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AHI-1 interacts with BCR-ABL and modulates BCR-ABL transforming activity and imatinib response of CML stem/progenitor cells.

Zhou LL, Zhao Y, Ringrose A, DeGeer D, Kennah E, Lin AE, Sheng G, Li XJ, Turhan A, Jiang X - J. Exp. Med. (2008)

Bottom Line: Conversely, RNAi-mediated suppression of AHI-1 in BCR-ABL-transduced lin(-)CD34(+) human cord blood cells and primary CML stem/progenitor cells reduces their growth autonomy in vitro.Interestingly, coexpression of Ahi-1 in BCR-ABL-inducible cells reverses growth deficiencies exhibited by BCR-ABL down-regulation and is associated with sustained phosphorylation of BCR-ABL and enhanced activation of JAK2-STAT5.Moreover, we identified an AHI-1-BCR-ABL-JAK2 interaction complex and found that modulation of AHI-1 expression regulates phosphorylation of BCR-ABL and JAK2-STAT5 in CML cells.

View Article: PubMed Central - PubMed

Affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver V5Z 1L3, BC, Canada.

ABSTRACT
Chronic myeloid leukemia (CML) represents the first human malignancy successfully treated with a tyrosine kinase inhibitor (TKI; imatinib). However, early relapses and the emergence of imatinib-resistant disease are problematic. Evidence suggests that imatinib and other inhibitors may not effectively eradicate leukemic stem/progenitor cells, and that combination therapy directed to complimentary targets may improve treatment. Abelson helper integration site 1 (Ahi-1)/AHI-1 is a novel oncogene that is highly deregulated in CML stem/progenitor cells where levels of BCR-ABL transcripts are also elevated. Here, we demonstrate that overexpression of Ahi-1/AHI-1 in murine and human hematopoietic cells confer growth advantages in vitro and induce leukemia in vivo, enhancing effects of BCR-ABL. Conversely, RNAi-mediated suppression of AHI-1 in BCR-ABL-transduced lin(-)CD34(+) human cord blood cells and primary CML stem/progenitor cells reduces their growth autonomy in vitro. Interestingly, coexpression of Ahi-1 in BCR-ABL-inducible cells reverses growth deficiencies exhibited by BCR-ABL down-regulation and is associated with sustained phosphorylation of BCR-ABL and enhanced activation of JAK2-STAT5. Moreover, we identified an AHI-1-BCR-ABL-JAK2 interaction complex and found that modulation of AHI-1 expression regulates phosphorylation of BCR-ABL and JAK2-STAT5 in CML cells. Importantly, this complex mediates TKI response/resistance of CML stem/progenitor cells. These studies implicate AHI-1 as a potential therapeutic target downstream of BCR-ABL in CML.

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AHI-1 mediates response/resistance of TKIs as assessed by overexpression or suppression of AHI-1 in BCR-ABL+ primitive CML cells. (A) Percentage of inhibition of CFC colonies generated in semisolid media ± IL-3 and IM (0–5 μM) from control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced BCR-ABL–inducible clonal cell lines. (B) Model of AHI-1–BCR-ABL–JAK2 complex regulation of constitutive activation of BCR-ABL and JAK2–STAT5 pathway, resulting in increased proliferation and reduced TKI response of CML stem/progenitor cells. (C) Percentage of inhibition of CFC generation in semisolid media with GF and IM (0–10 μM) from control K562, AHI/sh4–transduced cells (suppression of AHI-1), overexpression of AHI-1 in AHI/sh4 cells, and AHI-1–transduced K562 cells. * indicates significantly different from K562 control cells. (D) Western analyses of cell lysates from the same cells ± IM (5 μM) for 6 h. Antibodies used are indicated. (E) Inhibition of CFCs in semisolid media with IM (5 μM), DS (100 nM), and NL (5 μM) in lin−CD34+ CML cells from IM responders (n = 3), nonresponders (n = 3), and blast crisis (n = 3) patient samples transduced with either a control vector or AHI-1/sh4 vector. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different between BCR-ABL–inducible cells and inducible cells cotransduced with Ahi-1, and between lin−CD34+ cells transduced with a control vector or transduced with the AHI-1/sh4 vector.
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fig9: AHI-1 mediates response/resistance of TKIs as assessed by overexpression or suppression of AHI-1 in BCR-ABL+ primitive CML cells. (A) Percentage of inhibition of CFC colonies generated in semisolid media ± IL-3 and IM (0–5 μM) from control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced BCR-ABL–inducible clonal cell lines. (B) Model of AHI-1–BCR-ABL–JAK2 complex regulation of constitutive activation of BCR-ABL and JAK2–STAT5 pathway, resulting in increased proliferation and reduced TKI response of CML stem/progenitor cells. (C) Percentage of inhibition of CFC generation in semisolid media with GF and IM (0–10 μM) from control K562, AHI/sh4–transduced cells (suppression of AHI-1), overexpression of AHI-1 in AHI/sh4 cells, and AHI-1–transduced K562 cells. * indicates significantly different from K562 control cells. (D) Western analyses of cell lysates from the same cells ± IM (5 μM) for 6 h. Antibodies used are indicated. (E) Inhibition of CFCs in semisolid media with IM (5 μM), DS (100 nM), and NL (5 μM) in lin−CD34+ CML cells from IM responders (n = 3), nonresponders (n = 3), and blast crisis (n = 3) patient samples transduced with either a control vector or AHI-1/sh4 vector. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different between BCR-ABL–inducible cells and inducible cells cotransduced with Ahi-1, and between lin−CD34+ cells transduced with a control vector or transduced with the AHI-1/sh4 vector.

