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ARTICLE |
CORRESPONDENCE Xiaoyan Jiang: xjiang{at}bccrc.ca
 Top ABSTRACT RESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Chronic myeloid leukemia (CML) is a clonal, multistep, multilineage myeloproliferative disorder. It is initiated and propagated by a rare population of CML stem cells that have acquired a BCR-ABL fusion gene (1, 2). The BCR-ABL fusion gene encodes a chimeric oncoprotein that displays constitutively elevated tyrosine kinase activity that drives CML pathogenesis (3, 4). These features deregulate cellular proliferation and apoptosis control through effects on multiple intracellular signaling pathways, including the Ras, phosphatidylinositol 3-kinase (PI3K), JAK–STAT, and NF-
B pathways (5, 6). Recently, imatinib mesylate (IM), which is an inhibitor of the BCR-ABL tyrosine kinase (4), has shown promise in treating CML patients (7–9). However, early relapses and IM-resistant disease have emerged as significant clinical problems in some IM-treated CML patients (10, 11). Relapses are frequently associated with mutations in the BCR-ABL kinase domain (10, 12, 13), accounting for 60–90% of relapses (11). Dasatinib (DS) and nilotinib (NL) are more recently produced small molecule inhibitors of the BCR-ABL–encoded kinase with greater potencies than IM and predicted broader effectiveness in patients with IM-resistant disease (14, 15). Recent studies have indicated that CML stem/progenitor cells in chronic phase patients are less responsive to IM and other tyrosine kinase inhibitors (TKIs), and that they are a critical target population for IM resistance (16–18). In addition, CML stem cells are genetically unstable and rapidly generate IM-resistant mutants in vitro (19). Thus, it is critical to identify other therapies targeting CML stem/progenitor cells to prevent acquisition of resistance. There is also an emerging imperative to develop complementary therapies that target downstream molecular events in the CML stem/progenitor cells of those patients who fail to achieve lasting remission with current treatments. Abelson helper integration site 1 (Ahi-1) is a novel gene that was identified by provirus insertional mutagenesis in v-abl–induced mouse pre–B cell lymphoma as a candidate cooperate oncogene (20). Mouse Ahi-1 encodes a unique protein with a SH3 domain, multiple SH3 binding sites, and a WD40-repeat domain, which are all known to be important mediators of protein–protein interactions, suggesting that the normal Ahi-1 protein has novel signaling activities and that its deregulation could affect specific cellular signaling pathways. Interestingly, the conserved human homologue (AHI-1) has an additional coiled–coil domain in its N-terminal region. Involvement of Ahi-1 in leukemogenesis is suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced mouse leukemias and lymphomas (20, 21). We recently demonstrated that Ahi-1/AHI-1 expression is regulated at multiple stages of hematopoiesis in a fashion that is highly conserved between mice and humans (22). Ahi-1/AHI-1 is expressed at its highest level in the most primitive hematopoietic cells and is rapidly down-regulated as cells begin to differentiate. Interestingly, marked deregulation of AHI-1 expression is seen in several human leukemic cell lines (22, 23), particularly in a CML cell line (K562) and in Philadelphia chromosome–positive (Ph+ BCR-ABL+) primary leukemic cells, but not Ph– cells, especially in highly enriched leukemic stem cells from patients with CML. In addition, levels of BCR-ABL transcripts are highly elevated in the same CML stem cell population (18, 24), suggesting that it may be important to cooperative activities of AHI-1 and BCR-ABL to generate a permanently expanding clone of deregulated stem cells at the early stage of leukemia development.
In this study, biological and molecular functions of Ahi-1/AHI-1 and its cooperative activities with BCR-ABL were extensively investigated in primitive mouse and human hematopoietic cells using several overexpression, suppression, and inducible model systems. We found that overexpression of Ahi-1 alone in primitive hematopoietic cells confers a proliferative advantage in vitro and induces a lethal leukemia in vivo; these effects are enhanced by BCR-ABL. Stable suppression of AHI-1 by small interfering RNA in BCR-ABL–transduced primitive human cord blood (CB) cells and primitive leukemic cells from CML patients reduces their growth autonomy in vitro. The regulatory role of Ahi-1/AHI-1 in mediating BCR-ABL transforming activities can be further explained by demonstration of a direct physical interaction between AHI-1 and BCR-ABL at endogenous levels in CML cells. This is associated with JAK2 and results in modulation of sensitivity to TKIs and differing levels of BCR-ABL tyrosine phosphorylation and JAK2–STAT5 activity in BCR-ABL+ CML cells where Ahi-1/AHI-1 expression is either coexpressed or inhibited.
