (Ambion) according to the manufacturer's instructions to remove any contaminating genomic DNA, and the absence of appreciable genomic DNA was confirmed by real-time PCR of the treated RNA. RNA was normalized by OD, and reverse transcription reaction was performed using a cDNA synthesis kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative real-time PCR analysis was performed using Sybr green Master Mix (Applied Biosystems) and analyzed using the ABI 7000 machine (Applied Biosystems). Gene-specific primers (see the next paragraph) were designed using Primer Express (Applied Biosystems), and gene expression levels were normalized using ß-actin as an internal control.
Primer sequences.
The following primer sequences were used: ß-actin, (forward) 5'–TCAGCAAGCAGGAGTACGATG-3' and (reverse) 5'–AACAGTCCGCCTAGAAGCACTT-3'; ICSBP (forward) 5'-TGGGCAGTTTTTAAAGGGAAGTT-3' and (reverse) 5'-ACAGCGTAACCTCGTCTTCCA-3'; 12/15-LO (forward) 5'-ACCCCACCGCCGATTTT and (reverse) 5'-AGCTTCGGACCCAGCATTT; and neomycin cassette (forward) 5'-TTGGGTGGAGAGGCTATTCG-3' and (reverse) 5'-AACACGGCGGCATCAGA-3'.
Statistical analysis.
One-way analysis of variance (for comparing more than two groups) and t-tests (for comparing two groups) were calculated using Prism software (GraphPad) according to the program's designation of the most statistically valid test for each experiment. The 
level was set at 0.05 for all tests; p-values below this were considered statistically significant. All error bars represent means ± SD.
Online supplemental material.
Fig. S1 shows comparable hematopoietic compartments to wild-type controls in young Alox15 mice. Fig. S2 depicts increased megakaryocytes and decreased red blood cell numbers in Alox15 mice. Fig. S3 shows myeloid infiltrates in the skin of moribund Alox15 mice. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20061444/DC1.
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Acknowledgments |
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These studies were funded by Public Health Service grants R01AI45813 and T32CA01940 (to E. Puré) and National Institutes of Health grant R01CA 91016 (to I.A. Blair), as well as a grant from the Pennsylvania Department of Health.
The authors have no conflicting financial interests.
Submitted: 7 July 2006
Accepted: 27 September 2006
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10), Asx Alox15 (n 
