Role of the p38 mitogen-activated protein kinase pathway in the generation of the effects of imatinib mesylate (STI571) in BCR-ABL-expressing cells.

Imatinib mesylate (STI571), a specific inhibitor of the BCR-ABL tyrosine kinase, exhibits potent antileukemic effects in vitro and in vivo. Despite the well established role of STI571 in the treatment of chronic myelogenous leukemia, the precise mechanisms by which inhibition of BCR-ABL tyrosine kinase activity results in generation of antileukemic responses remain unknown. In the present study we provide evidence that treatment of CML-derived BCR-ABL-expressing leukemia cells with STI571 results in activation of the p38 mitogen-activated protein (MAP) kinase signaling pathway. Our data indicate that STI571 induces phosphorylation of the p38 and activation of its kinase domain, in KT-1 cells and other BCR-ABL-expressing cell lines. We also identify the kinases MAP kinase-activated protein kinase-2 and Msk1 as two downstream effectors of p38, activated during inhibition of BCR-ABL activity by STI571. Importantly, pharmacological inhibition of p38 reverses the growth inhibitory effects of STI571 on primary leukemic colony-forming unit granulocyte/macrophage progenitors from patients with CML. Altogether, our data establish that activation of the p38 MAP kinase signaling cascade plays an important role in the generation of the effects of STI571 on BCR-ABL-expressing cells. They also suggest that, in addition to activation of mitogenic pathways, BCR-ABL promotes leukemogenesis by suppressing the function of growth inhibitory signaling cascades.

The introduction of STI571 (imatinib mesylate) in the treatment of chronic myelogenous leukemia (CML) 1 has been a major medical advance for the management of this disease and has provided an important paradigm for the development of tyrosine kinase inhibitors in the treatment of human malignancies (1)(2)(3)(4)(5)(6)(7). CML is a clonal myeloproliferative disorder of stem cells, whose hallmark is the expression of the BCR-ABL oncoprotein in the leukemic cells. BCR-ABL is the protein product of the bcr-abl oncogene, which results from the fusion of bcr to c-abl, as a consequence of the reciprocal translocation between chromosomes 9 and 22 (8 -10). The abnormal BCR-ABL tyrosine kinase is constitutively activated and plays a critical role in the pathogenesis of CML (11,12), likely via phosphorylation of multiple downstream protein targets, resulting in activation of mitogenic cellular pathways (13).
STI571 is an ABL tyrosine kinase inhibitor (14) that selectively inhibits the kinase activity of the abnormal BCR-ABL protein and suppresses the growth of BCR-ABL-expressing leukemic progenitors (14,15). Extensive clinical studies have established the efficacy of this agent in the treatment of CML patients, including patients in the chronic phase of the disease, as well as patients in the accelerated or blast phases of this malignancy (1)(2)(3)(4)(5)(6)(7). The recent approval of STI571 by the Food and Drug Administration for the treatment of CML and its introduction in clinical oncology practice has had a dramatic impact on the way this leukemia is managed. Importantly, it has provided a paradigm for the development of specific therapeutic approaches for other leukemias and malignancies where tyrosine kinases are known to play pathogenetic roles.
Previous studies have shown that STI571 acts by binding the inactive form of the Abl kinase domain of BCR-ABL (reviewed in Ref. 7), and the structural basis for the autoinhibition of the c-Abl tyrosine kinase has been solved (16,17). Because BCR-ABL activates multiple downstream mitogenic effectors and anti-apoptotic pathways (7,13), it is presumed that the antineoplastic effects of imatinib mesylate are primarily mediated by the blockade of BCR-ABL-generated mitogenic signals. However, the downstream targets of BCR-ABL, whose inhibition accounts for the antileukemic effects of STI571, remain to be precisely defined. Another potential mechanism by which STI571 exhibits antileukemic activity may involve the restoration of function of growth inhibitory pathways that may be abnormally suppressed by BCR-ABL. It is of interest that overexpression of BCR-ABL has been shown to suppress gene tran-scription in response to IFN␣ (18), a cytokine to which CML cells exhibit unique sensitivity in vitro and in vivo (reviewed in Refs. 19 and 20). Furthermore, recent studies have indicated that overexpression of BCR-ABL in embryonic stem cell-derived hematopoietic progenitors suppresses the p38 MAP kinase pathway (21), whose function is required for IFN␣-dependent gene transcription (22)(23)(24) and generation of growth inhibitory responses (25).
