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Departments of Medicine (M.R., T.K.N., S.G.), Biochemistry (S.G., P.D.), and Pharmacology (S.G.), Virginia Commonwealth University, School of Medicine, Richmond, Virginia
Received for publication December 8, 2006.
Accepted for publication June 26, 2007.
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Abstract |
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). Bcr/Abl is a constitutively active tyrosine kinase that signals downstream to multiple survival signaling pathways, including signal transducer and activator of transcription (STAT) 5, mitogen-activated protein kinase kinase-1/2 (MEK1/2)/extracellular signal-regulated kinase-1/2 (ERK1/2), and nuclear factor-
B, which collectively confer on CML cells a survival advantage (Van Etten, 2004). CML treatment has been revolutionized by the development of Bcr/Abl kinase inhibitors, of which imatinib mesylate (IM; Gleevec) is the prototype. IM traps Bcr/Abl in an inactive configuration and potently inhibits Bcr/Abl and other kinases, including c-Kit and PDGF (Buchdunger et al., 2000). Despite its success in chronic phase disease (Druker et al., 2001), patients with CML eventually become refractory to IM through various mechanisms, including diminished drug uptake, bcr/abl amplification and/or increased expression of Bcr/Abl, or the development/pre-existence of kinase domain mutants that prevent drug binding (Walz and Sattler, 2006). The latter mechanism is most commonly encountered in IM-refractory patients (Walz and Sattler, 2006). To circumvent this problem, novel second-generation Bcr/Abl kinase inhibitors (e.g., AMN107, nilotinib and BMS-354825, dasatinib) have been developed that are more potent than IM and are active against most mutations in the phosphorylation loop or ATP-binding site (e.g., E255K, M351T), rendering cells resistant to IM (Talpaz et al., 2006). However, they are ineffective against mutations in the "gatekeeper" region (i.e., T315I) (Talpaz et al., 2006). Therefore, the development of new strategies to eradicate such cells represents a high priority.
The Raf pathway, which activates MEK1/2/ERK1/2, is frequently dysregulated in human cancer (Davies et al., 2002; Rajagopalan et al., 2002). Attention has recently focused on the multikinase inhibitor sorafenib (BAY 43-9006), originally developed as a specific inhibitor of C-Raf and B-Raf (Lyons et al., 2001). However, sorafenib inhibits multiple other kinases, including VEGFR-2, VEGFR-3, PDGFR-
, Flt3, and c-Kit (Wilhelm et al., 2004). Sorafenib is well-tolerated when administered with continuous dosing on a 200-mg, twice-daily schedule (Awada et al., 2005; Strumberg et al., 2005) and inactivates ERK1/2 at these doses. It is noteworthy that steady-state sorafenib plasma levels of 15 to 20 µM have been reported (Awada et al., 2005; Strumberg et al., 2005). Several groups, including our own, have reported that sorafenib potently induces apoptosis in human leukemia cells, including Bcr/Abl+ leukemias, through down-regulation of Mcl-1 (Rahmani et al., 2005a; Yu et al., 2005). Mcl-1, a multidomain member of the Bcl-2 family, promotes the survival of malignant human hematopoietic cells, including multiple myeloma and leukemia cells (Moulding et al., 2000; Derenne et al., 2002). The mechanism by which sorafenib down-regulates Mcl-1 expression involves translation inhibition, a phenomenon associated with dephosphorylation of the eIF4E translation initiation factor (Rahmani et al., 2005a). It is noteworthy that sorafenib-mediated down-regulation of Mcl-1 is independent of MEK1/2/ERK1/2 (Rahmani et al., 2005a; Yu et al., 2005), suggesting that the proapoptotic effects of sorafenib involve actions other than disruption of Raf and downstream signaling pathways.