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RESULTS |
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TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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To investigate effects of overexpression of Ahi-1 on the ability of transduced cells to induce leukemias in vivo, we injected transduced cells into sublethally irradiated NOD/SCID–β2 microglobulin (β2m)–/– mice. Strikingly, mice injected intravenously with Ahi-1–transduced BaF3 cells had a lethal leukemia within 70 d (5 x 106 cells/per mouse; Fig. 1 A). Disease latency was shortened to 40 d with BCR-ABL–transduced cells alone. Leukemogenic activity was further increased by introduction of cotransduced Ahi-1 and BCR-ABL cells, producing a latency of 26 d (P < 0.05; Fig. 1 A). Mice injected with either parental BaF3 cells or vector control cells had no evidence of disease after 120 d. Leukemic mice injected with either Ahi-1 or BCR-ABL–transduced cells developed splenomegaly and hepatomegaly, with 50–90% of YFP+/Ahi-1+, GFP+/BCR-ABL+, or both YFP+GFP+ cells detectable in these tissues (Fig. 1 B). As expected, larger spleens and livers were observed in mice injected with both Ahi-1 and BCR-ABL–transduced cells (Fig. 1 B and Table I). Interestingly, despite the apparently homogeneous pro–B cell phenotype of BaF3 cells transplanted, the leukemias generated from Ahi-1–transduced cells revealed multilineage features that included the production of Gr-1+Mac-1+ (myeloid), Ter119+ (erythroid), B220+ (B-lineage), and CD4+CD8+ (T-lineage; Fig. 1 C), suggesting that overexpression of Ahi-1 induces abnormal differentiation (including lineage switching) in hematopoietic cells. This was also observed in mice injected with Ahi-1 and BCR-ABL cotransduced cells. In addition, YFP+/Ahi-1+ or GFP+/BCR-ABL+ cells purified from BM cells of diseased mice showed increased proliferation and reduced apoptosis compared with control BaF3 cells; these effects were enhanced in Ahi-1– and BCR-ABL–cotransduced cells (Fig. S2 available at http://www.jem.org/cgi/content/full/jem.20072316/DC1). Thus, overexpression of Ahi-1 alone in IL-3–dependent hematopoietic cells has strong transforming activity in vitro and in vivo, and this is additive with the effects of BCR-ABL.
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15%) were already GF independent (Fig. 2, C and D). After 4 wk in long-term culture-initiating cell (LTC-IC) assays, which are used to measure stem cell activities in vitro, the Sca-1+lin–Ahi-1+ cells produced 3-fold more CFCs than control cells (P < 0.01; Fig. 2 E). All of these endpoints (proliferative activity, GF dependence, and CFC output in LTC-IC assays) are also perturbed by BCR-ABL transduction. In cells cotransduced with Ahi-1 and BCR-ABL, all of these effects were further enhanced compared with cells transduced with either BCR-ABL alone (2–4-fold) or Ahi-1 alone (3–6-fold; P < 0.01; Fig. 2). Thus, overexpression of Ahi-1 alone deregulates proliferative control of primitive mouse hematopoietic cells, and these effects can be enhanced by BCR-ABL.
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-AHI-1-IRES-YFP lentivirus in K562 cells resulted in sharply increased colony-forming ability compared with control cells (1.5–2-fold; P < 0.05; Fig. 3, B and C). Importantly, restored expression of AHI-1 both at RNA and protein levels in AHI/sh4 cells reversed growth deficiencies exhibited by suppression of AHI-1 (Fig. 3, A–C).
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Suppression of AHI-1 expression in primitive BCR-ABL–transduced human CB cells and primary CML stem/progenitor cells reduces their growth autonomy
We next evaluated the effects of down-regulation of AHI-1 in BCR-ABL–transduced primitive human CB stem/progenitor cells (Lin–CD34+) using lentiviral-mediated RNA interference. Q-RT-PCR analysis indicated that endogenous AHI-1 expression was suppressed by 40–50% when the shRNA construct (AHI/sh4) was introduced (Fig. 4 A).