Mentions: To determine whether AHI-1–BCR-ABL–JAK2 interaction complex may mediate IM sensitivity/resistance of BCR-ABL+ cells, BCR-ABL–transduced inducible BaF3 cells and cells cotransduced with Ahi-1 were treated with various doses of IM (0–10 μm). As expected, BCR-ABL–transduced cells showed a significant reduction in CFC output in response to IM treatment in the presence and absence of IL-3 (∼90% inhibition with 5 μM IM + IL-3 and 1 μM IM − IL-3; Fig. 9 A). Strikingly, BaF3 cells cotransduced with Ahi-1 and BCR-ABL showed no response to IM and produced as many CFCs in the presence of IL-3 as were produced by the same cells without IM treatment (Fig. 9 A, left). Moreover, cotransduced cells also displayed greater resistance to IM in CFC output in the absence of IL-3, although these cells were more sensitive to IM treatment than those in the presence of IL-3 (Fig. 9 A, right). These results indicate that Ahi-1 is capable of overcoming IM-induced growth suppression in BCR-ABL+ cells when IL-3 signaling is activated in these cells.


AHI-1 interacts with BCR-ABL and modulates BCR-ABL transforming activity and imatinib response of CML stem/progenitor cells.

Zhou LL, Zhao Y, Ringrose A, DeGeer D, Kennah E, Lin AE, Sheng G, Li XJ, Turhan A, Jiang X - J. Exp. Med. (2008)

AHI-1 mediates response/resistance of TKIs as assessed by overexpression or suppression of AHI-1 in BCR-ABL+ primitive CML cells. (A) Percentage of inhibition of CFC colonies generated in semisolid media ± IL-3 and IM (0–5 μM) from control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced BCR-ABL–inducible clonal cell lines. (B) Model of AHI-1–BCR-ABL–JAK2 complex regulation of constitutive activation of BCR-ABL and JAK2–STAT5 pathway, resulting in increased proliferation and reduced TKI response of CML stem/progenitor cells. (C) Percentage of inhibition of CFC generation in semisolid media with GF and IM (0–10 μM) from control K562, AHI/sh4–transduced cells (suppression of AHI-1), overexpression of AHI-1 in AHI/sh4 cells, and AHI-1–transduced K562 cells. * indicates significantly different from K562 control cells. (D) Western analyses of cell lysates from the same cells ± IM (5 μM) for 6 h. Antibodies used are indicated. (E) Inhibition of CFCs in semisolid media with IM (5 μM), DS (100 nM), and NL (5 μM) in lin−CD34+ CML cells from IM responders (n = 3), nonresponders (n = 3), and blast crisis (n = 3) patient samples transduced with either a control vector or AHI-1/sh4 vector. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different between BCR-ABL–inducible cells and inducible cells cotransduced with Ahi-1, and between lin−CD34+ cells transduced with a control vector or transduced with the AHI-1/sh4 vector.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2571939&req=5

