In Vitro Cellular Activity

In vitro studies using purified enzymes expressed as bacterial fusion proteins or immunoprecipitations of intact proteins showed that Glivec potently inhibited all of the Abl tyrosine kinases, including cellular Abl (c-Abl), v-Abl, the oncogenic form contained in the Abelson murine leukemia virus, and Bcr-Abl (Table 1) (14-16). STI571 is an ATP-competitive inhibitor of Abl with a K; value of 85 nm (17). Extended profiling against various serine/threonine and tyrosine kinases revealed that the compound was also an inhibitor of the platelet-derived growth factor (PDGF) receptor and c-KIT tyrosine kinases, devoid of activity against most other kinases. The selective inhibitory activity of Glivec was also demonstrated at the cellular level (18-20; Table 1). The compound inhibited the constitutively activated fusion forms of Abl, such as the p210Bcr-Abl (14), p185Bcr-Abl (15,16), and Tel-Abl (15) tyrosine kinases with IC50 values between 0.1 and 0.35 ^M. The inhibition of autophosphorylation of

Table 1

Inhibition of Protein Kinases by Glivec

Enzyme

In vitro substrate phosphorylation IC50 [hM]

Cellular tyrosine phosphorylation IC50 [hM]

Enzyme

c-Abl

0.17 ± 0.023; 0.025a

p210Bcr-Abl

0.025a

0.25

p185Bcr-Abl

0.025a

0.25

TEL-Abl

0.35

PDGFRP

0.87 ± 0.012

0.1

TEL-PDGF-RP

0.15

c-Kit

0.56 ± 0.092

0.1

FGF-R1

>10

c-Fms and v-Fms

>10

VEGFR1 (Flt-1)

>10

VEGFR2 (Kdr)

>10

>10

Flt-3

>10

>10

Flt-4

5.7 ± 1.1

EGFR (HER1)

>100

>100

ErbB2 (HER2)

>10

>10

ErbB4 (HER4)

>10

IGF-IR

>10

>100

Insulin receptor

>10

>100

c-Met

>10

Tie-2 (Tek)

>10

Jak-2

>100a

>100

c-Fgr

>100

Lck

9.0

c-Lyn

>100

Syk (TPK-IIB)

>100

c-Src

>10

Akt (PKB)

>10

Cdk1/cyclin B

>10

Jnk2

>10

p38 MAPK

>10

PDK1

>10

PKA

>10

PKCa, p1, p2, y, 5, e, Z, or n

>10

PPK

>10

Protein kinase CK-1, CK-2

>10

c-Raf-1

0.97 ± 0.16

>10

aIC50 was determined in immunocomplex assays. Data represent the mean ± SEM drug concentrations causing a 50% reduction in kinase activity (IC50 value; ||M). PDGFR, platelet-derived growth factor receptor; FGFR1, fibroblast growth factor receptor 1; VEGFR, vascular endothelial aIC50 was determined in immunocomplex assays. Data represent the mean ± SEM drug concentrations causing a 50% reduction in kinase activity (IC50 value; ||M). PDGFR, platelet-derived growth factor receptor; FGFR1, fibroblast growth factor receptor 1; VEGFR, vascular endothelial

Bcr-Abl was closely related to the antiproliferative activity of Glivec. Incubation with submicromolar concentrations of Glivec selectively induced apoptosis in Bcr-Abl-positive cell lines and also induced cell killing in primary leukemia cells from Ph chromosome-positive CML and acute lymphoblastic leukemia patients, whereas Ph chromosome-negative cells were not affected (14,16,21-24). The IC50 values for inhibition of the KU812 and MC3 Bcr-Abl-positive CML blast crisis cell lines were 0.1-0.3 ^M (24). Selective inhibition of CML colony formation by Glivec has been demonstrated. At concentrations of 1 ^M, the compound selectively inhibited colony formation from peripheral blood and bone marrow from Ph-positive CML patients, with a 92-98% decrease in Bcr-Abl-positive colonies but little effect on normal hematopoiesis (14,22). The findings were confirmed by assessing the effects of Glivec on proliferation of peripheral blood progenitors under stroma-dependent long-term culture (LTC) conditions (25).

Fundamental phenotypic features in Bcr-Abl-positive cells involve resistance to apoptosis, enhanced proliferation, and altered adhesion properties. The impact of Glivec on some known downstream signaling molecules of Bcr-Abl has been examined. A link between constitutive activation of signal transducer and activator of transcription (STAT) 5 and enhanced viability of Bcr-Abl-transformed cells has been demonstrated (26,27). Glivec had a profound inhibitory effect on STAT5 activation in vitro and in vivo (26-28). Furthermore, inhibition of the Bcr-Abl kinase activity by Glivec in Bcr-Abl-expressing cell lines and fresh leukemic cells from CML patients induced apoptosis by suppressing the capacity of STAT5 to activate the expression of the antiapoptotic protein Bcl-xL (27). The adapter molecule CrkL is a prominent target of Bcr-Abl, and its tyrosine phosphorylation has been a useful marker of Bcr-Abl kinase activity (29). As expected, a decrease in tyrosine phosphorylation of CrkL has been observed in Glivec-treated cell lines and has also served as an indicator of Bcr-Abl kinase activity in patients (see Section 4).

There is increasing evidence that cell cycle regulation is disturbed in Bcr-Abl-positive cells; however, the underlying molecular mechanisms are poorly understood. Recently, Bcr-Abl has been shown to promote cell cycle progression and activate cyclin-dependent kinases by interfering with the regulation of the cell cycle inhibitory protein p27 (30). Glivec prevented downregulation of p27 levels in Bcr-Abl-expressing cells (30,31).

The effects of Glivec on cytoskeletal changes and adhesion have been investigated using Bcr-Abl-transfected fibroblasts (32). Glivec was shown to restore normal architecture and to increase adhesion in this model of Bcr-Abl expression.

Table 1 (Continued) growth factor receptor; EGFR, epidermal growth factor receptor; ErbB, oncogene B of Avian Erythroblastosis virus; HER, human EGF receptor family; IGF-IR, insulin-like growth factor receptor I; TPK, tyrosine-protein kinase; PKB, protein kinase B; Jnk2, c-Jun N-terminal kinase 2; MAPK, mitogen-activated protein kinase; PDK1, 3-phosphoinositide-dependent protein kinase-1; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PPK, phosphorylase kinase; CK, casein kinase.

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