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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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Elevated AHI-1 transcript levels in CML stem/progenitor cells and reduced CFC production in the same cells when AHI-1 expression is suppressed. (A) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in lin−CD34+ normal bone marrow (NBM) and lin−CD34+ CML cells from IM responders, nonresponders, and blast crisis patients. (B) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in transduced lin−CD34+ normal BM and CML cells (three IM responders, three nonresponders, and three blast crisis patients) ± suppression of AHI-1 expression, including cells transduced with a control vector (Ctrl) or a AHI/sh4 vector. (C) The yield of BFU-E, CFU-GM, and CFU-GEMM colonies from primary CD34+lin− CML cells transduced with a control vector or AHI/sh4 vector. (D) The appearance of CFC colonies produced in semisolid cultures from control and AHI/sh4-transduced cells from an IM nonresponder patient. Bar, 100 μm. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between transduced control cells and cells transduced with AHI/sh4 vector.
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fig5: Elevated AHI-1 transcript levels in CML stem/progenitor cells and reduced CFC production in the same cells when AHI-1 expression is suppressed. (A) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in lin−CD34+ normal bone marrow (NBM) and lin−CD34+ CML cells from IM responders, nonresponders, and blast crisis patients. (B) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in transduced lin−CD34+ normal BM and CML cells (three IM responders, three nonresponders, and three blast crisis patients) ± suppression of AHI-1 expression, including cells transduced with a control vector (Ctrl) or a AHI/sh4 vector. (C) The yield of BFU-E, CFU-GM, and CFU-GEMM colonies from primary CD34+lin− CML cells transduced with a control vector or AHI/sh4 vector. (D) The appearance of CFC colonies produced in semisolid cultures from control and AHI/sh4-transduced cells from an IM nonresponder patient. Bar, 100 μm. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between transduced control cells and cells transduced with AHI/sh4 vector.

Mentions: To further elucidate the role of AHI-1 in primary CML stem/progenitor cells, AHI-1 transcript levels were evaluated in pretreatment lin−CD34+ cells from 16 chronic phase patients with subsequent clinical responses to IM therapy (6 responders and 10 nonresponders) and from 7 patients in blast crisis. Increased levels of AHI-1 expression were observed in lin−CD34+ CML stem/progenitor cells from all patient samples studies (2.5–18-fold; P < 0.04; Fig. 5 A), compared with lin−CD34+ normal BM cells. Interestingly, lin−CD34+ cells from IM nonresponders expressed higher levels of AHI-1 transcripts compared with the same cells isolated from IM responders (P = 0.03; Fig. 5 A). Lin−CD34+ cells from blast crisis patients also expressed relatively higher levels of AHI-1 transcripts (P < 0.03, compared with IM responders). A lentiviral-mediated AHI-1 shRNA construct was directly transduced into lin−CD34+ stem/progenitor cells isolated from three IM responders, three IM nonresponders, and three blast crisis patients. Lentiviral-mediated suppression of AHI-1 expression resulted in a reduction in CFC output by ∼28–60% in lin−CD34+ CML cells from all patient samples studied (with AHI-1 expression partially suppressed (∼40–55%; Fig. 5, B and C). CFC colonies formed by CML cells with suppression of AHI-1 were found to be smaller than those resulting from cells transduced with a control vector (Fig. 5 D). It was interesting that more significant reduction in CFC numbers was observed in transduced primary CML cells from IM nonresponders and blast crisis compared with IM responders (Fig. 5 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)

Elevated AHI-1 transcript levels in CML stem/progenitor cells and reduced CFC production in the same cells when AHI-1 expression is suppressed. (A) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in lin−CD34+ normal bone marrow (NBM) and lin−CD34+ CML cells from IM responders, nonresponders, and blast crisis patients. (B) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in transduced lin−CD34+ normal BM and CML cells (three IM responders, three nonresponders, and three blast crisis patients) ± suppression of AHI-1 expression, including cells transduced with a control vector (Ctrl) or a AHI/sh4 vector. (C) The yield of BFU-E, CFU-GM, and CFU-GEMM colonies from primary CD34+lin− CML cells transduced with a control vector or AHI/sh4 vector. (D) The appearance of CFC colonies produced in semisolid cultures from control and AHI/sh4-transduced cells from an IM nonresponder patient. Bar, 100 μm. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between transduced control cells and cells transduced with AHI/sh4 vector.
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Related In: Results  -  Collection

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

fig5: Elevated AHI-1 transcript levels in CML stem/progenitor cells and reduced CFC production in the same cells when AHI-1 expression is suppressed. (A) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in lin−CD34+ normal bone marrow (NBM) and lin−CD34+ CML cells from IM responders, nonresponders, and blast crisis patients. (B) Q-RT-PCR analysis of the levels of human AHI-1 transcripts relative to GAPDH in transduced lin−CD34+ normal BM and CML cells (three IM responders, three nonresponders, and three blast crisis patients) ± suppression of AHI-1 expression, including cells transduced with a control vector (Ctrl) or a AHI/sh4 vector. (C) The yield of BFU-E, CFU-GM, and CFU-GEMM colonies from primary CD34+lin− CML cells transduced with a control vector or AHI/sh4 vector. (D) The appearance of CFC colonies produced in semisolid cultures from control and AHI/sh4-transduced cells from an IM nonresponder patient. Bar, 100 μm. Values shown are the mean ± SEM of triplicate measurements. * indicates significant difference between transduced control cells and cells transduced with AHI/sh4 vector.
Mentions: To further elucidate the role of AHI-1 in primary CML stem/progenitor cells, AHI-1 transcript levels were evaluated in pretreatment lin−CD34+ cells from 16 chronic phase patients with subsequent clinical responses to IM therapy (6 responders and 10 nonresponders) and from 7 patients in blast crisis. Increased levels of AHI-1 expression were observed in lin−CD34+ CML stem/progenitor cells from all patient samples studies (2.5–18-fold; P < 0.04; Fig. 5 A), compared with lin−CD34+ normal BM cells. Interestingly, lin−CD34+ cells from IM nonresponders expressed higher levels of AHI-1 transcripts compared with the same cells isolated from IM responders (P = 0.03; Fig. 5 A). Lin−CD34+ cells from blast crisis patients also expressed relatively higher levels of AHI-1 transcripts (P < 0.03, compared with IM responders). A lentiviral-mediated AHI-1 shRNA construct was directly transduced into lin−CD34+ stem/progenitor cells isolated from three IM responders, three IM nonresponders, and three blast crisis patients. Lentiviral-mediated suppression of AHI-1 expression resulted in a reduction in CFC output by ∼28–60% in lin−CD34+ CML cells from all patient samples studied (with AHI-1 expression partially suppressed (∼40–55%; Fig. 5, B and C). CFC colonies formed by CML cells with suppression of AHI-1 were found to be smaller than those resulting from cells transduced with a control vector (Fig. 5 D). It was interesting that more significant reduction in CFC numbers was observed in transduced primary CML cells from IM nonresponders and blast crisis compared with IM responders (Fig. 5 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