Interestingly, BCR-ABL–transduced CB cells expanded rapidly in the presence or absence of GFs as expected, but the growth of the BCR-ABL–transduced cells with suppression of AHI-1 was significantly suppressed (2.2 x 106 versus 4.8 x 105 cells with GF; 106 versus 5 x 103 cells without GF; P < 0.01; n = 3; Fig. 4 B). Similarly, BCR-ABL–transduced CB cells with suppression of AHI-1 had up to 10-fold less CFC output compared with BCR-ABL–transduced CB cells (P < 0.01; Fig. 4 C). A similar reduction of CFC output was observed in the absence of GFs (data not shown). Notably, burst-forming-units-erythroids (BFU-Es) were especially increased in BCR-ABL–transduced CB cells compared with control cells, which remained predominantly myeloid as previously reported (110 ± 8 versus 18 ± 3; P < 0.01; Fig. 4 C) (25). Suppression of AHI-1 expression in BCR-ABL-transduced CB cells significantly reduced myeloid CFC (BFU-E and colony-forming units-granulocyte-macrophage [CFU-GM]) output, as well as primitive myeloid progenitors (colony-forming units-granulocyte-erythrocite-monocyte-megakaryocyte [CFU-GEMM]), suggesting that AHI-1 may play a role in regulation of overproduction of myeloid cells in CML.
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28–60% in lin–CD34+ CML cells from all patient samples studied (with AHI-1 expression partially suppressed (40–55%; Fig. 5, B and C). CFC colonies formed by CML cells with suppression of AHI-1 were found to be smaller than those resulting from cells transduced with a control vector (Fig. 5 D). It was interesting that more significant reduction in CFC numbers was observed in transduced primary CML cells from IM nonresponders and blast crisis compared with IM responders (Fig. 5 C).
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50% detectable Annexin V+ apoptotic cells, Fig. 6 B, top right), while the same cells showed a marked increase in cell expansion in the absence of both IL-3 and Dox (no down-regulation of BCR-ABL, Fig. 6 A, left). Similarly, down-regulation of BCR-ABL expression completely inhibited CFC generation in semisolid cultures in the absence of IL-3 (Fig. 6, C and D). Interestingly, introduction of Ahi-1 into cells with inhibited BCR-ABL expression under these stringent conditions enabled them to grow continuously in liquid suspension culture, to have less Annexin V+ apoptotic cells, and to produce more factor-independent CFCs than cells transduced with BCR-ABL alone (4 x 105 versus 103 cells after 48 h in liquid culture and 100 CFCs versus 0 in the CFC assay; P < 0.01; Fig. 6). These results were consistently observed in two individual clonal cell lines. In the presence of IL-3 and Dox, BCR-ABL–transduced BaF3 cells showed a significant reduction in CFC production; addition of IL-3 cannot rescue inhibitory effects caused by suppression of BCR-ABL expression. This can be enhanced significantly by cotransduction of Ahi-1 (10–15-fold; P < 0.01; Fig. 6 C). Collectively, these results demonstrate the ability of Ahi-1 to immediately reverse in vitro growth deficiencies resulting from down-regulation of BCR-ABL in vitro, and provide direct evidence of the regulatory role of Ahi-1 in BCR-ABL–mediated transformation.
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80% inhibition; P < 0.01; Fig. 7, B and C). In all cases, BCR-ABL phosphorylation was not completely suppressed after 24 h in culture with addition of Dox. These results were consistently observed in two individual cotransduced cell lines. Similarly, BCR-ABL protein expression was also suppressed to a lesser degree in cotransduced cells than in cells transduced with BCR-ABL only, and Ahi-1 protein expression was found to be higher in cotransduced cells than in cells transduced with BCR-ABL only.
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B p65 (at Ser 563 and Ser 468), and Src (at Tyr 416) in BCR-ABL–inducible cells and Ahi-1–cotransduced BCR-ABL–inducible cells compared with control BaF3 cells (Fig. 8, A and B).
Interestingly, phosphorylation of most downstream proteins was down-regulated when BCR-ABL expression was inhibited by Dox, but sustained phosphorylation of JAK2 and STAT5 was consistently observed in cotransduced cells in the presence of IL-3 and Dox (Fig. 8 A, lanes 6 and 8 compared with lane 4). In the absence of IL-3, phosphorylation of JAK2 and STAT5 was reduced in cotransduced cells when BCR-ABL expression was suppressed. A similar, albeit less pronounced, finding of sustained phosphorylation of Src was also observed, particularly in Ahi-1 + BCR-ABL-1 cells in the presence of IL-3. These results suggest that Ahi-1 may play a regulatory role in mediation of BCR-ABL activity associated with enhanced activation of JAK2 and STAT5 through the IL-3 signaling pathway.
