BCR-ABL independence and LYN kinase overexpression in chronic myelogenous leukemia cells selected for resistance to STI571

Clinical studies have shown that the tyrosine kinase inhibitor STI571 effectively controls BCR-ABL-positive chronic myelogenous leukemia (CML). However, disease progression while on STI571 therapy has been reported, suggesting de novo or intrinsic resistance to BCR-ABL-targeted therapy. To investigate possible mediators of acquired STI571 resistance, K562 cells resistant to 5 microM STI571 (K562-R) were cloned and compared to the parental cell population. K562-R cells had reduced BCR-ABL expression and limited activation of BCR-ABL signaling cascades (Stat 5, CrkL, MAPK). STI571 failed to activate caspase cascades or to suppress expression of survival genes (bcl-xL) in resistant cells. Gene sequencing and tyrosine kinase activity measurements demonstrated that K562-R cells retained wild-type and active BCR-ABL tyrosine kinase that was inhibitable by in vitro incubation with STI571, suggesting that BCR-ABL was not coupled to proliferation or survival of K562-R cells. The src-related kinase LYN was highly overexpressed and activated in K562-R cells, and its inhibition reduced proliferation and survival of K562-R cells while having limited effects of K562 cells. Specimens taken from patients with advanced CML that progressed on STI571 therapy also were analyzed for LYN kinase expression, and they were found to be elevated to a level similar to that of K562-R cells. Comparison of samples from patients taken prior to and following STI571 failure suggested that expression and/or activation of LYN/HCK occurs during disease progression. Together, these results suggest that acquired STI571 resistance may be associated with BCR-ABL independence and mediated in part through overexpression of other tyrosine kinases.     

Restoration of sensitivity to STI571 in STI571-resistant chronic myeloid leukemia cells.

STI571 induces sustained hematologic remission in patients with chronic myeloid leukemia (CML) in chronic phase. However, in advanced phases, especially blast crisis, the leukemia usually becomes resistant within months. It has been investigated whether resistance to STI571 is stable and immutable or whether it can be reversed in selected CML cell lines. Withdrawal of STI571 for varying lengths of time from cultures of 3 resistant lines (K562-r, KCL22-r, and Baf/BCR-ABL-r1) did not restore sensitivity to the inhibitor. In contrast, LAMA84-resistant cells experienced a sharp reduction in survival and proliferation during the first week of STI571 withdrawal but recovered thereafter. Moreover, when left off the inhibitor for 2 months or longer, this cell line reacquired sensitivity to STI571. It is hypothesized, therefore, that patients who have become resistant to the drug may respond again if STI571 therapy is temporarily interrupted.     

Establishment of a murine model for therapy-treated chronic myelogenous leukemia using the tyrosine kinase inhibitor STI571.

The murine bone marrow retroviral transduction and transplantation model of chronic myelogenous leukemia (CML) imperfectly mimics human CML because the murine CML-like disease causes death of all animals from an overwhelming granulocytosis within 3 to 4 weeks. In this report, mice reconstituted with P210(BCR/ABL)-transduced bone marrow cells received posttransplantation therapy with either the tyrosine kinase inhibitor STI571 or placebo. Compared with the rapidly fatal leukemia of placebo-treated animals, 80% of the STI571-treated mice were alive on day 74, with marked improvement in peripheral white blood counts and splenomegaly. There was decreased tyrosine phosphorylation of STAT5, Shc, and Crk-L in leukemic cells from STI571-treated animals, consistent with STI571-mediated inhibition of the Bcr/Abl tyrosine kinase in vivo. In some STI571-treated animals Bcr/Abl messenger RNA and protein expression were markedly increased. In contrast to the polyclonal leukemia of placebo-treated mice, STI571-treated murine CML was generally oligoclonal, suggesting that STI571 eliminated or severely suppressed certain leukemic clones. None of the STI571-treated mice were cured of the CML-like myeloproliferative disorder, however, and STI571-treated murine CML was transplanted to secondary recipients with high efficiency. These results demonstrate the utility of this murine model of CML in the evaluation of novel therapeutic agents against Bcr/Abl-induced leukemias. This improved murine chronic-phase CML model may be a useful tool for the study of STI571 resistance, CML progression, and the anti-CML immune response.     

