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Herein we describe the in vitro and in vivo activity of FK228 (Romidepsin), an inhibitor of class I HDACs, in counteracting and radiosensitizing embryonal (ERMS, fusion-negative) and alveolar (ARMS, fusion-positive) rhabdomyosarcoma (RMS). RH30 (ARMS, fusion-positive) and RD (ERMS, fusion-negative) cell lines and human multipotent mesenchymal stromal cells (HMSC) were used. Flow cytometry analysis, RT-qPCR, western blotting and enzymatic assays were performed. Irradiation was delivered by using an x-6 MV photon linear accelerator. FK228 (1.2 mg/kg) in vivo activity, combined or not with radiation therapy (2 Gy), was assessed in murine xenografts. Compared to HMSC, RMS expressed low levels of class I HDACs. In vitro, FK228, as single agents, reversibly downregulated class I HDACs expression and activity and induced oxidative stress, DNA damage and a concomitant growth arrest associated with PARP-1-mediated transient non-apoptotic cell death. Surviving cells upregulated the expression of cyclin A, B, D1, p27, Myc and activated PI3K/Akt/mTOR and MAPK signaling, known to be differently involved in cancer chemoresistance. Interestingly, while no radiosensitizing effects were detected, in vitro or in vivo, on RD cells, FK228 markedly radiosensitized RH30 cells by impairing antioxidant and DSBs repair pathways in vitro. Further, FK228 when combined with RT in vivo significantly reduced tumor mass in mouse RH30 xenografts. FK228 did not show antitumor activity as a single agent whilst its combination with RT resulted in radiosensitization of fusion-positive RMS cells, thus representing a possible strategy for the treatment of the most aggressive RMS subtype. [ABSTRACT FROM AUTHOR]

This study describes the in vitro and in vivo activity of PXD-101 (Belinostat), a novel hydroxamic acid-type pan-HDACs inhibitor characterized by a larger safety and efficacy, on myogenic-derived PAX3/FOXO1 fusion protein positive (RH30) or negative (RD) expressing rhabdomyosarcoma (RMS) cell lines. PXD-101 at low doses efficiently inhibited HDACs activity and counteracted the transformed phenotype of RMS by inducing growth arrest and apoptosis, affecting cancer stem cells population and inducing differentiation in RD. Notably, PXD-101 induced oxidative stress promoting DNA damages and affected the ability of RMS to assemble mitotic spindle. PXD-101 radiosensitized by inducing G2 cell cycle growth arrest, enhancing the radiation's ability to induce ROS accumulation and compromising both the ability of RMS to detoxify from ROS and to repair DNA damage. PXD-101 transcriptionally and post-transcriptionally affected c-Myc expression, key master regulator of rhabdomyosarcomagenesis and RMS radioresistance. All in vitro data were corroborated by in vivo experiments showing the cytostatic effects of PXD-101 when used alone and at low dose and its ability to promote the RT-induced killing of RMS. Taken together, our data confirm that altered HDACs activity plays a key role in RMS genesis and suggest PXD-101 as a valid therapeutic strategy particularly in combination with RT. [ABSTRACT FROM AUTHOR]

The role of histone deacetylase (HDAC) 4 and 6 in glioblastoma (GBM) radioresistance was investigated. We found that tumor samples from 31 GBM patients, who underwent temozolomide and radiotherapy combined treatment, showed HDAC4 and HDAC6 expression in 93.5% and 96.7% of cases, respectively. Retrospective clinical data analysis demonstrated that high-intensity HDAC4 and/or HDAC6 immunostaining was predictive of poor clinical outcome. In vitro experiments revealed that short hairpin RNA-mediated silencing of HDAC4 or HDAC6 radiosensitized U87MG and U251MG GBM cell lines by promoting DNA double-strand break (DSBs) accumulation and by affecting DSBs repair molecular machinery. We found that HDAC6 knock-down predisposes to radiation therapy-induced U251MG apoptosis- and U87MG autophagy-mediated cell death. HDAC4 silencing promoted radiation therapy-induced senescence, independently by the cellular context. Finally, we showed that p53WT expression contributed to the radiotherapy lethal effects and that HDAC4 or HDAC6 sustained GBM stem-like radioresistant phenotype. Altogether, these observations suggest that HDAC4 and HDAC6 are guardians of irradiation-induced DNA damages and stemness, thus promoting radioresistance, and may represent potential prognostic markers and therapeutic targets in GBM. [ABSTRACT FROM AUTHOR]