In the present study we examined whether STI571-dependent suppression of leukemic cell growth is in part mediated by restoration of activation of the p38 MAP kinase. Our studies establish that pharmacological inhibition of p38 reverses the suppressive effects of STI571 on primary leukemic CFU-GM progenitors, indicating that activation of this signaling cascade is essential for the antileukemic effects of imatinib mesylate. In other studies we identify the p38-dependent kinases Map-KapK-2 and Msk1 as putative downstream mediators of the STI571-activated form of p38. Altogether, our studies strongly suggest that reversal of the inhibitory effects of BCR-ABL on the activation of the p38 MAP kinase pathway plays a critical role in the induction of STI571 responses in CML cells.

MATERIALS AND METHODS
Cells and Reagents-The CML-derived KT-1 cell line was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics. The EM3 and EM2 cell lines were also grown in RPMI 1640 -10% fetal bovine serum. IFN␣ was provided by Hoffman Laroche. STI571 was provided by Novartis. Antibodies against the phosphorylated forms of p38 and Msk1 were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against p38 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and BD Transduction Laboratories. A monoclonal antibody against phosphotyrosine (4G-10) was obtained from Upstate Biotechnology Inc. Antibodies against phospho-Erk1/2 and an anti-Erk antibody were obtained from Cell Signaling Technology (Beverly MA). The p38 MAP kinase inhibitors SB203580 and SB202190 and the MEK kinase inhibitor PD098059 were purchased from Calbiochem Inc.
Cell Lysis and Immunoblotting-The cells were treated with 1 M of STI571 for the indicated times and lysed as described previously (26,27). Immunoprecipitations and immunoblotting using an ECL method were performed as described previously (26,27).
Isolation of Peripheral Blood Granulocytes-Informed consent was obtained from patients with CML, according to the guidelines established by the Institutional Review Board of Northwestern University Medical School. Polymorphonuclear leukocytes were separated from peripheral venous blood using the mono-poly resolving medium (ICN Biomedicals, Aurora, OH), as described previously (25). Briefly, after centrifugation at 300 ϫ g for 30 min at room temperature, the polymorphonuclear band was transferred into an individual tube. The cells were washed with culture medium and were subsequently resuspended in culture medium prior to treatment with STI571.
In Vitro Kinase Assays-In vitro kinase assays to detect the activation of the MapKapK-2 by STI571 were performed as described previously (28,29). To examine the induction of Msk1 kinase activity, cell lysates from STI571-treated cells were immunoprecipitated with either an anti-Msk1 antibody or control nonimmune RIgG, and in vitro kinase assays were performed as described previously (24). The values were calculated by subtracting nonspecific activity, detected in RIgG immunoprecipitates, from kinase activity detected in anti-Msk1 immunoprecipitates.
Hematopoietic Progenitor Cell Assays-The effects of STI571 on the growth of hematopoietic progenitors from patients with CML was determined in methylcellulose assays as described previously (30,31). Briefly, bone marrow aspirate specimens were obtained under local anesthesia from patients with CML, after obtaining Institutional Review Board-approved informed consent. Bone marrow mononuclear cells were separated by Ficoll-Hypaque sedimentation, and the cells were cultured in a methylcellulose mixture containing hematopoietic growth factors (30,31) in the presence or absence of STI571 (1 M) and/or pharmacological inhibitors of MAP kinases. The concentrations of the different MAP kinase inhibitors that were used were 10 M for SB202190, 10 M for SB203580, and 2 M for PD98059, as in previous studies (29 -31, 71). In patients where bone marrow aspirate was not available, peripheral blood was collected after obtaining informed consent, and peripheral blood hematopoietic progenitors were separated and used for the clonogenic assays in methylcellulose. CFU-GM from leukemic samples were scored on day 14 of culture.

