MS-275 (Entinostat) Promotes Radio-Sensitivity in PAX3-FOXO1 Rhabdomyosarcoma Cells

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7-FOXO1 (fusion-positive, FP) while fusion-negative (FN)-RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio-resistant. HDAC inhibitors (HDACi) radio-sensitize different cancer cells types. Thus, we evaluated MS−275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS−275 reversibly hampered cell survival in vitro in FN-RMS RD (RASmut) and irreversibly in FP-RMS RH30 cell lines down-regulating cyclin A, B, and D1, up-regulating p21 and p27 and reducing ERKs activity, and c-Myc expression in RD and PI3K/Akt/mTOR activity and N-Myc expression in RH30 cells. Further, MS−275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co-treatment increased DNA damage repair inhibition and reactive oxygen species formation, down-regulated NRF2, SOD, CAT and GPx4 anti-oxidant genes and improved RT ability to induce G2 growth arrest. MS−275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT-unresponsive RH30 xenografts when combined with radiation. Thus, MS−275 could be considered as a radio-sensitizing agent for the treatment of intrinsically radio-resistant PAX3-FOXO1 RMS.

Radioresistance in rhabdomyosarcomas: Much more than a question of dose

“…No preclinical and/or clinical data related to the use of directly targeting redox proteins drugs have been collected on RMS. However, several pieces of evidence suggest the use of drugs able to increase ROS beyond targeting redox proteins (137)(138)(139). On this regard, ROS generation has been identified as a mediator of histone deacetylase (HDAC) inhibitor (HDACi)-induced cell death (161), and the combination of HDACi with RT brings to RMS radiosensitization through increased ROS accumulation (137)(138)(139).…”

“…Ferroptosis, induced by excessive lipid peroxidation that leads to Fe 3+ accumulation-induced oxidative stress, is mediated by SLC11A2 and negatively regulated by GSH/GPx4 cascade (172). RMS is resistant to apoptosis (173) and necrosis (174), including from RT, as we have shown in preclinical in vitro and in vivo models (130, [137][138][139]175). The tumor suppressor p53, a master promoter of apoptosis (176) and programmed necrosis (177), is frequently mutated in ERMS (178) and downregulated in ARMS (179).…”

“…Thus, it seems unlikely that DNA-PKcs targeting will not lead to an RMS radiosensitization. Notably, several molecules have been identified as upstream regulators of DDR in RMS including ERKs ( 126 , 129 ), DNA methyltransferases 3A (DNMT3A) and DNMT3B ( 130 ), BET proteins ( 131 ), ephrin-A2 ( 132 ), caveolin-1 (CAV-1) ( 128 ), nuclear factor erythroid 2–related factor 2 (NRF-2) ( 133 ), c-Myc ( 116 ), SNAI2 ( 134 ), FAK ( 135 ), androgen receptor ( 136 ), and HDAC ( 137 – 139 ). Thus, another strategy to target DDR could be inhibiting these upstream molecules.…”

“…Increasing oxidative stress has been shown to efficiently kill RMS ( 154 ). RMS antioxidant response is finely regulated by molecular epigenetic mechanisms ( 137 – 139 ), known to be critical regulator of adaptive responses to stress ( 155 , 156 ), including RT ( 157 ). Interestingly, the ability of RMS to detoxify from ROS increases in parallel with the acquisition of a more radioresistant phenotype ( 109 ), suggesting that, for these cells, ROS detoxification is critical to survive to radiation.…”

“…Therefore, identifying the molecules and epigenetic reprogramming pathways used by cancer cells could lead to the development of promising targeted therapies able of weakening the different mechanisms of radioresistance, also in the context of RMS. Indeed, targeting specific DNMTs or HDACs has been demonstrated to reverse RMS phenotype, counteracting stemness and inducing radiosensitization ( 137 – 139 ). Specifically, our group has recently demonstrated the overexpression of DNMT3A and DNMT3B in ERMS primary tumor biopsies ( 251 ) and highlighted the synergic impact of DNMT3A or DNMT3B silencing and irradiation on viability and aggressiveness of RMS cells, suggesting that DNMT inhibitors could have a clinical application in combination with standard RT in the clinical management of patients with RMS.…”