Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1 μM) and the Src inhibitor PP2 (10 μM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca²⁺ transients were reduced by imatinib and/or PP2. Ca²⁺ transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca²⁺ transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCα catalytic activity and PKCα co-immunoprecipitated with Bcr/Abl.Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca²⁺ influx was reduced by complexing extracellular Ca²⁺ with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca²⁺ transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.     

Bcr - Abl-mediated resistance to apoptosis is independent of PI 3-kinase activity

The Bcr - Abl tyrosine kinase is responsible for the oncogenic phenotype observed in Philadelphia chromosome-positive leukemia and induces resistance to apoptotic cell death in a variety of cell types. Recent evidence supports the hypothesis that these two properties of Bcr - Abl are derived from cooperative but distinct signaling pathways. Phosphatidylinositol 3-kinase (PI3K), which has been suggested to associate with and become activated by Bcr - Abl, has been shown to be required for Bcr - Abl-mediated cell growth. Also, PI3K has been implicated in resistance to apoptosis induced by some growth factors. We therefore examined the role of PI 3-kinase in the anti-apoptotic effect of Bcr - Abl. First, we confirmed that expression of p185(bcr - abl) in HL-60 cells, which renders these cells resistant to apoptosis, induces tyrosine phosphorylation of the p85 subunit of PI3K. Consistent with this result, we observed a 20-fold increase in PI3K activity upon immunoprecipitation of tyrosine-phosphorylated proteins from cells expressing Bcr - Abl versus control cells. Nevertheless, treatment of HL-60.p185(bcr - abl) cells with wortmannin, a potent inhibitor of PI3K, eliminated PI3K activity but did not interfere with the resistance of these cells to apoptosis. Similar results were obtained with the CML line K562 and with the BaF3.p185 (bcr - abl) line. We conclude that while PI3K participates in the anti-apoptotic response mediated by some growth factors and also seems to be important for the growth of Bcr-Abl-positive cells, it does not play any role in Bcr - Abl-mediated resistance to apoptosis.     

The Bcr–Abl kinase regulates the actin cytoskeleton via a GADS/Slp-76/Nck1 adaptor protein pathway

Bcr–Abl is the transforming principle underlying chronic myelogenous leukaemia (CML). Here, we use a functional interaction proteomics approach to map pathways by which Bcr–Abl regulates defined cellular processes. The results show that Bcr–Abl regulates the actin cytoskeleton and non-apoptotic membrane blebbing via a GADS/Slp-76/Nck1 adaptor protein pathway. The binding of GADS to Bcr–Abl requires Bcr–Abl tyrosine kinase activity and is sensitive to the Bcr–Abl inhibitor imatinib, while the GADS/Slp-76 and Slp-76/Nck interactions are tyrosine phosphorylation independent. All three adaptor proteins co-localize with cortical actin in membrane blebs. Downregulation of each adaptor protein disrupts the actin cytoskeleton and membrane blebbing in a similar fashion and similar to imatinib. These findings highlight the importance of protein interaction dependent adaptor protein pathways in oncogenic kinase signaling.     

The decreased susceptibility of Bcr/Abl targets to NK cell-mediated lysis in response to imatinib mesylate involves modulation of NKG2D ligands, GM1 expression, and synapse formation

Chronic myeloid leukemia is a clonal multilineage myeloproliferative disease of stem cell origin characterized by the presence of the Bcr/Abl oncoprotein, a constitutively active tyrosine kinase. In previous studies, we have provided evidence that Bcr/Abl overexpression in leukemic cells increased their susceptibility to NK-mediated lysis by different mechanisms. In the present study, using UT-7/9 cells, a high level Bcr/Abl transfectant of UT-7 cells, we show that the treatment of Bcr/Abl target by imatinib mesylate (IM), a specific Abl tyrosine kinase inhibitor, hampers the formation of the NK/target immunological synapse. The main effect of IM involves an induction of surface GM1 ganglioside on Bcr/Abl transfectants that prevents the redistribution of MHC-related Ag molecules in lipid rafts upon interaction with NK cells. IM also affects cell surface glycosylation of targets, as assessed by binding of specific lectins resulting in the subsequent modulation of their binding to lectin type NK receptor, particularly NKG2D. In addition, we demonstrate that the tyrosine kinase activity repression results in a decrease of MHC-related Ags-A/B and UL-16-binding protein expression on Bcr/Abl transfectants UT-7/9. We show that NKG2D controls the NK-mediated lysis of UT-7/9 cells, and IM treatment inhibits this activating pathway. Taken together, our results show that the high expression of Bcr/Abl in leukemic cells controls the expression of NKG2D receptor ligands and membrane GM1 via a tyrosine kinase-dependent mechanism and that the modulation of these molecules by IM interferes with NK cell recognition and cytolysis of the transfectants.     

