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CLEAR CELL SARCOMA AND OTHER CLEAR CELL SARCOMA AND OTHER TRANSLOCATION-ASSOCIATED TRANSLOCATION-ASSOCIATED

SARCOMAS ARE HIGHLY SENSITIVE SARCOMAS ARE HIGHLY SENSITIVE TO HISTONE DEACETYLASE TO HISTONE DEACETYLASE

INHIBITOR MS-275INHIBITOR MS-275Suzanne Liu, Margaret A Knowling, Paul Clarkson, Suzanne Liu, Margaret A Knowling, Paul Clarkson,

Joanna M Lubieniecka, Hongwei Cheng, Joanna M Lubieniecka, Hongwei Cheng, and Torsten O Nielsenand Torsten O Nielsen

University of British Columbia and BC Cancer Agency, University of British Columbia and BC Cancer Agency, Vancouver, CanadaVancouver, Canada

HISTONESmodulate chromatin

structureH2A, H2B, H3, H4 = core nucleosome

“open” chromatin = transcriptionally active

condensed “closed” chromatin = silenced

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

• 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

New paper that fits nicely - StatusConclusions - Needs for SS (+ other sarcs)Acknowledgements

HDACmSin3A

TLE recruits, assembles repressor complex

SYT

TLE: a transcriptional corepressor

β-catenin

SSX

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

• 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

Annexin V + PI apoptotic assay of Depsipeptide in synovial sarcoma

Early apoptotic

NecroticAdvanced apoptotic

Fuji cell line, monolayer culture

SYO-1 cell line, spheroid (3D) culture

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

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.”

MS-275a 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; 23:3912-22] • dose-limiting side effects = nausea, fatigue

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

Cytotoxicity of MS-275 in KAO clear cell sarcoma cells after 24, 48, and 72 h (MTT assay)

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Cytotoxicity of MS-275 in SU-CCS-1 clear cell sarcoma cells after 24 and 72 hours (MTT assay)

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Cytotoxicity effects of HDACis on Human Mesenchymal Stem Cells

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

72h MTT assay

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

Cell line Disease IC50 (uM)

DTC1 clear cell sarcoma 0.28KAO clear cell sarcoma 0.38

SYO-1 synovial sarcoma 0.44SU-CCS-1 clear cell sarcoma 0.63SKNMC Ewing sarcoma 0.79DSRCT desmo. small round cell 1.12MLS402 myxoid liposarcoma 1.23

A549 lung carcinoma 3.55SW480 colon carcinoma 4.67MCF7 breast carcinoma 4.79Hs68 normal fibroblasts > 10hMSC 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

3D spheroid cultures: flow cytometry apoptosis assay

untreated doxorubicin 1 uM

MS-275 1 uMMS-275 0.1 uM MS-275 10 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

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

DTC clear cell sarcoma: untreated DTC clear cell sarcoma: MS-275

“Green genes” suppressed in sarcomas . . .

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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

Effect of depsipeptide on EGR1 expression in Fuji

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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.

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).

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)

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HDAC inhibitors in clear cell sarcoma

Effect of MS-275 on EWS-WT1

transcription in JN-DSRCT-

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HDAC inhibitors in other translocation-associated sarcomas

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[FK228] ng/mL x 24h [FK228] ng/mL x 24hsynovial sarcoma: depsipeptide knocks SYT-SSX down to undetectable in two cell lines

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

p21CDKN1 mRNA levels (gene known to be induced by HDACi

treatment) in same cells with same tx

HDAC inhibitors in myxoid liposarcoma

FUS-DDIT3

CDKN1

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

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 – CTEPExpression profile data: M van de Rijn