Refining targeted therapies in chronic myeloid leukemia: development and application of nilotinib, a step beyond imatinib.
ABSTRACT: The BCR-ABL kinase inhibitor imatinib mesylate is currently the standard therapy for patients with chronic myeloid leukemia (CML). Despite the remarkable results achieved with imatinib for the treatment of CML, the emergence of resistance to this drug has become a significant problem. Mutations within the ABL kinase domain have been identified as the main mechanism of resistance to imatinib. Other mechanisms include genomic amplification of BCR-ABL and modulation of drug efflux or influx transporters. Several strategies have been developed to overcome the problem of imatinib resistance, including dose escalation of imatinib, combination treatments, or novel targeted agents. Nilotinib is a tyrosine kinase inhibitor 30-fold more potent than imatinib, active against a wide range of mutant clones, except T315I. Phase I-II trials of nilotinib showed high activity in imatinib-resistant CML and Ph+ acute lymphoblastic leukemia. We here review the development of nilotinib and the activity of this agent in CML patients and in other forms of sensitive neoplasms.
Project description:Imatinib inhibits Bcr-Abl, the oncogenic tyrosine kinase that causes chronic myeloid leukemia. The second-line inhibitors nilotinib and dasatinib are effective in patients with imatinib resistance resulting from Bcr-Abl kinase domain mutations. Bcr-Abl(T315I), however, is resistant to all Abl kinase inhibitors in clinical use and is emerging as the most frequent cause of salvage therapy failure. SGX393 is a potent inhibitor of native and T315I-mutant Bcr-Abl kinase that blocks the growth of leukemia cell lines and primary hematopoietic cells expressing Bcr-Abl(T315I), with minimal toxicity against Bcr-Abl-negative cell lines or normal bone marrow. A screen for Bcr-Abl mutants emerging in the presence of SGX393 revealed concentration-dependent reduction in the number and range of mutations. Combining SGX393 with nilotinib or dasatinib preempted emergence of resistant subclones, including Bcr-Abl(T315I). These findings suggest that combination of a T315I inhibitor with the current clinically used inhibitors may be useful for reduction of Bcr-Abl mutants in Philadelphia chromosome-positive leukemia.
Project description:The advent of tyrosine kinase inhibitor (TKI) therapy has considerably improved the survival of patients suffering chronic myelogenous leukemia (CML). Indeed, inhibition of BCR-ABL by imatinib, dasatinib or nilotinib triggers durable responses in most patients suffering from this disease. Moreover, resistance to imatinib due to kinase domain mutations can be generally circumvented using dasatinib or nilotinib, but the multi-resistant T315I mutation that is insensitive to these TKIs, remains to date a major clinical problem. In this line, ponatinib (AP24534) has emerged as a promising therapeutic option in patients with all kinds of BCR-ABL mutations, especially the T315I one. However and surprisingly, the effect of ponatinib has not been extensively studied on imatinib-resistant CML cell lines. Therefore, in the present study, we used several CML cell lines with different mechanisms of resistance to TKI to evaluate the effect of ponatinib on cell viability, apoptosis and signaling. Our results show that ponatinib is highly effective on both sensitive and resistant CML cell lines, whatever the mode of resistance and also on BaF3 murine B cells carrying native BCR-ABL or T315I mutation. We conclude that ponatinib could be effectively used for all types of TKI-resistant patients.
Project description:BACKGROUND: BCR-ABL kinase domain mutations are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CML) patients. Recent studies indicate the presence of pre-existing BCR-ABL mutations in a higher percentage of CML patients when CD34+ stem/progenitor cells are investigated using sensitive techniques, and these mutations are associated with imatinib resistance and disease progression. However, such studies were limited to smaller number of patients. METHODS: We investigated BCR-ABL kinase domain mutations in CD34+ cells from 100 chronic-phase CML patients by multiplex allele-specific PCR and sequencing at diagnosis. Mutations were re-investigated upon manifestation of imatinib resistance using allele-specific PCR and direct sequencing of BCR-ABL kinase domain. RESULTS: Pre-existing BCR-ABL mutations were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8-48), all patients with pre-existing BCR-ABL mutations exhibited imatinib resistance. Of the 68 patients without pre-existing BCR-ABL mutations, 24 developed imatinib resistance; allele-specific PCR and BCR-ABL kinase domain sequencing detected mutations in 22 of these patients. All 32 patients with pre-existing BCR-ABL mutations had the same mutations after manifestation of imatinib-resistance. In imatinib-resistant patients without pre-existing BCR-ABL mutations, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients except T315I and Y253F mutations responded to imatinib dose escalation. CONCLUSION: Pre-existing BCR-ABL mutations can be detected in a substantial number of chronic-phase CML patients by sensitive allele-specific PCR technique using CD34+ cells. These mutations are associated with imatinib resistance if affecting drug binding directly or indirectly. After the recent approval of nilotinib, dasatinib, bosutinib and ponatinib for treatment of chronic myeloid leukemia along with imatinib, all of which vary in their effectiveness against mutated BCR-ABL forms, detection of pre-existing BCR-ABL mutations can help in selection of appropriate first-line drug therapy. Thus, mutation testing using CD34+ cells may facilitate improved, patient-tailored treatment.
