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Stem cell and kinase activity-independent pathway in resistance of leukaemia to BCR-ABL kinase inhibitors.

Li S, Li D - J. Cell. Mol. Med. (2007 Nov-Dec)

Bottom Line: BCR-ABL tyrosine kinase inhibitors, such as imatinib (Gleevec) are highly effective in treating human Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) in chronic phase but not in terminal acute phase; acquired drug resistance caused mainly by the development of BCR-ABL kinase domain mutations prevents cure of the leukaemia.This type of drug resistance that is unrelated to BCR-ABL kinase domain mutations is caused by the insensitivity of leukaemic stem cells to kinase inhibitors such as imatinib and dasatinib, and by activation of a newly-identified signalling pathway involving SRC kinases that are independent of BCR-ABL kinase activity for activation.Apart from BCR-ABL and SRC kinases, stem cell pathways must also be targeted for curative therapy of Ph+ leukaemia.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, ME, USA. shaoguang.li@jax.org

ABSTRACT
BCR-ABL tyrosine kinase inhibitors, such as imatinib (Gleevec) are highly effective in treating human Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) in chronic phase but not in terminal acute phase; acquired drug resistance caused mainly by the development of BCR-ABL kinase domain mutations prevents cure of the leukaemia. In addition, imatinib is ineffective in treating Ph+ B-cell acute lymphoblastic leukaemia (B-ALL) and CML blast crisis, even in the absence of the kinase domain mutations. This type of drug resistance that is unrelated to BCR-ABL kinase domain mutations is caused by the insensitivity of leukaemic stem cells to kinase inhibitors such as imatinib and dasatinib, and by activation of a newly-identified signalling pathway involving SRC kinases that are independent of BCR-ABL kinase activity for activation. This SRC pathway is essential for leukaemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Apart from BCR-ABL and SRC kinases, stem cell pathways must also be targeted for curative therapy of Ph+ leukaemia.

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Downstream signalling molecules activated by SRC kinases. (A) SRC kinases activate β-catenin in BCR-ABLexpressing leukaemic cells. Protein lysates from parental and BCR-ABL-expressing BaF/3 pre-B cells were analysed by Western blotting using anti-β-catenin and -β- actin antibodies. (B) v-SRC activates β-catenin. Protein lysates from parental and v-SRC-expressing BaF/3 pre-B cells were analysed by Western blotting using anti-c-SRC, -β-catenin, -β-actin antibodies. (C) Lack of LYN, HCK and FGR causes reduction of β-catenin activation in BCRABL- expressing leukaemic cells. Protein lysates from BCR-ABL-transformed wild-type (WT) and Lyn−/−Hck−/−Fgr−/− bone marrow cells were analysed by Western blotting using anti-β-catenin and β-actin antibodies. (D) Inhibition of SRC kinase activity reduces β-catenin activation in BCR-ABL-expressing leukaemic cells. Protein lysates from P210 BCR-ABL-T315I transformed WT bone marrow cells treated with different concentrations of dasatinib were analysed by Western blotting using anti-phospho-tyrosine (p-Tyr), c-ABL (ABL), β-catenin, active SRC kinase (SRCY416), and β-actin antibodies.
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fig01: Downstream signalling molecules activated by SRC kinases. (A) SRC kinases activate β-catenin in BCR-ABLexpressing leukaemic cells. Protein lysates from parental and BCR-ABL-expressing BaF/3 pre-B cells were analysed by Western blotting using anti-β-catenin and -β- actin antibodies. (B) v-SRC activates β-catenin. Protein lysates from parental and v-SRC-expressing BaF/3 pre-B cells were analysed by Western blotting using anti-c-SRC, -β-catenin, -β-actin antibodies. (C) Lack of LYN, HCK and FGR causes reduction of β-catenin activation in BCRABL- expressing leukaemic cells. Protein lysates from BCR-ABL-transformed wild-type (WT) and Lyn−/−Hck−/−Fgr−/− bone marrow cells were analysed by Western blotting using anti-β-catenin and β-actin antibodies. (D) Inhibition of SRC kinase activity reduces β-catenin activation in BCR-ABL-expressing leukaemic cells. Protein lysates from P210 BCR-ABL-T315I transformed WT bone marrow cells treated with different concentrations of dasatinib were analysed by Western blotting using anti-phospho-tyrosine (p-Tyr), c-ABL (ABL), β-catenin, active SRC kinase (SRCY416), and β-actin antibodies.

