Overcoming kinase resistance in chronic myeloid leukemia

Imatinib is a small-molecule inhibitor of BCR-ABL tyrosine kinase activity, with proven efficacy and tolerability. Despite imatinib's activity, the development of resistance, whether BCR-ABL dependent or independent, is a concern. BCR-ABL-dependent resistance is commonly a result of mutations in the BCR-ABL gene, which can induce a structural predisposition towards the active conformation of the protein, resulting in a shift in the equilibrium of BCR-ABL from inactive, which imatinib binds, to active, which imatinib is unable to bind. BCR-ABL gene amplification may play a role in the development of imatinib resistance in patients with CML. There are a number of BCR-ABL-independent mechanisms of imatinib resistance, including the efflux protein multidrug resistance protein-1, of which imatinib is a substrate. Another mechanism may be the development of alternative pathways of disease progression, leading to less reliance on BCR-ABL; indeed, the SRC family tyrosine kinases LYN and HCK have been frequently implicated in treatment resistance and progression of CML. Clearly, imatinib resistance requires the development of other treatment options. Dasatinib, with increased binding potency (325-fold greater potency than imatinib for wild-type BCR-ABL), inhibition of both the active and inactive formation of BCR-ABL, and targeting of SRC family kinases, is the only agent approved for the treatment of patients with imatinib-resistant or -intolerant CML and Ph+ ALL. Dasatinib is highly active in all phases of these diseases, and is active in the majority of imatinib-resistant mutations, with the exception of T315I. The development of agents that effectively inhibit T315I mutations suggests that future treatment options will include combination therapy.     

Growth arrest of BCR-ABL positive cells with a sequence-specific polyamide-chlorambucil conjugate

Chronic myeloid leukemia (CML) is characterized by the presence of a constitutively active Abl kinase, which is the product of a chimeric BCR-ABL gene, caused by the genetic translocation known as the Philadelphia chromosome. Imatinib, a selective inhibitor of the Bcr-Abl tyrosine kinase, has significantly improved the clinical outcome of patients with CML. However, subsets of patients lose their response to treatment through the emergence of imatinib-resistant cells, and imatinib treatment is less durable for patients with late stage CML. Although alternative Bcr-Abl tyrosine kinase inhibitors have been developed to overcome drug resistance, a cocktail therapy of different kinase inhibitors and additional chemotherapeutics may be needed for complete remission of CML in some cases. Chlorambucil has been used for treatment of B cell chronic lymphocytic leukemia, non-Hodgkin's and Hodgkin's disease. Here we report that a DNA sequence-specific pyrrole-imidazole polyamide-chlorambucil conjugate, 1R-Chl, causes growth arrest of cells harboring both unmutated BCR-ABL and three imatinib resistant strains. 1R-Chl also displays selective toxicities against activated lymphocytes and a high dose tolerance in a murine model.     

Imatinib can act as an Allosteric Activator of Abl Kinase

Imatinib is an ATP-competitive inhibitor of Bcr-Abl kinase and the first drug approved for chronic myelogenous leukemia (CML) treatment. Here we show that imatinib binds to a secondary, allosteric site located in the myristoyl pocket of Abl to function as an activator of the kinase activity. Abl transitions between an assembled, inhibited state and an extended, activated state. The equilibrium is regulated by the conformation of the αΙ helix, which is located nearby the allosteric pocket. Imatinib binding to the allosteric pocket elicits an αΙ helix conformation that is not compatible with the assembled state, thereby promoting the extended state and stimulating the kinase activity. Although in wild-type Abl the catalytic pocket has a much higher affinity for imatinib than the allosteric pocket does, the two binding affinities are comparable in Abl variants carrying imatinib-resistant mutations in the catalytic site. A previously isolated imatinib-resistant mutation in patients appears to be mediating its function by increasing the affinity of imatinib for the allosteric pocket, providing a hitherto unknown mechanism of drug resistance. Our results highlight the benefit of combining imatinib with allosteric inhibitors to maximize their inhibitory effect on Bcr-Abl.     