RESULTS
We initially examined whether treatment of KT-1cells with imatinib mesylate results in phosphorylation of the p38 MAP kinase. The initial concentration of STI571 that was used for these experiments was 1 M, because this concentration ap- Equal amounts of total cell lysates (100 g/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p38 MapK. B, the blot shown in A was stripped and reprobed with an antibody against p38 MapK to control for protein loading. C, EM2 cells were incubated with STI571 (1 M) for different time points as indicated. Equal amounts of total cell lysates (100 g/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p38 MapK. D, the blot shown in C was stripped and reprobed with an antibody against p38 to control for protein loading. E, EM3 cells were incubated with STI571 (1 M) for different time points as indicated. Equal amounts of total cell lysates (100 g/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p38 MapK. F, the blot shown in E was stripped and reprobed with an antibody against p38 to control for protein loading. G, KT-1 cells were incubated for the indicated times in the presence or absence of 1 ⌴ of STI571 as indicated. Equal amounts of total cell lysates (100 g/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody against that recognizes the phosphorylated forms of Erk1 and Erk2. H, the blot shown in G was stripped and reprobed with an anti-Erk1/2 antibody, to control for protein loading.
pears to be clinically relevant and has been previously shown to block growth of BCR-ABL-expressing cell lines and CML-derived hematopoietic progenitors (1,64,72). KT-1 cells were incubated in the presence or absence of STI571. The cells were subsequently lysed, and equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antiphospho-p38 antibody. Some weak base-line phosphorylation of p38 was detectable in untreated cells, and such phosphorylation was strongly increased after STI571 treatment (Fig. 1, A  and B). Such phosphorylation of p38 was also seen in the EM2 (Fig. 1, C and D) and EM3 (Fig. 1, E and F) cell lines, which also express BCR-ABL (32). On the other hand and consistent with previous reports (41), STI571 treatment induced dephosphorylation of Erk1/2 kinases (Fig. 1, G and H), which are activated by BCR-ABL (41). The phosphorylation of p38 in response to STI571 treatment of cells was dose-dependent, with the signal maximizing at 1 M concentration of STI571 (Fig. 2, A and B), at which concentration there was also strong inhibition of cell proliferation of KT-1 cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Fig. 2C).
To directly examine whether STI571-dependent phosphorylation of p38 results in activation of its kinase domain, in vitro kinase assays were carried out on p38 immunoprecipitates from STI571-treated KT-1 cells. Treatment of KT-1 cells with STI571 resulted in strong induction of p38 MAP kinase activity, evidenced by the phosphorylation of ATF-2 used as an exogenous substrate (Fig. 3, A and B), demonstrating that STI571-dependent phosphorylation of p38 results in functional activation of its kinase domain. As expected, treatment of KT-1 cells with STI571 also resulted in dephosphorylation of BCR-ABL (Fig. 3, C and D), suggesting that activation of p38 in response to STI571 results from reversal of BCR-ABL-generated inhibitory effects on p38 activity. Consistent with this, STI571 did not induce phosphorylation/activation of p38 in the U-266 (Fig. 4, A and B) and Molt-4 (Fig. 4, C and D) hemato- The cells were lysed, and the total cell lysates were immunoprecipitated (IP) with an antibody against p38. Immunoprecipitated proteins were subjected to in vitro kinase assays, using ATF-2 as an exogenous substrate. The proteins were resolved by SDS-PAGE and transferred to Immobilon. The phosphorylated form of ATF-2 was detected by autoradiography. B, The membrane shown in A was subsequently immunoblotted with an antibody against phospho-ATF-2. C, KT-1 cells were treated with STI571 (1 M) for the indicated times. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an anti-phospho-ABL antibody to detected phosphorylated BCR-ABL. D, the blot shown in C was stripped and reprobed with an antibody against ABL, to detect BCR-ABL. poietic cell lines that do not express BCR-ABL. On the other hand, STI571-induced phosphorylation of p38 in primary peripheral blood granulocytes from a CML patient (Fig. 4, A and  B), suggesting that such phosphorylation occurs under physiological conditions and may play an important role in the generation of the effects of STI571 in CML.
