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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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Overexpression of Ahi-1 rescues growth suppression induced by the inhibition of BCR-ABL expression in BCR-ABL–inducible BaF3 cells. (A) Growth of control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced, BCR-ABL–inducible clonal lines without IL-3 ± Dox (+Dox = suppression of BCR-ABL expression). Viable cell numbers were determined by hematocytometer counts of trypan blue–excluding cells. (B) Annexin V-PE/7-AAD staining of BCR-ABL–inducible cells and Ahi-1–transduced BCR-ABL–inducible cells (Ahi-1 + B/A-2) after culture without IL-3 ± Dox for 24 and 48 h. Percentages of Annexin V+ cells are indicated. (C) CFC colonies produced in semisolid media ± IL-3 and Dox from the same cells as shown in A. (D) The appearance of GF-independent CFC colonies in Ahi-1 and BCR-ABL–cotransduced cells without IL-3 ± Dox. Bar, 250 μm. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL inducible cells alone and inducible cells cotransduced with Ahi-1.
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fig6: Overexpression of Ahi-1 rescues growth suppression induced by the inhibition of BCR-ABL expression in BCR-ABL–inducible BaF3 cells. (A) Growth of control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced, BCR-ABL–inducible clonal lines without IL-3 ± Dox (+Dox = suppression of BCR-ABL expression). Viable cell numbers were determined by hematocytometer counts of trypan blue–excluding cells. (B) Annexin V-PE/7-AAD staining of BCR-ABL–inducible cells and Ahi-1–transduced BCR-ABL–inducible cells (Ahi-1 + B/A-2) after culture without IL-3 ± Dox for 24 and 48 h. Percentages of Annexin V+ cells are indicated. (C) CFC colonies produced in semisolid media ± IL-3 and Dox from the same cells as shown in A. (D) The appearance of GF-independent CFC colonies in Ahi-1 and BCR-ABL–cotransduced cells without IL-3 ± Dox. Bar, 250 μm. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL inducible cells alone and inducible cells cotransduced with Ahi-1.

Mentions: To further investigate potential regulatory roles of Ahi-1 in mediating BCR-ABL–transforming activities, we evaluated cooperative effects of Ahi-1 in a BCR-ABL–transduced BaF3 cell line in which the level of expression of p210BCR-ABL can be variably down-regulated by exposure to doxycycline (Dox) (26). Reduction in BCR-ABL protein expression in the presence of Dox results in a corresponding decrease in GF independence of BaF3 cells both in liquid suspension cultures and in semisolid cultures in vitro (Fig. 6). As shown in Fig. 6 (A and B), cell growth and survival was dramatically reduced when Dox was added to culture at 48 h (>90% inhibition of proliferation, Fig. 6 A, right; ∼50% detectable Annexin V+ apoptotic cells, Fig. 6 B, top right), while the same cells showed a marked increase in cell expansion in the absence of both IL-3 and Dox (no down-regulation of BCR-ABL, Fig. 6 A, left). Similarly, down-regulation of BCR-ABL expression completely inhibited CFC generation in semisolid cultures in the absence of IL-3 (Fig. 6, C and D). Interestingly, introduction of Ahi-1 into cells with inhibited BCR-ABL expression under these stringent conditions enabled them to grow continuously in liquid suspension culture, to have less Annexin V+ apoptotic cells, and to produce more factor-independent CFCs than cells transduced with BCR-ABL alone (∼4 × 105 versus 103 cells after 48 h in liquid culture and ∼100 CFCs versus 0 in the CFC assay; P < 0.01; Fig. 6). These results were consistently observed in two individual clonal cell lines. In the presence of IL-3 and Dox, BCR-ABL–transduced BaF3 cells showed a significant reduction in CFC production; addition of IL-3 cannot rescue inhibitory effects caused by suppression of BCR-ABL expression. This can be enhanced significantly by cotransduction of Ahi-1 (10–15-fold; P < 0.01; Fig. 6 C). Collectively, these results demonstrate the ability of Ahi-1 to immediately reverse in vitro growth deficiencies resulting from down-regulation of BCR-ABL in vitro, and provide direct evidence of the regulatory role of Ahi-1 in BCR-ABL–mediated transformation.