Effects of the Bcr/abl kinase inhibitors STI571 and adaphostin (NSC 680410) on chronic myelogenous leukemia cells in vitro.

The adenosine triphosphate binding-site-directed agent STI571 and the tyrphostin adaphostin are undergoing evaluation as bcr/abl kinase inhibitors. The current study compared the effects of these agents on the survival of K562 cells, bcr/abl-transduced FDC-P1 cells, and myeloid progenitors from patients with chronic myelogenous leukemia (CML) compared with healthy donors. Treatment of K562 cells with 10 microM adaphostin resulted in decreased p210(bcr/abl) polypeptide levels in the first 6 hours, followed by caspase activation and accumulation of apoptotic cells in less than 12 hours. By 24 hours, 90% of the cells were apoptotic and unable to form colonies. In contrast, 20 microM STI571 caused rapid inhibition of bcr/abl autophosphorylation without p210(bcr/abl) degradation. Although this was followed by the inhibition of Stat5 phosphorylation and the down-regulation of Bcl-x(L) and Mcl-1, only 7% +/- 3% and 25% +/- 9% of cells were apoptotic at 16 and 24 hours, respectively. Instead, the cytotoxic effects of STI571 became more pronounced with prolonged exposure, with IC90 values greater than 20 microM and 1.0 +/- 0.6 microM after 24 and 48 hours, respectively. Consistent with these results, 24-hour adaphostin exposure inhibited CML granulocyte colony-forming units (CFU-G) (median IC50, 12 microM) but not normal CFU-G (median IC50, greater than 20 microM), whereas 24-hour STI571 treatment had no effect on CML or normal CFU-G. Additional experiments revealed that STI571-resistant K562 cells remained sensitive to adaphostin. Moreover, the combination of STI571 + adaphostin induced more cytotoxicity in K562 cells and in CML CFU-G than either agent alone did. Collectively, these results identify adaphostin as a mechanistically distinct CML-selective agent that retains activity in STI571-resistant cell lines.     

Treatment of chronic myelogenous leukemia with the tyrosine kinase inhibitor STI571 results in marked regression of bone marrow fibrosis.

Morphologic bone marrow changes in patients with BCR-ABL-positive chronic myelogenous leukemia (CML) were investigated during treatment with the tyrosine kinase inhibitor STI571. Bone marrow trephine biopsy specimens from 23 pretreated patients with CML were examined morphologically and by morphometry before and 6 weeks and 3 months after the initiation of STI571 therapy (Glivec, Novartis, Basel, Switzerland). Bone marrow changes during treatment showed a quantitative normalization of erythropoiesis, a marked reduction of granulopoiesis, and a significant decrease in megakaryocytes with the reappearance of normal-sized forms. Furthermore, a significant regression of bone marrow fibrosis was observed in patients with initial fibrosis (P <.000,000,001). These results may expand the profile of STI571 and may offer novel therapeutic possibilities in diseases with bone marrow fibrosis.     

Imatinib mesylate (STI571) therapy for Philadelphia chromosome-positive chronic myelogenous leukemia in blast phase