A Bcr/Abl-independent, Lyn-dependent form of imatinib mesylate (STI-571) resistance is associated with altered expression of Bcl-2

The relationship between the Src kinase Lyn and Bcl-2 expression was examined in chronic myelogenous leukemia cells (K562 and LAMA84) displaying a Bcr/Abl-independent form of imatinib mesylate resistance. K562-R and LAMA-R cells that were markedly resistant to induction of mitochondrial dysfunction (e.g. loss of mitochondrial membrane potential, Bax translocation, cytochrome c, and apoptosis-inducing factor release) and apoptosis by imatinib mesylate exhibited a pronounced reduction in expression of Bcr/Abl, Bcl-x(L), and STAT5 but a striking increase in levels of activated Lyn. Whereas basal expression of Bcl-2 protein was very low in parental cells, imatinib-resistant cells displayed a marked increase in Bcl-2 mRNA and/or protein levels. Treatment of LAMA-R cells with the Src kinase inhibitor PP2 significantly reduced Lyn activation as well as Bcl-2 mRNA and protein levels. Transient or stable transfection of LAMA84 or K562 cells with a constitutively active Lyn (Y508F), but not with a kinase-dead mutant (K275D), significantly increased Bcl-2 protein expression and protected cells from lethality of imatinib mesylate. Ectopic expression of Bcl-2 protected K562 and LAMA84 cells from imatinib mesylate- and PP2-mediated lethality. Conversely, interference with Bcl-2 function by co-administration of the small molecule Bcl-2 inhibitor HA14-1 or down-regulation of Bcl-2 expression by small interfering RNA or antisense strategies significantly increased mitochondrial dysfunction and apoptosis induced by imatinib mesylate and the topoisomerase inhibitor VP-16 in LAMA-R cells. In marked contrast, these interventions had little effect in parental LAMA84 cells that display low basal levels of Bcl-2. Together, these findings indicate that activation of Lyn in leukemia cells displaying a Bcr/Abl-independent form of imatinib mesylate resistance plays a functional role in Bcl-2 up-regulation and provide a theoretical basis for the development of therapeutic strategies targeting Bcl-2 in such a setting.     

Drug repurposing for chronic myeloid leukemia: in silico and in vitro investigation of DrugBank database for allosteric Bcr-Abl inhibitors

Chronic myeloid leukemia (CML) is caused by chromosomal rearrangement resulting in the expression of Bcr-Abl fusion protein with deregulated Abl tyrosine kinase activity. Approved drugs – imatinib, dasatinib, nilotinib, and ponatinib – target the ATP-binding site of Abl kinase. Even though these drugs are initially effective, long-term usefulness is limited by the development of resistance. To overcome this problem, targeting the allosteric site of Abl kinase, which is remote from the ATP-binding site is found to be a useful strategy. In this study, structure-based and ligand-based virtual screening methods were applied to narrow down possible drugs (from DrugBank database) that could target the allosteric site of Abl kinase. Detailed investigations of the selected drugs in the allosteric site of Abl kinase, using molecular dynamics and steered molecular dynamics simulation shows that gefitinib, an EGFR inhibitor approved for the treatment of lung cancer, could bind effectively to the allosteric site of Bcr-Abl. More interestingly, gefitinib was found to enhance the ability of imatinib to bind at the ATP-binding site of Bcr-Abl kinase. Based on the in silico findings, gefitinib was tested in combination with imatinib in K562 CML cell line using MTT cell proliferation assay and found to have a synergistic antiproliferative activity. Further detailed mechanistic study could help to unravel the full potential of imatinib – gefitinib combination for the treatment of CML.     

A Src-like inactive conformation in the abl tyrosine kinase domain

The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML). The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif is flipped by approximately 180 degrees with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP-peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR-Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain.     