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AHI-1 regulates response of BCR-ABL+ primitive CML cells to TKIs
To determine whether AHI-1–BCR-ABL–JAK2 interaction complex may mediate IM sensitivity/resistance of BCR-ABL+ cells, BCR-ABL–transduced inducible BaF3 cells and cells cotransduced with Ahi-1 were treated with various doses of IM (0–10 µm). As expected, BCR-ABL–transduced cells showed a significant reduction in CFC output in response to IM treatment in the presence and absence of IL-3 (90% inhibition with 5 µM IM + IL-3 and 1 µM IM – IL-3; Fig. 9 A).
Strikingly, BaF3 cells cotransduced with Ahi-1 and BCR-ABL showed no response to IM and produced as many CFCs in the presence of IL-3 as were produced by the same cells without IM treatment (Fig. 9 A, left). Moreover, cotransduced cells also displayed greater resistance to IM in CFC output in the absence of IL-3, although these cells were more sensitive to IM treatment than those in the presence of IL-3 (Fig. 9 A, right). These results indicate that Ahi-1 is capable of overcoming IM-induced growth suppression in BCR-ABL+ cells when IL-3 signaling is activated in these cells.
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2-fold; P < 0.01) in comparison to K562 control cells (Fig. 9 C). Conversely, suppression of AHI-1 (AHI/sh4) resulted in increased sensitivity to IM, particularly in the presence of a low concentration of IM (1 µM). Strikingly, restored expression of AHI-1 by overexpression of an AHI-1 construct in AHI/sh4 cells (suppression of AHI-1) restored IM resistance to AHI/sh4 cells (Fig. 9 C). Western analysis revealed increased tyrosine-phosphorylated BCR-ABL, JAK2, and STAT5 in K562 cells with AHI-1 overexpression and reduced levels of these phosphorylated proteins when AHI-1 expression is suppressed (Fig. 9 D). Interestingly, phosphorylated BCR-ABL, JAK2, and STAT5 levels could be restored in the AHI-1/sh4 cells when AHI-1 construct was reintroduced into the same cells. Importantly, expression of AHI-1 not only modulates phosphorylation of BCR-ABL, JAK2, and STAT5 in BCR-ABL+ K562 cells, but also regulates protein expression of these genes, as demonstrated by significantly enhanced expression of these proteins when AHI-1 is overexpressed, reduced expression when AHI-1 is suppressed, and restored expression in AHI-1–suppressed cells where AHI-1 expression has been rescued by introduction of an AHI-1 construct (Fig. 9 D). These results further support our findings in the BCR-ABL–inducible system that AHI-1 plays a critical role in mediation of BCR-ABL and JAK2–STAT5 activities.
We next assessed sensitivity of lin–CD34+ CML stem/progenitor cells, with and without suppression of AHI-1 expression, to the TKIs IM, DS, and NL. Cells were obtained from three IM responders, three IM nonresponders, and three blast crisis patients with partial suppression of AHI-1 expression in transduced CML cells (40–50%) as shown in Fig. 5 B. Interestingly, in all cases, lin–CD34+ CML cells were more sensitive to DS treatment (100 nM) than to IM (5 µM) or NL (5 µM), as assessed by their ability to generate CFCs (Fig. 9 E), whereas lin–CD34+ cells with suppression of AHI-1 expression, particularly cells from the clinically IM-resistant and blast crisis patients, were more sensitive to all three inhibitors (2-fold; P < 0.05). Collectively, these data suggest that AHI-1 plays an important role in modulating sensitivity to IM and other selective BCR-ABL TKIs in BCR-ABL+ CML cells.