The p38 MAP kinase has been previously shown to be activated by IFN␣ in BCR-ABL-expressing cells and to mediate the generation of the antiproliferative effects of IFN␣ on CML progenitors (25). There is also evidence that STI571 and IFN␣ exhibit synergistic effects on the suppression of growth of CML cells (38 -40). The fact that treatment of CML-derived cell lines with STI571 resulted in activation of the p38 MAP kinase raised the possibility that STI571 may be modulating other Type I IFN-signaling pathways, as well. To determine whether other IFN-regulated pathways are modulated by STI571 treatment of CML cells, experiments were performed in which the effects of STI571 on the IFN-dependent activation of STAT proteins were evaluated. As shown in Fig. 5, treatment of cells with STI571 did not induce tyrosine phosphorylation of STAT1 (Fig. 5, A and B), STAT3 (Fig. 5, C and D), or STAT5 (Fig. 5, E  and F), nor did it have any effects on the IFN␣-dependent phosphorylation of these STAT proteins (Fig. 5). Consistent with this, STI571 did not affect the formation of STAT-contain-

FIG. 5. STI571 does not exhibit regulatory effects on the activation of STAT proteins and the IFN␣-dependent formation of ISGF3 and SIF DNA-binding complexes.
A, KT-1 cells were incubated in the presence or absence of the indicated concentrations of STI571 (STI) for 60 min, as indicated. The cells were subsequently treated with IFN␣ as indicated, and after cell lysis, the proteins were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of STAT1on tyrosine 701. B, the blot shown in A was stripped and reprobed with an anti-STAT1 antibody to control for loading. C, KT-1 cells were incubated in the presence or absence of the indicated concentrations of STI571 for 60 min, as indicated. The cells were subsequently treated with IFN␣ as indicated, and after cell lysis, the proteins were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of STAT3 on tyrosine 705. D, the blot shown in C was stripped and reprobed with an anti-STAT1 antibody, to control for loading. E, KT-1 cells were incubated in the presence or absence of the indicated concentrations of STI571 for 60 min, as indicated. The cells were subsequently treated with IFN␣ as indicated, and after cell lysis, the lysates were immunoprecipitated (IP) with an antibody against STAT5. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with an antiphosphotyrosine antibody. F, the blot shown in E was stripped and reprobed with an anti-STAT5 antibody, to control for loading. G and H, actively growing KT-1 cells were preincubated for 30 min with 1 M STI571 as indicated. The cells were subsequently treated with 10,000 IU of IFN␣ in the continuous presence or absence of STI571 as indicated. The nuclear extracts were reacted with 40,000 cpm of a 32 P-labeled IFN-stimulated response element probe (G) or a 32 P-labeled SIS-inducible element probe (H), and the complexes were resolved by native gel electrophoresis and visualized by autoradiography. WB, Western blot.
ing DNA-binding complexes that bind to IFN-stimulated response element or IFN-␥-activated site elements, as shown by gel shift assays (Fig. 5, G and H). Thus, STI571 selectively activates the p38 MAP kinase pathway in CML cells, whereas it does not appear to have any effects on the activation of IFN␣-regulated STAT pathways.
To better understand the mechanisms by which the p38 MAP kinase pathway may participate in the generation of STI571dependent responses, we sought to identify p38-regulated downstream effector kinases that may be activated in an STI571-inducible manner. The kinase MapKapK-2 is activated in response to stress signals, as well as during engagement of the Type I IFN receptor in BCR-ABL-expressing cells, and mediates p38-dependent functional responses (24). We determined whether this kinase is activated in response to STI571 treatment of BCR-ABL cells by performing in vitro kinase assays in anti-MapKapK-2 immunoprecipitates from STI571treated cell lysates. Treatment of KT-1 (Fig. 6A) or EM3 (Fig.  6B) cells with STI571 resulted in strong activation of the Map-KapK-2 kinase, evidenced by the phosphorylation of Hsp-25 in the in vitro kinase assays. Such activation was blocked by pretreatment of cells with SB203580, indicating that it occurs downstream of p38 (Fig. 6, A and B). In a similar manner, STI571 treatment of the cells resulted in phosphorylation/activation of Msk1 (Fig. 6, C and D), a kinase that acts as a downstream effector for p38, and whose function has been implicated in the regulation of stress-induced nuclear histone phosphorylation, chromatin remodeling, and immediate early gene expression (33)(34)(35)(36)(37). To examine whether STI571-dependent phosphorylation of Msk1 results in activation of its kinase domain, experiments were performed in which lysates from STI571-treated cells were immunoprecipitated with an anti-Msk1 antibody, and in vitro kinase assays were carried out on the immunoprecipitates. STI571 treatment resulted in induction of Msk1 kinase activity (Fig. 6E). Such activation was blocked by pretreatment of cells with SB203580 (Fig. 6E), strongly suggesting that Msk1 acts as a downstream effector of the STI571-activated p38 MAP kinase.
