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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-275

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

Suzanne 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 0.28

KAO clear cell sarcoma 0.38

SYO-1 synovial sarcoma 0.44

SU-CCS-1 clear cell sarcoma 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

> 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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EWS-ATF1

HDAC inhibitors in clear cell sarcoma

Effect of MS-275 on EWS-WT1

transcription in JN-DSRCT-

1 cells

HDAC inhibitors in other translocation-associated sarcomas

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SYT-SSX2

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SYO-1 Fuji

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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