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DISCUSSION |
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TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Identification of an AHI-1–BCR-ABL–JAK2 interaction complex raises many interesting questions about the nature of the interaction, how it is regulated, and how it contributes to malignant transformation, altered BCR-ABL signaling, and IM response/resistance of CML stem/progenitor cells. Particularly notable is the observation that coexpression of Ahi-1 in BCR-ABL–inducible cells can rescue GF-independent cell growth that is inhibited by down-regulation of BCR-ABL. Interestingly, this renewed GF independence with introduction of Ahi-1 appears to be regulated by sustained phosphorylation of BCR-ABL, rather than its continual expression, as these effects were observed in cotransduced cells where BCR-ABL expression was inducibly suppressed in vitro (Fig. 7 B). These results suggest that physical interaction between Ahi-1 and BCR-ABL may stabilize a protein–protein interaction complex that enables constitutively active BCR-ABL tyrosine kinase activity and further alters specific downstream BCR-ABL signaling pathways that deregulate cellular proliferation and apoptosis control (Fig. 9 B). This is further supported by identification of JAK2 as an associated protein in this interaction complex and observation of enhanced activity of the JAK2–STAT5 pathway in BCR-ABL–inducible cells with cotransduction of Ahi-1 in the presence of GF stimulation with IL-3 (Fig. 8). Interestingly, all these findings can be replicated significantly in a human CML cell line system in which changes in AHI-1 expression are found to modulate tyrosine phosphorylation and protein expression of BCR-ABL and JAK2–STAT5 (Fig. 9 D). It is known that BCR-ABL signaling closely mimics signaling pathways of cytokine receptors and that both IL-3/GM-CSF receptor activation and the BCR-ABL oncoprotein can induce a tyrosine phosphorylation cascade of which numerous proteins, including JAK2 and STAT5, are common substrates (5, 6, 31). Interestingly, BCR-ABL–expressing cells have many similarities to cells induced by IL-3 stimulation (32) or cells with forced IL-3 overexpression (33, 34). Early studies have shown that BCR-ABL can interact with the common β chain of IL-3/GM-CSF receptor and constitutively activate JAK2 (35). In particular, increased phosphorylation of STAT5, which was previously thought to be an immediate function of the BCR-ABL oncoprotein, has now been shown in primary CML CD34+ progenitor cells to occur largely as a consequence of BCR-ABL–induced activation of IL-3 autostimulation, leading to activation of STAT5 (36). It has been reported that targeted disruption of the STAT5A and STAT5B genes reduces myeloid progenitor numbers, suggesting a nonredundant role for STAT5 in primitive normal hematopoiesis (37). Recent studies further suggest that autocrine production of GM-CSF may contribute to IM and NL-resistance in BCR-ABL+ progenitors through activation of JAK2 and STAT5 pathway (38), and inhibition of STAT5 expression by a shRNA approach significantly reduced colony formation of CD34+ CML progenitor cells in vitro (39). In addition, JAK2 is known to interact with the C-terminal region of BCR-ABL, and recent studies further show that mouse hematopoietic cells transformed by the T315I mutant of BCR-ABL can be induced to undergo apoptosis by a JAK2 inhibitor (AG490) (40–42). Collectively, these results indicate that activation of the JAK2–STAT5 pathway in CML stem/progenitors is likely to be an important mechanism contributing to responses to BCR-ABL–targeted therapies and identification of Ahi-1/AHI-1 as a novel mediator involved in this pathway suggests AHI-1 alone or in combination with JAK2 and STAT5, as potential additional therapeutic targets.
The most revealing result presented here is the finding that expression of BCR-ABL was consistently associated with an up-regulation of endogenous Ahi-1/AHI-1 transcripts and an increase in Ahi-1/AHI-1 protein expression in several overexpression and inducible experimental systems. Up-regulated AHI-1 expression has been noted in K562 cells, a cell line derived from a patient with CML in blast crisis (22). In particular, modulation of AHI-1 expression in K562 cells regulates BCR-ABL and JAK2–STAT5 activities, as described above. Importantly, AHI-1 is highly deregulated in primary CML stem/progenitor cells where levels of BCR-ABL transcripts are also highly elevated, suggesting that deregulated expression of BCR-ABL and AHI-1 may be clinically relevant to their cooperative activities that result in a permanently expanding clone of deregulated stem cells during leukemia development. Indeed, our study further demonstrates that highly elevated levels of AHI-1 in CML stem/progenitor cells from IM nonresponders and blast crisis patients correlate with their relative resistance to TKIs. Similarly, a potential mechanism for a physical interaction between