In subsequent studies, we sought to identify the functional relevance of imatinib-dependent activation of p38 in BCR-ABLexpressing cells. We determined whether activation of p38 MAP kinase is required for the generation of inhibitory effects of STI571 on primary leukemic progenitors from patients with chronic myelogenous leukemia. For this purpose, the effect of SB203580 on the suppression of bone marrow-or peripheral blood-derived leukemic CFU-GM progenitors was evaluated in clonogenic assays in methylcellulose. Samples from different patients with CML were evaluated. STI571 inhibited colony formation of myeloid progenitors from all patients with CML studied (Fig. 7). Concomitant treatment of cells with SB203580 partially reversed the growth inhibitory effects of STI571 on the leukemic progenitors (Fig. 7, A and B), indicating that restoration of activation of p38 activity is essential for the induction of an antileukemic response. Similarly, another p38 inhibitor, SB202190, also reversed STI571-dependent suppression of leukemic CFU-GM progenitors (Fig. 7, C and D). On the other hand, the MEK/Erk inhibitor PD98059 did not reverse such effects and, on the contrary, slightly enhanced the suppression of leukemic CFU-GM growth (Fig. 7D).

FIG. 6. STI571-dependent activation of MapKapK-2 and Msk-1.
A, KT-1 cells were preincubated for 30 min at 37 C in the presence or absence of SB203580 (10 M) as indicated. The cells were subsequently incubated in the presence or absence of STI571 (STI, 1 M) for 30 min as indicated in the continuous presence or absence of SB203580. The cells were subsequently lysed; the cell lysates were immunoprecipitated (IP) with an antibody against MapKapK-2; and in vitro kinase assays were carried out on the immunoprecipitates using Hsp25 as an exogenous substrate. The proteins were analyzed by SDS-PAGE, and the phosphorylated form of Hsp25 was detected by autoradiography. B, EM3 cells were preincubated for 30 min at 37 C in the presence or absence of SB203580 (10 M) as indicated. The cells were subsequently incubated in the presence or absence of STI571 (1 M) for 30 min as indicated in the continuous presence or absence of SB203580. The cells were subsequently lysed; the cell lysates were immunoprecipitated with an antibody against MapKapK-2, and in vitro kinase assays were carried out on the immunoprecipitates using Hsp25 as an exogenous substrate. The proteins were analyzed by SDS-PAGE, and the phosphorylated form of Hsp25 was detected by autoradiography. C, KT-1 cells were incubated with STI571 for the indicated times. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of Msk-1 on serine 376. D, the blot shown in C was stripped and reprobed with an anti-tubulin antibody to control for protein loading. E, KT-1 cells were preincubated in the presence or absence of SB203580 for 60 min as indicated. The cells were subsequently treated with STI571 for 30 min, in the continuous absence or presence of SB203580. The cell lysates were immunoprecipitated with an anti-Msk1 antibody, and in vitro kinase assays were performed on the immunoprecipitates. The means Ϯ S.E. of two independent experiments are shown.

DISCUSSION
The remarkable clinical efficacy of STI571 in the treatment of CML has provided an important paradigm for the development of pharmacological agents that specifically target tyrosine kinases that regulate survival and growth of malignant cells. STI571 exhibits its activity by blocking the function of the abnormal BCR-ABL tyrosine kinase, which causes the malignant transformation in CML, and is currently the first line of treatment for patients with this leukemia. Despite the substantial advances in this research area over the last few years, the precise mechanisms and pathways that regulate induction of STI571 responses after blocking the kinase activity of BCR-ABL are not known. STI571 has been shown to regulate the phosphorylation and/or activation of several proteins that are direct or indirect downstream targets for the kinase activity of BCR-ABL. These include the adaptor protein CrkL (41)(42)(43), the multisite docking proteins CBL (43) and IRS-1 (44), the Shc oncogenic protein (42), the Erk kinase (41), the Akt kinase (45), and the forkhead transcription factor FKHRL1 (46). Apparently, the inhibition of mitogenic signals induced by BCR-ABL plays a major role in the induction of the antileukemic effects of imatinib mesylate. However, it is possible that reversal of BCR-ABL-mediated suppression of pathways that mediate growth inhibitory signals is also required for the antineoplastic effects of STI571 in chronic myelogenous leukemia.