fig9: AHI-1 mediates response/resistance of TKIs as assessed by overexpression or suppression of AHI-1 in BCR-ABL+ primitive CML cells. (A) Percentage of inhibition of CFC colonies generated in semisolid media ± IL-3 and IM (0–5 μM) from control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced BCR-ABL–inducible clonal cell lines. (B) Model of AHI-1–BCR-ABL–JAK2 complex regulation of constitutive activation of BCR-ABL and JAK2–STAT5 pathway, resulting in increased proliferation and reduced TKI response of CML stem/progenitor cells. (C) Percentage of inhibition of CFC generation in semisolid media with GF and IM (0–10 μM) from control K562, AHI/sh4–transduced cells (suppression of AHI-1), overexpression of AHI-1 in AHI/sh4 cells, and AHI-1–transduced K562 cells. * indicates significantly different from K562 control cells. (D) Western analyses of cell lysates from the same cells ± IM (5 μM) for 6 h. Antibodies used are indicated. (E) Inhibition of CFCs in semisolid media with IM (5 μM), DS (100 nM), and NL (5 μM) in lin−CD34+ CML cells from IM responders (n = 3), nonresponders (n = 3), and blast crisis (n = 3) patient samples transduced with either a control vector or AHI-1/sh4 vector. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different between BCR-ABL–inducible cells and inducible cells cotransduced with Ahi-1, and between lin−CD34+ cells transduced with a control vector or transduced with the AHI-1/sh4 vector.
Mentions: To determine whether AHI-1–BCR-ABL–JAK2 interaction complex may mediate IM sensitivity/resistance of BCR-ABL+ cells, BCR-ABL–transduced inducible BaF3 cells and cells cotransduced with Ahi-1 were treated with various doses of IM (0–10 μm). As expected, BCR-ABL–transduced cells showed a significant reduction in CFC output in response to IM treatment in the presence and absence of IL-3 (∼90% inhibition with 5 μM IM + IL-3 and 1 μM IM − IL-3; Fig. 9 A). Strikingly, BaF3 cells cotransduced with Ahi-1 and BCR-ABL showed no response to IM and produced as many CFCs in the presence of IL-3 as were produced by the same cells without IM treatment (Fig. 9 A, left). Moreover, cotransduced cells also displayed greater resistance to IM in CFC output in the absence of IL-3, although these cells were more sensitive to IM treatment than those in the presence of IL-3 (Fig. 9 A, right). These results indicate that Ahi-1 is capable of overcoming IM-induced growth suppression in BCR-ABL+ cells when IL-3 signaling is activated in these cells.

Bottom Line: Conversely, RNAi-mediated suppression of AHI-1 in BCR-ABL-transduced lin(-)CD34(+) human cord blood cells and primary CML stem/progenitor cells reduces their growth autonomy in vitro.Interestingly, coexpression of Ahi-1 in BCR-ABL-inducible cells reverses growth deficiencies exhibited by BCR-ABL down-regulation and is associated with sustained phosphorylation of BCR-ABL and enhanced activation of JAK2-STAT5.Moreover, we identified an AHI-1-BCR-ABL-JAK2 interaction complex and found that modulation of AHI-1 expression regulates phosphorylation of BCR-ABL and JAK2-STAT5 in CML cells.

View Article: PubMed Central - PubMed

Affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver V5Z 1L3, BC, Canada.

ABSTRACT
Chronic myeloid leukemia (CML) represents the first human malignancy successfully treated with a tyrosine kinase inhibitor (TKI; imatinib). However, early relapses and the emergence of imatinib-resistant disease are problematic. Evidence suggests that imatinib and other inhibitors may not effectively eradicate leukemic stem/progenitor cells, and that combination therapy directed to complimentary targets may improve treatment. Abelson helper integration site 1 (Ahi-1)/AHI-1 is a novel oncogene that is highly deregulated in CML stem/progenitor cells where levels of BCR-ABL transcripts are also elevated. Here, we demonstrate that overexpression of Ahi-1/AHI-1 in murine and human hematopoietic cells confer growth advantages in vitro and induce leukemia in vivo, enhancing effects of BCR-ABL. Conversely, RNAi-mediated suppression of AHI-1 in BCR-ABL-transduced lin(-)CD34(+) human cord blood cells and primary CML stem/progenitor cells reduces their growth autonomy in vitro. Interestingly, coexpression of Ahi-1 in BCR-ABL-inducible cells reverses growth deficiencies exhibited by BCR-ABL down-regulation and is associated with sustained phosphorylation of BCR-ABL and enhanced activation of JAK2-STAT5. Moreover, we identified an AHI-1-BCR-ABL-JAK2 interaction complex and found that modulation of AHI-1 expression regulates phosphorylation of BCR-ABL and JAK2-STAT5 in CML cells. Importantly, this complex mediates TKI response/resistance of CML stem/progenitor cells. These studies implicate AHI-1 as a potential therapeutic target downstream of BCR-ABL in CML.

Show MeSH
Related in: MedlinePlus