Ahi-1/AHI-1 and BCR-ABL is revealed based on their molecular structures (20, 43), which are compatible with specific protein–protein interactions. Ahi-1 may interact with BCR-ABL through its SH3 domain or its SH3 binding sites (i.e., the SH3 domain of one protein interacting with the SH3 binding sites of the other) or through the SH2 domain of BCR-ABL if Ahi-1 is tyrosine phosphorylated (Ahi-1 contains two potential tyrosine phosphorylation sites). In addition, Ahi-1 may bind to a SH2-containing protein that is a substrate of BCR-ABL, thus forming a complex, as it is known that BCR-ABL is extensively tyrosine-phosphorylated, providing numerous, potential docking sites for SH2 domain-containing proteins (44). Moreover, Ahi-1 may interact with multiple domains of BCR-ABL, as demonstrated by other BCR-ABL–interacting proteins (45). Although more detailed structural mapping will be required to define specific protein–protein interactions between Ahi-1 and BCR-ABL, in association with JAK2, our findings that coexpression of Ahi-1 in BCR-ABL–transduced cells can completely rescue IM-induced suppression of cell growth in the presence of IL-3 suggests that Ahi-1 may not be a direct substrate of the BCR-ABL tyrosine kinase, but rather a modular protein that forms a stable protein interaction complex with other tyrosine phosphorylated proteins to mediate IL-3–dependent BCR-ABL and JAK2–STAT5 activities. In addition, this protein interaction complex seems to be disrupted by suppression of tyrosine phosphorylation of BCR-ABL by IM. Studies are underway to define how AHI-1 specifically interacts with BCR-ABL and JAK2. It was interesting to note that enhanced phosphorylation of Src was observed in Ahi-1 coexpressed BCR-ABL–inducible cells in the presence of IL-3 (Fig. 8), suggesting that other kinases are also activated with stimulation of IL-3 when BCR-ABL and AHI-1 are coexpressed. This finding also explains the observation that CML progenitor cells with suppression of AHI-1 experienced greater inhibition of CFC generation in response to dasatinib, a more potent TKI that also inhibits Src activity (14).
In conclusion, our studies demonstrate that Ahi-1/AHI-1 is a novel BCR-ABL interacting protein that is also physically associated with JAK2. This AHI-1–BCR-ABL–AK2 complex seems to modulate BCR-ABL–transforming activity and TKI response/resistance of CML stem/progenitor cells through the IL-3–dependent BCR-ABL and JAK2–STAT5 pathway. These results suggest that a more promising potential therapeutic approach would be the combined suppression of BCR-ABL tyrosine kinase activity, Ahi-1/AHI-1 expression, and JAK2–STAT5 signaling.
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MATERIALS AND METHODS |
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TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-IRES-YFP lentiviral vector (with substitution of eGFP with YFP from an EF1-IRES-eGFP lentiviral vector; P. Leboulch, Massachusetts Institute of Technology, Boston, MA) between AscI and PacI sites. To generate the AHI sh4 lentiviral construct, the H1 promoter-driven small hairpin against human AHI-1 was released from a modified pSUPER AHI sh4 construct (23) and inserted into an EF1-IRES-YFP lentiviral vector. A similar subcloning procedure was performed to insert only the H1 promoter into the BamHI site of the EF1-IRES-YFP lentiviral vector as control. All constructs were verified by restriction enzyme digestion analysis and DNA sequencing.
Lentiviral production was performed after a previously described procedure (47). The titers of these concentrated viruses were measured with HeLa cells as 1 x 108 infectious U/ml for the BCR-ABL virus and 2 x 108 infectious U/ml for control, AHI sh4, and AHI-1 viruses. Helper-free retrovirus was obtained by transfecting ecotropic Phoenix packaging cells (48) cultured in DMEM plus 10% fetal calf serum using the calcium-phosphate precipitation method, as previously described (46).
Human primary cells and FACS sorting.
Peripheral blood was obtained from 23 CML patients who had elevated white blood cell counts (105 x 109 cells/liter to 420 x 109 cells/liter) and had not been treated with any inhibitors, 10 of whom subsequently developed IM resistance. CB cells were obtained from mothers undergoing cesarean delivery of healthy, full-term infants. In all cases, informed consent was obtained, and the procedures used, including the Declaration of Helsinki protocol, were approved by the Research Ethics Board of the University of British Columbia (Vancouver).
Lin–CD34+ CB cells from six individuals were isolated using the EasySep cell separation kit (Stem Cell Technologies) according to the manufacturer's instruction. The purity of the enriched cells was measured by FACS as >95%. FACS isolation of BaF3 cells, Sca-1+lin– mouse BM cells, and K562 cells transduced with GFP, YFP, or GFP and YFP using a FACStar Plus or FACSVantage (BD Biosciences) was previously described (46).
Transduction of murine and human hematopoetic cells.
