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Mol Cell Biol. Feb 2000; 20(4): 1179–1186.

The Survival Function of the Bcr-Abl Oncogene Is Mediated by Bad-Dependent and -Independent Pathways: Roles for Phosphatidylinositol 3-Kinase and Raf


The Bcr-Abl tyrosine kinase constitutively activates cytokine signal transduction pathways that stimulate growth and prevent apoptosis in hematopoietic cells. The antiapoptotic action of interleukin-3 (IL-3) has been linked to a signaling pathway which inactivates the proapoptotic protein Bad by phosphorylation through kinases such as Akt and Raf. Here we report also that expression of Bcr-Abl leads to phosphorylation of Bad in hematopoietic cells. Bad phosphorylation induced by Bcr-Abl is kinase dependent, requires phosphatidylinositol 3-kinase (PI3-kinase), and mitochondrial targeting of Raf, and occurs independently of Erk. The ability of Bcr-Abl to confer cytokine-independent survival to hematopoietic cells was compromised by inhibitors of PI3-kinase, as well as by a dominant negative form of Raf targeted to the mitochondria. Furthermore, when the capacity of Bcr-Abl to phosphorylate Bad was completely blocked by dominant negative Raf, a subpopulation of cells remained viable, providing evidence for Bad-independent survival pathways. This alternative survival pathway remained PI3-kinase dependent. Finally, Bcr-Abl, but not IL-3, inhibited the proapoptotic activity of overexpressed Bad. We conclude that the antiapoptotic function of Bcr-Abl is mediated through pathways involving PI3-kinase and Raf and that survival can occur in the absence of Bad phosphorylation.

Chronic myelogenous leukemia (CML) is a hematopoietic disorder which shows features of enhanced myeloid cell survival early during the chronic phase and uncontrolled mitogenesis during late-stage blast crisis. The initiating event in CML is the Philadelphia chromosome translocation which creates a constitutively active cytoplasmic thymidine kinase (TK) encoded by the Bcr-Abl fusion gene (21). Bcr-Abl induces mitogenesis in fibroblast and hematopoietic cell transformation models (reviewed in reference 31) and protects cells from apoptosis induced by numerous stimuli including cytokine withdrawal, DNA damage, and Fas activation (2, 3, 12, 2426). Similar to studies of cytokine and TK receptors, mutational analysis of Bcr-Abl suggests that signals responsible for protection from apoptosis may be separable from those responsible for transformation (7). Bcr-Abl activates Ras (23), Raf (27, 41), Myc (6, 35), Stat (5, 18, 38), Jun (32), phosphatidylinositol 3-kinase (PI3-kinase) (39, 40), and Akt (39) but not Erk (19). Of these, Ras (36), Raf (41), Myc (35), PI3-kinase (39, 40), Akt (39), and c-Jun N-terminal kinase (11) and its substrate Jun (32) are critical for transforming activity. The mechanism for the antiapoptotic effect of Bcr-Abl is less clear, and the connections between Bcr-Abl and the apoptosis machinery are just beginning to be appreciated.

Mitochondria are known to play a central role in the control of apoptosis (reviewed in reference 15a); therefore, there has been intense interest in defining the pathways responsible for transmitting survival-promoting signals from cell surface receptors to the mitochondria. One such pathway leads to inactivation of the proapoptotic Bcl-2 family protein Bad through activation of kinases such as Akt (9, 10) or Raf (43, 46). Nonphosphorylated Bad binds to Bcl-XL, inhibits its antiapoptotic function, and promotes cell death (44). Survival signals mediated by cytokines such as interleukin-3 (IL-3), nerve growth factor (NGF), or insulin-like growth factor 1 promote phosphorylation of Bad through a PI3-kinase/Akt-dependent pathway (9, 10). The consequence of Bad phosphorylation on two residues (Ser112 and Ser136) is inhibition of binding to Bcl-XL and sequestration in the cytosol by the phosphoserine-interacting protein 14-3-3 (46). Bad phosphorylation at serine 136 is mediated by Akt. While the identities of kinases responsible for phosphorylation of Bad at other residues remain elusive, recent evidence strongly point to the role of protein kinase A (PKA) for phosphorylation of Bad at residue 112 (16).