Molecular abnormalities caused by the hybrid Bcr-Abl gene are causally associated with the development and progression of Philadelphia chromosome-positive (Ph(+)) chronic myelogenous leukemia (CML). Imatinib mesylate (STI571), a specific Bcr-Abl tyrosine-kinase signal-transduction inhibitor, has shown encouraging activity in phase I and II studies of CML. Here, we describe the use of imatinib mesylate to treat 75 patients in blast-phase CML (median age, 53 years; 65 with nonlymphoid and 10 with lymphoid blasts), and compare the results with those of a historical control group treated with standard cytarabine-based therapy. Imatinib mesylate was given as oral doses at 300 to 1000 mg per day and was the first salvage therapy for 47 patients. The objective response rate was 52% (39 of 75 patients: 16 had complete and 3 had partial hematologic response; 12 had hematologic improvement; 7 returned to second chronic phase; and 1 had a complete response in extramedullary blastic disease). Response rates were not different between nonlymphoid and lymphoid groups. The cytogenetic response rate was 16% (12 patients: 5 complete, 3 partial [Ph(+) below 35%], and 4 minor [Ph(+), 34% to 90%]). The estimated median overall survival was 6.5 months; the estimated 1-year survival was 22%. Response to therapy (landmark analysis at 8 weeks) was associated with survival prolongation. Compared with standard cytarabine combinations, imatinib mesylate therapy was less toxic and produced a higher response rate (55% versus 29%, P =.001), longer median survival (7 versus 4 months, P =.04), and lower 4-week induction mortality (4% versus 15%, P =.07). Imatinib mesylate is currently being tested in combination with other drugs to improve the prognosis for blast-phase CML.     

Successful treatment of extramedullary blast crisis of chronic myelogenous leukemia with imatinib mesylate (STI571)

We describe a patient with chronic myelogenous leukemia (CML) who developed extramedullary blast crisis, and was successfully treated with imatinib mesylate (STI571). A 42-year-old man had been diagnosed with chronic phase Philadelphia chromosome (Ph)-positive CML and treated with interferon-alpha. He achieved partial cytogenetic response. Two years after the diagnosis, he presented with superficial lymphadenopathy in his neck and supraclavicular regions. Lymph node biopsy disclosed the infiltration of myeloblasts. Although the patient's bone marrow was without increasing blasts at that time, cytogenetic response was no longer observed. STI571 at a dose of 600 mg/day was initiated, and led to the complete disappearance of lymphadenopathy within a month and also to major cytogenetic response in the bone marrow (90% Ph-negative metaphases). Subsequently, the patient underwent allogeneic bone marrow transplantation from an HLA-matched unrelated donor and was in complete remission without evidence of extramedullary disease 12 months after transplantation.     

Differential regulation of the p70 S6 kinase pathway by interferon alpha (IFNalpha) and imatinib mesylate (STI571) in chronic myelogenous leukemia cells

The precise mechanisms by which imatinib mesylate (STI571) and interferon alpha (IFNalpha) exhibit antileukemic effects are not known. We examined the effects of IFNs or imatinib mesylate on signaling pathways regulating initiation of mRNA translation in BCR-ABL-expressing cells. Treatment of IFN-sensitive KT-1 cells with IFNalpha resulted in phosphorylation/activation of mammalian target of rapamycin (mTOR) and downstream activation of p70 S6 kinase. The IFN-activated p70 S6 kinase was found to regulate phosphorylation of S6 ribosomal protein, which regulates translation of mRNAs with oligopyrimidine tracts in the 5'-untranslated region. In addition, IFNalpha treatment resulted in an mTOR- and/or phosphatidyl-inositol 3'(PI 3') kinase-dependent phosphorylation of 4E-BP1 repressor of mRNA translation on sites that are required for its deactivation and dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. In contrast to the effects of IFNs, imatinib mesylate suppressed p70 S6 kinase activity, consistent with inhibition of BCR-ABL-mediated activation of the mTOR/p70 S6 kinase pathway. Moreover, the mTOR inhibitor rapamycin enhanced the suppressive effects of imatinib mesylate on primary leukemic granulocyte macrophage-colony-forming unit (CFU-GM) progenitors from patients with chronic myelogenous leukemia (CML). Taken altogether, our data demonstrate that IFNs and imatinib mesylate differentially regulate PI 3' kinase/mTOR-dependent signaling cascades in BCR-ABL-transformed cells, consistent with distinct effects of these agents on pathways regulating mRNA translation. They also support the concept that combined use of imatinib mesylate with mTOR inhibitors may be an appropriate future therapeutic strategy for the treatment of CML.     