BM cells from 6–10-wk-old C57BL/6 mice were harvested 4 d after injection of 150 mg/kg 5-FU. Low-density cells were isolated and cultured for 48 h in serum-free medium (SFM) supplemented with BIT (StemCell Technologies), to which 100 ng/ml mouse Steel Factor (SF; STEMCELL Technologies), 10 ng/ml mouse IL-3 (StemCell Technologies), and 10 ng/ml human IL-6 (Cangene) were also added. The BM cells, as well as IL-3–dependent BaF3 cells, were then collected and resuspended at 5 x 105 cells/ml in VCS diluted 1:3 in DMEM containing 15% FCS, protamine sulfate (5 µg/ml), and SF, IL-3, and IL-6 at the same concentrations as described above. BaF3 cells were cultured with IL-3 only. The cells were then transferred to Petri dishes that had been precoated with fibronectin (Sigma-Aldrich) and preloaded with virus as previously described (46).
CD34+ CB cells were prestimulated with a cocktail of cytokines and transduced with virus as previously described (25). BCR-ABL–transduced cells were sorted for GFP+ cells after 3 d of culture and sorted GFP+BCR-ABL+ cells were retransduced with either control or AHI/sh4 virus. Cells transduced with only control or AHI/sh4 were cultured for 6 d before FACS sorting. The GFP+YFP+ cells (BCR-ABL+AHI-1/sh4+) were FACS-sorted after 3 d of expansion. Lin–CD34+ CML cells were prestimulated and transduced with either control or AHI/sh4 virus in a similar manner to that described for CB cells. After 3–4 d of expansion, YFP+CD34+ cells were FACS sorted.
Cell culture.
FACS-sorted cells were cultured in SFM with or without GFs. At various time points, viable cell numbers were determined by hemocytometer counts of trypan blue-excluding cells. For single cells sorting, YFP+–transduced K562 cells were sorted into the wells of a 96 well plate by FACS. Subsequent clone formation (presence of 
2 refractile cells) and clone size (total number of refractile cells) in wells seeded with single viable (PI–) cells was assessed by direct visual inspection using an inverted microscope.
Proliferation assays.
Aliquots of 2 x 104 cells were cultured in triplicate in round bottom wells of 96-well Falcon plates in 100 µl media per well. The cells were incubated for 24 and 48 h at 37°C. The amount of tritiated [3H]thymidine incorporated during a 4-h pulse of the culture was measured. In brief, 1 µCi of tritiated [3H]thymidine was added to each well. 4 h later, the cells were harvested onto a membrane using a Skatron instruments combi Harvester (LKB Wallace-PerkinElmer). The amount of tritiated [3H]thymidine was measured with a LKB Betaplate scintillation counter.
Apoptosis analysis.
The apoptosis analysis was performed using an Apoptosis Detection kit (BD Biosciences) following the manufacturer's instruction. In brief, aliquots of 1 x 105 cells were washed twice with PBS, resuspended in 100 µl binding buffer, and incubated with 5 µl of 7-amino-actinomycin D and 5 µl of PE-conjugated Annexin V antibody at room temperature in the dark for 15 min. 400 µl binding buffer was added for FACS analysis.
Progenitor assays.
CFCs and LTC-ICs for mouse BM cells were assayed using culture media from Stem Cell Technologies. Colony counts were performed, using standard scoring criteria, on the basis of the ability to produce colonies containing a minimum of 20 cells after 12 to 14 d of incubation (49). LTCs were set up and maintained using preestablished irradiated mouse marrow LTC feeder layers and weekly half medium changes of LTC medium as previously described (46). CFC assays for transduced K562 cells, human CB cells and CML cells were performed in FCS-containing methylcellulose medium (StemCell Technologies) in the presence or absence of GFs ± imatinib (0.1–10 µM), dasatinib (10–150 nM), and NL (0.1–10 µM).
Q-RT-PCR.
Total RNA was extracted from aliquots of cells using the Absolutely RNA Microprep kit (Stratagene) or TRIzol (Invitrogen), and RT reactions were performed as previously described (46). Real-time PCR was performed using 25 µl of 2X SYBR Green PCR Master Mix (Applied Biosystems), 1 µl of 20 pM of specific primers, 1–2 µl cDNA, and water to a final volume of 50 µl, as previously described (19). Specific forward and reverse primers to detect total expression levels were as follows: AHI-1, 5'-GCCGAGATAGCCCGGTTTATC-3' and 5'-TCAGTTCGGTGAATGTAAACTCC-3'; BCR-ABL, 5'-CATTCCGCTGACCATCAATAAG-3' and 5'-GATGCTACTGGCCGCTGAAG-3'; GAPDH, 5'-CCCATCACCATCTTCCAGGAG-3' and 5'-CTTCTCCATGGTGGTGAAGACG-3'. Optimal conditions for the amplification of these gene products and for normalization of the Ct values of the gene of interest to that of GAPDH were previously described (19).