The PI3-kinase/Akt/Bad pathway represents a well-established and important bridge between the extracellular survival signal and modulators of mitochondrially initiated apoptosis. However, evidence exists that the survival function of cytokines cannot be fully explained by this single mechanism. For example, IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) activate similar signaling pathways in hematopoietic cells through a common β subunit, yet GM-CSF alone can prolong survival in the absence of PI3-kinase or PKB activity, with Bad phosphorylation status remaining unperturbed (37). Similar results have been observed in primary neurons, where NGF can maintain survival in the absence of PI3-kinase activity (29). In addition, activation of Akt does not necessarily result in phosphorylation of Bad. The cytokine IL-4 clearly activates the PI3-kinase/Akt pathway without induction of Bad phosphorylation (37). These observations collectively point to the importance of Akt-independent as well as Bad-independent survival pathways in response to cytokines.

Because Bcr-Abl activates many of the same signaling pathways as cytokines such as IL-3 and GM-CSF, we investigated the role of Bad phosphorylation as a mediator of the Bcr-Abl survival function. We find that Bad is phosphorylated in cells expressing Bcr-Abl in a kinase-dependent fashion. This phosphorylation requires PI3-kinase, Ras, and Raf. Similarly, the ability of Bcr-Abl to promote survival of hematopoietic cells in the absence of IL-3 requires PI3-kinase and mitochondrial targeting of Raf. Interestingly, some Bcr-Abl-expressing hematopoietic cells expressing dominant negative mitochondrion-targeted Raf can survive despite complete inhibition of Bad phosphorylation, revealing a Bad-independent survival pathway. Finally, we have observed that Bcr-Abl counteracts the proapoptotic function of Bad by allowing stable overexpression of Bad in hematopoietic cells whereas IL-3 does not. These findings collectively establish novel mechanisms for the antiapoptotic function of Bcr-Abl in hematopoietic cells via Bad-dependent as well as Bad-independent pathways.


Cells and plasmids.

293 human embryonic kidney cells were grown in Dulbecco modified Eagle medium supplemented with 10% calf serum (HyClone), penicillin, streptomycin, and glutamine. FL5.12 IL-3-dependent hematopoietic cells (46) were grown in RPMI medium with 10% fetal bovine serum (Omega Scientific), penicillin-streptomycin, glutamine, and 10% WEHI-3 supernatant as a source of IL-3. For Bad phosphorylation studies, FL5.12 cells were infected with retrovirus expressing Flag-Bad or hemagglutinin (HA) epitope-tagged Bad and selected in puromycin or G418. The retroviral expression vectors pSRαMSV Bcr-Abl tkNeo (p185 form) and pSRα RasN17 tkNeo have been previously described (36). pSRαMSV DN Erk1 tkNeo was constructed by subcloning a NotI fragment containing the HA epitope-tagged kinase-inactive mutant of Erk1 (K72R) from pCEP4 (13) into pSRαMSVtkNeo. pCDNA3 DN M-Raf-1 and pCDNA3 HA-Bad have been previously described (43). DN M-Raf-1 (a kinase-inactive dominant negative mutant of Raf-1 fused with the transmembrane domain from the yeast outer mitochondrial membrane protein Mas70) was subcloned into pSRαMSVtkNeo by PCR. The DN Akt plasmid has been described previously (42).

Bad phosphorylation and CAT assays.