Molecular mechanisms of resistance to STI571 in chronic myeloid leukemia

Therapeutic use of the recently FDA-approved drug STI571 has been successful in the treatment of Philadelphia chromosome-positive leukemias. STI571 is a small molecule inhibitor with activity against BCR-ABL, the deregulated tyrosine kinase responsible for initiation and maintenance of the disease in the chronic phase of chronic myeloid leukemia (CML). Clinical trials demonstrated the ability of STI571 to induce remissions in patients with chronic phase CML with only rare relapses after 18 months of follow-up. However, in patients with more advanced stages of disease, responses to STI571 were less common and often transient. Studies investigating the molecular mechanisms of resistance to this novel compound have progressed rapidly and point to the continued importance of BCR-ABL in disease maintenance even at its latest stages. Here the authors review recent work aimed at elucidating the nature of STI51 resistance.     

甲磺酸伊马替尼在慢性粒细胞白血病异基因造血干细胞移植治疗前的应用

目的:动态观察甲磺酸伊马替尼(商品名:格列卫)在2例加速期BCRABL阳性的慢性粒细胞白血病(CML)患者异基因外周血造血干细胞移植(AlloPBSCT)前的应用。方法:应用PCR技术,对接受甲磺酸伊马替尼的BCRABL阳性的CML患者选择不同时间定性检测bcr/abl基因,同时进行骨髓形态学及染色体分析。结果:①甲磺酸伊马替尼治疗1个月后2例均达到血液学完全缓解,病例2达到细胞遗传学完全缓解;②移植后中性粒细胞>0.5×109/L,血小板>20×109/L病例1为13d,病例2为20d;③病例1于+32d血型转为供者型,病例2于+40d供受细胞稳定嵌合,目前2例患者血液学持续完全缓解。结论:化疗可以动员G0期bcr/abl阳性克隆进入增殖周期,表现对甲磺酸伊马替尼敏感,在CML尤其是进入加速期、急变期的患者行PBSCT前应用甲磺酸伊马替尼,有助于体内的bcr/abl白血病细胞清除,达到肿瘤净化作用。     

亲缘异基因造血干细胞移植与STI571治疗慢性粒细胞白血病的临床对比研究

目的：评价对比亲缘异基因造血干细胞移植与酪氨酸激酶抑制剂STI571治疗慢性粒细胞白血病的有效性及安全性。方法：90例慢性粒细胞白血病慢性期患者，分为2组，其中亲缘异基因造血干细胞移植组23例，均采用经典或改良BuCy2方案预处理，短程甲氨蝶呤联合环孢素A（MTX＋CsA）方案预防移植物抗宿主病（GVHD）。STI571组67例，每天应用STI571 400mg，每周复查血常规，每3个月进行骨髓象及细胞遗传学检查，根据血象和骨髓象调整剂量。结果：观察截止时，移植组和STI571组获得细胞遗传学完全缓解率分别为100％和60％（P〈0．01），但移植组和STI571组的2年生存率分别为77．03％和83．33％，2组患者生存率比较差异无统计学意义（P〉0．05）。结论：与异基因造血干细胞移植相比，STI571治疗慢性粒细胞白血病患者治疗相关并发症较少较轻，但获得细胞遗传学完全缓解率较低。     

Efficacy of imatinib mesylate (STI571) treatment for a patient with rheumatoid arthritis developing chronic myelogenous leukemia

We report on an 80-year-old man with rheumatoid arthritis (RA) who presented with chronic myelogenous leukemia (CML). Five years after the onset of RA, the CML diagnosis was made. The patient was treated for CML with 300 mg of imatinib mesylate (STI; signal transduction inhibitor 571) for 8 weeks. Laboratory tests showed that the C-reactive protein level, percentage of cells exhibiting the Philadelphia chromosome (Ph1), WBC count, and Lansbury index for RA all dropped respectively from 7.5 mg/dl to 1.0 mg/dl, 74.9% to 1%, 25, 100/l to 9900/l, and 51% to 14%. Administration of imatinib mesylate is felt to be effective in treating not only CML but also RA in the active stage.     

