Imatinib induces mitochondria‐dependent apoptosis of the Bcr‐Abl‐positive K562 cell line and its differentiation toward the erythroid lineage <sup>1</sup> (2003)

Imatinib has emerged as the lead compound for clinical development against chronic myelogenous leukemia (CML). Imatinib induces apoptosis of CML cells, but how the drug kills them is only partially understood. This study was conducted to 1) analyze the effect of imatinib on apoptosis and differentiation of the Bcr-Abl-positive K562 cell line and 2) determine the influence that apoptosis and differentiation have on each other in this appropriate model. A 36 h exposure of K562 cells to 1 µM imatinib induced DNA fragmentation (Fig. 1A), indicating that cells underwent an apoptotic program in the presence of the inhibitor. Imatinib induces caspase 3, 8, and 9 activity after 24–36 h (Fig. 1B, C). Apoptosis was also evidenced by the disappearance of the zymogenic form of caspase 3 and caspase 9, reflecting their activation, and by the cleavage of poly-ADP-ribose polymerase (Fig. 1C). DiOC6 was next used to monitor the disruption of mitochondrial potential (ΔΨm). Imatinib induced a decrease of DiOC6 staining in >58% of K562 cells after 48 h of incubation, confirming its effect is integrated at the mitochondrial level. Expression of p210Bcr-Abl and p145Abl diminished after 36 h of incubation with imatinib (Fig. 1C), suggesting that Bcr-Abl and Abl were degraded in cells treated with the therapeutic agent. To further characterize the mechanisms involved in the proapoptotic action of imatinib, we analyzed its effect on the main signaling pathways regulated by Bcr-Abl. Imatinib abolished ERK1/2 basal activation and to a lesser extend JNK activation, but failed to affect basal AKT and p38 phosphorylation. By contrast, PMA increased ERK1/2 activation but also enhanced p90RSK, JNK, and AKT activation. Although imatinib was found to abolish ERK1/2 activation, it failed to affect PMA-mediated activation of ERK1/2, RSK, JNK, and AKT. In the presence of PMA, K562 cells underwent megakaryocytic differentiation. Indeed, large cells with prominent dense granules were visualized, reflecting megakaryocytic differentiation (Fig. 2Ab). The proapoptotic effect of imatinib was visualized by the loss of viability of K562 cells, but careful examination of the cell culture at a higher magnification showed the presence of a significant number of small viable cells (Fig. 2Ac, arrows). When cells were cultured with a combination of imatinib and PMA, imatinib-induced cell death was abolished and megakaryocytic differentiation occurred (Fig. 2Ad). The hypothesis that K562 cells underwent erythroid differentiation upon imatinib treatment was confirmed by a reduction of cell viability and an increase in benzidine staining (Fig. 2Ac, B). After 48 h in the presence of imatinib, 77% of K562 cells died by apoptosis whereas >80% of the remaining cells differentiated toward the erythroid lineage (Fig. 2Ac, B). In the presence of imatinib, mRNA encoding glycophorin A, the Kell blood group antigen, and CD36 increased, indicating that K562 cells had undergone erythroid differentiation. Moreover, imatinib increased the steady-state levels of Gfi-1B, a transcription factor involved in erythroid differentiation, and decreased the expression of c-fos, Egr-1, and Fli-1, thought to be involved in megakaroycytic differentiation and up-regulated by PMA. Imatinib down-regulated expression of antiapoptotic proteins, including Bcl-x, Bcl-2, and Mcl-1, and increased that of the proapoptotic BH3-only member Bim-L, in agreement with its ability to induce mitochondria-dependent apoptosis of K562 cells. PMA induced GATA-2 transcripts and inhibited glycophorin A and CD36 expression, as cells differentiated toward the megakaryocytic lineage (Fig. 2C). In the presence of both effectors, the effects of imatinib were blocked and cells proceeded along the megakaryocytic pathway (Fig. 2Ad, C). We used a cDNA array approach to assess the effect of imatinib and PMA on gene expression. Imatinib increased the expression of reelin, PTX3 (pentraxin), GRB14, GAB1, transferin receptor, and Alox5 and decreased the expression of VEGF-R1, IGF-R1 c-Fos, EGR1, cyclin D2, α3, α5, and β7 integrins, CAD-12, and I-CAM. Most but not all of the genes comodulated by PMA and imatinib behave in an opposite way. Indeed PMA induced VEGF-R1, IGF-R1, c-fos, EGR1, integrins α3, α5, β7, CAD-12, and ICAM-1 and decreased expression of PTX3, GAB1, transferrin receptor, and Alox5. We show that imatinib-induced apoptosis of the Bcr-Abl-positive cell line K562 is mitochondrion dependent and requires caspase 3, 8, and 9 activation. Caspase activation upon imatinib treatment of K562 cells was accompanied by cleavage of Bcr-Abl. Thus, imatinib may mediate its proapoptotic effect at two different levels by 1) a rapid and sustained inhibition of Bcr-Abl kinase activity leading to inactivation of survival pathways and 2) inducing long-term activation of caspases responsible for the degradation and inactivation of Bcr-Abl tyrosine kinase. Here we show that the main effect of imatinib is to abolish ERK1/2, STAT-5, and JNK constitutive activation in K562 cells. Thus, the proapototic effect of imatinib could be explained by a rapid and sustained inhibition of Bcr-Abl leading to abrogation of survival pathways such as Erk1/2, STAT5, and JNK, followed by induction of mitochondria-dependent apoptosis (Fig. 3, left). If our interpretation is correct, maintaining a high level of Erk1/2 activation should result in inhibition of imatinib-induced apoptosis. This is indeed the case since cotreatment of K562 cells with imatinib and PMA abrogates imatinib-induced ΔΨm dissipation and caspase activation. PMA-induced protection against imatinib-mediated apoptosis correlates with the restoration of Erk1/2 and AKT activation (Fig. 3). Finally, the modulation of Bcl-2 family member expression may represent part of the mechanism by which imatinib reverses the resistance of CML cells to apoptosis. Within 48–72 h in culture with imatinib, a significant number of K562 cells presents erythroid-like features. The ability of early hematopoietic cells to undertake specific differentiation steps toward different lineages is a tightly regulated process that requires specific transcription factors. The observation that imatinib can increase Gfi-1B and repress Fli-1 expression, respectively, agrees well with its ability to induce erythroid differentiation. Thus, besides triggering K562 apoptosis, imatinib also mediated their differentiation along the erythroid lineage. In our study, we searched for genes that may participate in imatinib-induced apoptotic and differentiation programs. When comparing untreated and imatinibtreated K562 cells, 20 of 847 genes were found to be modulated. Most of these genes were regulated in an opposite way by PMA and imatinib, suggesting they may be linked specifically to the differentiation programs, since erythroid and megakaryocytic differentiation are thought to be mutually exclusive. In each case, we identified several genes never before shown to be regulated or to play a role in K562 cell differentiation. Imatinib increased expression of the transferrin receptor, 5-lipooxygenase, GAB1, GRB14, PREF-1, and the serine protease reelin. Increased expression of reelin is particularly intriguing. Reelin is a matrix serine protease secreted by specialized neurons during development. Reelin serves as a ligand for the ApoE2 and VLDL receptors, triggering a signaling cascade that guides neurons to their correct position within developing nervous system. This is the first observation that reelin may be expressed and secreted by hematopoietic cells. In conclusion, we have established for the first time the requirements for imatinib-induced cell death and erythroid differentiation in CML cells and identified new genes potentially involved in this processes. Further investigation of this novel imatinib-induced pattern of gene expression may offer insight into the mechanisms of hematopoietic precursor erythroid differentiation and apoptosis that may have implications for the design of future anti-Bcr-Abl therapeutic strategies.