FL5.12 cells expressing Bad and/or Bcr-Abl were cultured for 2 h in the absence of IL-3 and then labeled with [32P]orthophosphate for 2 more h. IL-3 was added for 15 min. Bad was immunoprecipitated with anti-Flag or anti-HA antibody and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using autoradiography or immunoblot analysis. In some experiments, wortmannin or LY294002 diluted in dimethyl sulfoxide (DMSO) was added 30 min prior to phosphate labeling to a final concentration of 0 (DMSO alone), 0.1, or 1.0 μM. 293 cells (70% confluent in 10-cm-diameter dishes) were transfected overnight with pCDNA3 HA-Bad expression plasmid with or without Bcr-Abl and/or the various dominant negative expression plasmids, using the calcium phosphate method. Cells were starved of serum for 24 h prior to harvesting at 48 h. Cell lysates were prepared in a buffer containing 20 mM HEPES (pH 7.4), 1% Triton X-100, 0.5% SDS, 100 μg of phenylmethylsulfonyl fluoride per ml, 10 μg of leupeptin per ml, 1 mM NaVO4, 25 mM β-glycerophosphate, and 10 mM NaF. Lysates were adjusted to 0.05% SDS and centrifuged at 25,000 × g to pellet DNA and nonsolubilized debris. Cell lysates were separated by SDS-PAGE (10% gel), and HA-Bad phosphorylation was monitored by Western blotting as previously described (43) with monoclonal antibody 12CA5 (Boehringer). Chloramphenicol acetyltransferase (CAT) assays were carried out as previously described (33), using the Ras-responsive reporter construct pB4X-CAT (28) and pEXV-Raf-CAAX expression plasmid (30) as an activator. Pan-Bad antibody was purchased from Santa Cruz Biotechnology, and phosphospecific Bad antibody was kindly provided by Michael Greenberg (Children's Hospital, Boston, Mass.).

Biological assays.

FL5.12 cells were washed three times and plated in medium without IL-3 for all IL-3 withdrawal experiments. The number of viable cells was measured by trypan blue exclusion, and apoptosis was documented by propidium iodide staining and fluorescence-activated cell sorting analysis. For wortmannin or LY294002 experiments, concentrations of 0 (DMSO alone), 0.1, and 1.0 μM were used. FL5.12 cells stably expressing DN M-Raf-1 were derived by retroviral infection and selection in the antibiotic G418 or puromycin. Expression of DN M-Raf-1 was confirmed with anti-C-terminal Raf-1 antibody (Santa Cruz). Bcr-Abl expression was measured using the anti-Abl monoclonal antibody pex5 (15). FL5.12 cell lines were infected with Bcr-Abl retrovirus for 2 days, washed in serum-free medium, and then plated at 200 cells/well into 96-well plates in complete medium with and without IL-3. Individual wells were examined daily by light microscopy and scored negative (−; <200 cells), positive (+; 200 to 1,000 cells), or strongly positive (++; >1,000 cells) based on the number of viable cells. The accuracy of this scoring system was validated by performing cell counts on randomly selected wells in a blinded fashion.


Bad is constitutively phosphorylated in hematopoietic cells expressing Bcr-Abl in the presence or absence of IL-3.

Since Bcr-Abl can replace the IL-3 requirement in a number of cytokine-dependent hematopoietic cell lines, we asked if Bcr-Abl activated a pathway leading to Bad phosphorylation in FL5.12 cells, an IL-3 dependent pre-B hematopoietic cell used previously to characterize the IL-3-induced phosphorylation of Bad (10, 46). Parental FL5.12 cells or FL5.12 cells expressing Bcr-Abl were starved of IL-3 for 2 h and then cultured in [32P]orthophosphate-containing medium in the presence or absence of IL-3, and the phosphorylation state of transfected Bad was examined by autoradiography following immunoprecipitation. Immunoblot analysis confirmed equivalent levels of immunoprecipitated Bad protein (Fig. (Fig.1A,1A, bottom panel). In parental cells, Bad phosphorylation was induced by exposure to IL-3 (Fig. (Fig.1A),1A), consistent with previous reports (10, 46). In Bcr-Abl-expressing cells, Bad remained phosphorylated even in the absence of IL-3 (Fig. (Fig.1A).1A). These results demonstrate that Bad is constitutively phosphorylated in hematopoietic cells expressing Bcr-Abl.

