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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 in Sca-1+lin− mouse stem/progenitor BM cells perturbs their in vitro proliferative activity and enhances the effects of BCR-ABL. (A) The levels of Ahi-1 transcripts relative to GAPDH from FACS-purified MIY (Sca-1+lin−YFP+), Ahi-1 (Sca-1+lin−YFP+), BCR-ABL (Sca-1+lin−GFP+), and Ahi-1 plus BCR-ABL (Sca-1+lin−YFP+GFP+) transduced primary mouse BM cells. (B) Growth of each FACS-purified population with GFs. Viable cell numbers were determined by hematocytometer counts of trypan blue–excluding cells. (C) Number of CFC colonies produced in semisolid cultures ± GF from the same FACS-purified mouse BM cells. (D) The appearance of GF-independent CFC colonies is shown. Bar, 250 μm. (E) The numbers of LTC-IC–derived CFCs produced from the same cells ± GF. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different from MIY control cells.
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fig2: Overexpression of Ahi-1 in Sca-1+lin− mouse stem/progenitor BM cells perturbs their in vitro proliferative activity and enhances the effects of BCR-ABL. (A) The levels of Ahi-1 transcripts relative to GAPDH from FACS-purified MIY (Sca-1+lin−YFP+), Ahi-1 (Sca-1+lin−YFP+), BCR-ABL (Sca-1+lin−GFP+), and Ahi-1 plus BCR-ABL (Sca-1+lin−YFP+GFP+) transduced primary mouse BM cells. (B) Growth of each FACS-purified population with GFs. Viable cell numbers were determined by hematocytometer counts of trypan blue–excluding cells. (C) Number of CFC colonies produced in semisolid cultures ± GF from the same FACS-purified mouse BM cells. (D) The appearance of GF-independent CFC colonies is shown. Bar, 250 μm. (E) The numbers of LTC-IC–derived CFCs produced from the same cells ± GF. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different from MIY control cells.

Mentions: To further investigate the role of Ahi-1 as a potential cooperating oncogene relevant to BCR-ABL–mediated transformation in primitive primary hematopoietic cells, we compared the biological behavior of primitive mouse hematopoietic cells from the BM of 5-FU–treated adult C57BL/6 mice after transduction with MSCV-Ahi-1-IRES-YFP and MSCV-BCR-ABL-IRES-GFP retroviruses, alone or in combination. Q-RT-PCR analysis of RNA from FACS-purified primitive Sca-1+lin−YFP+ (Ahi-1+), Sca-1+lin−GFP+ (BCR-ABL+), and Sca-1+lin−YFP+GFP+ (Ahi-1+BCR-ABL+)–transduced BM cells showed that Ahi-1 transcripts were 40-fold higher in Ahi-1–transduced cells compared with cells transduced with control vector (MIY). Elevated endogenous Ahi-1 expression was also observed in cells transduced with BCR-ABL alone as compared with control cells (8-fold; P < 0.01; Fig. 2 A). 5 d after transduction, the rate of expansion of the Sca-1+lin−YFP+ (Ahi-1+) cells in liquid cultures was approximately twofold higher than in cultures initiated with control cells (Fig. 2 B). Ahi-1–transduced Sca-1+lin− cells produced a slightly greater number of colony-forming cells (CFCs) than MIY-transduced control cells in semisolid cultures in the presence of growth factors (GFs), and some of the Ahi-1+ CFCs (∼15%) were already GF independent (Fig. 2, C and D). After 4 wk in long-term culture-initiating cell (LTC-IC) assays, which are used to measure stem cell activities in vitro, the Sca-1+lin−Ahi-1+ cells produced 3-fold more CFCs than control cells (P < 0.01; Fig. 2 E). All of these endpoints (proliferative activity, GF dependence, and CFC output in LTC-IC assays) are also perturbed by BCR-ABL transduction. In cells cotransduced with Ahi-1 and BCR-ABL, all of these effects were further enhanced compared with cells transduced with either BCR-ABL alone (2–4-fold) or Ahi-1 alone (3–6-fold; P < 0.01; Fig. 2). Thus, overexpression of Ahi-1 alone deregulates proliferative control of primitive mouse hematopoietic cells, and these effects can be enhanced by BCR-ABL.


