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HSP90 inhibitor AUY922 induces cell death by disruption of the Bcr-Abl, Jak2 and HSP90 signaling network complex in leukemia cells.

Tao W, Chakraborty SN, Leng X, Ma H, Arlinghaus RB - Genes Cancer (2015)

Bottom Line: Co-IP results showed that HSP90 directly bound to Bcr-Abl, Jak2, Stat 3 and Akt.Tyrosine phosphorylation of Bcr-Abl showed a dose-dependent decrease in 32Dp210T315I following AUY922 treatment for 16h.Our results showed that Bcr-Abl and Jak2 form HMWNC with HSP90 in CML cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
The Bcr-Abl protein is an important client protein of heat shock protein 90 (HSP90). We evaluated the inhibitory effects of the HSP90 ATPase inhibitor AUY922 on 32D mouse hematopoietic cells expressing wild-type Bcr-Abl (b3a2, 32Dp210) and mutant Bcr-Abl imatinib (IM)-resistant cell lines. Western blotting results of fractions from gel filtration column chromatography of 32Dp210 cells showed that HSP90 together with Bcr-Abl, Jak2 Stat3 and several other proteins co-eluted in peak column fractions of a high molecular weight network complex (HMWNC). Co-IP results showed that HSP90 directly bound to Bcr-Abl, Jak2, Stat 3 and Akt. The associations between HSP90 and Bcr-Abl or Bcr-Abl kinase domain mutants (T315I and E255K) were interrupted by AUY922 treatment. Tyrosine phosphorylation of Bcr-Abl showed a dose-dependent decrease in 32Dp210T315I following AUY922 treatment for 16h. AUY922 also markedly inhibited cell proliferation of both IM-sensitive 32Dp210 (IC50 =6 nM) and IM-resistant 32Dp210T315I cells (IC50 ≈6 nM) and human KBM-5R/KBM-7R cell lines (IC50 =50 nM). AUY922 caused significant G1 arrest in 32Dp210 cells but not in T315I or E255K cells. AUY922 efficiently induced apoptosis in 32Dp210 (IC50 =10 nM) and T315I or E255K lines with IC50 around 20 to 50 nM. Our results showed that Bcr-Abl and Jak2 form HMWNC with HSP90 in CML cells. Inhibition of HSP90 by AUY922 disrupted the structure of HMWNC, leading to Bcr-Abl degradation, nhibiting cell proliferation and inducing apoptosis. Thus, inhibition of HSP90 is a powerful way to inhibit not only IM-sensitive CML cells but also IM-resistant CML cells.

No MeSH data available.


Related in: MedlinePlus

AUY922 caused cell cycle arrest in G1 phase and induced apoptosis in both IM-sensitive and resistant mouse leukemic cellsA, The percentage of G1 phase cells from 32Dp210, 32Dp210T315I and 32Dp210E255K cells treated with 5 nM AUY922 for 16 h. B and C, Apoptosis of the same cells under 48 h treatment with either IM (5 and 10μM) (panel B) or AUY922 (10, 20, 50,100 and 200 nM) (panel C).
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Figure 4: AUY922 caused cell cycle arrest in G1 phase and induced apoptosis in both IM-sensitive and resistant mouse leukemic cellsA, The percentage of G1 phase cells from 32Dp210, 32Dp210T315I and 32Dp210E255K cells treated with 5 nM AUY922 for 16 h. B and C, Apoptosis of the same cells under 48 h treatment with either IM (5 and 10μM) (panel B) or AUY922 (10, 20, 50,100 and 200 nM) (panel C).

Mentions: HSP90 is reported to be involved in cell cycle regulation by regulating cell cycle associated proteins [39,40]. Thus, we wanted to examine whether AUY922 affected the cell cycle. PI staining showed that an increase of 20% of 32Dp210 cells presented in G1 phase after 5nM AUY922 for 24 h (Fig. 4A), which indicated AUY922 induced G1 arrest in 32Dp210 cells. However, the cell cycle of IM-resistant 32Dp210T315I and 32Dp210E255K cells did not respond to AUY922, as G1 phase only slightly increased under the same treatment (Fig. 4A). We also didn't observe the changes in other cell cycle phases (e.g. G2 and S phase) of the IM-resistant cells after AUY treatment. These data showed that AUY922 caused G1 phase arrest in wt Bcr-Abl cells but had less effect on Bcr-Abl mutant cells, suggesting the regulation mechanism of cell cycle by kinase domain mutants of Bcr-Abl appears to differ from that of wt Bcr-Abl.