FIG. 1
Bad is constitutively phosphorylated in hematopoietic cells expressing Bcr-Abl in the presence or absence of IL-3. (A) FL5.12 cells infected with retrovirus expressing Bcr-Abl or the parental vector (Neo) were superinfected with retrovirus expressing ...

We confirmed these findings in a transient transfection model. Phosphorylated Bad migrates more slowly in SDS-polyacrylamide gels and is easily detected as a band shift in Western blots (43, 46). 293 cells were cotransfected with plasmids expressing HA-Bad and either Bcr-Abl or a control vector, and lysates were analyzed after 48 h by Western blotting using an anti-HA antibody. In control cells transfected with HA-Bad alone, two bands representing phosphorylated (upper band) and unphosphorylated (lower band) forms are visible (Fig. (Fig.1b,1b, lane 1). Consistent with the effects observed in hematopoietic cells, Bcr-Abl significantly increased the level of hyperphosphorylated Bad (Fig. (Fig.1B,1B, lane 2). This effect requires the TK activity of Bcr-Abl since a kinase-inactive mutant of Bcr-Abl (15) failed to induce phosphorylation of Bad despite levels of protein expression comparable to those for wild-type Bcr-Abl (Fig. (Fig.1B,1B, lane 3). These results confirm and extend the finding in hematopoietic cells that Bcr-Abl activates a pathway leading to Bad phosphorylation.

Bcr-Abl phosphorylation of Bad on serine 136 is PI3-kinase and Akt dependent.

The serine/threonine kinase Akt is known to phosphorylate Bad on serine 136 through a signaling pathway involving PI3-kinase (9, 10). Since Bcr-Abl can activate a PI3-kinase/Akt pathway (39), we tested whether Bad is phosphorylated on serine 136, using a phosphospecific antibody for this residue in hematopoietic cells expressing Bcr-Abl (9). In parental FL5.12 cells starved of IL-3 for 3 h, endogenous Bad protein was visualized with an antiserum that detects phosphorylated or unphosphorylated protein (anti-Bad) (Fig. (Fig.2A,2A, bottom). Using the phosphospecific anti-Bad 136 antiserum, Bad could not be detected after IL-3 withdrawal, indicating that Bad was no longer phosphorylated on serine 136 (Fig. (Fig.2A,2A, lane 1). However, in cells expressing Bcr-Abl, Bad was detected with both antisera, indicating constitutive phosphorylation of Bad at serine 136 (Fig. (Fig.2A,2A, lane 2).

FIG. 2
Bcr-Abl-mediated Bad phosphorylation occurs on Ser136 in a PI3-kinase-dependent manner. (A) Parental or Bcr-Abl-expressing FL5.12 cells were starved of IL-3 for 3 h. Bad protein was examined in whole-cell lysates by immunoblotting using a phosphospecific ...

We investigated the pathway which leads to phosphorylation of Bad at Ser136 in Bcr-Abl-expressing FL5.12 cells and in transient transfection assays of 293 cells. Increasing doses of the PI3-kinase inhibitor LY294002 or wortmannin caused a 10-fold reduction in the level of Bad phosphorylation in Bcr-Abl-expressing hematopoietic cells (Fig. (Fig.2B)2B) and in 293 cells cotransfected with Bcr-Abl and Bad (data not shown). To directly assess the role of the serine/threonine kinase Akt, we also overexpressed a dominant negative mutant (Akt R197M) along with Bcr-Abl and Bad. It should be noted that this kinase-inactive mutant of Akt becomes phosphorylated in response to PI3-kinase activation since it retains residues Ser473 and Thr308. As expected, the PI3-kinase pathway was active in these cells, as measured by the level of 32P incorporation in the Akt R197M mutant immunoprecipitated with an HA antibody (Fig. (Fig.2C).2C). We find that Bcr-Abl-mediated Bad phosphorylation was inhibited by expression of DN Akt. We conclude that Bad is phosphorylated in Bcr-Abl-expressing cells through the PI3-kinase/Akt pathway at residue 136. These results confirm and extend previous reports implicating the PI3-kinase/Akt pathway in Bcr-Abl function (39).

