In vitro cytotoxic effects of a tyrosine kinase inhibitor STI571 in combination with commonly used antileukemic agents

The BCR/ABL tyrosine kinase has been implicated in the pathogenesis of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL). STI571 is a novel anticancer agent that selectively inhibits the BCR/ABL tyrosine kinase. The cytotoxic effects of STI571 were studied in combination with antileukemic agents against Ph(+) leukemia cell lines, KU812, K-562, TCC-S, and TCC-Y. The cells were exposed to STI571 and to other agents simultaneously for 5 or 7 days. Cell growth inhibition was determined by MTT assay. The cytotoxic effects in combinations at the inhibitory concentration of 80% level were evaluated by the isobologram. STI571 produced synergistic effects with recombinant and natural alpha-interferons in 2 of 3 and 3 of 3 cell lines, respectively. STI571 produced additive effects with hydroxyurea, cytarabine, homoharringtonine, doxorubicin, and etoposide in all 4 cell lines. STI571 with 4-hydroperoxy-cyclophosphamide, methotrexate, or vincristine produced additive, antagonistic, and synergistic effects in 3 of 4 cell lines, respectively. These findings suggest that the simultaneous administration of STI571 with other agents except methotrexate would be advantageous for cytotoxic effects against Ph(+) leukemias. Among them, the simultaneous administration of STI571 and alpha-interferons or vincristine would be highly effective against Ph(+) leukemias and these combinations would be worthy of clinical trials. In contrast, the simultaneous administration of STI571 with methotrexate would have little therapeutic efficacy. Although there are gaps between in vitro studies and clinical trials, the present findings provide useful information for the establishment of clinical protocols involving STI571. (Blood. 2001;97:1999-2007)     

Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor

STI 571 (formerly known as CGP 57148B) is a known inhibitor of the c-abl, bcr-abl, and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases. This compound is being evaluated in clinical trials for the treatment of chronic myelogenous leukemia. We sought to extend the activity profile of STI 571 by testing its ability to inhibit the tyrosine kinase activity of c-kit, a receptor structurally similar to PDGFR. We treated a c-kit expressing a human myeloid leukemia cell line, M-07e, with STI 571 before stimulation with Steel factor (SLF). STI 571 inhibited c-kit autophosphorylation, activation of mitogen-activated protein (MAP) kinase, and activation of Akt without altering total protein levels of c-kit, MAP kinase, or Akt. The concentration that produced 50% inhibition for these effects was approximately 100 nmol/L. STI 571 also significantly decreased SLF-dependent growth of M-07e cells in a dose-dependent manner and blocked the antiapoptotic activity of SLF. In contrast, the compound had no effect on MAP kinase activation or cellular proliferation in response to granulocyte-macrophage colony-stimulating factor. We also tested the activity of STI 571 in a human mast cell leukemia cell line (HMC-1), which has an activated mutant form of c-kit. STI 571 had a more potent inhibitory effect on the kinase activity of this mutant receptor than it did on ligand-dependent activation of the wild-type receptor. These findings show that STI 571 selectively inhibits c-kit tyrosine kinase activity and downstream activation of target proteins involved in cellular proliferation and survival. This compound may be useful in treating cancers associated with increased c-kit kinase activity.     

The selective tyrosine kinase inhibitor,STI571, inhibits growth of anaplastic thyroid cancer cells

To establish a molecular targeting therapy for anaplastic thyroid carcinomas, we studied the effect of the specific tyrosine kinase inhibitor, STI571, on anaplastic thyroid cancer cell lines highly expressing c-ABL ARO (mutated p53) and FRO (undetectable p53). These lines showed marked inhibition of cell growth after treatment with STI571. In contrast, the growth of papillary thyroid cancer cell lines that harbor wild-type p53 and have low levels of c-ABL was not affected by STI571. Fluorescent-activated cell sorting analysis revealed that STI571 treatment increased the fraction of FRO and ARO cells in S and G(2)/M phases, respectively, indicating induction of S and G(2)/M transition arrest. These changes were accompanied by inhibition of c-ABL phosphorylation/activation and increased expression of p21(cip1) in FRO and p27(kip1) in both FRO and ARO cells. Treatment with STI571 also led to reduction of cyclin A, B1, and CDC2 levels. The growth of FRO cells implanted into immunocompromised mice was significantly inhibited by STI571. Taken together, these results suggest that selective suppression of c-ABL activity by STI571 may represent a potential anticancer strategy for p53-mutated undifferentiated thyroid carcinomas.     

