Introduction: Soft tissue and bone sarcomas are rare malignancies of mesenchymal origin that account for about 1% of all adult solid tumors. Surgical resection is the first treatment option but is heavily hampered by delayed diagnosis. Due to few therapeutic treatments available, novel and efficacious therapy is urgently needed. Histone deacetylase inhibitors (HDACi) are emerging as a prominent class of therapeutic agents for several cancers; however, little is known about HDACi activity in sarcomas. By using human sarcoma models, we studied the therapeutic activity of ITF2357, a novel histone deacetylase inhibitor, as an anti-cancer agent alone or in combination with doxorubicin, on both wild-type and mutated p53-bearing sarcoma cell lines.

Materials and methods: A wide panel of sarcoma cell lines, including both established and patient-derived sarcoma from different histotypes, were used. Western Blot analysis was performed to evaluate the effect of ITF2357 on histone and non-histone protein acetylation and expression of potential downstream targets. Cell viability was tested by MTT and CellTiterGlo assays. Apoptosis induction and cell cycle perturbation were assessed by flow cytometry and Western Blot analysis. Autophagy was assessed by analysis of autophagosome formation in EGFP-LC3B expressing cells and Western Blot analysis. Pharmacological interaction between ITF2357 and Doxorubicin was assessed by conservative isobologram analysis. In vivo efficacy of drug combination was evaluated in nude mice bearing sarcoma xenografts.

Results: In sarcoma cell lines, HDAC inhibition induced H3 histone and alpha-tubulin acetylation, cell proliferation inhibition and activation of apoptotic program in a dose-dependent manner. ITF2357 cytotoxicity was independent on p53 status, despite its ability to inhibit the expression of mutated p53 protein. Moreover, ITF2357 induced a canonical-autophagic process, which protects sarcoma cells from apoptotic cell death and enhances cell survival. The combination of doxorubicin and ITF2357 was found to be highly synergistic in terms of cell proliferation inhibition and was related to the engagement of the apoptotic mitochondrial pathway. Noteworthy, treatment with both drugs also showed synergistic inhibition of cell viability in a doxorubicin-resistant osteosarcoma line, as compared with either agent alone. The effect of the drug combination was corroborated in sarcoma stem cells obtained from sarcoma patients. In vivo experiments in mouse xenograft models confirmed that co-treatment increased both inhibition of tumor growth and apoptosis, as compared with either agent alone.

Conclusions: Overall, HDAC inhibition in sarcoma may provide an effective therapeutic modality capable of overcoming the doxorubicin resistance, and thus may lead to more durable responses in the clinic.

Citation Format: Maria Grazia Tupone, Daniela Trisciuoglio, Marianna Desideri, Marta Di Martile, Simonetta Buglioni, Barbara Antoniani, Rossella Loria, Rita Falcioni, Michele Milella, Virginia Ferraresi, Gemma Di Pompo, Nicola Baldini, Roberto Biagini, Donatella Del Bufalo. Enhancement of doxorubicin cytotoxicity by histone deacetylase inhibition in human sarcoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4721.