Bcr-Abl-mediated Bad phosphorylation is Raf and Ras dependent but Erk independent.

We examined the role of the Ras/Raf/Erk signal transduction pathway in the phosphorylation signal from Bcr-Abl to Bad since transformation by Bcr-Abl is dependent on activation of the Ras pathway (15, 28, 36). Coexpression of a dominant negative mutant of Ras diminished the effect of Bcr-Abl on Bad phosphorylation (Fig. (Fig.3A,3A, lane 2). To determine whether mitochondrial targeting of Raf is required for Bcr-Abl-mediated phosphorylation of Bad, cells were cotransfected with DN M-Raf-1. This mutant localizes to mitochondria, blocks endogenous Raf activity at the mitochondrial surface, and interferes with the antiapoptotic activity of IL-3 (43). DN M-Raf-1 completely blocked the effect of Bcr-Abl on Bad phosphorylation (Fig. (Fig.3A,3A, lane 3), whereas a kinase-inactive dominant negative mutant of Erk2 (DN Erk) (13) did not (Fig. (Fig.3A,3A, lane 4). DN Erk did, however, inhibit the activation of an Erk-dependent reporter gene by membrane-targeted Raf-CAAX (Fig. (Fig.3B).3B). These results are consistent with Bcr-Abl activating a Ras-dependent, Raf-dependent, Erk-independent mitochondrial pathway leading to Bad phosphorylation.

FIG. 3
Bcr-Abl induces the phosphorylation of the Bcl-2 family member BAD in a Ras- and Raf-dependent but Erk-independent manner. (A) 293 cells were cotransfected with HA-Bad plus Bcr-Abl and Neo (lane 1), DN Ras (lane 2), DN M-Raf-1 (lane 3), or DN Erk (lane ...

Inhibition of Bad phosphorylation impairs the survival function of Bcr-Abl in hematopoietic cells.

It has previously been established that Bcr-Abl protects hematopoietic cells from apoptosis due to cytokine withdrawal (12) and induces cytokine-independent growth (8, 17). To further examine the importance of Bad phosphorylation in Bcr-Abl survival function, FL5.12 cells were monitored for their survival and growth upon acute infection with a retroviral vector harboring DN M-Raf-1. The biological effect of Bcr-Abl was measured in a single-step quantitative assay by plating Bcr-Abl-infected cells into 96-well tissue culture plates in the absence of IL-3. Each well was examined daily by light microscopy and scored as negative, positive, or strongly positive (see Materials and Methods) for 10 consecutive days. Of note, DN M-Raf-1 did not affect the survival or growth of FL5.12 cells in the presence of IL-3 (Fig. (Fig.4A),4A), indicating that expression of this gene is not generally toxic to cells. In the absence of Bcr-Abl, no viable cells were observed after 48 h postplating, whereas 100% of wells containing parental FL5.12 cells infected with Bcr-Abl scored strongly positive by day 6 (Fig. (Fig.4A).4A). In contrast, coinfection of DN M-Raf-1 blocked the survival function of Bcr-Abl in close to 50% of the wells (Fig. (Fig.4A,4A, column 1). In the remaining half, the number of viable cells was clearly decreased.

FIG. 4
Bcr-Abl confers a survival signal to FL5.12 cells that requires mitochondrial targeting of Raf. Populations of FL5.12 cells expressing Neo or DN M-Raf-1 were infected with Bcr-Abl or control retrovirus (Neo) for 48 h and plated at 200 cells/well in microtiter ...