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.     

Modification of the clonogenic capacity of bone marrow cells from normal individuals by the tyrosine kinase inhibitor STI571

STI571 shows clinical activity in the treatment of chronic myelogenous leukemia, Philadelphia positive acute lymphoblastic leukemia and gastrointestinal stromal tumors. Resistance of normal progenitor cells to STI571 is essential when determining the optimal therapeutic window for patients with cancer without bone marrow involvement, and for patients with chronic myeloid leukemia who achieved complete cytogenetic remission. The effect of graded concentrations of STI571 on the clonogenic potential of normal fresh bone marrow hematopoetic progenitor cells from 13 normal individuals was analyzed. It was shown that lower concentrations of STI571 (0.1 and 0.5 microM/l) may increase colony numbers, whereas higher concentrations (>1 microM/l) may reduce normal colony formation.     

Treatment with STI571, a tyrosine kinase inhibitor, for gastrointestinal stromal tumor with peritoneal dissemination and multiple liver metastases

Background. Gastrointestinal stromal tumors (GISTs) are usually refractory to standard chemotherapeutic agents. We successfully treated a patient with a tyrosine kinase inhibitor (STI571) for GIST with peritoneal dissemination and liver metastases. Methods. In a 32-year-old man presenting with abdominal pain from diffuse peritonitis, a GIST and associated perforated small intestine were resected. Multiple liver metastases were present. After therapies with microwave coagulation, ethanol injection, and local and systemic antineoplastic drugs (fluorouracil, cisplatin, tegafur-uracil, and tegafur) failed, investigational treatment with a tyrosine kinase inhibitor was initiated (STI571, 300mg, p.o. daily). Results. Anorexia and abdominal fullness resolved within a few days. At 24 days after initiation, positron emission tomography showed a remarkable decrease in the abdominal uptake of [¹⁸F] fluorodeoxyglucose. Adverse effects of STI571, including mild alopecia and anemia, were minimal. Conclusions. The tyrosine kinase inhibitor STI571 may be effective against GISTs.     

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)     

Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines

The tyrosine kinase activity of the Bcr/Abl oncogene is required for transformation of hematopoietic cells. The tyrosine kinase inhibitor STI571 (formerly called CGP57148B, Novartis Pharmaceuticals) inhibits BCR/ABL, TEL/ABL, and v-ABL kinase activity and inhibits growth and viability of cells transformed by any of these ABL oncogenes. Here we report the generation of 2 BCR/ABL-positive cell lines that have developed partial resistance to STI571. BCR/ABL-transformed Ba/F3 hematopoietic cells and Philadelphia-positive human K562 cells were cultured in gradually increasing concentrations of STI571 over a period of several months to generate resistant lines. Resistant Ba/F3.p210 cells were found to have an increase in Bcr/Abl messenger RNA, amplification of the Bcr/Abl transgene, and a greater than tenfold increase in the level of BCR/ABL protein. In contrast to Ba/F3.p210 cells, drug-resistant K562 cells did not undergo detectable amplification of the BCR/ABL gene, although they displayed a 2-fold to 3-fold increase in p210BCR/ABL protein. The addition of STI571 to both resistant Ba/F3. p210 and K562 cells resulted in a rapid reduction of tyrosine phosphorylation of cellular proteins, similar to that observed for nonresistant cells. However, the inhibition of kinase activity was transient and partial and was not accompanied by apoptosis. The results suggest that resistance to STI571 may be multifactorial. Increased expression of the target protein BCR/ABL was observed in both lines, and resulted from oncogene amplification in one line. However, altered drug metabolism, transport, or other related mechanisms may also contribute to drug resistance.     

Effect of a tyrosine kinase inhibitor STI571 in a patient with hepatic metastases from a duodenal gastrointestinal stromal tumor

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors. The molecular etiology is the result of mutations in the c-Kit gene. The mutant c-Kit proteins, which are activated without a stem cell factor, contribute to the tumor development. STI571 selectively inhibits c-Kit, BCR-ABL, and PDGFR tyrosine kinases. Based on this potential to inhibit critical c-Kit function in GISTs, case studies have reported effective outcomes following treatment with STI571. This case report describes a highly effective use of STI571 in a 54-year-old woman with multiple liver metastases from a GIST originating in the duodenum.     

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.     

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.     

