Abstract 3699: Histone deacetylase inhibitor ITF2357 induces apoptosis and increases doxorubicin cytotoxicity in preclinical models of human sarcoma
Authors: Tupone Maria Grazia , Martile Marta Di , Desideri Marianna , Buglioni Simonetta , Antoniani Barbara , Mastroiorio Carlotta , Falcioni Rita , Ferraresi Virginia , Baldini Nicola , Biagini Roberto , Milella Michele , Trisciuoglio Daniela , Bufalo Donatella Del 
Published: August 2018

Sarcomas are rare malignancies with generally poor prognosis, for which current therapies have shown limited efficacy. In order to improve the prognosis of patients, there is an urgent medical need to identify new molecular targets that can be exploited for the development of innovative treatment approaches. Inhibition of histone deacetylases (HDACs) has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer, and HDAC inhibitors (HDACi) have been reported as potent and specific anticancer molecules in preclinical and clinical studies. Here we investigated the molecular and functional effects of a new generation HDACi, ITF2357, in preclinical models of soft tissue and bone sarcomas. A wide panel of sarcomas cells, both established and patient derived cell lines, were used. For in vitro experiments Western Blot, RT-PCR, flow cytometry and conservative isobologram analysis have been performed. In vivo efficacy of ITF2357/doxorubucin combination was evaluated in nude mice bearing sarcoma xenografts. Using in vitro and in vivo sarcoma models, we demonstrate that ITF2357 decreases cell viability, activates apoptosis and increases the growth inhibitory efficacy of doxorubicin. The molecular mechanisms of action of ITF2357-mediated cell death implied the activation of mitochondrial apoptosis, as attested by the increase of pro-apoptotic BH3-only proteins, and both caspase and bcl-2 dependent cell death. We also found that ITF2357 transcriptionally upregulated the pro-apoptotic BH3-only genes Bax, Puma, Noxa and BIM in cells harbouring mutant or wild type p53, thus indicating a p53 independent mechanism. Both bcl-2 overexpression and caspases inhibition strongly reduced apoptosis, corroborating the evidence that activation of mitochondrial pathway occurs upstream and before caspases activation. We also found that ITF2357 induces a canonical autophagic process, and that inhibition of autophagy increased apoptosis induced by ITF2357, indicating that in our models autophagy shows a prosurvival effect. We also reported that ITF2357 induced FOXO1/3a up-regulation, FOXO proteins nuclear accumulation, and transcriptional activation of FOXO-target genes, such as BH3-only proteins Puma, Noxa and Bim. Knockdown experiments demonstrated that reduction of both FOXO1 and 3a did not inhibit the induction of Bim expression in response to ITF2357, and consequently did not protect sarcoma cells against ITF2357-induced apoptosis, independently from p53 status. Notably, ITF2357 synergized with doxorubicin to induce in vitro cell death and to reduce in vivo tumor growth. Therefore ITF2357 may represent an important therapeutic agent against human sarcoma regardless p53 status, and the pharmacological combination of ITF2357 with doxorubicin has the potential to enhance sensitization in different preclinical models of sarcoma.

Citation Format: Maria Grazia Tupone, Marta Di Martile, Marianna Desideri, Simonetta Buglioni, Barbara Antoniani, Carlotta Mastroiorio, Rita Falcioni, Virginia Ferraresi, Nicola Baldini, Roberto Biagini, Michele Milella, Daniela Trisciuoglio, Donatella Del Bufalo. Histone deacetylase inhibitor ITF2357 induces apoptosis and increases doxorubicin cytotoxicity in preclinical models of human sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3699.