To further examine this population, stable lines were derived from FL5.12 cells coexpressing DN M-Raf-1 and Bcr-Abl. Growth of these cells was shown to be five- to sevenfold slower than that of cells solely expressing Bcr-Abl (Fig. (Fig.5A).5A). Furthermore, no increased rate of apoptosis was detected in this subpopulation, as judged by trypan blue staining (data not shown). Therefore, overexpression of DN M-Raf-1 impaired the growth but not the survival of this subpopulation of Bcr-Abl-expressing cells. Interestingly, within limits of detection of our assay, no phosphorylated Bad was detected in the six independent stable lines expressing Bcr-Abl and DN M-Raf-1 (Fig. (Fig.5B5B and data not shown). This result suggests the existence of a survival pathway in hematopoietic cells that can bypass the need to phosphorylate and deactivate Bad.

FIG. 5
A subpopulation of hematopoietic cells coexpressing Bcr-Abl and DN M-Raf-1 remain viable in the absence of Bad phosphorylation. Stable FL5.12 hematopoietic cell lines were derived from Bcr-Abl-expressing cells after retroviral infection with DN M Raf-1. ...

Inhibition of PI3-kinase interferes with the survival function of Bcr-Abl.

To determine the effects of the PI3-kinase/Akt pathway on growth and survival in the FL5.12 hematopoietic model, we measured the viability of parental or Bcr-Abl-expressing cells starved of IL-3 in the presence of various concentrations of LY294002 or wortmannin. As expected, about 50% of parental cells were dead within 24 h of IL-3 withdrawal (Fig. (Fig.6A).6A). Bcr-Abl-expressing FL5.12 cells proliferated in the absence of IL-3, and this growth was inhibited by LY294002 (Fig. (Fig.6B)6B) and wortmannin (data not shown). LY294002 similarly blocked the growth of stable lines escaping the inhibitory effect of DN M-Raf-1 on Bcr-Abl (Fig. (Fig.6C).6C). We conclude that Bcr-Abl-induced survival functions are PI3-kinase dependent and include Bad-dependent as well as Bad-independent pathways.

FIG. 6
The PI3-kinase inhibitor LY294002 impairs the cytokine-independent growth Bcr-Abl-expressing hematopoietic cells. Parental (A), Bcr-Abl-expressing (B), or Bcr-Abl/DN M-Raf-1-expressing (C) FL5.12 cells were cultured with the PI3-kinase inhibitor LY294002 ...

To explore the potential connections between the Raf and PI3-kinase pathways, we expressed a dominant active form of mitochondrial Raf (DA M-Raf-1) in FL5.12 hematopoietic cells. Prior work has established that DA M-Raf-1 induces Bad phosphorylation and delays apoptosis in hematopoietic cells (43). In our hands, DA M-Raf-1 also extended survival of FL5.12 cells for at least 24 h after IL-3 withdrawal (Fig. (Fig.7).7). This survival was independent of PI3-kinase since wortmannin doses up to 1 μM did not significantly affect viability. Taken together with the DN M-Raf-1 data, these findings lead us to conclude that M-Raf-1 can rescue the proapoptotic consequences of PI3-kinase inhibition and that phosphorylation of Bad at mitochondria is sufficient but not necessary for survival.

FIG. 7
Mitochondrial targeting of Raf confers survival in FL5.12 cells independently of PI3-kinase activity. FL5.12 cells infected with retrovirus expressing DA M-Raf-1 (FL:M.RAF) were subjected to drug selection for 14 days in the presence of IL-3. The viability ...

Bcr-Abl allows cells to retain high-level expression of Bad protein.