Current information is lacking concerning the activity of sorafenib against imatinib mesylate-resistant CML cells. Resistance to imatinib mesylate has been characterized as either Bcr/Abl-dependent (Donato et al., 2003; Walz and Sattler, 2006) or Bcr/Abl-independent (Donato et al., 2003; Dai et al., 2004). For example, mutations in the Bcr/Abl kinase domain (e.g., T315I) induce steric changes in the kinase domain that prevent drug binding and reduce or abrogate activity (Gorre et al., 2001); nevertheless, cells remain dependent on Bcr/Abl for survival. A logical approach to the eradication of such cells would be the use of alternative small molecules to inhibit mutant Bcr/Abl. Indeed, certain aurora kinase inhibitors (e.g., VX-680) inhibit Bcr/Abl displaying the T315I mutation and effectively kill highly IM-resistant cells (Young et al., 2006). On the other hand, IM resistance associated with loss of Bcr/Abl, and increased activity of the Src kinase Lyn has been described previously (Donato et al., 2003; Dai et al., 2004). Because such cells have lost Bcr/Abl-dependence, their elimination may require interruption of other survival pathways. The present studies were prompted by a desire to determine whether sorafenib triggers apoptosis in various IM-resistant Bcr/Abl+ leukemic cells and to elucidate mechanisms underlying these actions. Our results indicate that sorafenib potently induces apoptosis in Bcr/Abl+ cells highly resistant to IM through diverse mechanisms, including expression of the T315I Bcr/Abl mutation. These events are associated with a rapid inactivation of STAT5, a transcriptional factor that plays a critical role in myeloid leukemia cells survival, and down-regulation of the antiapoptotic multidomain Bcl-2 family member Mcl-1.
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Materials and Methods |
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). STI571-resistant K562 cells designated K562-STI-R were generated by culturing cells in progressively higher concentrations of STI571. These cells exhibit an STI571 IC50 value 
15-fold greater than parental cells. K562 cells ectopically overexpressing constitutively active STAT5 (Flag-tagged pMX-STAT5A-N642H) and their control empty vector pMX-neo were described previously (Rahmani et al., 2005b). K562 cells, which display a marked reduction in Bcr/abl protein levels (K562-Bcr/Abl–), have been described in detail previously (Dai et al., 2004). Ba/F3 cells expressing wild-type Bcr/Abl (Bcr/Abl-wt) and Bcr/Abl bearing 3 major clinically relevant mutations (E255K, T315I, and M351T) have been described previously (La Rosée et al., 2002).
K562 cells stably expressing short hairpin RNA (shRNA) directed against Bim were generated as follows: Two complementary DNA oligonucleotides containing the targeted sequence reported previously (Malhi et al., 2006; 5'-AATTACCAAGCAGCCGAAGAC-3') were synthesized, annealed, and cloned into pSUPER.retro.neo vector (Oligoengine, Seattle, WA) using standard techniques. An shRNA directed against GFP (ggttatgtacaggaacgca) obtained from Ambion (Austin, TX) was cloned into the pSUPER.retro.neo as described above and served as a control of Bim shRNA-expressing cells. The constructs were verified by DNA sequencing and transfected into K562 cells using Amaxa nucleofector. Stable clones were selected in the presence of 400 µg/ml G418 and screened for reduced Bim expression by Western blot analysis.
Isolation of Patient-Derived Leukemic CD34+ Mononuclear Cells. Bone marrow cells were obtained with informed consent during routine diagnostic procedures from four patients with CML who were treated with IM and displayed progressive disease. Bcr/Abl mutational analysis was performed on two of these samples and did not reveal known mutations. These studies were sanctioned by the Investigational Review Board of Virginia Commonwealth University/Medical College of Virginia. Mononuclear cells were isolated by Ficoll-Hypaque density gradient separation. Mononuclear cells from patients with CML were enriched for CD34+ cells using a Miltenyi microbead separation system (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer's protocol. Leukemic CD34+ mononuclear cells were then diluted into RPMI 1640 medium containing 10% fetal calf serum at a concentration of 106 cells/ml and exposed to drugs as described in the case of continuously cultured cell lines.
Reagents. Sorafenib was provided by Bayer Pharmaceuticals Corporation (West Haven, CT) and the National Cancer Institute, National Institutes of Health (Bethesda, MD). It was dissolved in dimethyl sulfoxide, and aliquots were maintained at –80°C.
