Bcr/Abl-based Novel Therapeutic Strategies In Chronic Myelogenous Leukemia Treatment

Chronic myelogenous leukemia (CML) is a clonal hematopoetic stem cell disorder with an annual incidence of one to two cases per 100,000 per year. CML is characterized by a reciprocal translocation of oncogene abl from the long arm of chromosome 9 to the long arm of chromosome 22 in the bcr region. The resulting bcr-abl fusion gene encodes a chimeric protein with strong tyrosine kinase activity. The formation of the Bcr/Abl tyrosine kinase renders CML resistant to traditional therapeutic approaches, thus Bcr/Abl becomes an important therapeutic target. Recently, the first Bcr/Abl tyrosine kinase inhibitor STI571 (Gleevec, imatinib mesylate), made by Novartis, Switzerland, has been presented as a revolutionary approach in CML treatment. STI571 becomes the first successful model in target-based cancer therapy. STI571 is now the first-line treatment for all newly diagnosed CML patients. Oral administration of STI571 results in a clinical response in more than 90% of CML patients, but the initial striking efficacy of this drug has been overshadowed by the development of clinical resistance. Patients with advanced phases of CML either fail to respond or quickly relapse following an initial response to STI571. Predominant mechanisms of resistance to STI571 include amplification, mutation, and loss of Bcr/Abl. Thus, it is important to search for additional approaches for the treatment of this malignancy.BCR/ABL has been found to activate multiple survival signaling pathways, including Ras/Raf/MEK, PI3K/Akt, JAK/STATs, c-Myc, Mcl-1, and Bcl-X_L, that regulate cell growth, survival, and adhesion/migration, and render CML cells highly resistant to most commonly employed cytotoxic drugs. Interruption of down stream signals of Bcr/Abl, such as Raf/MEK/MAPK, may enhance the sensitivity of STI571.Based on this hypothesis, interactions between the kinase inhibitor STI571 and MEK/MAPK cascade have been examined in human myeloid leukemia cells (K562 and LAMA 84) that express the Bcr-Abl kinase. Exposure of K562 cells to concentrations of STI571 that minimally induced apoptosis (e.g., approximately 200 nM) resulted in early suppression (i.e., at 6 h) of p42/44 MAPK phosphorylation followed at later intervals (i.e.,> or =24 h) by a marked increase in p42/44 MAPK phosphorylation/activation. Coadministration of a nontoxic concentration of the MEK1/2 inhibitor PD184352 (5 microM) prevented STI571-mediated activation of p42/44 MAPK. Cells exposed to STI571 in combination with PDl84352 for 48 h demonstrated a very dramatic increase in mitochondrial dysfunction (e.g., loss of DeltaPsim and cytosolic cytochrome c release) associated with procaspase-3 activation, poly(ADP-ribose) polymerase cleavage, and the appearance of the characteristic morphological features of apoptosis. Similar results were obtained using other pharmacological MEK1/2 inhibitors (e.g., PD 98059 and U0126) as well as another leukemic cell line that expresses Bcr-Abl (e.g., LAMA 84). However, synergistic induction of apoptosis by STI571 and PDl 84352 was not observed in human myeloid leukemia cells that do not express the Bcr-Abl kinase (e.g., HL-60 and U937) nor in normal human peripheral blood mononuclear cells. Synergistic potentiation of STI571-mediated lethality by PDl84352 was associated with multiple perturbations in signaling and apoptotic regulatory pathways, including caspase-dependent down-regulation of Bcr-Abl and Bcl-2; caspase-independent down-regulation of Bcl-x(L) and Mcl-1; activation of JNK, p38 MAPK, and p34(cdc2); and diminished phosphorylation of Stat5 and CREB. Significantly, co-exposure to PDl84352 strikingly increased the lethality of a pharmacologically achievable concentration of STI571 (i.e., 1-2 microM) in resistant K562 cells expressing marked increases in Bcr-Abl protein levels. Together, these findings raise the possibility that treatment of Bcr-Abl-expressing cells with STI571 elicits a cytoprotective MAPK activation response and that interruption of the latter pathway (e.g., by pharmacological MEK1/2 inhibitors) is associated with a highly synergistic induction of mitochondrial damage and apoptosis. They also indicate that in the case of Bcr-Abl-positive cells, simultaneous interruption of two signal transduction pathways may represent an effective antileukemic strategy.Recently, histone deacetylase inhibitors (HDIs) have emerged as a potentially promising new class of