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1 CLEAR CELL SARCOMA AND OTHER TRANSLOCATION-ASSOCIATED SARCOMAS ARE HIGHLY SENSITIVE TO HISTONE DEACETYLASE INHIBITOR MS-275 Suzanne Liu, Margaret A Knowling,

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Presentation on theme: "1 CLEAR CELL SARCOMA AND OTHER TRANSLOCATION-ASSOCIATED SARCOMAS ARE HIGHLY SENSITIVE TO HISTONE DEACETYLASE INHIBITOR MS-275 Suzanne Liu, Margaret A Knowling,"— Presentation transcript:

1 1 CLEAR CELL SARCOMA AND OTHER TRANSLOCATION-ASSOCIATED SARCOMAS ARE HIGHLY SENSITIVE TO HISTONE DEACETYLASE INHIBITOR MS-275 Suzanne Liu, Margaret A Knowling, Paul Clarkson, Joanna M Lubieniecka, Hongwei Cheng, and Torsten O Nielsen University of British Columbia and BC Cancer Agency, Vancouver, Canada Suzanne Liu, Margaret A Knowling, Paul Clarkson, Joanna M Lubieniecka, Hongwei Cheng, and Torsten O Nielsen University of British Columbia and BC Cancer Agency, Vancouver, Canada

2 HISTONES modulate chromatin structure H2A, H2B, H3, H4 = core nucleosome “open” chromatin = transcriptionally active condensed “closed” chromatin = silenced

3 HISTONE MODIFICATIONS: the “epigenetic code” acetylation, methylation, phosphorylation, poly- ADP ribosylation, ubiquitinylation, sumoylation: especially amino-terminal tails of H3 and H4 on outside of nucleosome acetylation of H3 and H4 amino-terminal lysines by HAT  open chromatin deacetylation by HDAC  condensed chromatin global hypo-acetylation of H4 is common in human tumours and occurs early in tumorigenesis

4 18 HDAC proteins, in 4 families mostly nuclear, in transcription factor complexes some alternate substrates: tubulin, p53, E2F1, NfKB, Hsp90, myoD1 Class 1 HDACs are overexpressed in colon, breast, prostate, gastric, and cervical carcinomas PML–RARα, PLZF–RARα and AML1–ETO fusion proteins induce leukaemia (AML), and Bcl-6 lymphoma (DLBCL), by recruiting HDAC- containing repressor complexes to target genes Histone deacetylases: enzymes altering chromatin structure and gene transcription to silence differentiation

5 New paper that fits nicely - Status Conclusions - Needs for SS (+ other sarcs) Acknowledgements HDAC mSin3A

6 TLE recruits, assembles repressor complex SYT TLE: a transcriptional corepressor β-catenin SSX

7 HDAC INHIBITORS 12 agents in clinical trials additional agents in development, including HDAC subtype -specific HDACi being used in combination with retinoids to treat AML/APL

8 mild side fx (thrombocytopenia, nausea; rarely cardiac fx) in vitro: induce p21 checkpoint and multiple apoptosis pathways; exact mechanism not clear. Some synergism with other anti- apoptotic agents in vitro: carcinoma cells 10x more sensitive than nontransformed HDAC INHIBITORS

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10 Annexin V + PI apoptotic assay of Depsipeptide in synovial sarcoma Early apoptotic Necrotic Advanced apoptotic Fuji cell line, monolayer culture SYO-1 cell line, spheroid (3D) culture

11 Apoptosis & necrosis visible in 3-D spheroid assays, greater than doxorubicin Relative sensitivity: synovial sarcoma ≥ MDAMB453 breast > MMRU melanoma, SW480 colon, A549 lung > PC-3 prostate, MCF7 breast > normal fibroblasts

12 Using HDACi FK228 (depsipeptide): “Results indicated that EWS-Fli1 deregulated histone acetylation through both the repression of histone acetyltransferase (HAT) and the enhancement of histone deacetylase (HDAC) activities in EFT cells” “Expression of EWS-Fli1 protein and mRNA were also inhibited by HDACIs. We suggest that HDACIs might inhibit the expression of EWS-FLI1 via the suppression of the EWS promoter activity.”