Assessment of Apoptosis. Apoptotic cells were routinely identified by Annexin V-fluorescein isothiocyanate staining as described previously (Rahmani et al., 2002). In brief, 105 cells were collected, washed in cold phosphate-buffered saline, and then resuspended in binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2) containing fluorescein-labeled annexin V (BD Pharmingen, San Diego, CA) and propidium iodide. Samples were incubated for 15 min and then analyzed by flow cytometer (FACScan; BD Biosciences, San Jose, CA).
Transient Transfection and Reporter Gene Assay. Transient transfection was performed using Amaxa nucleofector (Koeln, Germany) as described previously (Rahmani et al., 2005b). To determine the transcriptional activity of STAT5, K562 cells were cotransfected with STAT5-luc or its control counterpart TA-luc plasmids encoding firefly luciferase (Panomix, Fremont, CA) and pRL-TK-luc plasmid encoding for Renilla reniformis luciferase using the Amaxa nucleofector. Cells were incubated for 6 h and then treated with indicated agents for an additional 16 h, after which activity of firefly and R. reniformis luciferases were measured using the Dual-Luciferase reporter assay system (Promega, Madison, WI). Values of firefly luciferase activity were normalized to those obtained for R. reniformis luciferase activity. Then the ratios obtained for STAT5-luc were divided by those obtained for TA-luc.
Immunoblotting. Immunoblotting was performed using whole-cell lysates prepared as described in detail previously (Rahmani et al., 2002). The primary antibodies used in this study were Mcl-1 (BD Pharmingen), poly(ADP-ribose) polymerase (PARP; BIOMOL Research Laboratories, Plymouth Meeting, PA), phospho-STAT5 (Tyr694) and phospho-eIF4E (Ser209; Cell Signaling Technology, Danvers, MA), and 
-tubulin (Calbiochem).
Statistical Analysis. The significance of differences between experimental conditions was determined using the Student's t test for unpaired observations.
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Results |
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; Yu et al., 2005). Attempts were therefore undertaken to determine whether sorafenib might be active against cells resistant to imatinib mesylate through various mechanisms. To this end, sorafenib dose-response curves were compared in three cell types: wild-type K562 cells, K562 cells cultured in progressively higher concentrations of IM as described previously (Yu et al., 2002) and that exhibited approximately a 4- to 5-fold increase in Bcr/Abl protein, and imatinib-resistant K562 cells that displayed a reduction in Bcr/Abl expression accompanied by an increase in Lyn activation (Dai et al., 2004) (Fig. 1A). The latter two cell types have been shown previously to exhibit a marked reduction in IM sensitivity (Yu et al., 2002; Dai et al., 2004). However, dose-response curves for sorafenib concentrations of 5 to 20 µM were identical for the three cell lines (P > 0.05 in each case; Fig. 1B). Thus, IM-resistant K562 cells displaying increased Bcr/Abl expression or the development of a Bcr/Abl-independent form of resistance remained fully sensitive to sorafenib.
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), each of the mutant cell lines was highly resistant to IM (1 µM) administered for 24 or 48 h (Fig. 2A). In contrast, there was little or no cross-resistance to sorafenib administered at concentrations of 2.5 to 20 µM for 24 (Fig. 2B) and 2.5 to 15 µM for 48 h (Fig. 2C). Consistent with these results, sorafenib (10 µM) exerted similar effects on PARP degradation in each of the cell lines after 24-h (Fig. 2D) or 48-h drug exposure (data not shown). These findings indicate that Bcr/Abl mutations conferring a high degree of resistance to IM, including the T315I mutation, which effectively protects cells from agents such as dasatinib and AMN107 (Talpaz et al., 2006; von Bubnoff et al., 2006), fail to attenuate sorafenib-induced apoptosis.
