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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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Lentiviral-mediated suppression of AHI-1 expression causes reduced proliferative activity and GF independence of BCR-ABL–transduced human CB cells. (A) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in transduced lin−CD34+ CB cells, including cells transduced with a control vector (Ctrl), AHI/sh4 cells (with suppression of AHI-1), BCR-ABL (B/A), and AHI/sh4 cotransduced cells (B/A + AHI/sh4). (B) Growth of each transduced population in suspension cultures ± GF for 12 d. Viable cell numbers were determined by hematocytometer counts of trypan blue-excluding cells. (C) Number of BFU-E, CFU-GM, and CFU-GEMM colonies produced in semisolid cultures from the same cells as shown in B. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL–transduced cells alone and cells cotransduced with BCR-ABL and AHI/sh4.
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fig4: Lentiviral-mediated suppression of AHI-1 expression causes reduced proliferative activity and GF independence of BCR-ABL–transduced human CB cells. (A) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in transduced lin−CD34+ CB cells, including cells transduced with a control vector (Ctrl), AHI/sh4 cells (with suppression of AHI-1), BCR-ABL (B/A), and AHI/sh4 cotransduced cells (B/A + AHI/sh4). (B) Growth of each transduced population in suspension cultures ± GF for 12 d. Viable cell numbers were determined by hematocytometer counts of trypan blue-excluding cells. (C) Number of BFU-E, CFU-GM, and CFU-GEMM colonies produced in semisolid cultures from the same cells as shown in B. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL–transduced cells alone and cells cotransduced with BCR-ABL and AHI/sh4.

Mentions: We next evaluated the effects of down-regulation of AHI-1 in BCR-ABL–transduced primitive human CB stem/progenitor cells (Lin−CD34+) using lentiviral-mediated RNA interference. Q-RT-PCR analysis indicated that endogenous AHI-1 expression was suppressed by 40–50% when the shRNA construct (AHI/sh4) was introduced (Fig. 4 A). Interestingly, BCR-ABL–transduced CB cells expanded rapidly in the presence or absence of GFs as expected, but the growth of the BCR-ABL–transduced cells with suppression of AHI-1 was significantly suppressed (2.2 × 106 versus 4.8 × 105 cells with GF; 106 versus 5 × 103 cells without GF; P < 0.01; n = 3; Fig. 4 B). Similarly, BCR-ABL–transduced CB cells with suppression of AHI-1 had up to 10-fold less CFC output compared with BCR-ABL–transduced CB cells (P < 0.01; Fig. 4 C). A similar reduction of CFC output was observed in the absence of GFs (data not shown). Notably, burst-forming-units-erythroids (BFU-Es) were especially increased in BCR-ABL–transduced CB cells compared with control cells, which remained predominantly myeloid as previously reported (110 ± 8 versus 18 ± 3; P < 0.01; Fig. 4 C) (25). Suppression of AHI-1 expression in BCR-ABL-transduced CB cells significantly reduced myeloid CFC (BFU-E and colony-forming units-granulocyte-macrophage [CFU-GM]) output, as well as primitive myeloid progenitors (colony-forming units-granulocyte-erythrocite-monocyte-megakaryocyte [CFU-GEMM]), suggesting that AHI-1 may play a role in regulation of overproduction of myeloid cells in CML.


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)

Lentiviral-mediated suppression of AHI-1 expression causes reduced proliferative activity and GF independence of BCR-ABL–transduced human CB cells. (A) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in transduced lin−CD34+ CB cells, including cells transduced with a control vector (Ctrl), AHI/sh4 cells (with suppression of AHI-1), BCR-ABL (B/A), and AHI/sh4 cotransduced cells (B/A + AHI/sh4). (B) Growth of each transduced population in suspension cultures ± GF for 12 d. Viable cell numbers were determined by hematocytometer counts of trypan blue-excluding cells. (C) Number of BFU-E, CFU-GM, and CFU-GEMM colonies produced in semisolid cultures from the same cells as shown in B. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL–transduced cells alone and cells cotransduced with BCR-ABL and AHI/sh4.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Lentiviral-mediated suppression of AHI-1 expression causes reduced proliferative activity and GF independence of BCR-ABL–transduced human CB cells. (A) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in transduced lin−CD34+ CB cells, including cells transduced with a control vector (Ctrl), AHI/sh4 cells (with suppression of AHI-1), BCR-ABL (B/A), and AHI/sh4 cotransduced cells (B/A + AHI/sh4). (B) Growth of each transduced population in suspension cultures ± GF for 12 d. Viable cell numbers were determined by hematocytometer counts of trypan blue-excluding cells. (C) Number of BFU-E, CFU-GM, and CFU-GEMM colonies produced in semisolid cultures from the same cells as shown in B. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between BCR-ABL–transduced cells alone and cells cotransduced with BCR-ABL and AHI/sh4.
Mentions: We next evaluated the effects of down-regulation of AHI-1 in BCR-ABL–transduced primitive human CB stem/progenitor cells (Lin−CD34+) using lentiviral-mediated RNA interference. Q-RT-PCR analysis indicated that endogenous AHI-1 expression was suppressed by 40–50% when the shRNA construct (AHI/sh4) was introduced (Fig. 4 A). Interestingly, BCR-ABL–transduced CB cells expanded rapidly in the presence or absence of GFs as expected, but the growth of the BCR-ABL–transduced cells with suppression of AHI-1 was significantly suppressed (2.2 × 106 versus 4.8 × 105 cells with GF; 106 versus 5 × 103 cells without GF; P < 0.01; n = 3; Fig. 4 B). Similarly, BCR-ABL–transduced CB cells with suppression of AHI-1 had up to 10-fold less CFC output compared with BCR-ABL–transduced CB cells (P < 0.01; Fig. 4 C). A similar reduction of CFC output was observed in the absence of GFs (data not shown). Notably, burst-forming-units-erythroids (BFU-Es) were especially increased in BCR-ABL–transduced CB cells compared with control cells, which remained predominantly myeloid as previously reported (110 ± 8 versus 18 ± 3; P < 0.01; Fig. 4 C) (25). Suppression of AHI-1 expression in BCR-ABL-transduced CB cells significantly reduced myeloid CFC (BFU-E and colony-forming units-granulocyte-macrophage [CFU-GM]) output, as well as primitive myeloid progenitors (colony-forming units-granulocyte-erythrocite-monocyte-megakaryocyte [CFU-GEMM]), suggesting that AHI-1 may play a role in regulation of overproduction of myeloid cells in CML.

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