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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: "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 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

2 modulate chromatin structure H2A, H2B, H3, H4 = core nucleosome
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 Histone deacetylases: enzymes altering chromatin structure and gene transcription to silence differentiation 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

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

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

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 HDAC INHIBITORS 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

9 shown last year

10 Annexin V + PI apoptotic assay of Depsipeptide in synovial sarcoma
Fuji cell line, monolayer culture Necrotic Advanced apoptotic Early apoptotic Depsipeptide SHIFT OF CELLS FROM THE VIABLE TO THE APOPTOTIC FRACTION WITHIN 24H AT A VERY LOW DOSE SYO-1 cell line, spheroid (3D) culture

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

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 FK228/depsipeptide MS-275 lipidic, delivered p.o.
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) MS-275 a new synthetic benzamide HDAC inhibitor inhibits Class 1 HDACs (HDAC 1 > HDAC 2, 3) 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.
similar also in DTC1 cells, a third line

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 SYO is even more sensitive than Fuji; I brought SL’s data but didn’t include it here. Doxo is toxic to Hs68, data brought but not included (file MS275-Hs68 by SL); these cells are resistant to depsi however.

17 bone marrow-derived mesenchymal stem cells
Cell line Disease IC50 (uM) DTC1 clear cell sarcoma 0.28 KAO 0.38 SYO-1 synovial sarcoma 0.44 SU-CCS-1 0.63 SKNMC Ewing sarcoma 0.79 DSRCT desmo. small round cell 1.12 MLS402 myxoid liposarcoma 1.23 A549 lung carcinoma 3.55 SW480 colon carcinoma 4.67 MCF7 breast carcinoma 4.79 Hs68 normal fibroblasts > 10 hMSC bone marrow-derived mesenchymal stem cells Melanoma not run with ms275 yet, only depsipeptide, where it fell between colon and breast carcinomas = 1 log less sensitive that CCS 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
method: Annexin V - propidium iodide untreated doxorubicin 1 uM 5% necrotic 3% 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 4% apoptotic 32% 65% 88% viable MS uM MS uM MS uM 11% 34% 26% Confirming results using a different assay and tells us it works in 3D model, better than doxorubicin 16% 49% 70% 72% 17% 4%

19 SU-CCS-1 clear cell sarcoma: untreated SU-CCS-1: after 24h MS-275
Also seen with KAO cells DTC clear cell sarcoma: untreated DTC clear cell sarcoma: MS-275

20 “Green genes” suppressed in sarcomas . . .

21 MEIS2 = conserved homeobox transcription regulator
MEIS2 = conserved homeobox transcription regulator. Negatively regulates BMP and sonic hedgehog in vertebrate limb development to express proximal rather than distal limb pattern 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 (a transcription factor controlling compartmental differentiation in limb development (Capdevila 1999),

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

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 HDAC inhibitors in clear cell sarcoma
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)

25 Effect of MS-275 on EWS-WT1 transcription in JN-DSRCT-1 cells
HDAC inhibitors in other translocation-associated sarcomas Effect of MS-275 on EWS-WT1 transcription in JN-DSRCT-1 cells Synovial sarcoma: 24 hour depsipeptide/FK228 at 1-10 uM → dose-dependent decrease of SYT-SSX2 to undetectable levels relative to β-actin control synovial sarcoma: depsipeptide knocks SYT-SSX down to undetectable in two cell lines SYO-1 Fuji [FK228] ng/mL x 24h [FK228] ng/mL x 24h 1 10 1 10 SYT-SSX2 b-actin

26 HDAC inhibitors in myxoid liposarcoma
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 FUS-DDIT3 CDKN1 p21CDKN1 mRNA levels (gene known to be induced by HDACi treatment) in same cells with same tx consistent with results of Sakimura paper mentioned earlier on EFT. Fits with FUS as a close homologue of EWS. Promoters of FUS, EWS, SYT all acetylated at baseline, unchanged by HDACs. So – likely a secondary effect? Or a TF is acetylated-inactivated. Also doing siRNA knockdowns of fusion oncogene mRNAs to see if recapitulates fx on differentiation gene (cause vs effect) – so far no dice, suggesting again effect on fusion oncogene is secondary not primary event

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. 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 FUNDING Terry Fox Foundation MSFHR, CIHR


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