Imatinib mesylate (STI571) inhibits growth of primitive malignant progenitors in chronic myelogenous leukemia through reversal of abnormally increased proliferation

Imatinib mesylate (STI571) is a promising new treatment for chronic myelogenous leukemia (CML). The effect of imatinib mesylate on primitive malignant progenitors in CML has not been evaluated, and it is not clear whether suppression of progenitor growth represents inhibition of increased proliferation, induction of apoptosis, or both. We demonstrated here that in vitro exposure to concentrations of imatinib mesylate usually achieved in patients (1-2 microM) for 96 hours inhibited BCR/ABL-positive primitive progenitors (6-week long-term culture-initiating cells [LTCICs]) as well as committed progenitors (colony-forming cells [CFCs]). No suppression of normal LTCICs and significantly less suppression of normal CFCs were observed. A higher concentration of imatinib mesylate (5 microM) did not significantly increase suppression of CML or normal LTCICs but did increase suppression of CML CFCs, and to a lesser extent, normal CFCs. Analysis of cell division using the fluorescent dye carboxyfluorescein diacetate succinimidyl ester indicated that imatinib mesylate (1-2 microM) inhibits cycling of CML primitive (CD34(+)CD38(-)) and committed (CD34(+)CD38(+)) progenitors to a much greater extent than normal cells. Conversely, treatment with 1 to 2 microM imatinib mesylate did not significantly increase the percentage of cells undergoing apoptosis. Although a higher concentration of imatinib mesylate (5 microM) led to an increase in apoptosis of CML cells, apoptosis also increased in normal samples. In summary, at clinically relevant concentrations, imatinib mesylate selectively suppresses CML primitive progenitors by reversing abnormally increased proliferation but does not significantly increase apoptosis. These results suggest that inhibition of Bcr-Abl tyrosine kinase by imatinib mesylate restores normal hematopoiesis by removing the proliferative advantage of CML progenitors but that elimination of all CML progenitors may not occur.     

Sensitivity to the abl inhibitor STI571 in fresh leukaemic cells obtained from chronic myelogenous leukaemia patients in different stages of disease

STI571 (CGP57148B) is an inhibitor of BCR/ABL, the cause of chronic myeloid leukaemia (CML). A difference exists between CML patients in chronic phase, in which responses to STI571are durable, and patients in blast crisis, who generally experience only transient responses. Leukaemic cells from six CML patients from whom samples could be obtained during chronic phase and at the time of blast crisis (BC) were compared for sensitivity to STI571, using an in vitro assay. BC samples showed a sensitivity similar to that obtained during chronic phase, suggesting that no substantial intrinsic resistance to STI571 was present in BC.     

The use of imatinib (STI571) in chronic myelod leukemia: some practical considerations

BACKGROUND AND OBJECTIVES: The new Abl tyrosine kinase inhibitor imatinib (imatinib mesylate, STI571) is very effective in the treatment of patients with chronic myeloid leukemia (CML). It induces complete hematologic control in >90% of patients with CML in chronic phase and preliminary data suggest that the incidence of complete cytogenetic remission may exceed 60% in previously untreated patients, though its capacity to prolong life in comparison with other agents has not yet been clearly established. It is also active in the management of patients in advanced phases of CML. INFORMATION SOURCES: We present here provisional indications for the use of imatinib in CML patients and give details of the dosage regimens we have used and side-effects we have encountered. STATE OF THE ART AND PERSPECTIVES: It is likely that the optimal usage of this important new agent will become better defined as more experience is gained.     

Comparison of effects of the tyrosine kinase inhibitors AG957, AG490, and STI571 on BCR-ABL--expressing cells, demonstrating synergy between AG490 and STI571