Cell-penetrating fusion peptides OD1 and OD2 interact with Bcr–Abl and influence the growth and apoptosis of K562 cells

The Bcr–Abl oncoprotein is the cause of chronic myelogenous leukemia (CML). Crystal structure analysis suggests that Bcr_30–63 is the core of the Bcr–Abl oligomerization interface for aberrant kinase activity; however, the precise role of other residues of Bcr_1–72 excluding Bcr_30–63 have not been evaluated. In this study, Bcr_30–63 was named OD2 and other residues of Bcr_1–72 were named OD1. Cytoplasmic transduction peptide (CTP) was used to carry molecules into cytoplasm. CTP-OD1 and CTP-OD2 fusion peptides were expressed from a cold-inducible expression system. Our results demonstrated that both fusion peptides could localize into the cytoplasm, specifically interact with the Bcr–Abl protein and further inhibit growth, induce apoptosis, and decrease the phosphorylation of Bcr–Abl in K562 cell lines. However, the viability of THP-1, a Bcr–Abl negative cell line, was unaffected. These results suggested that CTP-OD1 and CTP-OD2 may be an attractive therapeutic option to inhibit the activation of Bcr–Abl kinase in CML.     

Induction of Heme Oxygenase-1 by Na+-H+ Exchanger 1 Protein Plays a Crucial Role in Imatinib-resistant Chronic Myeloid Leukemia Cells

Resistance toward imatinib (IM) and other BCR/ABL tyrosine kinase inhibitors remains troublesome in the treatment of advanced stage chronic myeloid leukemia (CML). The aim of this study was to estimate the reversal effects of down-regulation of Na⁺/H⁺ exchanger 1 (NHE1) on the chemoresistance of BCR-ABL-positive leukemia patients'' cells and cell lines. After treatment with the specific NHE1 inhibitor cariporide to decrease intracellular pH (pH_i), the heme oxygenase-1 (HO-1) levels of the K562R cell line and cells from IM-insensitive CML patients decreased. HO-1, as a Bcr/Abl-dependent survival molecule in CML cells, is important for the resistance to tyrosine kinase inhibitors in patients with newly diagnosed CML or IM-resistant CML. Silencing PKC-β and Nrf-2 or treatment with inhibitors of p38 pathways obviously blocked NHE1-induced HO-1 expression. Furthermore, treatment with HO-1 or p38 inhibitor plus IM increased the apoptosis of the K562R cell line and IM-insensitive CML patients'' cells. Inhibiting HO-1 enhanced the activation of caspase-3 and poly(ADP-ribose) polymerase-1. Hence, the results support the anti-apoptotic role of HO-1 induced by NHE1 in the K562R cell line and IM-insensitive CML patients and provide a mechanism by which inducing HO-1 expression via the PKC-β/p38-MAPK pathway may promote tumor resistance to oxidative stress.     

IGF-I-induced oligodendrocyte progenitor proliferation requires PI3K/Akt, MEK/ERK, and Src-like tyrosine kinases

Insulin-like growth factor-I (IGF-I) is required for the growth of oligodendrocytes, although the underlying mechanisms are not fully understood. Our aim was to investigate the role of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase kinase (MEK1), and Src family tyrosine kinases in IGF-I-stimulated proliferation of oligodendrocyte progenitors. IGF-I treatment increased the proliferation of cultured oligodendrocyte progenitors as determined by measuring incorporation of [(3)H]-thymidine and bromodeoxy-uridine (BrdU). IGF-I stimulated a transient phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK1) and extracellular signal-regulated kinases (ERK1/2) (targets of MEK1), as well as a rapid and sustained activation of Akt (a target of PI3K). Furthermore, inhibitors of PI3K (LY294002 and Wortmannin), MEK1 (PD98059 and U0126), and Src family tyrosine kinases (PP2) decreased IGF-I-induced proliferation, and blocked ERK1/2 activation. LY294002, Wortmannin and PP2 also blocked Akt activation. To further determine whether Akt is required for IGF-I stimulated oligodendrocyte progenitor proliferation, cultures were infected with adenovirus vectors expressing dominant-negative mutants of Akt or treated with pharmacological inhibitors of Akt. All treatments reduced IGF-I-induced oligodendrocyte progenitor proliferation. Our data indicate that stimulation of oligodendrocyte progenitor proliferation by IGF-I requires Src-like tyrosine kinases as well as the PI3K/Akt and MEK1/ERK signaling pathways.     

c‐Jun blocks cell differentiation but not growth inhibition or apoptosis of chronic myelogenous leukemia cells induced by STI571 and by histone deacetylase inhibitors