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 in Sca-1+lin− mouse stem/progenitor BM cells perturbs their in vitro proliferative activity and enhances the effects of BCR-ABL. (A) The levels of Ahi-1 transcripts relative to GAPDH from FACS-purified MIY (Sca-1+lin−YFP+), Ahi-1 (Sca-1+lin−YFP+), BCR-ABL (Sca-1+lin−GFP+), and Ahi-1 plus BCR-ABL (Sca-1+lin−YFP+GFP+) transduced primary mouse BM cells. (B) Growth of each FACS-purified population with GFs. Viable cell numbers were determined by hematocytometer counts of trypan blue–excluding cells. (C) Number of CFC colonies produced in semisolid cultures ± GF from the same FACS-purified mouse BM cells. (D) The appearance of GF-independent CFC colonies is shown. Bar, 250 μm. (E) The numbers of LTC-IC–derived CFCs produced from the same cells ± GF. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different from MIY control cells.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571939&req=5

fig2: Overexpression of Ahi-1 in Sca-1+lin− mouse stem/progenitor BM cells perturbs their in vitro proliferative activity and enhances the effects of BCR-ABL. (A) The levels of Ahi-1 transcripts relative to GAPDH from FACS-purified MIY (Sca-1+lin−YFP+), Ahi-1 (Sca-1+lin−YFP+), BCR-ABL (Sca-1+lin−GFP+), and Ahi-1 plus BCR-ABL (Sca-1+lin−YFP+GFP+) transduced primary mouse BM cells. (B) Growth of each FACS-purified population with GFs. Viable cell numbers were determined by hematocytometer counts of trypan blue–excluding cells. (C) Number of CFC colonies produced in semisolid cultures ± GF from the same FACS-purified mouse BM cells. (D) The appearance of GF-independent CFC colonies is shown. Bar, 250 μm. (E) The numbers of LTC-IC–derived CFCs produced from the same cells ± GF. Values shown are the mean ± SEM of triplicate measurements. * indicates significantly different from MIY control cells.
Mentions: To further investigate the role of Ahi-1 as a potential cooperating oncogene relevant to BCR-ABL–mediated transformation in primitive primary hematopoietic cells, we compared the biological behavior of primitive mouse hematopoietic cells from the BM of 5-FU–treated adult C57BL/6 mice after transduction with MSCV-Ahi-1-IRES-YFP and MSCV-BCR-ABL-IRES-GFP retroviruses, alone or in combination. Q-RT-PCR analysis of RNA from FACS-purified primitive Sca-1+lin−YFP+ (Ahi-1+), Sca-1+lin−GFP+ (BCR-ABL+), and Sca-1+lin−YFP+GFP+ (Ahi-1+BCR-ABL+)–transduced BM cells showed that Ahi-1 transcripts were 40-fold higher in Ahi-1–transduced cells compared with cells transduced with control vector (MIY). Elevated endogenous Ahi-1 expression was also observed in cells transduced with BCR-ABL alone as compared with control cells (8-fold; P < 0.01; Fig. 2 A). 5 d after transduction, the rate of expansion of the Sca-1+lin−YFP+ (Ahi-1+) cells in liquid cultures was approximately twofold higher than in cultures initiated with control cells (Fig. 2 B). Ahi-1–transduced Sca-1+lin− cells produced a slightly greater number of colony-forming cells (CFCs) than MIY-transduced control cells in semisolid cultures in the presence of growth factors (GFs), and some of the Ahi-1+ CFCs (∼15%) were already GF independent (Fig. 2, C and D). After 4 wk in long-term culture-initiating cell (LTC-IC) assays, which are used to measure stem cell activities in vitro, the Sca-1+lin−Ahi-1+ cells produced 3-fold more CFCs than control cells (P < 0.01; Fig. 2 E). All of these endpoints (proliferative activity, GF dependence, and CFC output in LTC-IC assays) are also perturbed by BCR-ABL transduction. In cells cotransduced with Ahi-1 and BCR-ABL, all of these effects were further enhanced compared with cells transduced with either BCR-ABL alone (2–4-fold) or Ahi-1 alone (3–6-fold; P < 0.01; Fig. 2). Thus, overexpression of Ahi-1 alone deregulates proliferative control of primitive mouse hematopoietic cells, and these effects can be enhanced by BCR-ABL.

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