anticancer drugs. In this regard, there is much evidence that disruption of leukemic cell maturation provides a potent apoptotic stimulus. Additionally, we have found that HDIs inactivate the Raf/MEK/MAPK cascade, and are activated in inducing apoptosis in Bcr/Abl positive cells. Thus HDIs share the same feature as MEK inhibitors (e.g., inhibition of MEK/MAPK cascade), so combining treatment of HDIs with STI571 may promote apoptosis in CML cells.Interactions between histone deacetylase inhibitors (HDIs) and the Bcr/Abl kinase inhibitor STI571 (Gleevec) have been examined in Bcr/Abl+ human leukemia cells (K562 and LAMA 84) sensitive and resistant to STI571. Co-treatment of K562 cells with 250 nM STI571 and 2.0 uM suberoylanilide hydroxamic acid (SAHA) for 24 hr, exposures that were minimally toxic alone, resulted in a marked increase inmitochondrial damage (e.g., cytochrome c, Smac/DIABLO, and AIF release), caspase activation, and apoptosis. Similar events were observed in other Bcr/Abl+ cells (i.e., LAMA 84), and in cells exposed to STI571 in combination with the HDI sodium butyrate (SB). Co-exposure of cells to HDIs in conjunction with STI571 resulted in multiple perturbations in signaling and cell cycle regulatory proteins, including down-regulation of Raf, phospho-MEK(mitogen-activated protein kinase kinase), phospho-ERK (extracellular-regulated kinase), cyclin Di, and Mcl-1, accompanied by dephosphorylation and cleavage of pRb, and a striking increase in phosphorylation of JNK. Co-exposure of Bcr/Abf cells to STI571 and SAHA also blocked SAHA-mediated induction of p21CIP1, and resulted in down-regulation of Bcr/Abl protein expression. STI571 and SAHA also interacted synergistically to induce apoptosis in STI571-resistant K562 and LAMA 84 cells that display increased Bcr/Abl protein expression. Lastly, inducible expression of a constitutively active MEK1/2 construct substantially attenuated SAHA/STI571 -mediated apoptosis in K562 cells, implicating that disruption of the Raf/MEK/ERK axis is a mechanism that may explain the synergistic antileukemic effects of this drug combination. Together, these findings indicate that combined exposure of Bcr/Abl+ cells to the kinase inhibitor STI571 and HDIs leads to diverse perturbations in signaling and cell cycle regulatory proteins, associated with a marked increase in mitochondrial damage and cell death. They also raise the possibility that this strategy may be effective in some Bcr/Abl+ cells that are resistant to STI571 through increased Bcr/Abl expression.However, the above strategy is not effective in the STI571 resistant CML cells with loss of Bcr/Abl. Further search for intensive strategies are necessary to overcome STI571-resistant cells with loss of Bcr/Abl. Logically, we may need to remove STI571 from the combination because the Bcr/Abl target of STI571 is lost in those cells.It has been reported that ROS reactive oxygen species (ROS) generation is the crucial mechanism of HDIs underlying their anticancer effects. Excessive production of ROS in the cell is known to induce apoptosis. The ability of ROS to inflict severe cellular damage and cause cell death provides an approach to kill cancer cells by imposing excessive ROS stress. ROS broadly interacts with signal transduction pathways, including MEK/MAPK, JNK, p38, Akt, p53, PKC, JAK and others. ROS generation has also been shown to play an important role in mediating apoptosis induced by cisplatin, bleomycin, UV irradiation, and PS-341 (Bortezomib), a proteosome inhibitor recently approved by FDA for treatment of multiple myeloma. We have found that ROS generation by PS-341 plays a central role in apoptosis induction in leukemia. It is responsible for activation of the stress-related JNK pathway coupled with inactivation of the cytoprotective ERK cascade, leading, in turn, to release of proapoptogenic proteins(e.g., cytochrome c) from the mitochondria and caspase activation. Considering the above evidence, the approach of combining HDIs with PS-341 may enhance ROS generation and result in synergism of apoptosis induction.Based on these predictions, interactions between the proteasome inhibitor bortezomib (Velcade?; previously known as PS-341) and HDIs have been examined in Bcr/Abl+ human leukemia cells (K562 and LAMA 84). Co-exposure of cells (24-48hr) to minimally toxic concentrations of bortezimib + either SAHA or sodium butyrate (SB) resulted in a striking increase in mitochondrial injury, caspase activation, and apoptosis, reflected