The constitutively active Bcr‐Abl tyrosine kinase plays a crucial role in chronic myelogenous leukemia (CML) pathogenesis. The Bcr‐Abl protein induces the upregulation of proto‐oncogene c‐Jun, which is involved in Bcr‐Abl transforming activity in Bcr‐Abl positive cells. Recent studies reported that c‐Jun inhibited hemoglobin synthesis in human CML cell line K562. However, c‐Jun also plays a critical role in cell proliferation and apoptosis. In this study, we investigated the physiological roles of c‐Jun in cell proliferation, apoptosis and erythroid differentiation of K562 cells. Firstly, we generated K562 cell lines stably overexpressing c‐Jun. These clones have the same proliferation rate as the parental cell line in general culture medium. Endogenous c‐Jun expression was analyzed to determine the effective concentration of STI571 for inhibiting Bcr‐Abl signaling. Western blots show that STI571 inhibited c‐Jun expression in a dose‐dependent manner, reaching a maximum inhibition at 1 µM. STI571 could inhibit c‐Jun expression in K562 cells, but not in c‐Jun‐overexpression cells. c‐Jun did not alter growth inhibition and apoptotic induction by STI571 treatment, but inhibited STI571‐induced erythroid differentiation. Moreover, c‐Jun did not alter growth inhibition and apoptotic induction by histone deacetylase (HDAC) inhibitors (apicidin, sodium butyrate, and MS275) treatment, but inhibited HDAC inhibitors‐induced erythroid differentiation. These results suggest that c‐Jun may modulate anticancer drugs‐induced cell differentiation but not growth inhibition and apoptosis in CML cells. J. Cell. Physiol. 218: 568–574, 2009. © 2008 Wiley‐Liss, Inc.     

Cytoprotective effect of imatinib mesylate in non-BCR-ABL-expressing cells along with autophagosome formation

Treatment with imatinib mesylate (IM) results in an increased viable cell number of non-BCR-ABL-expressing cell lines by inhibiting spontaneous apoptosis. Electron microscopy revealed an increase of autophagosomes in response to IM. IM attenuated the cytotoxic effect of cytosine arabinoside, as well as inhibiting cell death with serum-deprived culture. Cytoprotection with autophagosome formation by IM was observed in various leukemia and cancer cell lines as well as normal murine embryonic fibroblasts (MEFs). Complete inhibition of autophagy by knockdown of atg5 in the Tet-off atg5^−/− MEF system attenuated the cytoprotective effect of IM, indicating that the effect is partially dependent on autophagy. However, cytoprotection by IM was not mediated through suppression of ROS production via mitophagy, ER stress via ribophagy, or proapoptotic function of ABL kinase. Although the target tyrosine kinase(s) of IM remains unclear, our data provide novel therapeutic possibilities of using IM for cytoprotection.     

The NH(2)-terminal coiled-coil domain and tyrosine 177 play important roles in induction of a myeloproliferative disease in mice by Bcr-Abl

Bcr-Abl, a fusion protein generated by t(9;22)(q34;q11) translocation, plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). It has been shown that Bcr-Abl contains multiple functional domains and motifs and can disrupt regulation of many signaling pathways and cellular functions. However, the role of specific domains and motifs of Bcr-Abl or of specific signaling pathways in the complex in vivo pathogenesis of CML is not completely known. We have previously shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder (MPD) in mice resembling human CML. We have also shown that the Abl kinase activity within Bcr-Abl is essential for Bcr-Abl leukemogenesis, yet activation of the Abl kinase without Bcr sequences is not sufficient to induce MPD in mice. In this study we investigated the role of Bcr sequences within Bcr-Abl in inducing MPD using this murine model for CML. We found that the NH(2)-terminal coiled-coil (CC) domain was both essential and sufficient, even though not efficient, to activate Abl to induce an MPD in mice. Interestingly, deletion of the Src homology 3 domain complemented the deficiencies of the CC-deleted Bcr-Abl in inducing MPD in mice. We further demonstrated that the Grb2 binding site at Y177 played an important role in efficient induction of MPD. These studies directly demonstrated the important roles of Bcr sequences in induction of MPD by Bcr-Abl.     

Changes in cell adhesivity and cytoskeleton‐related proteins during imatinib‐induced apoptosis of leukemic JURL‐MK1 cells

The fusion protein Bcr–Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr–Abl protein used in front‐line CML therapy, on the adhesivity of JURL‐MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL‐MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr–Abl inhibition and secondary changes related to the apoptosis. Imatinib treatment caused a transient increase in JURL‐MK1 cell adhesivity to fibronectin, possibly due to the switch off of Bcr–Abl activity. Subsequently, we observed a number of changes including a decrease in cell adhesivity, F‐actin decomposition, reduction of integrin β1, CD44, and paxillin expression levels and a marked increase in cofilin phophorylation at Ser3. These events were generally related to the proceeding apoptosis but they differed in their sensitivity to the individual caspase inhibitors. J. Cell. Biochem. 111: 1413–1425, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.     