Two different point mutations in ABL gene ATP-binding domain conferring primary imatinib resistance in a chronic myeloid leukemia (CML) patient: A case report

Imatinib (Gleevec) is the effective therapy for BCR-ABL positive CML patients. Point mutations have been detected in ATP-binding domain of ABL gene which disturbs the binding of Gleevec to this target leading to resistance. Detection of mutations is helpful in clinical management of imatinib resistance. We established a very sensitive (ASO) PCR to detect mutations in an imatinib-resistant CML patient. Mutations C944T and T1052C were detected which cause complete partial imatinib resistance, respectively. This is the first report of multiple point mutations conferring primary imatinib resistance in same patient at the same time. Understanding the biological reasons of primary imatinib resistance is one of the emerging issues of pharmacogenomics and will be helpful in understanding primary resistance of molecularly-targeted cancer therapies. It will also be of great utilization in clinical management of imatinib resistance. Moreover, this ASO-PCR assay is very effective in detecting mutations related to imatinib resistance. Published: July 1, 2004.     

Advances in the structural biology, design and clinical development of Bcr-Abl kinase inhibitors for the treatment of chronic myeloid leukaemia

The constitutively activated Abl tyrosine kinase domain of the chimeric Bcr-Abl oncoprotein is responsible for the transformation of haematopoietic stem cells and the symptoms of chronic myeloid leukaemia (CML). Imatinib targets the tyrosine kinase activity of Bcr-Abl and is a first-line therapy for this malignancy. Although highly effective in chronic phase CML, patients who have progressed to the advanced phase of the disease frequently fail to respond to imatinib or develop resistance to therapy and relapse. This is often due to the emergence of clones expressing mutant forms of Bcr-Abl, which exhibit a decreased sensitivity towards inhibition by imatinib. Considerable progress has recently been made in understanding the structural biology of Abl and the molecular basis for resistance, facilitating the discovery and development of second generation drugs designed to combat mutant forms of Bcr-Abl. The first of these compounds to enter clinical development were BMS-354825 (BristolMyersSquibb) and AMN107 (Novartis Pharma) and, from Phase I results, both of these promise a breakthrough in the treatment of imatinib-resistant CML. Recent advances with these and other promising classes of new CML drugs are reviewed.     

Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)

Imatinib was the first BCR-ABL-targeted agent approved for the treatment of patients with chronic myeloid leukemia (CML) and confers significant benefit for most patients; however, a substantial number of patients are either initially refractory or develop resistance. Point mutations within the ABL kinase domain of the BCR-ABL fusion protein are a major underlying cause of resistance. Of the known imatinib-resistant mutations, the most frequently occurring involve the ATP-binding loop (P-loop). In vitro evidence has suggested that these mutations are more oncogenic with respect to other mutations and wild type BCR-ABL. Dasatinib and nilotinib have been approved for second-line treatment of patients with CML who demonstrate resistance (or intolerance) to imatinib. Both agents have marked activity in patients resistant to imatinib; however, they have differential activity against certain mutations, including those of the P-loop. Data from clinical trials suggest that dasatinib may be more effective vs. nilotinib for treating patients harboring P-loop mutations. Other mutations that are differentially sensitive to the second-line tyrosine kinase inhibitors (TKIs) include F317L and F359I/V, which are more sensitive to nilotinib and dasatinib, respectively. P-loop status in patients with CML and the potency of TKIs against P-loop mutations are key determinants for prognosis and response to treatment. This communication reviews the clinical importance of P-loop mutations and the efficacy of the currently available TKIs against them.     

Effects of carboxyamidotriazole on in vitro models of imatinib-resistant chronic myeloid leukemia

Although imatinib mesylate (IM) has revolutionized the treatment of chronic myeloid leukemia (CML), some patients develop resistance with progression of leukemia. Alternative or additional targeting of signaling pathways deregulated in bcr-abl-driven CML cells may provide a feasible option for improving clinical response and overcoming resistance. In this study, we show that carboxyamidotriazole (CAI), an orally bioavailable calcium influx and signal transduction inhibitor, is equally effective in inhibiting the proliferation and bcr-abl dependent- and independent-signaling pathways in imatinib-resistant CML cells. CAI inhibits phosphorylation of cellular proteins including STAT5 and CrkL at concentrations that induce apoptosis in IM-resistant CML cells. The combination of imatinib and CAI also down-regulated bcr-abl protein levels. Since CAI is already available for clinical use, these results suggest that it may be an effective addition to the armamentarium of drugs for the treatment of CML.     

