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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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Knockdown or overexpression of AHI-1 in human K562 cells mediates their transforming activity in vitro and in vivo. (A) Q-RT-PCR analysis of the levels of AHI-1 transcripts relative to GAPDH in FACS-purified RPG vector–transduced K562 cells, AHI-1/sh4 cells (with suppression of AHI-1), AHI/sh4 + AHI-1 cells (coexpression of AHI-1 in AHI-1/sh4 cells), and AHI-1 cells (AHI-1 overexpressed cells, top). Western analysis (bottom) of AHI-1 expression in the same transduced cells with an anti–AHI-1 antibody. (B) The numbers of CFC colonies produced in semisolid cultures ± GF (IL-3, GM-CSF, SF) in the same transduced cells. (C) The appearance of CFC colonies produced in semisolid cultures ± GF as shown in B. Bar, 250 μm. (D) Percentage of single control K562 cells or AHI/sh4 cells generating clones (top) and clone size distributions obtained from these cells after being cultured for 7 d (bottom). (E) NOD/SCID-β2M−/− mice were injected subcutaneously with 107 control K562 and transduced cells. Tumor volume is expressed as mean ± SEM areas of each group (n = 4). (F) The appearance of tumors generated by the same cells as shown in E. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different from K562 or RPG control cells.
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fig3: Knockdown or overexpression of AHI-1 in human K562 cells mediates their transforming activity in vitro and in vivo. (A) Q-RT-PCR analysis of the levels of AHI-1 transcripts relative to GAPDH in FACS-purified RPG vector–transduced K562 cells, AHI-1/sh4 cells (with suppression of AHI-1), AHI/sh4 + AHI-1 cells (coexpression of AHI-1 in AHI-1/sh4 cells), and AHI-1 cells (AHI-1 overexpressed cells, top). Western analysis (bottom) of AHI-1 expression in the same transduced cells with an anti–AHI-1 antibody. (B) The numbers of CFC colonies produced in semisolid cultures ± GF (IL-3, GM-CSF, SF) in the same transduced cells. (C) The appearance of CFC colonies produced in semisolid cultures ± GF as shown in B. Bar, 250 μm. (D) Percentage of single control K562 cells or AHI/sh4 cells generating clones (top) and clone size distributions obtained from these cells after being cultured for 7 d (bottom). (E) NOD/SCID-β2M−/− mice were injected subcutaneously with 107 control K562 and transduced cells. Tumor volume is expressed as mean ± SEM areas of each group (n = 4). (F) The appearance of tumors generated by the same cells as shown in E. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different from K562 or RPG control cells.

Mentions: To investigate directly whether deregulated expression of AHI-1 contributes to BCR-ABL–mediated transformation of human CML, knockdown or overexpression of AHI-1 in K562 cells, a cell line derived from a patient with CML and characterized by highly increased expression of AHI-1 (22), was performed, with confirmed down-regulation of AHI-1 in suppressed cells (AHI/sh4) and overexpression of AHI-1 in overexpressed cells (P < 0.01; Fig. 3 A). Interestingly, suppression of AHI-1 expression reduced GF independence in semisolid cultures (up to 5-fold; P < 0.01; Fig. 3 B) compared with cells transduced with control vector (RPG). Colonies formed by AHI-1–suppressed cells were much smaller than those of control cells (Fig. 3 C). Also, the ability to form a clone from a single cell was 2-fold less than that of the controls without GFs in liquid suspension cultures (P < 0.05; Fig. 3 D). In contrast, overexpression of human AHI-1 by transduction of EF1α-AHI-1-IRES-YFP lentivirus in K562 cells resulted in sharply increased colony-forming ability compared with control cells (1.5–2-fold; P < 0.05; Fig. 3, B and C). Importantly, restored expression of AHI-1 both at RNA and protein levels in AHI/sh4 cells reversed growth deficiencies exhibited by suppression of AHI-1 (Fig. 3, A–C).