by caspase-3 and -8 cleavage and PARP degradation. These events were accompanied by down-regulation of the Raf-1/MEK/ERK pathway as well as diminished expression of Bcr/Abl and cyclin Di, cleavage of p21CIP1 and pRb, and induction of the stress-related kinases JNK and p38 MAPK. Transient transfection of cells with a constitutively active MEK construct significantly protected them from bortezimib /SAHA-mediated lethality. Co-administration of bortezimib and SAHA resulted in increased ROS generation and diminished NF-kB activation; moreover, the free radical scavenger L-N-acetylcyteine (LNAC) blocked bortezimib /SAHA-related ROS generation, induction of JNK and p21CIP1, and apoptosis. Lastly, this regimen potently induced apoptosis in STI571 (Gleevec)-resistant K562 cells with amplification of Bcr/Abl, and CD34+ mononuclear cells obtained from a patient with STI571 -resistant disease, as well as in Bcr/Abl" cells (e.g., Jurkat, NB4, KM-H2). Together, these findings raise the possibility that combined proteasome/HDI may represent a novel therapeutic strategy in a broad spectrum of cancers, including refractory hematologic malignancies with Bcr/Abl amplification and loss, as well as other tumors (e.g. lymphoma).Collectively, the studies demonstrate that approaches by combining novel target-based agents not only result in apoptosis synergisms, but also overcome clinic relevant resistant malignancies. Lastly, it is very interesting to know whether apoptosis synergism is induced by combining target-based agents with chemotherapeutic agents.PI3K/Akt/mTOR is a very attractive pathway for target-based therapy. Several agents targeting this cascade, such as Akt inhibitor, and mTOR inhibitors (e.g., rapamycin, CCI-779) have been used in clinic trials. Fladarabine is one of the most active cytotoxic drugs in leukemia treatment. We attempt to examine whether inhibition of PI3K/Akt/mTOR promotes Fludarabine-induced apoptosis.We have investigated the effects of PI3K/AktmTOR pathway on the response of human leukemia cells to fludarabine. Inhibition of PI3K/Akt pathway with a selective inhibitor (eg. LY294002, or wortmannin) in leukemic cells markedly potentiated fludarabine-induced apoptosis. Inhibition of the PI3K/Akt downstream target mTOR, by rapamycin also significantly enhanced fludarabine-induced apoptosis. The co-treatmentof fludarabine/LY294002 resulted in significant attenuation in the levels of both phospho-Erkl/2 and phospho-Akt, as well as a marked increase in the level of phospho-JNK. The broad spectrum caspase inhibitor BOC-D-fmk markedly blocked fludarabine/LY-induced apoptosis, had no effect on cytochrome c release to the cytosol, and did abrogate caspase and PARP cleavage. This indicates that mitochondrial dysfunction is upstream of the caspase cascade. Moreover, constitutive activation of the MEK/ERK pathway completely blocked apoptosis induced by the combination of fludarabine/LY294002. Additionally, either constitutive activation of Akt or blockage of the JNK pathway significantly diminished apoptosis induced by the combination. Collectively, these findings demonstrate that inactivation of MAPK, Akt, and activation of the JNK pathway contributes to the induction of apoptosis induced by fludarabine/LY. Comparatively, MAPK inactivation plays a crucial role in fludarabine/LY-induced apoptosis. These results strongly suggest that combining fludarabine with an inhibitor of the PI3K/Akt/mT0R pathway may represent a novel therapeutic strategy for hematological malignancies. This study also indicates that inhibition of cell survival pathways by target-based agents can enhance cytotoxic agent-induced apoptosis.Taken together, the preceding findings demonstrate that numerous of survival pathways are activated in transformed cells and form signal transduction networks. Thus, even though an agent is very effective in targeting a specific survival pathway, it may not be an effective cancer treatment as other pathways may compensate allowing cell survival, so only interrupting one or a few of survival signals might not be enough to lead cancer cells into apoptosis, especially in refractory cancer cells. Effective cancer therapies may need to interrupt all of the crucial survival pathways, so approaches combining approximate agents might be effective to reach this aim. Present results also suggest a model in which the cell's fate depends on the balance between anti-apoptotic cytoprotective signals and pro-apoptotic stress-related signals.