13 MS-275 a new synthetic benzamide HDAC inhibitor inhibits Class 1 HDACs (HDAC 1 > HDAC 2, 3) FK228/depsipeptide Class 1 (HDAC 1, 2) > Class 2 inhibitor cardiotoxicity seen in recent trials (V-tach, prolonged QT, one death: Shah MH et al Clin Cancer Res 2006;12:3997) lipidic, delivered p.o. long half-life (2-3 days)  q week dosing schedules safe in humans [Ryan QC et al. JCO 2005; 23: ] dose-limiting side effects = nausea, fatigue

14 MS-275 causes dose- and time- dependent killing of clear cell sarcoma cell lines.

15 Same assay: MS-275 is not toxic to bone marrow- derived human mesenchymal stem cells, whereas doxorubicin and depsipeptide/FK228 are

16 Hs68 normal fibroblasts are also resistant to MS-275, whereas synovial sarcoma and DSRCT are sensitive

17 Cell lineDiseaseIC 50 (uM) DTC1clear cell sarcoma0.28 KAOclear cell sarcoma0.38 SYO-1synovial sarcoma0.44 SU-CCS-1clear cell sarcoma0.63 SKNMCEwing sarcoma0.79 DSRCTdesmo. small round cell1.12 MLS402myxoid liposarcoma1.23 A549lung carcinoma3.55 SW480colon carcinoma4.67 MCF7breast carcinoma4.79 Hs68normal fibroblasts> 10 hMSC bone marrow-derived mesenchymal stem cells > 10 Relative effectiveness of HDAC inhibitor MS-275 against sarcoma, carcinoma and nonmalignant cell lines in 72h MTT assays

18 3D spheroid cultures: flow cytometry apoptosis assay untreateddoxorubicin 1 uM MS uM MS uMMS uM Shown: 48h effects on KAO clear cell sarcoma. Similar fx seen on SU-CCS-1 and SYO-1 synovial sarcoma cell lines, fx starting at 24h 5% necrotic 4% apoptotic 88% viable 3% 32% 65% 11% 34%26% 16%49%70% 72%17% 4% method: Annexin V - propidium iodide

19 SU-CCS-1 clear cell sarcoma: untreated SU-CCS-1: after 24h MS-275 DTC clear cell sarcoma: untreatedDTC clear cell sarcoma: MS-275

20 “Green genes” suppressed in sarcomas...

21 HDAC inhibitors induce MEIS2 expression in synovial sarcoma cells (Fuji) by qRT- PCR. Expression change quantified relative to vehicle (0.1% ethanol) -treated cells. Curcumin = negative control (HAT inhibitor). Bars = standard error of triplicate exp’ts. MEIS2 = conserved homeobox transcription regulator. Negatively regulates BMP and sonic hedgehog in vertebrate limb development to express proximal rather than distal limb pattern

22 Similar results also with MEF2C (a transcription factor regulating myogenesis): undetectable at baseline, readily seen after 6h MS-275 or FK228/depsipeptide HDAC inhibitors induce EGR1 expression in synovial sarcoma cells by qRT-PCR in a dose- and time- dependent fashion EGR1 = Zn-finger transcription factor; tumor suppressor in fibrosarcoma cells.

23 ChIP assay: the HDACi Depsipeptide/FK228 increases acetylation of histones in the MEIS2 promoter. Curcumin = negative control. Input = total chromatin; rabbit IgG=anti rabbit Ig antibody immunoprecipitations (negative control).

24 Effect of 1 uM MS-275 on the transcription of EWS-ATF1 in KAO clear cell sarcoma, by quantitative RT-PCR (primers spanning fusion site) HDAC inhibitors in clear cell sarcoma

25 Effect of MS-275 on EWS-WT1 transcription in JN-DSRCT- 1 cells HDAC inhibitors in other translocation-associated sarcomas SYT-SSX2  -actin SYO-1Fuji [FK228] ng/mL x 24h synovial sarcoma: depsipeptide knocks SYT-SSX down to undetectable in two cell lines

26 Effect of MS-275 (1 uM) on transcription of FUS- DDIT3 in myxoid liposarcoma cell line MLS402 qPCR relative to untreated control = 100%; equal loading of template p21 CDKN1 mRNA levels (gene known to be induced by HDACi treatment) in same cells with same tx HDAC inhibitors in myxoid liposarcoma FUS-DDIT3 CDKN1

27 FUTURE DIRECTIONS generating expression profiles before & after HDACi treatment synergism with Hsp90 inhibitors further preclinical study: xenograft models for CCS; metastatic model monitored by in vivo imaging

28 Conclusion: HDAC inhibitors induce growth inhibition, apoptosis, and differentiation in translocation- associated sarcoma cell lines. FUNDING Terry Fox Foundation MSFHR, CIHR Cell lines: AL Epstein, C Poremba, A Kawai, K Nagashima, J Nishio, P Aman, N Mandahl Experimental drugs: NCI – CTEP Expression profile data: M van de Rijn


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