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 tyrosine kinase inhibitor STI571, like interferon-alpha, preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors from patients with chronic myeloid leukemia

OBJECTIVE: To determine whether the compound STI571 (formerly known as CGP571418B), a selective inhibitor of the protein tyrosine kinase (PTK) activity of ABL and BCR-ABL proteins, preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors (CFU-GM) from patients with chronic myeloid leukemia while sparing normal CFU-GM and to compare responses of CML and normal cells with STI571 and IFN-alpha. MATERIALS AND METHODS: Chronic phase CML and normal CFU-GM were grown with and without STI571, IFN-alpha, or the two agents in combination. Colonies were plucked and replated in 96-well microtiter plates. Secondary colonies were scored, and the results were expressed as the area-under-the-curve (AUC) of the distribution of secondary colony numbers per primary CFU-GM. This value gives an overall measure of the replating ability or amplification of the original CFU-GM population. RESULTS: STI571 selectively inhibits the formation of granulocyte-macrophage colony-forming cells (CFU-GM) from CML patients. It also significantly inhibits the amplification of CML CFU-GM (p = 0.002) as measured by secondary colony formation after replating primary CFU-GM colonies. In contrast, amplification of normal CFU-GM was enhanced (p = 0.001) at low concentrations (0.1 microM) of STI571 with a return to baseline at 10 microM STI571. Addition of interferon (IFN)-alpha to STI571 abolished the increase in normal CFU-GM amplification seen with either agent alone. There was a highly significant correlation between the in vitro response to STI571 and the in vitro response to IFN-alpha (r = 0.74 for CML cells, and 0.77 for normal cells). CONCLUSION: We conclude that STI571, like IFN-alpha, preferentially suppresses amplification of CML CFU-GM while sparing normal CFU-GM.     

Ph(+) acute lymphoblastic leukemia resistant to the tyrosine kinase inhibitor STI571 has a unique BCR-ABL gene mutation

The tyrosine kinase inhibitor STI571 is a promising agent for the treatment of advanced Philadelphia chromosome positive (Ph(+)) acute lymphoblastic leukemia (ALL), but resistance develops rapidly in most patients after an initial response. To identify mechanisms of resistance to STI571, 30 complementary DNAs (including 9 matched samples) obtained from the bone marrow of individuals with Ph(+) ALL were analyzed by direct sequencing of a 714-base pair region of ABL encoding for the adenosine triphosphate (ATP)-binding site and the kinase activation loop. A single point mutation was found at nucleotide 1127 (GI6382056) resulting in Glu255Lys. This mutation occurred in 6 of 9 patients (67%) following their treatment with STI571 but not in the samples from patients before beginning treatment with STI571. Glu255Lys is within the motif important for forming the pocket of the ATP-binding site in ABL and it is highly conserved across species. In conclusion, Ph(+) ALL samples resistant to STI571 have a unique mutation Glu255Lys of BCR-ABL.     

Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance

Targeting the tyrosine kinase activity of Bcr-Abl with STI571 is an attractive therapeutic strategy in chronic myelogenous leukemia (CML). A few CML cell lines and primary progenitors are, however, resistant to this compound. We investigated the mechanism of this resistance in clones of the murine BaF/3 cells transfected with BCR-ABL and in 4 human cell lines from which sensitive (s) and resistant (r) clones were generated by various methods. Although the resistant cells were able to survive in the presence of STI571, their proliferation was approximately 30% lower than that of their sensitive counterparts in the absence of the compound. The concentration of STI571 needed for a 50% reduction in viable cells after a 3-day exposure was on average 10 times higher in the resistant (2-3 micromol/L) than in the sensitive (0.2-0.25 micromol/L) clones. The mechanism of resistance to STI571 varied among the cell lines. Thus, in Baf/BCR-ABL-r, LAMA84-r, and AR230-r, there was up-regulation of the Bcr-Abl protein associated with amplification of the BCR-ABL gene. In K562-r, there was no Bcr-Abl overexpression, but the IC(50) for the inhibition of Bcr-Abl autophosphorylation was increased in the resistant clones. Sequencing of the Abl kinase domain revealed no mutations. The multidrug resistance P-glycoprotein (Pgp) was overexpressed in LAMA84-r, indicating that at least 2 mechanisms of resistance operate in this cell line. KCL22-r showed neither Bcr-Abl up-regulation nor a higher threshold for tyrosine kinase inhibition by STI571. We conclude that BCR-ABL-positive cells can evade the inhibitory effect of STI571 by different mechanisms, such as Bcr-Abl overexpression, reduced intake mediated by Pgp, and, possibly, acquisition of compensatory mutations in genes other than BCR-ABL.