HSP90 inhibitor AUY922 induces cell death by disruption of the Bcr-Abl, Jak2 and HSP90 signaling network complex in leukemia cells.

Tao W, Chakraborty SN, Leng X, Ma H, Arlinghaus RB - Genes Cancer (2015)

AUY922 caused cell cycle arrest in G1 phase and induced apoptosis in both IM-sensitive and resistant mouse leukemic cellsA, The percentage of G1 phase cells from 32Dp210, 32Dp210T315I and 32Dp210E255K cells treated with 5 nM AUY922 for 16 h. B and C, Apoptosis of the same cells under 48 h treatment with either IM (5 and 10μM) (panel B) or AUY922 (10, 20, 50,100 and 200 nM) (panel C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362481&req=5

Figure 4: AUY922 caused cell cycle arrest in G1 phase and induced apoptosis in both IM-sensitive and resistant mouse leukemic cellsA, The percentage of G1 phase cells from 32Dp210, 32Dp210T315I and 32Dp210E255K cells treated with 5 nM AUY922 for 16 h. B and C, Apoptosis of the same cells under 48 h treatment with either IM (5 and 10μM) (panel B) or AUY922 (10, 20, 50,100 and 200 nM) (panel C).
Mentions: HSP90 is reported to be involved in cell cycle regulation by regulating cell cycle associated proteins [39,40]. Thus, we wanted to examine whether AUY922 affected the cell cycle. PI staining showed that an increase of 20% of 32Dp210 cells presented in G1 phase after 5nM AUY922 for 24 h (Fig. 4A), which indicated AUY922 induced G1 arrest in 32Dp210 cells. However, the cell cycle of IM-resistant 32Dp210T315I and 32Dp210E255K cells did not respond to AUY922, as G1 phase only slightly increased under the same treatment (Fig. 4A). We also didn't observe the changes in other cell cycle phases (e.g. G2 and S phase) of the IM-resistant cells after AUY treatment. These data showed that AUY922 caused G1 phase arrest in wt Bcr-Abl cells but had less effect on Bcr-Abl mutant cells, suggesting the regulation mechanism of cell cycle by kinase domain mutants of Bcr-Abl appears to differ from that of wt Bcr-Abl.

Bottom Line: Co-IP results showed that HSP90 directly bound to Bcr-Abl, Jak2, Stat 3 and Akt.Tyrosine phosphorylation of Bcr-Abl showed a dose-dependent decrease in 32Dp210T315I following AUY922 treatment for 16h.Our results showed that Bcr-Abl and Jak2 form HMWNC with HSP90 in CML cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
The Bcr-Abl protein is an important client protein of heat shock protein 90 (HSP90). We evaluated the inhibitory effects of the HSP90 ATPase inhibitor AUY922 on 32D mouse hematopoietic cells expressing wild-type Bcr-Abl (b3a2, 32Dp210) and mutant Bcr-Abl imatinib (IM)-resistant cell lines. Western blotting results of fractions from gel filtration column chromatography of 32Dp210 cells showed that HSP90 together with Bcr-Abl, Jak2 Stat3 and several other proteins co-eluted in peak column fractions of a high molecular weight network complex (HMWNC). Co-IP results showed that HSP90 directly bound to Bcr-Abl, Jak2, Stat 3 and Akt. The associations between HSP90 and Bcr-Abl or Bcr-Abl kinase domain mutants (T315I and E255K) were interrupted by AUY922 treatment. Tyrosine phosphorylation of Bcr-Abl showed a dose-dependent decrease in 32Dp210T315I following AUY922 treatment for 16h. AUY922 also markedly inhibited cell proliferation of both IM-sensitive 32Dp210 (IC50 =6 nM) and IM-resistant 32Dp210T315I cells (IC50 ≈6 nM) and human KBM-5R/KBM-7R cell lines (IC50 =50 nM). AUY922 caused significant G1 arrest in 32Dp210 cells but not in T315I or E255K cells. AUY922 efficiently induced apoptosis in 32Dp210 (IC50 =10 nM) and T315I or E255K lines with IC50 around 20 to 50 nM. Our results showed that Bcr-Abl and Jak2 form HMWNC with HSP90 in CML cells. Inhibition of HSP90 by AUY922 disrupted the structure of HMWNC, leading to Bcr-Abl degradation, nhibiting cell proliferation and inducing apoptosis. Thus, inhibition of HSP90 is a powerful way to inhibit not only IM-sensitive CML cells but also IM-resistant CML cells.

No MeSH data available.


Related in: MedlinePlus