Imatinib and Nilotinib Reverse Multidrug Resistance in Cancer Cells by Inhibiting the Efflux Activity of the MRP7 (ABCC10)

Background

One of the major mechanisms that could produce resistance to antineoplastic drugs in cancer cells is the ATP binding cassette (ABC) transporters. The ABC transporters can significantly decrease the intracellular concentration of antineoplastic drugs by increasing their efflux, thereby lowering the cytotoxic activity of antineoplastic drugs. One of these transporters, the multiple resistant protein 7 (MRP7, ABCC10), has recently been shown to produce resistance to antineoplastic drugs by increasing the efflux of paclitaxel. In this study, we examined the effects of BCR-Abl tyrosine kinase inhibitors imatinib, nilotinib and dasatinib on the activity and expression of MRP7 in HEK293 cells transfected with MRP7, designated HEK-MRP7-2.

Methodology and/or Principal Findings

We report for the first time that imatinib and nilotinib reversed MRP7-mediated multidrug resistance. Our MTT assay results indicated that MRP7 expression in HEK-MRP7-2 cells was not significantly altered by incubation with 5 µM of imatinib or nilotinib for up to 72 hours. In addition, imatinib and nilotinib (1-5 µM) produced a significant concentration-dependent reversal of MRP7-mediated multidrug resistance by enhancing the sensitivity of HEK-MRP7-2 cells to paclitaxel and vincristine. Imatinib and nilotinib, at 5 µM, significantly increased the accumulation of [³H]-paclitaxel in HEK-MRP7-2 cells. The incubation of the HEK-MRP7-2 cells with imatinib or nilotinib (5 µM) also significantly inhibited the efflux of paclitaxel.

Conclusions

Imatinib and nilotinib reverse MRP7-mediated paclitaxel resistance, most likely due to their inhibition of the efflux of paclitaxel via MRP7. These findings suggest that imatinib or nilotinib, in combination with other antineoplastic drugs, may be useful in the treatment of certain resistant cancers.     

Enhancement of imatinib-induced apoptosis of BCR/ABL-expressing cells by nutlin-3 through synergistic activation of the mitochondrial apoptotic pathway

The BCR/ABL tyrosine kinase inhibitor imatinib is highly effective for treatment of chronic myeloid leukemia (CML) and Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). However, relapses with emerging imatinib-resistance mutations in the BCR/ABL kinase domain pose a significant problem. Here, we demonstrate that nutlin-3, an inhibitor of Mdm2, inhibits proliferation and induces apoptosis more effectively in BCR/ABL-driven Ton.B210 cells than in those driven by IL-3. Moreover, nutlin-3 drastically enhanced imatinib-induced apoptosis in a p53-dependent manner in various BCR/ABL-expressing cells, which included primary leukemic cells from patients with CML blast crisis or Ph+ ALL and cells expressing the imatinib-resistant E255K BCR/ABL mutant. Nutlin-3 and imatinib synergistically induced Bax activation, mitochondrial membrane depolarization, and caspase-3 cleavage leading to caspase-dependent apoptosis, which was inhibited by overexpression of Bcl-XL. Imatinib did not significantly affect the nutlin-3-induced expression of p53 but abrogated that of p21. Furthermore, activation of Bax as well as caspase-3 induced by combined treatment with imatinib and nutlin-3 was observed preferentially in cells expressing p21 at reduced levels. The present study indicates that combined treatment with nutlin-3 and imatinib activates p53 without inducing p21 and synergistically activates Bax-mediated intrinsic mitochondrial pathway to induce apoptosis in BCR/ABL-expressing cells.     

