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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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Sustained tyrosine phosphorylation and protein expression of P210BCR-ABL in BCR-ABL–inducible BaF3 cells cotransduced with Ahi-1. (A) Q-RT-PCR analysis of the levels of BCR-ABL (left) and Ahi-1 (right) transcripts relative to GAPDH in control BaF3, BCR-ABL inducible cells and two Ahi-1-transduced BCR-ABL inducible clonal lines cultured without IL-3 ± Dox for 24 h. (B) Western analyses of cell lysates from the same cells for 24 h. Antibodies used are indicated. (C) Tyrosine phosphorylation and protein expression of p210BCR-ABL and Ahi-1 relative to actin, as compared with BCR-ABL–transduced cells alone. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL–inducible cells alone and the inducible cells cotransduced with Ahi-1.
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fig7: Sustained tyrosine phosphorylation and protein expression of P210BCR-ABL in BCR-ABL–inducible BaF3 cells cotransduced with Ahi-1. (A) Q-RT-PCR analysis of the levels of BCR-ABL (left) and Ahi-1 (right) transcripts relative to GAPDH in control BaF3, BCR-ABL inducible cells and two Ahi-1-transduced BCR-ABL inducible clonal lines cultured without IL-3 ± Dox for 24 h. (B) Western analyses of cell lysates from the same cells for 24 h. Antibodies used are indicated. (C) Tyrosine phosphorylation and protein expression of p210BCR-ABL and Ahi-1 relative to actin, as compared with BCR-ABL–transduced cells alone. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL–inducible cells alone and the inducible cells cotransduced with Ahi-1.

Mentions: To determine whether coexpression of Ahi-1 in BCR-ABL–inducible BaF3 cells influences protein expression and tyrosine kinase activity of BCR-ABL, and candidate downstream signaling that reflects enhanced transforming phenotypes observed, we performed Q-RT-PCR and Western blot analyses in these cells in the presence and absence of Dox. As expected, BCR-ABL transcripts were highly expressed without Dox and down-regulated in the presence of Dox in the BCR-ABL–inducible cells after 24–48 h in culture (Fig. 7 A). Elevated BCR-ABL transcript levels were again observed in BCR-ABL– and Ahi-1–cotransduced cells generated from two individual cells lines (3–6-fold; P < 0.01). As expected, Ahi-1 transcripts were highly elevated in cotransduced cells when compared with control BaF3 cells (>50-fold; P < 0.01; Fig. 7 A). Strikingly, tyrosine phosphorylation of p210BCR-ABL could not sufficiently be suppressed in Ahi-1- and BCR-ABL–cotransduced cells, as compared with BCR-ABL–transduced cells alone in the presence of the same amount of Dox (30–40% inhibition versus ∼80% inhibition; P < 0.01; Fig. 7, B and C). In all cases, BCR-ABL phosphorylation was not completely suppressed after 24 h in culture with addition of Dox. These results were consistently observed in two individual cotransduced cell lines. Similarly, BCR-ABL protein expression was also suppressed to a lesser degree in cotransduced cells than in cells transduced with BCR-ABL only, and Ahi-1 protein expression was found to be higher in cotransduced cells than in cells transduced with BCR-ABL only.


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)

Sustained tyrosine phosphorylation and protein expression of P210BCR-ABL in BCR-ABL–inducible BaF3 cells cotransduced with Ahi-1. (A) Q-RT-PCR analysis of the levels of BCR-ABL (left) and Ahi-1 (right) transcripts relative to GAPDH in control BaF3, BCR-ABL inducible cells and two Ahi-1-transduced BCR-ABL inducible clonal lines cultured without IL-3 ± Dox for 24 h. (B) Western analyses of cell lysates from the same cells for 24 h. Antibodies used are indicated. (C) Tyrosine phosphorylation and protein expression of p210BCR-ABL and Ahi-1 relative to actin, as compared with BCR-ABL–transduced cells alone. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL–inducible cells alone and the inducible cells cotransduced with Ahi-1.
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Related In: Results  -  Collection

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fig7: Sustained tyrosine phosphorylation and protein expression of P210BCR-ABL in BCR-ABL–inducible BaF3 cells cotransduced with Ahi-1. (A) Q-RT-PCR analysis of the levels of BCR-ABL (left) and Ahi-1 (right) transcripts relative to GAPDH in control BaF3, BCR-ABL inducible cells and two Ahi-1-transduced BCR-ABL inducible clonal lines cultured without IL-3 ± Dox for 24 h. (B) Western analyses of cell lysates from the same cells for 24 h. Antibodies used are indicated. (C) Tyrosine phosphorylation and protein expression of p210BCR-ABL and Ahi-1 relative to actin, as compared with BCR-ABL–transduced cells alone. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL–inducible cells alone and the inducible cells cotransduced with Ahi-1.
Mentions: To determine whether coexpression of Ahi-1 in BCR-ABL–inducible BaF3 cells influences protein expression and tyrosine kinase activity of BCR-ABL, and candidate downstream signaling that reflects enhanced transforming phenotypes observed, we performed Q-RT-PCR and Western blot analyses in these cells in the presence and absence of Dox. As expected, BCR-ABL transcripts were highly expressed without Dox and down-regulated in the presence of Dox in the BCR-ABL–inducible cells after 24–48 h in culture (Fig. 7 A). Elevated BCR-ABL transcript levels were again observed in BCR-ABL– and Ahi-1–cotransduced cells generated from two individual cells lines (3–6-fold; P < 0.01). As expected, Ahi-1 transcripts were highly elevated in cotransduced cells when compared with control BaF3 cells (>50-fold; P < 0.01; Fig. 7 A). Strikingly, tyrosine phosphorylation of p210BCR-ABL could not sufficiently be suppressed in Ahi-1- and BCR-ABL–cotransduced cells, as compared with BCR-ABL–transduced cells alone in the presence of the same amount of Dox (30–40% inhibition versus ∼80% inhibition; P < 0.01; Fig. 7, B and C). In all cases, BCR-ABL phosphorylation was not completely suppressed after 24 h in culture with addition of Dox. These results were consistently observed in two individual cotransduced cell lines. Similarly, BCR-ABL protein expression was also suppressed to a lesser degree in cotransduced cells than in cells transduced with BCR-ABL only, and Ahi-1 protein expression was found to be higher in cotransduced cells than in cells transduced with BCR-ABL only.

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