Mentions: Activation of β-catenin in the Wnt signalling pathway has been shown to play a role in self-renewal of HSCs and proliferation of tumour cells [33, 34, 53]. As mentioned above, this molecule is activated in granulocyte-macrophage progenitors of advanced phase CML patients and is involved in self-renewal ability of these cells in vitro[36]. The striking therapeutic effect of dasatinib, but not imatinib, on B-ALL [19] suggests that inhibition of SRC kinases not only played a key role in killing the highly proliferating leukaemic cells, but also may have an inhibitory effect on leukaemic stem cells, which would diminish the contribution of these stem cells to the disease. Therefore, we investigated whether SRC kinases are involved in activation of β-catenin in BCR-ABLexpressing cells.We observed that β-catenin is activated in the BCR-ABL-expressing mouse pre-B cell line (BaF/3) (Fig. 1A). To test whether activated SRC kinases directly activate β-catenin, we expressed v- SRC in BaF/3 cells. v-SRC activated β-catenin (Fig. 1B), suggesting that normal SRC kinases activated by BCR-ABL may activate β-catenin directly. To provide supporting evidence for this hypothesis, we first compared the levels of β-catenin expression in BCR-ABL-expressing pre-B leukaemic cells in the presence and absence of the SRC kinases Lyn, Hck and Fgr. We have previously shown that, while the lack of Lyn, Hck and Fgr causes a severe defect in the development of B-ALL, BCR-ABL-transduced Lyn−/−Hck−/−Fgr−/− bone marrow cells can grow under Whitlock-Witte culture conditions at high cell density [11]. Using these conditions to test the role of the three SRC kinases in β-catenin activation, we found that the level of β-catenin activation in Lyn−/−Hck−/−Fgr−/− leukaemic cells was lower than that in wild-type leukaemic cells (Fig. 1C).To test whether inhibition of SRC kinases by dasatinib down-regulates β-catenin activation, we treated BCR-ABL-T315I-expressing pre-B leukaemic cells with dasatinib. β-catenin activation was inhibited by dasatinib at 100 nM, and this was associated with complete inhibition of SRC activation (Fig. 1D). These results link SRC kinases to β- catenin activation, implying that Src kinase may impact B-ALL stem cell functions through activation of the downstream signalling molecule β-catenin.


Stem cell and kinase activity-independent pathway in resistance of leukaemia to BCR-ABL kinase inhibitors.

Li S, Li D - J. Cell. Mol. Med. (2007 Nov-Dec)

Downstream signalling molecules activated by SRC kinases. (A) SRC kinases activate β-catenin in BCR-ABLexpressing leukaemic cells. Protein lysates from parental and BCR-ABL-expressing BaF/3 pre-B cells were analysed by Western blotting using anti-β-catenin and -β- actin antibodies. (B) v-SRC activates β-catenin. Protein lysates from parental and v-SRC-expressing BaF/3 pre-B cells were analysed by Western blotting using anti-c-SRC, -β-catenin, -β-actin antibodies. (C) Lack of LYN, HCK and FGR causes reduction of β-catenin activation in BCRABL- expressing leukaemic cells. Protein lysates from BCR-ABL-transformed wild-type (WT) and Lyn−/−Hck−/−Fgr−/− bone marrow cells were analysed by Western blotting using anti-β-catenin and β-actin antibodies. (D) Inhibition of SRC kinase activity reduces β-catenin activation in BCR-ABL-expressing leukaemic cells. Protein lysates from P210 BCR-ABL-T315I transformed WT bone marrow cells treated with different concentrations of dasatinib were analysed by Western blotting using anti-phospho-tyrosine (p-Tyr), c-ABL (ABL), β-catenin, active SRC kinase (SRCY416), and β-actin antibodies.
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Related In: Results  -  Collection