In the current report we provide the first evidence that the p38 MAP kinase pathway is activated during STI571 treatment of BCR-ABL-expressing cells. Our data demonstrate that STI571 induces phosphorylation of the p38 MAP kinase and activation of its kinase domain. We also demonstrate that MapKapK-2 and Msk1 are activated in a p38-dependent manner during STI571 treatment of CML cells. The latter findings provide further evidence that imatinib mesylate induces functional activation of p38, resulting in downstream signaling events that regulate p38-mediated biological responses. Most importantly, our findings directly establish that inhibition of p38 MAP kinase activity reverses the growth inhibitory effects of STI571 on primary CML-derived leukemic progenitors. This is demonstrated by studies using the pyridinyl imidazole compounds SB203580 and SB202190, whose high selectivity and specificity for the p38 MAP kinase has been established by mutagenesis studies and x-ray crystallography (47)(48)(49)(50)(51). In contrast, inhibition of the MEK/Erk pathway, using the PD98059 pharmacological inhibitor, enhances the inhibitory effects of STI571 on the growth of primary leukemic CFU-GM progenitors. These findings are in agreement with a previous study demonstrating that MEK kinase inhibitors enhance the effects of STI571 on BCR-ABL-expressing cell lines (52) and further support the concept that combined use of STI571 with MEK inhibitors may prove useful in the future treatment of CML. The members of the p38 family of kinases (53-58) are activated in response to stress and regulate signals essential for important biological responses, such as phosphorylation of transcription factors and gene transcription, cellular differentiation, cytokine production, and apoptosis (53)(54)(55)(56)(57)(58). The p38 MAP kinase pathway is activated by IFN␣ and appears to play an important in the regulation of gene transcription by the Type I IFN receptor and the induction of interferon responses (22)(23)(24)59). This is of particular interest, because IFN␣ is a cytokine that has important clinical activity in the treatment of CML, and prior to the introduction of STI571, it was the pharmacological agent of choice for patients with this leukemia (reviewed in Refs. 60 and 61). Our previous studies have shown that IFN␣ activates the p38 MAP kinase cascade in BCR-ABLexpressing cells and that pharmacological inhibition of such activation reverses the growth inhibitory effects of this cytokine on bone marrow-derived leukemic progenitors from patients with CML (25). Taken together with the results of the current study, these findings suggest that the p38 pathway is a common effector for the generation of the antileukemic effects of IFN␣ and STI571 in CML cells. They also further support the hypothesis that one of the mechanisms by which BCR-ABL promotes leukemogenesis may be suppression of the p38 pathway and are consistent with the observed suppression of p38 in embryonic cell-derived hematopoietic progenitors in which BCR-ABL is exogenously expressed (21).
There is now accumulating evidence that resistance to the effects of STI571 occurs in vitro and in vivo (6,7,62,63). The most common mechanism of resistance appears to result from point mutations of the kinase domain of BCR-ABL, which result in decrease or abrogation of the affinity of STI571 (reviewed in Ref. 6). However, additional mechanisms of resistance, unrelated to point mutation of BCR-ABL, also occur and may play a role in the development of clinical refractoriness to the drug. For instance, recent studies have implicated overexpression of the Lyn and/or Hck kinases of the Src family in the development of BCR-ABL independence (65), whereas other studies have demonstrated that MDR1 gene overexpression in CML cells also accounts for STI571 resistance (66). Based on our findings, the function of p38 and its downstream effectors are essential for the generation of an antileukemic response by STI571. It is therefore possible that resistance to imatinib may also result from defects on the activation of the p38 MAP kinase pathway in certain cases of CML, and this should be directly examined in future studies. It should be also pointed out that pharmacological inhibitors of p38 are currently under development for the treatment of rheumatoid arthritis, bronchial asthma, and other inflammatory diseases (67)(68)(69)(70), based on their well documented ability to decrease production of proinflammatory cytokines. Our findings raise the possibility that concomitant use of STI571 with such pharmacological inhibitors may abrogate the antitumor effects of STI571 in vivo. Perhaps, pharmacological inhibitors of the p38 pathway should be avoided in the design and development of future trials including STI571 in CML.