Defining the Mechanism of Action of Rigosertib in Pediatric Cancers

我们已经进行了几项研究，以确定rigosertib在RAS突变的神经母细胞瘤和横纹肌肉瘤中的作用机制。一些证据表明，rigosertib在这些细胞类型中不是作为RAS模拟物发挥作用，而是作为微管破坏稳定剂发挥作用。首先，rigosertib在含有野生型RAS的儿科癌细胞株中与在含有突变型RAS的儿科癌细胞株中同样有效。其次，rigosertib诱导M期停滞，而RAS信号的抑制剂通常导致G1期停滞。我们进一步表明，用rigosertib治疗会导致有丝分裂纺锤体缺陷，并减少微管乙酰化，这与微管稳定性的降低是一致的。Rigosertib还减少了RMS和NB细胞微管中微管蛋白的含量。然而，rigosertib在横纹肌肉瘤和神经母细胞瘤的异种移植模型中无效，可能是因为该药物对肿瘤的渗透性较差。Rigosertib对不同组织学类型的儿科癌细胞具有不同的磷酸化蛋白质组学作用。特别是，rigosertib诱导横纹肌肉瘤细胞ERK和AKT的磷酸化，但降低神经母细胞瘤细胞系的ERK和AKT的磷酸化。我们使用纳米链分析表明，在RAS突变的横纹肌肉瘤和神经母细胞瘤中，4EBP1在RNA和蛋白质水平的表达都降低。目前的工作旨在确定这种下降发生的机制。除了这项临床前工作外，我们还编写了一份儿科患者Rigosertib I期试验的方案。然而，由于在动物模型中缺乏疗效，我们将不会继续进行这项临床试验。目前的工作旨在确定与立格司替布有协同作用的药物，以便我们可以提高这种药物在儿童实体瘤中的疗效。

We have conducted several studies to identify the mechanism of action of rigosertib in Ras-mutated neuroblastoma and rhabdomyosarcoma. Several lines of evidence suggest that rigosertib is not functioning as a Ras mimetic and is instead functioning as a microtubule destabilizing agent in these cell types. First, rigosertib is equally as effective in pediatric cancer cell lines with wild type Ras as it is in pediatric cancer cell lines with mutant Ras. Second, rigosertib induces an M-phase arrest, while inhibitors of Ras signaling generally cause an arrest in G1. We further show that treatment with rigosertib induces mitotic spindle defects and decreases tubulin acetylation, consistent with a decrease in microtubule stability. Rigosertib also decreases the fraction of tubulin incorporated in microtubules in RMS and NB cells. However, rigosertib is ineffective in xenograft models of rhabdomyosarcoma and neuroblastoma, possibly due to poor tumor penetration of the drug. Rigosertib has different phospho-proteomic effects on pediatric cancer cell lines of different histologies. In particular, rigosertib induces ERK and AKT phosphorylation in rhabdomyosarcoma cells but decreases ERK and AKT phosphorylation in neuroblastoma cell lines. We used a nanostring assay to show that in both Ras-mutated rhabdomyosarcoma and neuroblastoma, expression of 4EBP1 at the RNA and protein level is decreased. Current work is aimed at identifying the mechanism by which this decrease occurs. In addition to this preclinical work, we have written a protocol for a phase I trial of rigosertib in pediatric patients. However, due to the lack of efficacy in animal models we will not pursue this clinical trial. Current work is aimed at identifying drugs synergisitic with rigosertib such that we might improve on the efficacy of this drug in pediatric solid tumors.