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). Furthermore, other Bcr/Abl kinase inhibitors have been shown to inactivate STAT5 (Huang et al., 2002; Fiskus et al., 2006). As a consequence, the effects of sorafenib on STAT5 activation were examined. As shown in Fig. 3A, exposure of K562 cells to sorafenib induced a rapid (i.e., within 2 h) inactivation of STAT5, which persisted throughout the entire exposure interval (16 h, a period in which 40% of cells were apoptotic, data not shown). Levels of total STAT5 decreased only slightly during this interval. In contrast, dephosphorylation of Crkl, a known target of Bcr/Abl (ten Hoeve et al., 1994), was only modest and was most prominent at later exposure intervals (e.g., 16 h). Further analysis comparing the effects of sorafenib and the MEK1/2 inhibitors U0126 and PD184352 on phosphorylation of ERK1/2 and STAT5 in K562 cells was then performed (Fig. 3B). Although 5 µM U0126 or PD184352 was more potent than sorafenib in diminishing ERK1/2 phosphorylation, neither U0126 nor PD184352 decreased STAT5 phosphorylation. Instead, these agents induced, if anything, a modest increase in STAT5 phosphorylation. Such findings are in accord with results of a very recent report suggesting that MEK1 negatively regulates Janus tyrosine kinase (JAK2) by enhancing phosphorylation at serine 523, an event associated with diminished STAT5 phosphorylation (Mazurkiewicz-Munoz et al., 2006). Furthermore, STAT5 transcriptional activity monitored by luciferase reporter assays revealed that sorafenib (7.5 µM) blocked STAT5 activity to a similar extent as 1 µM IM (Fig. 3C), whereas U0126 or PD184352 failed to diminish STAT5 activity. Together, these findings suggest that sorafenib triggers a rapid dephosphorylation and inactivation of STAT5 and that this phenomenon operates independently of the MEK1/2/ERK1/2 pathway.
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STAT5 Inactivation in Bcr/Abl+ Cells by Sorafenib Plays a Functional Role in Lethality. To determine the functional significance of these findings, K562 cells were stably transfected with plasmids encoding a constitutively active form of STAT5 (pMX-STAT5A-N642H). In this construct, the asparagine residue at position 642 was mutated to histidine, a mutation known to be associated with high DNA binding and transactivation activities (Ariyoshi et al., 2000). Two clones expressing the constitutively active STAT5, designated K562 cl4 and K562 cl18, were isolated (Fig. 3D, top). It is noteworthy that these clones were significantly more resistant to sorafenib-mediated lethality when administered at either 7.5 or 10 µM(P < 0.05 or 0.02; Fig. 3D, bottom). Together, these findings suggest that inactivation of STAT5 contributes functionally to sorafenib-mediated lethality in Bcr/Abl+ cells. They also raise the possibility that STAT5 inactivation may represent a marker for sorafenib activity in these cells.
Sorafenib Induces Mcl-1 Down-Regulation, STAT5 Inactivation, and eIF4E Dephosphorylation in Imatinib Mesylate-Resistant Cells Exhibiting Bcr/Abl Kinase Mutations. To determine whether these events also occurred in Bcr/Abl+ cells expressing various Bcr/Abl mutants, wild-type and mutant Ba/F3 cells were exposed to 10 µM sorafenib for 2 to 24 h, after which expression of phospho-STAT5 was monitored. Expression of the antiapoptotic protein Mcl-1, a downstream target of STAT5 (Aichberger et al., 2005) that we have also shown to be down-regulated at the translational level by sorafenib (Rahmani et al., 2005a), was monitored in parallel. As shown in Fig. 4, A through D, 10 µM sorafenib rapidly (e.g., within 2 h) induced inactivation of STAT5 in each of the cell lines. Although a slight rebound phenomenon was observed in E255K cells, activity in all cases was essentially abrogated after 24 h. In addition, Mcl-1 expression was also rapidly down-regulated in cells expressing wild-type or each of the Bcr/Abl mutant proteins and was largely complete after 8 h of exposure. Furthermore, a rapid and pronounced dephosphorylation of the translation initiation factor eIF4E was observed in Ba/F3-expressing wild type and mutant Bcr/Abl after sorafenib exposure. Thus, sorafenib rapidly and potently inactivated STAT5, dephosphorylated eIF4E, and down-regulated Mcl-1 expression in cells expressing mutant forms of Bcr/Abl, which confer marked resistance to IM.