STI571 (formerly CGP57148) and AG957 are small molecule inhibitors of the protein tyrosine kinase (PTK) p145(abl) and its oncogenic derivative p210(bcr-abl). AG490 is an inhibitor of the PTK Janus kinase 2 (JAK2). No direct comparison of these inhibitors has previously been reported, so this study compared their effects on factor-dependent FDC-P1, 32D, and MO7e cells and their p210(bcr-abl)-expressing factor-independent derivatives. STI571 was a more potent inhibitor of (3)H-thymidine incorporation in p210(bcr-abl)-expressing cells than was AG957, and it showed superior discrimination between inhibitory effects on parental cell lines and effects on their p210(bcr-abl)-expressing derivatives. Assays performed with and without growth factor demonstrated that STI571 but not AG957 reversed the p210(bcr-abl)-driven factor independence of cell lines. p210(bcr-abl)-expressing cells were less sensitive to AG490 than to AG957 or STI571. However, for p210(bcr-abl)-expressing clones from all 3 cell lines, synergistic inhibition was demonstrated between STI571 and concentrations of AG490 with no independent inhibitory effect. Inhibition of nucleic acid synthesis with AG957 treatment was associated with reduced cell numbers, reduced viability, and small pyknotic apoptotic cells. At concentrations of STI571 that reversed the p210(bcr-abl) factor-independent phenotype, STI571 treatment and growth factor deprivation together were sufficient to induce apoptosis. This study concludes that, for the cell lines studied, (1) STI571 is a more potent and more selective inhibitor of a p210(bcr-abl)-dependent phenotype than AG957; (2) AG490 synergizes with STI571 to enhance its inhibitory effect on p210(bcr-abl)-driven proliferation; and (3) the combination of p210(bcr-abl)-tyrosine kinase inhibition and growth factor signal withdrawal can be sufficient to induce apoptotic death of transformed cells. (Blood. 2001;97:2008-2015)     

Effects of tyrosine kinase inhibitor STI571 on human mast cells bearing wild-type or mutated c-kit

OBJECTIVE: STI571 is a tyrosine kinase inhibitor which inhibits the kinase activity of kit, the receptor for stem cell factor (SCF). Because activating mutations of c-kit affecting codon 816 are associated with human mast cell neoplasms, we determined whether STI571 exerted a similar cytotoxic effect on neoplastic and normal human mast cells. METHODS: We investigated the effect of addition of STI571 in increasing concentrations (0.01 to 10 micromolar) to two HMC-1 human mast cell leukemia cell lines carrying two different activating c-kit mutations in codons 816 or 560, as well as the effect of the drug on short-term bone marrow cultures obtained from patients who carry a mutated codon 816 or wild-type c-kit. RESULTS: STI571 failed to inhibit the growth of HMC-1(560,816) cells bearing a codon 816 mutation but effectively suppressed the proliferation of HMC-1(560) carrying c-kit with the wild-type codon 816. STI571 did not induce preferential killing of neoplastic bone marrow mast cells in short-term cultures from patients bearing a codon 816 c-kit mutation. In contrast, STI571 caused a dramatic reduction in mast cells in patients without codon 816 c-kit mutations. CONCLUSION: These results suggest that STI571, while effectively killing mast cells with wild-type c-kit, did not show preferential cytotoxicity to neoplastic human mast cells and thus may not be effective in the treatment of human systemic mastocytosis associated with codon 816 c-kit mutations.     

Reversal of bone marrow angiogenesis in chronic myeloid leukemia following imatinib mesylate (STI571) therapy

The effect of imatinib mesylate (imatinib) therapy on angiogenesis and myelofibrosis was investigated and compared with interferon (IFN) and hydroxyurea (HU) in 98 patients with newly diagnosed Philadelphia chromosome-positive/BCR-ABL(+) (Ph(+)/BCR-ABL(+)) chronic myeloid leukemia in first chronic phase and no other pretreatment. By means of immunostaining (CD34) and morphometry, a relationship between microvessel frequency and fiber density was detectable in initial bone marrow (BM) biopsies and sequential examinations after at least 8 months of therapy. First-line monotherapy with imatinib induced a significant reduction (normalization in comparison with controls) of microvessels and reticulin fibers. In most patients, decrease in BM vascularity was associated with a complete cytogenetic response. A significant anti-angiogenic effect was also observed after HU treatment, contrasting with IFN administration or combination regimens (IFN plus HU). In conclusion, our data support the anti-angiogenic capacity of imatinib by normalization of vascularity. In contrast, hematologic response following IFN treatment is independent from BM angiogenesis.