|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
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.
|A novel role for histone deacetylase 6 in the regulation of the tolerogenic STAT3/IL-10 pathway in APCs.|
Cheng, F; Lienlaf, M; Wang, HW; Perez-Villarroel, P; Lee, C; Woan, K; Rock-Klotz, J; Sahakian, E; Woods, D; Pinilla-Ibarz, J; Kalin, J; Tao, J; Hancock, W; Kozikowski, A; Seto, E; Villagra, A; Sotomayor, EM
Journal of immunology (Baltimore, Md. : 1950)
APCs are critical in T cell activation and in the induction of T cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them histone deacetylases (HDACs) have emerged as key participants. HDAC6, one of the members of this family of enzymes, has been shown to be involved in regulation of inflammatory and immune responses. In this study, to our knowledge we show for the first time that genetic or pharmacologic disruption of HDAC6 in macrophages and dendritic cells results in diminished production of the immunosuppressive cytokine IL-10 and induction of inflammatory APCs that effectively activate Ag-specific naive T cells and restore the responsiveness of anergic CD4(+) T cells. Mechanistically, we have found that HDAC6 forms a previously unknown molecular complex with STAT3, association that was detected in both the cytoplasmic and nuclear compartments of the APC. By using HDAC6 recombinant mutants we identified the domain comprising amino acids 503-840 as being required for HDAC6 interaction with STAT3. Furthermore, by re-chromatin immunoprecipitation we confirmed that HDAC6 and STAT3 are both recruited to the same DNA sequence within the Il10 gene promoter. Of note, disruption of this complex by knocking down HDAC6 resulted in decreased STAT3 phosphorylation--but no changes in STAT3 acetylation--as well as diminished recruitment of STAT3 to the Il10 gene promoter region. The additional demonstration that a selective HDAC6 inhibitor disrupts this STAT3/IL-10 tolerogenic axis points to HDAC6 as a novel molecular target in APCs to overcome immune tolerance and tips the balance toward T cell immunity.
|c-Abl activates janus kinase 2 in normal hematopoietic cells.|
Tao, W; Leng, X; Chakraborty, SN; Ma, H; Arlinghaus, RB
The Journal of biological chemistry
Jak2 is involved in cytokine growth factor-stimulated signal transduction, but the mechanism of its activation is largely unknown. Here, we investigated Jak2 activation in a normal hematopoietic cell line, 32D mouse myeloid cells. The bimolecular fluorescence complementation studies showed that c-Abl formed a stable complex with Jak2 in live cells. Co-immunoprecipitation results showed that c-Abl bound to the βc chain of IL-3/IL-5/GM-CSF receptors. The kinase activities of both c-Abl and Jak2 were stimulated by IL-3 in 32D cells. Decreasing c-Abl protein expression in 32D cells by inducible shRNA decreased Jak2 activity and resulted in the failure of Jak2 activation in response to IL-3. Treatment of IL-3 and serum-starved 32D cells with 1 μM imatinib mysylate inhibited IL-3 stimulated kinase activities of both c-Abl and Jak2. In addition, the kinase-deficient Bcr-Abl mutant (p210K1172R) was defective for activation of Jak2 in 32D cells and impaired IL-3 independent growth, which was rescued by overexpression of c-Abl (+Abl). IL-3 efficiently inhibited apoptosis of 32Dp210K/R+Abl cells induced by imatinib mysylate but not Jak2 kinase inhibitor TG101209. In summary, our findings provide evidence that the kinase function of c-Abl and its C-terminal CT4 region is crucial for its interaction with Jak2 and its activation. c-Abl kinase activity induced by IL-3 is required for IL-3-stimulated Jak2 and Jak1 activation. Our findings reveal a novel regulatory role of c-Abl in Jak2 activation induced by IL-3 cytokine growth factor in 32D hematopoietic cells.