Blockade of the ERK or PI3K-Akt signaling pathway enhances the cytotoxicity of histone deacetylase inhibitors in tumor cells resistant to gefitinib or imatinib

Deregulated activation of protein tyrosine kinases, such as the epidermal growth factor receptor (EGFR) and Abl, is associated with human cancers including non-small cell lung cancer (NSCLC) and chronic myeloid leukemia (CML). Although inhibitors of such activated kinases have proved to be of therapeutic benefit in individuals with NSCLC or CML, some patients manifest intrinsic or acquired resistance to these drugs. We now show that, whereas blockade of either the extracellular signal-regulated kinase (ERK) pathway or the phosphatidylinositol 3-kinase (PI3K)-Akt pathway alone induced only a low level of cell death, it markedly sensitized NSCLC or CML cells to the induction of apoptosis by histone deacetylase (HDAC) inhibitors. Such enhanced cell death induced by the respective drug combinations was apparent even in NSCLC or CML cells exhibiting resistance to EGFR or Abl tyrosine kinase inhibitors, respectively. Co-administration of a cytostatic signaling pathway inhibitor may contribute to the development of safer anticancer strategies by lowering the required dose of cytotoxic HDAC inhibitors for a variety of cancers.     

Bcr-Abl with an SH3 deletion retains the ability To induce a myeloproliferative disease in mice, yet c-Abl activated by an SH3 deletion induces only lymphoid malignancy

The bcr-abl oncogene plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). The fusion of Bcr sequences to Abl constitutively activates the Abl protein tyrosine kinase. We have recently shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces in mice a myeloproliferative disease resembling human CML and that Abl kinase activity is essential for Bcr-Abl to induce a CML-like myeloproliferative disease. However, it is not known if activation of the Abl kinase alone is sufficient to induce a myeloproliferative disease. In this study, we examined the role of the Abl SH3 domain of Bcr-Abl in induction of myeloproliferative disease and tested whether c-Abl activated by SH3 deletion can induce a CML-like disease. We found that Bcr-Abl with an Abl SH3 deletion still induced a CML-like disease in mice. In contrast, c-Abl activated by SH3 deletion induced only lymphoid malignancies in mice and did not stimulate the growth of myeloid colonies from 5-fluorouracil-treated bone marrow cells in vitro. These results indicate that Bcr sequences in Bcr-Abl play additional roles in inducing myeloproliferative disease beyond simply activating the Abl kinase domain and that functions of the Abl SH3 domain are either not required or redundant in Bcr-Abl-induced myeloproliferative disease. The results also suggest that the type of hematological neoplasm induced by an abl oncogene is influenced not only by what type of hematopoietic cells the oncogene is targeted into but also by the intrinsic oncogenic properties of the particular abl oncogene. In addition, we found that DeltaSH3 c-Abl induced less activation of Akt and STAT5 than did Bcr-Abl, suggesting that activation of these pathways plays a critical role in inducing a CML-like disease.     

Anti‐leukemic effect of PI3K inhibition on chronic myeloid leukemia (CML) cells: shedding new light on the mitigating effect of c‐Myc and autophagy on BKM120 cytotoxicity

The success in the identification of BCR/ABL tyrosine kinase role in the pathogenesis of chronic myeloid leukemia (CML) went as far as to find a path to cure this leukemia; however, compensatory activation of leukomogenic signals get across the message that the small molecule inhibitors of oncogenic pathways, along with tyrosine kinase inhibitors, might be a beneficial approach in CML treatment. The results of the present study showed that the abrogation of the phosphoinositide 3‐kinase (PI3K) pathway using pan‐PI3K inhibitor BKM120 exerted a cytotoxic effect against CML‐derived K562 cells through both the induction of p21‐mediated G2/M arrest and the stimulation of apoptosis. Notably, the apoptotic effect of the inhibitor was further confirmed by the molecular analysis showing that BKM120 significantly increased the expression of pro‐apoptotic genes. To the best of our knowledge, the involvement of autophagy in resistance to BKM120 has not been yet described and our study suggests for the first time that the elevation of autophagy‐related genes might serve as a compensatory pathway to cease the anti‐leukemic effect of BKM120 in K562; since we found a reinforced anti‐survival event when the cells were treated with BKM120 in combination with autophagy inhibitor. In conclusion, the results of the present study showed that the abrogation of PI3K using BKM120 might be a befitting approach in CML treatment, either as a single agent or in a combined‐modal strategy; however, further evaluations including clinical trials and in vivo investigations are demanded to ascertain the safety and the efficacy of the inhibitor in treatment strategies.