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fig01: Downstream signalling molecules activated by SRC kinases. (A) SRC kinases activate β-catenin in BCR-ABLexpressing leukaemic cells. Protein lysates from parental and BCR-ABL-expressing BaF/3 pre-B cells were analysed by Western blotting using anti-β-catenin and -β- actin antibodies. (B) v-SRC activates β-catenin. Protein lysates from parental and v-SRC-expressing BaF/3 pre-B cells were analysed by Western blotting using anti-c-SRC, -β-catenin, -β-actin antibodies. (C) Lack of LYN, HCK and FGR causes reduction of β-catenin activation in BCRABL- expressing leukaemic cells. Protein lysates from BCR-ABL-transformed wild-type (WT) and Lyn−/−Hck−/−Fgr−/− bone marrow cells were analysed by Western blotting using anti-β-catenin and β-actin antibodies. (D) Inhibition of SRC kinase activity reduces β-catenin activation in BCR-ABL-expressing leukaemic cells. Protein lysates from P210 BCR-ABL-T315I transformed WT bone marrow cells treated with different concentrations of dasatinib were analysed by Western blotting using anti-phospho-tyrosine (p-Tyr), c-ABL (ABL), β-catenin, active SRC kinase (SRCY416), and β-actin antibodies.
Mentions: Activation of β-catenin in the Wnt signalling pathway has been shown to play a role in self-renewal of HSCs and proliferation of tumour cells [33, 34, 53]. As mentioned above, this molecule is activated in granulocyte-macrophage progenitors of advanced phase CML patients and is involved in self-renewal ability of these cells in vitro[36]. The striking therapeutic effect of dasatinib, but not imatinib, on B-ALL [19] suggests that inhibition of SRC kinases not only played a key role in killing the highly proliferating leukaemic cells, but also may have an inhibitory effect on leukaemic stem cells, which would diminish the contribution of these stem cells to the disease. Therefore, we investigated whether SRC kinases are involved in activation of β-catenin in BCR-ABLexpressing cells.We observed that β-catenin is activated in the BCR-ABL-expressing mouse pre-B cell line (BaF/3) (Fig. 1A). To test whether activated SRC kinases directly activate β-catenin, we expressed v- SRC in BaF/3 cells. v-SRC activated β-catenin (Fig. 1B), suggesting that normal SRC kinases activated by BCR-ABL may activate β-catenin directly. To provide supporting evidence for this hypothesis, we first compared the levels of β-catenin expression in BCR-ABL-expressing pre-B leukaemic cells in the presence and absence of the SRC kinases Lyn, Hck and Fgr. We have previously shown that, while the lack of Lyn, Hck and Fgr causes a severe defect in the development of B-ALL, BCR-ABL-transduced Lyn−/−Hck−/−Fgr−/− bone marrow cells can grow under Whitlock-Witte culture conditions at high cell density [11]. Using these conditions to test the role of the three SRC kinases in β-catenin activation, we found that the level of β-catenin activation in Lyn−/−Hck−/−Fgr−/− leukaemic cells was lower than that in wild-type leukaemic cells (Fig. 1C).To test whether inhibition of SRC kinases by dasatinib down-regulates β-catenin activation, we treated BCR-ABL-T315I-expressing pre-B leukaemic cells with dasatinib. β-catenin activation was inhibited by dasatinib at 100 nM, and this was associated with complete inhibition of SRC activation (Fig. 1D). These results link SRC kinases to β- catenin activation, implying that Src kinase may impact B-ALL stem cell functions through activation of the downstream signalling molecule β-catenin.

Bottom Line: BCR-ABL tyrosine kinase inhibitors, such as imatinib (Gleevec) are highly effective in treating human Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) in chronic phase but not in terminal acute phase; acquired drug resistance caused mainly by the development of BCR-ABL kinase domain mutations prevents cure of the leukaemia.This type of drug resistance that is unrelated to BCR-ABL kinase domain mutations is caused by the insensitivity of leukaemic stem cells to kinase inhibitors such as imatinib and dasatinib, and by activation of a newly-identified signalling pathway involving SRC kinases that are independent of BCR-ABL kinase activity for activation.Apart from BCR-ABL and SRC kinases, stem cell pathways must also be targeted for curative therapy of Ph+ leukaemia.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, ME, USA. shaoguang.li@jax.org

ABSTRACT
BCR-ABL tyrosine kinase inhibitors, such as imatinib (Gleevec) are highly effective in treating human Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) in chronic phase but not in terminal acute phase; acquired drug resistance caused mainly by the development of BCR-ABL kinase domain mutations prevents cure of the leukaemia. In addition, imatinib is ineffective in treating Ph+ B-cell acute lymphoblastic leukaemia (B-ALL) and CML blast crisis, even in the absence of the kinase domain mutations. This type of drug resistance that is unrelated to BCR-ABL kinase domain mutations is caused by the insensitivity of leukaemic stem cells to kinase inhibitors such as imatinib and dasatinib, and by activation of a newly-identified signalling pathway involving SRC kinases that are independent of BCR-ABL kinase activity for activation. This SRC pathway is essential for leukaemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Apart from BCR-ABL and SRC kinases, stem cell pathways must also be targeted for curative therapy of Ph+ leukaemia.

Show MeSH
Related in: MedlinePlus