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. 2009 Sep 1;69(17):6941-50.
doi: 10.1158/0008-5472.CAN-08-4004. Epub 2009 Aug 25.

Inhibitors of deacetylases suppress oncogenic KIT signaling, acetylate HSP90, and induce apoptosis in gastrointestinal stromal tumors

Thomas Mühlenberg  1 , Yixiang Zhang, Andrew J Wagner, Florian Grabellus, James Bradner, Georg Taeger, Hauke Lang, Takahiro Taguchi, Martin Schuler, Jonathan A Fletcher, Sebastian Bauer
Affiliations

Inhibitors of deacetylases suppress oncogenic KIT signaling, acetylate HSP90, and induce apoptosis in gastrointestinal stromal tumors

Thomas Mühlenberg et al. Cancer Res. .

Abstract

Gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT or platelet-derived growth factor receptor A (PDGFRA), and treatment with the tyrosine kinase inhibitor imatinib yields responses in the majority of patients VSports手机版. However, most patients develop secondary resistance, which is associated with a dismal prognosis. Histone deacetylase inhibitors (HDACI) have been shown to enhance imatinib activity in imatinib-resistant chronic myelogenous leukemia. Against this background, we explored whether HDACI might provide an alternative therapeutic strategy to KIT/PDGFRA kinase inhibitors in GIST. Inhibition of cell proliferation by HDACI was seen in KIT-positive but not in KIT-negative GIST cell lines, suggesting that HDACI activity is mainly conferred by targeting oncogenic KIT. KIT activity, expression, and activation of downstream pathways were strongly inhibited by several HDACI (SAHA, LBH589, VPA, trichostatin A, and NaButyrate). SAHA and LBH589 induced apoptosis in KIT-positive GIST, and strong synergism with imatinib was observed at low concentrations of SAHA and LBH589. Mechanistically, treatment with HDACI reduced KIT mRNA transcript levels and led to strong acetylation of HSP90, interfering with its activity as KIT chaperone. These results provide preclinical evidence for a disease-specific effect of HDACI in KIT-positive GIST, which could translate into therapeutic activity. .

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"VSports注册入口" Figures

Figure 1
Figure 1
A: CellTiter-Glo ATP-based viability assays for SAHA in imatinib-sensitive (GIST882, GIST-T1) and imatinib-resistant (GIST48, GIST48B, GIST62, GIST522) cell lines. All cells were treated with the indicated concentrations and assessed after 3 days of treatment, with the data normalized to DMSO-only controls. x = 0 is DMSO-only treated. Lines, KIT-positive cell lines; dotted lines, KIT-negative cell lines, points, mean of quadruplicate cultures; bars, SD. B: Treatment with DMSO, IM or SAHA alone and a combination of SAHA 2.5 μM and IM 500 nM in 4 GIST cell lines. C: Treatment of other, KIT-negative, IM-resistant soft tissue sarcoma cell lines with SAHA.
Figure 2
Figure 2
A: Induction of apoptosis represented by amount of activated caspases 3 and 7 as measured by a luminescence based assay (Caspase-Glo®). Indicated concentrations of SAHA were assayed alone and in combination with IM (500 nM) in KIT-positive GIST after 24h and 48h of incubation. DMSO was vehicle control. Bars, mean of quadruplicate cultures with SD represent multitudes of DMSO-only values. B: Immunoblotting of apoptosis markers (cleaved Caspase 3 and PARP), p21 and acetylation of histone H3, after 18h of treatment with increasing doses of SAHA, IM (500 nM), and a combination of SAHA (2 μM) and IM (500 nM) in IM-sensitive (GIST-T1 and GIST882) and IM-resistant (GIST48 and GIST48B) GIST cell lines. Actin served as control for equal protein loading.
Figure 3
Figure 3
Western Blot analyses of SAHA effects on KIT and KIT-dependent signaling pathways. A: Dose-response study after 18h of incubation with increasing doses of SAHA, IM (500 nM), and a combination (S + IM) of SAHA (2 μM) and IM (500 nM). In KIT-negative GIST48B, untreated GIST882-lysate served as positive control for KIT-staining. B: Time course study in GIST882. Cells treated for indicated intervals with SAHA 5 μM.
Figure 4
Figure 4
Effects of various HDACI on KIT-positive GIST. A, B: Viability studies (72h) for different HDACI in GIST882 and GIST48 C: Combination treatments of HDACI with IM 500 nM in GIST48 after 3 days of treatment D: Immunoblot studies of different HDACI on oncogenic KIT signaling and Histone acetylation in GIST882.
Figure 5
Figure 5
Effects of different inhibitors on KIT mRNA and Protein levels A, B: Quantitative Real Time RT-PCR evaluation of KIT mRNA in KIT-positive GIST. Time course study (4, 8 and 12 hours of treatment) with IM 500 nM and 2 concentrations of SAHA (2 μM and 5 μM) (A). Comparison of KIT-inhibitor IM, HSP90-inhibitor 17-AAG and HDAC-inhibitors SAHA and LBH589 on KIT-RNA levels after 12h of treatment (B). Values were normalized to the DMSO value for each point in time. C: Western Blot studies of GIST882 treated with 100 μM cycloheximide (CHX) from 3h to 24h.
Figure 6
Figure 6
Immunoprecipitation of HSP90 (A), HDAC6 (B) and KIT (C) from GIST882 cell lysates treated with DMSO and SAHA 5 μM for 12 hours. D: Staining of whole cell lysates (Inputs) for the indicated proteins.
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"VSports注册入口" References

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