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). Given the finding that sorafenib down-regulates Mcl-1, the possibility that Bim might play a functional role in sorafenib-mediated lethality seemed plausible. To test this hypothesis, K562 cells were stably transfected with constructs encoding for shRNA directed against Bim. As shown in Fig. 5A, two clones exhibiting significant knockdown of Bim proteins were used. It is noteworthy that cells in which Bim was knocked down were significantly more resistant to sorafenib-mediated lethality than their control counterparts (Fig. 5B). These findings suggest that Bim plays a functional role in sorafenib-mediated lethality in K562 cells.
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Discussion |
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). Moreover, patients in accelerated or blast-phase CML are relatively refractory to this agent. The development of second-generation Bcr/Abl kinase inhibitors such as nilotinib and dasatinib represent a significant advance, because in addition to their enhanced activity, such agents remain active against leukemia cells bearing most Bcr/Abl mutations, including those residing in the activation loop and ATP-binding domain (Talpaz et al., 2006; Walz and Sattler, 2006). However, these agents are unable to bind to Bcr/Abl-exhibiting mutations in the gatekeeper region (e.g., T315I) (O'Hare et al., 2005; Talpaz et al., 2006; von Bubnoff et al., 2006), raising the possibility that cells expressing this or related mutations will be selected for during therapy with second-generation kinase inhibitors. Indeed, the appearance of this and related mutations has been observed in preliminary trials involving these agents (Talpaz et al., 2006; von Bubnoff et al., 2006). Therefore, a search for alternative strategies capable of eradicating cells bearing such mutations is clearly justified.
Several groups, including our own, have reported that sorafenib potently induced apoptosis in human leukemia cells, including Bcr/Abl+ leukemias, through a mechanism involving down-regulation of Mcl-1 (Rahmani et al., 2005a; Yu et al., 2005). Furthermore, we demonstrated that this process stemmed from inhibition of Mcl-1 translation, a process associated with diminished phosphorylation of the eIF4E translation initiation factor. Recent studies suggest that Mcl-1 cooperates with Bcl-xL to tether the proapoptotic proteins Bak and Bim, a multidomain and BH3-only domain protein, respectively, and prevent their activation (Gomez-Bougie et al., 2005; Willis et al., 2005). Because Bcl-xL is a well-described downstream target of Bcr/Abl, it is tempting to speculate that interventions that down-regulate both Mcl-1 and Bcl-xL might act through this mechanism. However, in our earlier study, no major changes in Bcl-xL protein levels were noted in sorafenib-treated cells, at least during the first 8 h of treatment, and there were no significant changes in the expression of antiapoptotic proteins other than Mcl-1(Rahmani et al., 2005a). Moreover, similar findings were observed in Bcr/Abl mutants (M. Rahmani and S. Grant, unpublished results), arguing against this possibility. The notion that sorafenib down-regulates Mcl-1 expression in Bcr/Abl+ leukemia cells by multiple mechanisms cannot be excluded. For example, Mcl-1 expression is regulated at the transcriptional level by STAT5 (Aichberger et al., 2005), a downstream target of Bcr/Abl that has been implicated in Bcr/Abl-related leukemogenesis (de Groot et al., 1999). In addition, previous studies demonstrated that sorafenib potently and rapidly diminishes the expression of Mcl-1 in Bcr/Abl leukemia cells primarily through translation inhibition (Rahmani et al., 2005a). Thus, although sorafenib-mediated disruption of Bcr/Abl and STAT5 function cannot be excluded as contributing factors in Mcl-1 down-regulation, it is likely that interference with Mcl-1 translation represents the predominant mode of action. The finding that knockdown of Bim significantly diminished sorafenib lethality in K562 cells argues strongly for a functional role of this proapoptotic Bcl-2 family member in sorafenib-mediated lethality. In this regard, the bulk of evidence indicates that Mcl-1 physically interacts with Bim and blocks its proapoptotic activity (Gomez-Bougie et al., 2005). It is therefore conceivable that down-regulation of Mcl-1 might lead to an increase in free Bim protein, thereby enhancing its proapoptotic activity. In addition, other studies have shown that Mcl-1 down-regulation by itself may be sufficient to trigger apoptosis in certain transformed cells (Moulding et al., 2000; Derenne et al., 2002). In any case, to the extent that Mcl-1 down-regulation occurs independently of Bcr/Abl, such a mechanism would be operative in cells resistant to IM through multiple mechanisms, including increased expression of Bcr/Abl, or diverse mutations in the kinase domain. Indeed, each of the resistant cell types examined in this study displayed roughly equivalent sensitivity to sorafenib-induced lethality. Moreover, sorafenib was equally effective in blocking eIF4E phosphorylation in imatinib-sensitive and -resistant cells, including those expressing the T315I mutation.