Immunoprecipitation and Western blot analyses.
Cells were lysed in PBS as previously described (19). For immunoprecipitation, protein extracts were diluted to 1 µg total protein/µl with PBS containing proteinase inhibitors, and specific antibodies were added and incubated at 4°C overnight. The immunocomplexes were then captured by protein G bead slurry, rocked, and separated from the beads by washing. The samples were then heated at 70°C for 10 min before Western blotting. Protein lysates were then separated on NuPAGE Novex bis-tris gels (Invitrogen), and the filters were probed with specific antibodies as previously described (19). Primary antibodies used in these studies included a monoclonal anti-ABL antibody (8E9; BD Biosciences), an antiphosphotyrosine antibody (4G10; Millipore), anti–phospho-JAK2 (Y1007/Y1008) and anti-JAK2 antibodies (Millipore) in transduced BaF3 cells, anti–phospho-JAK2 (Y1007/Y1008) and anti-JAK2 antibodies (Cell Signaling Technology) in transduced K562 cells, anti–phospho-Src (Tyr 416) and anti-Src antibodies (Cell Signaling Technology), anti–phospho-Akt and anti-Akt antibodies (Cell Signaling Technology), anti–phospho-STAT5 (Tyr694) and anti-STAT5 antibodies (Cell Signaling Technology), anti–phospho-NF-B p65 and anti- NF-B p65 antibodies (Cell Signaling Technology), polyclonal anti–Ahi-1 antibody (C-terminal) (23), C-terminal monoclonal anti–Ahi-1 antibody (C-mAhi-1 M5) generated by a standard procedure as previously described (50), and polyclonal N-terminal AHI-1 antibody, IMX-3395 (IMGENEX). Relative expression levels were normalized to expression levels of human actin (Sigma-Aldrich) after quantification with ImageQuant software. The peptide competition assay was completed as per an online protocol (http://www.rockland-inc.com/commerce/misc/methods) by Rockland Immunochemicals, Inc.
Animals.
NOD/SCID-β2m–/– mice were bred and maintained in microisolator cages and provided with autoclaved food and water. Mice were irradiated at 8–10 wk of age with 350 cGy 137Cs 
-rays. Cells were injected into mice intravenously a few hours after they had been irradiated. Mice were monitored daily for signs of weight loss or lethargy. Peripheral blood was collected every 1–2 wk, and leukocytes were analyzed by FACS as previously described (46). For tumor formation studies, 10 million cells were injected subcutaneously into the left and right flank of mice, and the mice were monitored daily for the tumor growth. The tumor volume was calculated from the greatest transverse (width) and longitudinal (length) diameter of the tumor using the formula: tumor volume = length x width2/2. All animal experiments were performed in the Animal Resource Centre of BC Cancer Research Centre, and the procedures used were approved by the Animal Care Committee of the University of British Columbia (Vancouver).
Statistical analysis.
Results are shown as the mean ± SEM of values obtained in independent experiments. Differences between groups were assessed using the Student's t test for paired samples.
Online supplemental material.
Fig. S1 shows that overexpression of Ahi-1 confers growth advantage on BaF3 cells and enhances effects of BCR-ABL with increased protein expression and tyrosine phosphorylation of p210BCR-ABL. Fig. S2 shows that Ahi-1 and BCR-ABL–transduced cells purified from BM cells of diseased mice have increased proliferative and antiapoptotic activities and these effects can be enhanced by BCR-ABL. The online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20072316/DC1.
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Acknowledgments |
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This work was supported by grants from the Cancer Research Society and in part by the Leukemia and Lymphoma Society of Canada (X. Jiang). Y. Zhao was a recipient of a Stem Cell Network Fellowship and a Leukemia and Lymphoma Society of Canada Fellowship and A. Ringrose was a recipient of a BC Cancer Studentship. D. DeGeer received a Medical Genetics Graduate Entrance Studentship from University of British Columbia and a BC Cancer Agency Recruitment Training Incentive Studentship. E. Kennah was a recipient of a Medical Genetics Graduate Entrance Studentship from University of British Columbia. X. Jiang is a Michael Smith Foundation for Health Research Scholar.
The authors have no conflicting financial interests.
Submitted: 29 October 2007
Accepted: 19 September 2008
© 2008 Zhou et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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