Overexpression of wild-type Bad in FL5.12 cells induces apoptosis (44). Mutations in the BH3 domain (Bcl-2 homology domain 2) of Bad impair apoptotic activity, supporting the concept that the proapoptotic function of Bad is most likely mediated by complex formation with other Bcl-2 family members such as Bcl-XL or Bcl-2, which leads to inhibition of their antiapoptotic function (20, 45). One prediction of our observation that Bcr-Abl survival pathways overcome the proapoptotic effects of Bad is that cells expressing Bcr-Abl should tolerate high levels of Bad protein expression. To test this hypothesis, we infected parental or Bcr-Abl-expressing FL5.12 cells with retroviruses expressing HA-Bad and then derived stable lines by selection in the antibiotic G418 in the presence of IL-3. Ectopic expression of HA-Bad was demonstrated by immunoblot analysis in both populations immediately after the infection (data not shown) and after 2 weeks of G418 selection (Fig. (Fig.8,8, Early). However, HA-Bad expression could no longer be detected after 4 weeks of passage in three independent experiments, despite continued selection in IL-3 and G418 (figure 8, Middle and Late). These results indicate selection against high-level expression of Bad, consistent with its proapoptotic function. The fact that this counterselection occurs even in the presence of cytokine argues that IL-3, which activates a pathway leading to Bad phosphorylation (10), is insufficient to overcome the Bad-mediated proapoptotic signal when the protein is expressed at high levels. Indeed, in previous reports of constitutive Bad overexpression in hematopoietic cells, investigators have overexpressed Bcl-2 in the same cells to counteract the effects of Bad overexpression (10, 46). In contrast to the parental line, Bcr-Abl-expressing FL5.12 cells maintained high-level HA-Bad expression throughout the period of drug selection (Fig. (Fig.8)8) and continued to do so for several months in culture (data not shown). These data indicate that Bcr-Abl blocks counterselection against high-level Bad expression whereas IL-3 does not. Taken together with the biochemical experiments showing PI3-kinase and Raf-dependent Bad phosphorylation, these data provide functional evidence for inactivation of Bad activity by Bcr-Abl.

FIG. 8
Bcr-Abl prevents selection against Bad overexpression in hematopoietic cells. Parental or Bcr-Abl-expressing FL5.12 cells were infected with retrovirus expressing HA-Bad/Neo and cultured in G418 to select for infected cells. The level of HA-Bad was measured ...


Although it is clear that cytokines and growth factors deliver signals to the apoptotic machinery that promote cell survival, the details of how these survival signals are delivered is not fully examined. To date, two general mechanisms have been implicated: modulation of the level of antiapoptotic proteins such as Bcl-2 and Bcl-XL (reviewed in reference 14) and inhibition of the activity of proapoptotic proteins such as Bad through posttranslational modifications (43, 46). Since Bcr-Abl protects hematopoietic cells from apoptosis in response to a wide range of stimuli including cytokine withdrawal, chemotherapy, irradiation, and Fas activation (2, 3, 12, 2426), it is likely to function at a distal point in the apoptosis pathway, perhaps involving Bcl-2 family members. Previous work has shown that some cell lines expressing Bcr-Abl have elevated levels of either Bcl-2 (34) or Bcl-XL (4). Here we show that Bcr-Abl also activates a pathway that leads to phosphorylation of Bad. These observations provide evidence for posttranslational modification of the apoptosis machinery as a mechanism of enhanced cell survival in CML. Once phosphorylated, the proapoptotic Bad protein is unable to interact with and inhibit Bcl-2 or Bcl-XL (46); therefore, the antiapoptotic effects of these proteins are unopposed. In conjunction with previously documented effects of Bcr-Abl on transcription of Bcl-2 (34) and Bcl-XL (4), the effects of Bad allow Bcr-Abl to effectively tilt the balance of competing death versus survival signals in favor of survival.

Our data further clarify the role of the role of the PI3-kinase/Akt pathway in the survival function of Bcr-Abl. Bcr-Abl induces Bad phosphorylation at residue 136, the consensus site for the kinase Akt. In addition, a dominant negative form of Akt as well as inhibitors of PI3-kinase effectively blocked Bcr-Abl-induced phosphorylation of Bad. The survival function of Bcr-Abl was also impaired in the presence of PI3-kinase inhibitors. These results are in agreement with established antiapoptotic mechanisms of cytokines such as IL-3. However, our findings also establish the existence of PI3-kinase-dependent, Bad-independent signaling pathways downstream of Bcr-Abl. Stable overexpression of DN M-Raf-1 in Bcr-Abl allowed us to isolate a subpopulation of cells which remained viable in the absence of detectable Bad phosphorylation. This result indicates that an alternative PI3-kinase-dependent pathway, also activated by Bcr-Abl, can bypass the need to deactivate Bad through phosphorylation. Certain cytokines such as IL-4 may also confer survival without affecting Bad phosphorylation (37).