The results of this study indicate, for the first time, that sorafenib induces a rapid and pronounced dephosphorylation of STAT5, a major survival transcription factor in myeloid leukemia cells (de Groot et al., 1999). This was associated with diminished STAT5 activity as observed in cells exposed to IM but in striking contrast to the actions of the MEK1/2 inhibitors U0126 and PD184352, which failed to diminish STAT5 phosphorylation or activity. This suggests that sorafenib inactivates STAT5 through a MEK1/2/ERK1/2-independent mechanism. Moreover, the capacity of constitutively active STAT5 to protect Bcr/Abl+ cells from sorafenib lethality argues that STAT5 inactivation plays a significant functional role in sorafenib-induced apoptosis. It is noteworthy that sorafenib down-regulated phospho-STAT5 levels in cells both sensitive and resistant to IM, including those bearing the T315I mutation. The finding that sorafenib rapidly and profoundly diminished STAT5 phosphorylation whereas the Bcr/Abl downstream target Crkl (ten Hoeve et al., 1994) was minimally affected suggests that sorafenib disrupts STAT5 signaling through a Bcr/Abl-independent mechanism. In this context, STAT5 is known to be phosphorylated by JAK2 and the nonreceptor tyrosine kinase Src, members of kinase families that lie downstream of multiple tyrosine kinase receptors including PDGFR and VEGFR, which are recognized targets of sorafenib (Wilhelm et al., 2004). It is therefore possible that sorafenib inactivates STAT5 through a mechanism involving the inhibition of PDGFR and VEGFR and their downstream kinases JAK2 and Src. However, the contribution of other mechanisms to this phenomenon cannot be excluded, and clearly additional studies are required to resolve these issues.
The bulk of evidence from this and our earlier study (Rahmani et al., 2005a) suggest that sorafenib acts independently of Bcr/Abl to induce apoptosis. We observed previously that sorafenib modestly diminished the expression of total and phospho-Bcr/Abl in wild-type CML cells (Rahmani et al., 2005a), effects that are likely to reflect a reduction in Bcr/Abl translation. The finding that sorafenib diminished phosphorylation of Crkl only modestly, and at relatively late intervals, argues against direct inhibition of Bcr/Abl as a primary mechanism of lethality. Thus, the actions of sorafenib stand in marked contrast to those of other tyrosine kinase inhibitors recently found to be active in IM-resistant leukemia cells. For example, the tyrphostin adaphostin (NSC680410) has been shown to inactivate/down-regulate Bcr/Abl in Bcr/Abl mutant cells, including those expressing T315I (Chandra et al., 2006). On the other hand, the lethality of adaphostin in these cells stems from the induction of oxidative damage (i.e., reactive oxygen species generation), a phenomenon that may be independent of effects on Bcr/Abl (Chandra et al., 2006). More recently, the aurora kinase inhibitor VX-680 has shown activity against patient-derived CML cells exhibiting the T315I mutation (Young et al., 2006). This capacity is believed to stem from the ability of VX-680 to bind to the active form of the T315I variant Bcr/Abl and to prevent phosphorylation of the activation loop (Young et al., 2006). The present results also differ sharply from those of a very recent report demonstrating that the growth of hematopoietic cells bearing constitutively active FIP1L1-PDGFR, the oncogenic kinase responsible for chronic eosinophilic leukemia (CEL) (Lierman et al., 2006), was extremely sensitive to sorafenib. It is noteworthy that sorafenib was also highly active against IM-resistant cells expressing the FIP1L1-PDGFR T647I mutation, which is similar to the T315I Bcr/Abl mutation (Lierman et al., 2006). In this setting, sorafenib acts directly on the oncogenic kinase to inhibit cell survival. In striking contrast, the present results suggest that in cells bearing Bcr/Abl mutations rendering them resistant to IM, sorafenib acts downstream and/or independently of the Bcr/Abl kinase rather than inhibiting it directly and is therefore able to kill cells resistant to second-generation Bcr/Abl kinase inhibitors such as dasatinib and nilotinib (e.g., those bearing the T315I mutation) (Talpaz et al., 2006; von Bubnoff et al., 2006).