Bcl6 gene-silencing facilitates PMA-induced megakaryocyte differentiation in K562 cells

Targeted therapy via imatinib appears to be a promising approach for chronic myeloid leukemia (CML) therapy. However, refractory and resistance to imatinib therapy has encouraged many investigators to get involved in development of new therapeutic agents such as Phorbol 12-myrestrat 13-acetate (PMA) for patients with CML. In that line, we attempted to investigate the chemosensitizing effect of PMA on the imatinib-resistant cells. Based on our western blot analyses, resistant K562 cells (K562R) showed high levels of FoxO3a and Bcl6 expressions which were not modulated by imatinib treatment. However, upon PMA treatment, the levels of both FoxO3a and Bcl6 were up-regulated among both the sensitive and the resistant cells and this treatment was associated with initiation of megakaryocytic differentiation of the cells. SiRNA-silencing of FoxO3a led to augmentation of megakaryocytic differentiation of the cells. Similarly, siRNA gene silencing of Bcl6 enhanced the differentiation and induced cell apoptosis among both types of cells. Regarding these results, it might be concluded that Bcl6 knockdown combined with PMA therapy could present a new therapeutical strategy for refractory CML patients to imatinib.     

Erythropoietin promotes resistance against the Abl tyrosine kinase inhibitor imatinib (STI571) in K562 human leukemia cells

Chronic myeloid leukemia is characterized by the Philadelphia chromosome translocation that causes expression of Bcr-Abl, a deregulated tyrosine kinase. Imatinib mesylate (STI571, Gleevec), a therapeutically used inhibitor of Bcr-Abl, causes apoptosis of Bcr-Abl-positive cells. In the leukemia cell line K562, we observed spontaneous resistance to imatinib at very low frequencies when cells were exposed to the drug (1 micro M) for more than 4 weeks. Surprisingly, in the presence of erythropoietin (Epo), K562 cells were temporarily able to sustain proliferation in the presence of imatinib, and imatinib-resistant clones could be isolated with high frequencies. From such imatinib-resistant, Epo-dependent clones, sublines could be established that were resistant to imatinib in the absence of Epo. Mitogen-activated protein (MAP) kinase activity was inhibited by imatinib treatment but could be partially restored by Epo. Inhibition of MAP kinase or phosphatidylinositol 3-kinase blocked the protective effect of Epo. The data suggest that K562 cells acquire factor dependency under imatinib/Epo treatment, allowing them to escape from imatinib-induced, immediate cell death. This pool of cells provides the basis for the outgrowth of imatinib-resistant clones of unlimited proliferative capacity. Thus, Epo, an endogenous regulator of hematopoiesis, promotes the development of resistance to imatinib.     

Imatinib-resistant CML cells have low ENT activity but maintain sensitivity to gemcitabine

Philadelphia chromosome-positive chronic myelogenus leukemia (CML) is widely treated with imatinib mesylate (imatinib), a potent inhibitor of the Bcr-Abl tyrosine kinase. However, resistance to this compound remains a concern. Current treatment approaches include combinations of imatinib with nucleoside analogs such as gemcitabine, which requires equilibrative nucleoside transporters (ENTs) for uptake, to overcome this resistance. Here we report that imatinib treatment decreased ENT1-dependent activity and mRNA expression. Although, imatinib-resistant cells showed decreased levels of both ENT1 and ENT2 activity and expression, these cells remained sensitive to gemcitabine, suggesting that nucleoside analogs can be used as adjunctive therapy.     

Targeting the kinase activity of the BCR-ABL fusion protein in patients with chronic myeloid leukemia

Imatinib mesylate is a major advance in the therapy of patients with chronic myelogenous leukemia (CML). Imatinib mesylate binds to the inactive conformation of BCR-ABL tyrosine kinase suppressing the Philadelphia chromosome positive clone in CML. Clinical studies have yielded impressive results in all phases of CML. With higher rates of complete cytogenetic response with imatinib, molecular monitoring of disease is now advisable in assessing response and determining prognosis. Emergence of resistance to imatinib may be manifest at the hematologic, cytogenetic, or molecular levels in patients who remain in chronic phase, or may be evidenced by the development of more advanced CML phases. Resistance and eventual clinical failure of imatinib occurs in most patients with blastic phase disease. Resistance may occur at the level of Bcr-Abl, with reduction or loss of imatinib effectiveness as a kinase inhibitor, or, despite retention of its inhibitory ability, with changes in the ability to deliver an effective dose at the cellular level, and/or, the leukemia becoming less dependent on Bcr-Abl. The various mechanisms underlying these differing, non-mutually exclusive, mechanisms of resistance must be understood to develop corresponding therapeutic remedies. We review the current data on imatinib in CML, the criteria for diagnosis of imatinib resistance, and the mechanisms that underlie such resistance in CML.     