The fact that a dominant negative mutant of Raf-1 targeted to mitochondria also blocks Bcr-Abl-induced Bad phosphorylation raises the question of how the Raf and the PI3-kinase/Akt pathways are connected. First, it is possible that Raf is part of the PI3-kinase/Akt/Bad pathway. Recent studies have reported that overexpression of the active form of Akt, in the absence of Bcr-Abl, can induce plasma membrane and mitochondrial Raf activation through a PKC-dependent pathway (22). Alternatively, the Raf and PI3-kinase/Akt pathways may transduce signals independently to common effector molecules such as Bad. Several lines of evidence suggest that Bad can be phosphorylated independently of PI3-kinase and Akt. Studies of the GM-CSF receptor demonstrate a PI3-kinase/Akt-independent pathway leading to phosphorylation of Bad (37). Raf can directly phosphorylate Bad even when the characterized phosphorylation sites Ser112 and Ser136 are mutated (J. C. Reed, unpublished data). Our studies of Bcr-Abl suggest that Raf and PI3-kinase exert their antiapoptotic activity through distinct but overlapping pathways. By isolating a population of Bcr-Abl-expressing cells which survive in the setting of Raf inhibition, we were able to demonstrate an effect of PI3-kinase inhibition that is distinct from Raf inhibition. In addition, we have shown that mitochondrion-targeted Raf can phosphorylate Bad and confer a survival signal in hematopoietic cells even in the setting of PI3-kinase inhibition. While further work is required to fully delineate these two signals, the collective data suggest that inhibition of both Raf and PI3-kinase may be required to fully impair the Bcr-Abl survival function. This model is consistent with prior work showing that multiple independent pathways contribute to the transformation activity of Bcr-Abl (1).

In addition to showing that the PI3-kinase and Raf pathways both play a role in Bcr-Abl signaling, our work also provides direct evidence of a functional link between Bcr-Abl and Bad. Specifically, we show that Bcr-Abl allows hematopoietic cells to retain high-level Bad expression. The fact that Bcr-Abl prevents counterselection against high-level Bad expression whereas IL-3 does not argues for important differences between IL-3-mediated and Bcr-Abl-mediated signaling. One possibility is that both signals function through the same pathway(s) but the magnitude of the Bcr-Abl signal is stronger, such that a greater pool of Bad protein becomes phosphorylated and inactivated. Alternatively, Bcr-Abl may activate additional pathways that lead to more efficient inactivation of Bad than IL-3 alone. Bcr-Abl may also modulate the levels of antiapoptotic proteins such as Bcl-2 or Bcl-XL.

In conjunction with recent evidence that survival signals mediated by receptor TKs such as those for NGF, platelet-derived growth factor, and insulin-like growth factor 1 lead to phosphorylation of Bad (9), our findings demonstrate that cytoplasmic TKs can also activate signaling pathways that directly affect the apoptotic machinery. Importantly, our results with Bcr-Abl establish that constitutive activation of these pathways can contribute to the neoplastic state. These findings have important therapeutic implications. Blockade of this survival pathway might halt the accumulation of myeloid cells that occurs in chronic-phase CML and increase their sensitivity to chemotherapeutic drugs.


We thank Michael Greenberg for antibody, Elizabeth Major for help with early phases of this work, Xinyi Wu for assistance with cloning, Lisa Dove for manuscript preparation, and David Chang, Chris Denny, Ke Shuai, and Owen Witte for comments.

This work was supported by grants from the American Cancer Society (C.L.S.), National Institutes of Health (C.L.S. and J.C.R.) and the James S. McDonnell Foundation (C.L.S.). H.G.W. is an AACR Research Fellow in Basic or Translational Research sponsored by the Sidney Kimmel Foundation for Cancer Research. C.L.S. is a Scholar of the Leukemia Society of America.


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