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)

Knockdown or overexpression of AHI-1 in human K562 cells mediates their transforming activity in vitro and in vivo. (A) Q-RT-PCR analysis of the levels of AHI-1 transcripts relative to GAPDH in FACS-purified RPG vector–transduced K562 cells, AHI-1/sh4 cells (with suppression of AHI-1), AHI/sh4 + AHI-1 cells (coexpression of AHI-1 in AHI-1/sh4 cells), and AHI-1 cells (AHI-1 overexpressed cells, top). Western analysis (bottom) of AHI-1 expression in the same transduced cells with an anti–AHI-1 antibody. (B) The numbers of CFC colonies produced in semisolid cultures ± GF (IL-3, GM-CSF, SF) in the same transduced cells. (C) The appearance of CFC colonies produced in semisolid cultures ± GF as shown in B. Bar, 250 μm. (D) Percentage of single control K562 cells or AHI/sh4 cells generating clones (top) and clone size distributions obtained from these cells after being cultured for 7 d (bottom). (E) NOD/SCID-β2M−/− mice were injected subcutaneously with 107 control K562 and transduced cells. Tumor volume is expressed as mean ± SEM areas of each group (n = 4). (F) The appearance of tumors generated by the same cells as shown in E. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different from K562 or RPG control cells.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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fig3: Knockdown or overexpression of AHI-1 in human K562 cells mediates their transforming activity in vitro and in vivo. (A) Q-RT-PCR analysis of the levels of AHI-1 transcripts relative to GAPDH in FACS-purified RPG vector–transduced K562 cells, AHI-1/sh4 cells (with suppression of AHI-1), AHI/sh4 + AHI-1 cells (coexpression of AHI-1 in AHI-1/sh4 cells), and AHI-1 cells (AHI-1 overexpressed cells, top). Western analysis (bottom) of AHI-1 expression in the same transduced cells with an anti–AHI-1 antibody. (B) The numbers of CFC colonies produced in semisolid cultures ± GF (IL-3, GM-CSF, SF) in the same transduced cells. (C) The appearance of CFC colonies produced in semisolid cultures ± GF as shown in B. Bar, 250 μm. (D) Percentage of single control K562 cells or AHI/sh4 cells generating clones (top) and clone size distributions obtained from these cells after being cultured for 7 d (bottom). (E) NOD/SCID-β2M−/− mice were injected subcutaneously with 107 control K562 and transduced cells. Tumor volume is expressed as mean ± SEM areas of each group (n = 4). (F) The appearance of tumors generated by the same cells as shown in E. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different from K562 or RPG control cells.
Mentions: To investigate directly whether deregulated expression of AHI-1 contributes to BCR-ABL–mediated transformation of human CML, knockdown or overexpression of AHI-1 in K562 cells, a cell line derived from a patient with CML and characterized by highly increased expression of AHI-1 (22), was performed, with confirmed down-regulation of AHI-1 in suppressed cells (AHI/sh4) and overexpression of AHI-1 in overexpressed cells (P < 0.01; Fig. 3 A). Interestingly, suppression of AHI-1 expression reduced GF independence in semisolid cultures (up to 5-fold; P < 0.01; Fig. 3 B) compared with cells transduced with control vector (RPG). Colonies formed by AHI-1–suppressed cells were much smaller than those of control cells (Fig. 3 C). Also, the ability to form a clone from a single cell was 2-fold less than that of the controls without GFs in liquid suspension cultures (P < 0.05; Fig. 3 D). In contrast, overexpression of human AHI-1 by transduction of EF1α-AHI-1-IRES-YFP lentivirus in K562 cells resulted in sharply increased colony-forming ability compared with control cells (1.5–2-fold; P < 0.05; Fig. 3, B and C). Importantly, restored expression of AHI-1 both at RNA and protein levels in AHI/sh4 cells reversed growth deficiencies exhibited by suppression of AHI-1 (Fig. 3, A–C).

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