AKR1C3 decreased CML sensitivity to Imatinib in bone marrow microenvironment via dysregulation of miR-379-5p

BackgroundDrug resistance is an important cause of death for most patients with chronic myeloid leukemia (CML). The bone marrow microenvironment is believed to be mainly responsible for resistance to BCR-ABL tyrosine kinase inhibitors. The mechanism involved, however, is still unclear.MethodsBioinformatic analysis from GEO database of AKR1C3 was utilized to identify the AKR1C3 expression in CML cells under bone marrow microenvironment. Western blot and qPCR were performed to detect the AKR1C3 expression in two CML cell lines K562 and KU812 cultured +/‐ bone microenvironment derived stromal cells. CCK-8, soft agar colony assay, and Annexin V/PI assay were performed to detect the sensitivity of CML cells (K562 and KU812) to Imatinib under a gain of or loss of function of AKR1C3 treatment. The CML murine model intravenous inoculated with K562-OE-vector and K562-OE-AKR1C3 cells were established to estimate the effect of AKR1C3 inhibitor Indomethacin on Imatinib resistance. The bioinformatic analysis of miRNA databases was used to predict the potential miRNAs targeting AKR1C3. And the luciferase assay was utilized to validate the target relationship between miR-379-5p and AKR1C3. And, the soft agar colony assay and Annexin V/PI were used to validate the effect of miR-379-5p in AKR1C3 induced Imatinib resistance.ResultsIn present study, we investigated AKR1C3 was highly expressed in CML under bone marrow microenvironment. AKR1C3 decreased Imatinib activity in K562 and KU812 cells, while inhibition of AKR1C3 could enhance Imatinib sensitivity in vitro study. Furthermore, murine model results showed combination use of AKR1C3 inhibitor Indomethacin effectively prolong mice survival, indicating that AKR1C3 is a promising target to enhance Imatinib treatment. Mechanically, AKR1C3 was found to be suppressed by miR-379-5p, which was down-expression in bone marrow microenvironment. Besides, we found miR-379-5p could bind AKR1C3 3’UTR but not degrade its mRNA level. Further, gain of miR-379-5p rescued the imatinib resistance induced by AKR1C3 overexpression in CML cells.ConclusionsAltogether, our study identifies a novel signaling regulation of miR-379-5p/AKR1C3/EKR axis in regulating IM resistance in CML cell, and provides a scientific base for exploring AKR1C3 as a biomarker in impeding IM resistance in CML.     

Exploiting mitochondrial dysfunction for effective elimination of imatinib-resistant leukemic cells

Challenges today concern chronic myeloid leukemia (CML) patients resistant to imatinib. There is growing evidence that imatinib-resistant leukemic cells present abnormal glucose metabolism but the impact on mitochondria has been neglected. Our work aimed to better understand and exploit the metabolic alterations of imatinib-resistant leukemic cells. Imatinib-resistant cells presented high glycolysis as compared to sensitive cells. Consistently, expression of key glycolytic enzymes, at least partly mediated by HIF-1α, was modified in imatinib-resistant cells suggesting that imatinib-resistant cells uncouple glycolytic flux from pyruvate oxidation. Interestingly, mitochondria of imatinib-resistant cells exhibited accumulation of TCA cycle intermediates, increased NADH and low oxygen consumption. These mitochondrial alterations due to the partial failure of ETC were further confirmed in leukemic cells isolated from some imatinib-resistant CML patients. As a consequence, mitochondria generated more ROS than those of imatinib-sensitive cells. This, in turn, resulted in increased death of imatinib-resistant leukemic cells following in vitro or in vivo treatment with the pro-oxidants, PEITC and Trisenox, in a syngeneic mouse tumor model. Conversely, inhibition of glycolysis caused derepression of respiration leading to lower cellular ROS. In conclusion, these findings indicate that imatinib-resistant leukemic cells have an unexpected mitochondrial dysfunction that could be exploited for selective therapeutic intervention.