In summary, the present findings suggest that as in the case of IM-resistant CEL cells (Lierman et al., 2006), CML cells resistant to IM may remain susceptible to sorafenib, albeit through a fundamentally different mechanism. Although sorafenib kills IM-resistant CEL cells by inhibiting FIP1L1-PDGFR bearing the T674I mutation that confers resistance, it seems to induce apoptosis in IM-resistant CML cells through a Bcr/Abl-independent mechanism. It is likely that activation of a distinct death pathway involving disruption of Mcl-1 translation and inhibition of STAT5 contributes significantly to this phenomenon. An important consideration is whether sorafenib will be able to eradicate CML stem cells postulated to account for disease recurrence after therapy. For example, imatinib mesylate has been shown to be relatively ineffective in eliminating such stem cells (Graham et al., 2002), and recent studies suggest that newer-generation kinase inhibitors (e.g., dasatinib) may also have limited activity against these cells (Copland et al., 2006). In this context, the dependence of hematopoietic stem cells on Mcl-1 for survival (Opferman et al., 2005) may be relevant. In any case, the present results suggest that investigation of sorafenib as an agent capable of eradicating IM-resistant CML cells, either alone or perhaps in combination with other agents, deserves further consideration. Therefore, studies addressing this issue are currently underway.
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Footnotes |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: CML, chronic myelogenous leukemia; IM, imatinib mesylate; ERK, extracellular regulated kinase; MEK, mitogen-activated extracellular-regulated kinase; STAT, signal transducer and activator of transcription; Mcl-1, myeloid cell leukemia-1; eIF4E, eukaryotic initiation factor 4E; Bim, Bcl-2-interacting mediator of cell death; shRNA, short hairpin RNA; PARP, poly(ADP-ribose) polymerase; JAK2, Janus tyrosine kinase; CEL, chronic eosinophilic leukemia; PDGFR, platelet-derived growth factor receptor; VEGFR, vascular endothelial growth factor receptor; BAY 43-9006, 4-(4-(3-(4-chloro-3-trifluoromethylphenyl)ureido)phenoxy)pyridine-2-carboxyllic acid methyamide-4-methylbenzenesulfonate; AMN107, 4-methyl-N-(3-(4-methylimidazol-1-yl)-5-(trifluoromethyl)phenyl)-3-((4-pyridin-3-ylpyrimidin-2-yl)amino)benzamide; BMS-354825, N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide; VX-680, cyclopropanecarboxylic acid N-(4-((4-(4-methylpiperazin-1-yl)-6-(5-methyl-2H-pyrazol-3-ylamino)pyrimidin-2-yl)sulfanyl)phenyl)amide; STI571, -(4-methyl-1-piperazinyl)-3'-((4-(3-pyridyl)-2-pyrimidinyl)amino)-p-tolu-p-toluidide; NSC680410, adaphostin; PD184352, 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene.
Address correspondence to: Dr. Steven Grant, Division of Hematology/Oncology, MCV Station Box 230, Virginia Commonwealth University, Richmond, VA 23298. E-mail: stgrant{at}hsc.vcu.edu
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