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)

Overexpression of Ahi-1 rescues growth suppression induced by the inhibition of BCR-ABL expression in BCR-ABL–inducible BaF3 cells. (A) Growth of control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced, BCR-ABL–inducible clonal lines without IL-3 ± Dox (+Dox = suppression of BCR-ABL expression). Viable cell numbers were determined by hematocytometer counts of trypan blue–excluding cells. (B) Annexin V-PE/7-AAD staining of BCR-ABL–inducible cells and Ahi-1–transduced BCR-ABL–inducible cells (Ahi-1 + B/A-2) after culture without IL-3 ± Dox for 24 and 48 h. Percentages of Annexin V+ cells are indicated. (C) CFC colonies produced in semisolid media ± IL-3 and Dox from the same cells as shown in A. (D) The appearance of GF-independent CFC colonies in Ahi-1 and BCR-ABL–cotransduced cells without IL-3 ± Dox. Bar, 250 μm. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL inducible cells alone and inducible cells cotransduced with Ahi-1.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Overexpression of Ahi-1 rescues growth suppression induced by the inhibition of BCR-ABL expression in BCR-ABL–inducible BaF3 cells. (A) Growth of control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced, BCR-ABL–inducible clonal lines without IL-3 ± Dox (+Dox = suppression of BCR-ABL expression). Viable cell numbers were determined by hematocytometer counts of trypan blue–excluding cells. (B) Annexin V-PE/7-AAD staining of BCR-ABL–inducible cells and Ahi-1–transduced BCR-ABL–inducible cells (Ahi-1 + B/A-2) after culture without IL-3 ± Dox for 24 and 48 h. Percentages of Annexin V+ cells are indicated. (C) CFC colonies produced in semisolid media ± IL-3 and Dox from the same cells as shown in A. (D) The appearance of GF-independent CFC colonies in Ahi-1 and BCR-ABL–cotransduced cells without IL-3 ± Dox. Bar, 250 μm. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL inducible cells alone and inducible cells cotransduced with Ahi-1.
Mentions: To further investigate potential regulatory roles of Ahi-1 in mediating BCR-ABL–transforming activities, we evaluated cooperative effects of Ahi-1 in a BCR-ABL–transduced BaF3 cell line in which the level of expression of p210BCR-ABL can be variably down-regulated by exposure to doxycycline (Dox) (26). Reduction in BCR-ABL protein expression in the presence of Dox results in a corresponding decrease in GF independence of BaF3 cells both in liquid suspension cultures and in semisolid cultures in vitro (Fig. 6). As shown in Fig. 6 (A and B), cell growth and survival was dramatically reduced when Dox was added to culture at 48 h (>90% inhibition of proliferation, Fig. 6 A, right; ∼50% detectable Annexin V+ apoptotic cells, Fig. 6 B, top right), while the same cells showed a marked increase in cell expansion in the absence of both IL-3 and Dox (no down-regulation of BCR-ABL, Fig. 6 A, left). Similarly, down-regulation of BCR-ABL expression completely inhibited CFC generation in semisolid cultures in the absence of IL-3 (Fig. 6, C and D). Interestingly, introduction of Ahi-1 into cells with inhibited BCR-ABL expression under these stringent conditions enabled them to grow continuously in liquid suspension culture, to have less Annexin V+ apoptotic cells, and to produce more factor-independent CFCs than cells transduced with BCR-ABL alone (∼4 × 105 versus 103 cells after 48 h in liquid culture and ∼100 CFCs versus 0 in the CFC assay; P < 0.01; Fig. 6). These results were consistently observed in two individual clonal cell lines. In the presence of IL-3 and Dox, BCR-ABL–transduced BaF3 cells showed a significant reduction in CFC production; addition of IL-3 cannot rescue inhibitory effects caused by suppression of BCR-ABL expression. This can be enhanced significantly by cotransduction of Ahi-1 (10–15-fold; P < 0.01; Fig. 6 C). Collectively, these results demonstrate the ability of Ahi-1 to immediately reverse in vitro growth deficiencies resulting from down-regulation of BCR-ABL in vitro, and provide direct evidence of the regulatory role of Ahi-1 in BCR-ABL–mediated transformation.

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