Project description:Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.
Project description:UNLABELLED:Tyrosine kinase inhibitors have revolutionized the treatment of several malignancies, converting lethal diseases in a manageable aspect. Imitanib, a small molecule ABL kinase inhibitor is a highly effective therapy for early phase chronic myeloid leukemia (CML), which has constitutively active ABL kinase activity owing to the over expression of the BCR-ABL fusion protein. But some patients develop imatinib resistance, particularly in the advanced phases of CML.The discovery of resistance mechanisms of imitanib; urge forward the development of second generation drugs. Nilotinib, a second generation drug is more potent inhibitor of BCR-ABL than imatinib. But nilotinib also develops dermatologic events and headache in patients. Large information about BCR-ABL structure and its inhibitors are now available. Based on the pharmacophore modeling approaches, it is possible to decipher the molecular determinants to inhibit BCR-ABL. We conducted a structure based and ligand based study to identify potent natural compounds as BCR-ABL inhibitor. First kinase inhibitors were docked with the receptor (BCR-ABL) and nilotinib was selected as a pharmacophore due its high binding efficiency. Eleven compounds were selected out of 1457 substances which have mutual pharmacopohre features with nilotinib. These eleven compounds were validated and used for docking study to find the drug like molecules. The best molecules from the final set of screening candidates can be evaluated in cell lines and may represent a novel class of BCR-ABL inhibitors. ABBREVIATIONS:CML - Chronic myeloid leukemia, PDGFR - Platelet derived growth factor receptor, TKI - Tyrosine kinase inhibitors.
Project description:Nilotinib is a second-generation tyrosine kinase inhibitor, designed to specifically inhibit break-point cluster region (BCR)-Abelson (ABL) and developed to treat chronic myeloid leukemia (CML) in patients showing a resistance to imatinib. We previously demonstrated that nilotinib-induced apoptosis was reduced by stem cell factor (SCF) addition. Here, the SCF-activated survival pathway was investigated. BCR-ABL expression was accompanied by the activation of the SCF receptor: c-KIT. Nilotinib inhibited this activation that was restored by SCF binding. Parallel variations were observed for mammaliam target of rapamycin (mTOR) kinase and mTOR complex 1 substrate S6K. The inhibition of mTORC1 restored the response of BCR-ABL cell lines to nilotinib in the presence of SCF. PI3K inhibition restored nilotinib-induced apoptosis. On hematopoietic progenitors from CML patient's bone marrows, mTORC1 inhibition also restored nilotinib sensitivity in the presence of SCF, confirming its involvement in SCF-activated survival pathway. However, this pathway seems not to be involved in the nilotinib-induced resistance of the CML stem cell population. Conversely, PI3K inhibition sensitized both CML progenitors and stem cells to nilotinib, suggesting that, downstream PI3K, two different kinase pathways are activated in CML progenitor and stem cell populations.
