Defining the Mechanism of Action of Rigosertib in Pediatric Cancers

我们进行了几项研究，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。几条证据表明，rigosertib不是作为Ras模拟物发挥作用，而是在这些细胞类型中作为微管去稳定剂发挥作用。首先，rigosertib在具有野生型Ras的儿科癌细胞系中与在具有突变型Ras的儿科癌细胞系中同样有效。其次，rigosertib诱导M期阻滞，而Ras信号传导抑制剂通常导致G1期阻滞。我们进一步表明，治疗与rigosertib诱导有丝分裂纺锤体缺陷和减少微管蛋白乙酰化，与微管稳定性下降一致。Rigosertib还降低了RMS和NB细胞中微管中微管蛋白的掺入分数。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，可能是由于药物的肿瘤渗透性差。Rigosertib对不同组织学的儿科癌细胞系具有不同的磷酸化蛋白质组学效应。特别是，rigosertib在横纹肌肉瘤细胞中诱导ERK和AKT磷酸化，但在神经母细胞瘤细胞系中降低ERK和AKT磷酸化。我们使用nanostring分析表明，在Ras突变的横纹肌肉瘤和神经母细胞瘤中，4EBP 1在RNA和蛋白质水平的表达降低。目前的工作旨在确定这种下降的机制。除了这项临床前工作外，我们还编写了一份在儿科患者中进行rigosertib I期试验的方案。然而，由于在动物模型中缺乏疗效，我们将不进行这项临床试验。目前的工作旨在确定与rigosertib协同的药物，以便我们可以改善这种药物在儿科实体瘤中的疗效。

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.