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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 down-regulated Bcr-Abl and its tyrosine phosphorylationA. 32Dp210, 32Dp210T315I, 32Dp210E255K and 32Dp210 F359V cells were treated with or without 10 nM AUY922 for 16h. Cell lysates were subjected for Western blotting with 8E9 and HSP90 antibody. B. The protein levels of Bcr-Abl (wt or mutants) were normalized by β actin and data were plotted in a histogram. C. 32Dp210T315I cells were treated with various doses of AUY922 for 16 h followed by Western blotting with indicated antibodies.
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Figure 2: AUY922 down-regulated Bcr-Abl and its tyrosine phosphorylationA. 32Dp210, 32Dp210T315I, 32Dp210E255K and 32Dp210 F359V cells were treated with or without 10 nM AUY922 for 16h. Cell lysates were subjected for Western blotting with 8E9 and HSP90 antibody. B. The protein levels of Bcr-Abl (wt or mutants) were normalized by β actin and data were plotted in a histogram. C. 32Dp210T315I cells were treated with various doses of AUY922 for 16 h followed by Western blotting with indicated antibodies.

Mentions: Next, we wanted to investigate whether wt Bcr-Abl cells (IM sensitive) or Bcr-Abl kinase domain mutant cells (IM resistant) responded differently to the treatment with the HSP90 inhibitor. To determine this, 32Dp210 and Bcr-Abl kinase domain mutant cells (T315I, E255K and F359V) were treated with 10 nM AUY922 for 16 h. Bcr-Abl protein levels were detected by Western blotting. As Fig. 2A and B showed, the total protein levels of mutated Bcr-Abl (T315I, E255K and F359V) dramatically decreased following AUY922 treatment whereas wt Bcr-Abl was only modestly decreased. HSP90 protein levels were only slightly reduced under the same treatment (Fig. 2A).


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 down-regulated Bcr-Abl and its tyrosine phosphorylationA. 32Dp210, 32Dp210T315I, 32Dp210E255K and 32Dp210 F359V cells were treated with or without 10 nM AUY922 for 16h. Cell lysates were subjected for Western blotting with 8E9 and HSP90 antibody. B. The protein levels of Bcr-Abl (wt or mutants) were normalized by β actin and data were plotted in a histogram. C. 32Dp210T315I cells were treated with various doses of AUY922 for 16 h followed by Western blotting with indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: AUY922 down-regulated Bcr-Abl and its tyrosine phosphorylationA. 32Dp210, 32Dp210T315I, 32Dp210E255K and 32Dp210 F359V cells were treated with or without 10 nM AUY922 for 16h. Cell lysates were subjected for Western blotting with 8E9 and HSP90 antibody. B. The protein levels of Bcr-Abl (wt or mutants) were normalized by β actin and data were plotted in a histogram. C. 32Dp210T315I cells were treated with various doses of AUY922 for 16 h followed by Western blotting with indicated antibodies.
Mentions: Next, we wanted to investigate whether wt Bcr-Abl cells (IM sensitive) or Bcr-Abl kinase domain mutant cells (IM resistant) responded differently to the treatment with the HSP90 inhibitor. To determine this, 32Dp210 and Bcr-Abl kinase domain mutant cells (T315I, E255K and F359V) were treated with 10 nM AUY922 for 16 h. Bcr-Abl protein levels were detected by Western blotting. As Fig. 2A and B showed, the total protein levels of mutated Bcr-Abl (T315I, E255K and F359V) dramatically decreased following AUY922 treatment whereas wt Bcr-Abl was only modestly decreased. HSP90 protein levels were only slightly reduced under the same treatment (Fig. 2A).

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