Limits...
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.

Show MeSH

Related in: MedlinePlus

Enhanced activation of JAK2 and STAT5 in BCR-ABL inducible BaF3 cells cotransduced with Ahi-1 and detection of a AHI-1–BCR-ABL–JAK2 interaction complex in human K562 cells. (A and B) Western blot analyses of cell lysates from control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced BCR-ABL–inducible clonal lines cultured in the presence (A) and absence (B) of IL-3 ± Dox for 24 h. Antibodies used are indicated. (C–H) Protein lysates of K562 cells (BCR-ABL+) and Hut 78 cells (BCR-ABL−) were used to immunoprecipitate AHI-1 (C, E-F, and H), ABL (D), and JAK2 (G), and proteins were detected by Western blotting with antibodies to ABL (C), AHI-1 (D, F, and G), JAK-2 (G), and phosphotyrosine (E and H) as indicated. In H, before immunoprecipitation, lysates were incubated for 1 h with the indicated AHI-1 peptide; unrelated peptide (unr.), no peptide (-), and antigenic peptide (+) or cells were incubated ± IM (5 μM) for 6 h before preparation of protein lysates.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Enhanced activation of JAK2 and STAT5 in BCR-ABL inducible BaF3 cells cotransduced with Ahi-1 and detection of a AHI-1–BCR-ABL–JAK2 interaction complex in human K562 cells. (A and B) Western blot analyses of cell lysates from control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced BCR-ABL–inducible clonal lines cultured in the presence (A) and absence (B) of IL-3 ± Dox for 24 h. Antibodies used are indicated. (C–H) Protein lysates of K562 cells (BCR-ABL+) and Hut 78 cells (BCR-ABL−) were used to immunoprecipitate AHI-1 (C, E-F, and H), ABL (D), and JAK2 (G), and proteins were detected by Western blotting with antibodies to ABL (C), AHI-1 (D, F, and G), JAK-2 (G), and phosphotyrosine (E and H) as indicated. In H, before immunoprecipitation, lysates were incubated for 1 h with the indicated AHI-1 peptide; unrelated peptide (unr.), no peptide (-), and antigenic peptide (+) or cells were incubated ± IM (5 μM) for 6 h before preparation of protein lysates.

Mentions: We next evaluated the effect of increased BCR-ABL expression and tyrosine phosphorylation of cotransduced cells on the activity of candidate signaling mechanisms. We observed increased levels of phosphorylation of JAK2, STAT5, NF-κB p65 (at Ser 563 and Ser 468), and Src (at Tyr 416) in BCR-ABL–inducible cells and Ahi-1–cotransduced BCR-ABL–inducible cells compared with control BaF3 cells (Fig. 8, A and B). Interestingly, phosphorylation of most downstream proteins was down-regulated when BCR-ABL expression was inhibited by Dox, but sustained phosphorylation of JAK2 and STAT5 was consistently observed in cotransduced cells in the presence of IL-3 and Dox (Fig. 8 A, lanes 6 and 8 compared with lane 4). In the absence of IL-3, phosphorylation of JAK2 and STAT5 was reduced in cotransduced cells when BCR-ABL expression was suppressed. A similar, albeit less pronounced, finding of sustained phosphorylation of Src was also observed, particularly in Ahi-1 + BCR-ABL-1 cells in the presence of IL-3. These results suggest that Ahi-1 may play a regulatory role in mediation of BCR-ABL activity associated with enhanced activation of JAK2 and STAT5 through the IL-3 signaling pathway.


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)

Enhanced activation of JAK2 and STAT5 in BCR-ABL inducible BaF3 cells cotransduced with Ahi-1 and detection of a AHI-1–BCR-ABL–JAK2 interaction complex in human K562 cells. (A and B) Western blot analyses of cell lysates from control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced BCR-ABL–inducible clonal lines cultured in the presence (A) and absence (B) of IL-3 ± Dox for 24 h. Antibodies used are indicated. (C–H) Protein lysates of K562 cells (BCR-ABL+) and Hut 78 cells (BCR-ABL−) were used to immunoprecipitate AHI-1 (C, E-F, and H), ABL (D), and JAK2 (G), and proteins were detected by Western blotting with antibodies to ABL (C), AHI-1 (D, F, and G), JAK-2 (G), and phosphotyrosine (E and H) as indicated. In H, before immunoprecipitation, lysates were incubated for 1 h with the indicated AHI-1 peptide; unrelated peptide (unr.), no peptide (-), and antigenic peptide (+) or cells were incubated ± IM (5 μM) for 6 h before preparation of protein lysates.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Enhanced activation of JAK2 and STAT5 in BCR-ABL inducible BaF3 cells cotransduced with Ahi-1 and detection of a AHI-1–BCR-ABL–JAK2 interaction complex in human K562 cells. (A and B) Western blot analyses of cell lysates from control BaF3, BCR-ABL–inducible cells, and two Ahi-1–transduced BCR-ABL–inducible clonal lines cultured in the presence (A) and absence (B) of IL-3 ± Dox for 24 h. Antibodies used are indicated. (C–H) Protein lysates of K562 cells (BCR-ABL+) and Hut 78 cells (BCR-ABL−) were used to immunoprecipitate AHI-1 (C, E-F, and H), ABL (D), and JAK2 (G), and proteins were detected by Western blotting with antibodies to ABL (C), AHI-1 (D, F, and G), JAK-2 (G), and phosphotyrosine (E and H) as indicated. In H, before immunoprecipitation, lysates were incubated for 1 h with the indicated AHI-1 peptide; unrelated peptide (unr.), no peptide (-), and antigenic peptide (+) or cells were incubated ± IM (5 μM) for 6 h before preparation of protein lysates.
Mentions: We next evaluated the effect of increased BCR-ABL expression and tyrosine phosphorylation of cotransduced cells on the activity of candidate signaling mechanisms. We observed increased levels of phosphorylation of JAK2, STAT5, NF-κB p65 (at Ser 563 and Ser 468), and Src (at Tyr 416) in BCR-ABL–inducible cells and Ahi-1–cotransduced BCR-ABL–inducible cells compared with control BaF3 cells (Fig. 8, A and B). Interestingly, phosphorylation of most downstream proteins was down-regulated when BCR-ABL expression was inhibited by Dox, but sustained phosphorylation of JAK2 and STAT5 was consistently observed in cotransduced cells in the presence of IL-3 and Dox (Fig. 8 A, lanes 6 and 8 compared with lane 4). In the absence of IL-3, phosphorylation of JAK2 and STAT5 was reduced in cotransduced cells when BCR-ABL expression was suppressed. A similar, albeit less pronounced, finding of sustained phosphorylation of Src was also observed, particularly in Ahi-1 + BCR-ABL-1 cells in the presence of IL-3. These results suggest that Ahi-1 may play a regulatory role in mediation of BCR-ABL activity associated with enhanced activation of JAK2 and STAT5 through the IL-3 signaling pathway.

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