Project description:Acquired point mutations within the BCR-ABL kinase domain represent a common mechanism of resistance to ABL inhibitor therapy in patients with chronic myeloid leukemia (CML). The BCR-ABL(T315I) mutant is highly resistant to imatinib, nilotinib, and dasatinib, and is frequently detected in relapsed patients. This critical gap in resistance coverage drove development of DCC-2036, an ABL inhibitor that binds the switch control pocket involved in conformational regulation of the kinase domain. We evaluated the efficacy of DCC-2036 against BCR-ABL(T315I) and other mutants in cellular and biochemical assays and conducted cell-based mutagenesis screens. DCC-2036 inhibited autophosphorylation of ABL and ABL(T315I) enzymes, and this activity was consistent with selective efficacy against Ba/F3 cells expressing BCR-ABL (IC(50): 19 nmol/L), BCR-ABL(T315I) (IC(50): 63 nmol/L), and most kinase domain mutants. Ex vivo exposure of CML cells from patients harboring BCR-ABL or BCR-ABL(T315I) to DCC-2036 revealed marked inhibition of colony formation and reduced phosphorylation of the direct BCR-ABL target CrkL. Cell-based mutagenesis screens identified a resistance profile for DCC-2036 centered around select P-loop mutations (G250E, Q252H, Y253H, E255K/V), although a concentration of 750 nmol/L DCC-2036 suppressed the emergence of all resistant clones. A decreased concentration of DCC-2036 (160 nmol/L) in dual combination with either nilotinib or dasatinib achieved the same zero outgrowth result. Further screens for resistance due to BCR-ABL compound mutations (two mutations in the same clone) identified BCR-ABL(E255V / T315I) as the most resistant mutant. Taken together, these findings support continued evaluation of DCC-2036 as an important new agent for treatment-refractory CML.
Project description:Chromosomal translocations generating the BCR-ABL oncogene cause chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia. The BCR-ABL(T315I) mutation confers drug resistance to FDA-approved targeted therapeutics imatinib mesylate, dasatinib, and nilotinib. We tested the ability of a recombinant yeast-based vaccine expressing the T315I-mutated BCR-ABL antigen to stimulate an anti-BCR-ABL(T315I) immune response. The yeast-based immunotherapy significantly reduced or eliminated BCR-ABL(T315I) leukemia cells from the peripheral blood of immunized animals and extended leukemia-free survival in a murine model of BCR-ABL(+) leukemia compared to animals receiving sham injection or yeast expressing ovalbumin. With immunization, leukemic cells harboring BCR-ABL(T315I) were selectively eliminated after challenge with a mixed population of BCR-ABL and BCR-ABL(T315I) leukemias. In summary, yeast-based immunotherapy represents a novel approach against the emergence of cancer drug resistance by the pre-emptive targeted ablation of tumor escape mutants.
Project description:Serine/threonine kinase Aurora A is essential for regulating mammalian cell division and is overexpressed in many types of human cancer. However, the role of Aurora A in chemoresistance of chronic myelogenous leukemia (CML) is not well understood. Using the KCL-22 cell culture model we have recently developed for studying mechanisms of CML acquired resistance, we found that Aurora A expression was partially reduced in these cells upon treatment with the tyrosine kinase inhibitor imatinib, which accompanied the acquisition of BCR-ABL mutation for imatinib resistance. Gene knockdown of BCR-ABL also reduced Aurora A expression, and conversely, Aurora A expression increased in hematopoietic progenitor cells after BCR-ABL expression. Inhibition of Aurora A induced apoptosis of CML cells with or without T315I BCR-ABL mutation and suppressed CML cell growth. Inhibition of Aurora A by gene knockdown or a highly specific small molecule inhibitor sensitized CML cells to imatinib treatment and effectively blocked acquisition of BCR-ABL mutations and KCL-22 cell relapse on imatinib, nilotinib or dasatinib. Our results show that Aurora A plays an important role for facilitating acquisition of BCR-ABL mutation and acquired resistance to tyrosine kinase inhibitors in the culture model and suggest that inhibition of Aurora A may provide an alternative strategy to improve CML treatment to overcome resistance.
Project description:The BCR-ABL T315I mutation causes resistance to imatinib, nilotinib and dasatinib in chronic myeloid leukemia. Forty BCR-ABL positive patients with imatinib resistance were analyzed for T315I mutated clones after six months on nilotinib or dasatinib treatment by quantitative allele-specific ligation polymerase chain reaction with a sensitivity of 0.05%. Ligation polymerase chain reaction revealed 10 patients with more than 10(-5) BCR-ABL(T315I%)/GUS (high levels), none of whom achieved major molecular response after 12 months, and a further 8 patients with 10(-5) or below BCR-ABL(T315I%)/GUS (low levels) who all achieved major molecular response (P<0.001). A second independent group showed molecular response in one of 12 patients with high levels and 5 of 8 patients with low levels (P=0.018). Combining the groups resulted in a sensitivity and specificity of 92.9% and 87.5%, respectively. We conclude that the quantitative level of mutant T315I allele is predictive of major molecular response at 12 months on second-line nilotinib or dasatinib treatment. www.clinicaltrials.gov: CT00109707, NCT00384228, CA180013, CA180005 CA180006.