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CLEAR CELL SARCOMA AND OTHER TRANSLOCATION-ASSOCIATED SARCOMAS ARE HIGHLY SENSITIVE TO HISTONE DEACETYLASE INHIBITOR MS-

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,

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CLEAR CELL SARCOMA AND OTHER TRANSLOCATION-ASSOCIATED SARCOMAS ARE HIGHLY SENSITIVE TO HISTONE DEACETYLASE INHIBITOR MS-

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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, Paul Clarkson, Joanna M Lubieniecka, Hongwei Cheng, and Torsten O NielsenUniversity 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. 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. 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. Annexin V + PI apoptotic assay of Depsipeptide in synovial sarcoma Fuji cell line, monolayer culture Necrotic Advanced apoptotic Early apoptotic SYO-1 cell line, spheroid (3D) culture

  10. 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

  11. 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.”

  12. 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) 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; 23:3912-22] • dose-limiting side effects = nausea, fatigue

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

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

  15. Hs68 normal fibroblasts are also resistant to MS-275, whereassynovial sarcomaandDSRCTare sensitive

  16. Relative effectiveness of HDAC inhibitor MS-275 against sarcoma, carcinoma and nonmalignant cell lines in 72h MTT assays

  17. 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-275 0.1 uM MS-275 10 uM MS-275 1 uM 34% 26% 11% 16% 49% 70% 72% 17% 4%

  18. 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

  19. “Green genes” suppressed in sarcomas . . .

  20. 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.

  21. 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

  22. 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).

  23. 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

  24. Effect of MS-275 on EWS-WT1 transcription in JN-DSRCT-1 cells HDAC inhibitors in other translocation-associated sarcomas SYO-1 Fuji synovial sarcoma: depsipeptide knocks SYT-SSX down to undetectable in two cell lines [FK228] ng/mL x 24h [FK228] ng/mL x 24h 0 1 10 0 1 10 SYT-SSX2 b-actin

  25. 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 HDAC inhibitors in myxoid liposarcoma FUS-DDIT3 CDKN1 p21CDKN1 mRNA levels (gene known to be induced by HDACi treatment) in same cells with same tx

  26. 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

  27. 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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