Defining the Mechanism of Action of Rigosertib in Pediatric Cancers

我们已经进行了几项研究来确定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模拟物起作用，而是作为微管不稳定剂起作用。首先，rigosertib对具有野生型Ras的儿童癌细胞系和具有突变型Ras的儿童癌细胞系同样有效。其次，rigosertib诱导m期阻滞，而Ras信号抑制剂通常导致G1期阻滞。我们进一步表明，用rigosertib治疗诱导有丝分裂纺锤体缺陷，降低微管蛋白乙酰化，与微管稳定性下降一致。Rigosertib还能降低RMS和NB细胞微管中微管蛋白的掺入比例。然而，rigosertib在横纹肌肉瘤和神经母细胞瘤的异种移植模型中是无效的，可能是由于药物的肿瘤渗透性差。Rigosertib对不同组织学的儿童癌细胞系具有不同的磷酸化蛋白质组学作用。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。我们使用纳米链实验显示，在ras突变的横纹肌肉瘤和神经母细胞瘤中，4EBP1在RNA和蛋白水平上的表达都降低了。目前的工作旨在查明这种减少发生的机制。除了这项临床前工作，我们还为rigosertib在儿科患者中的I期试验撰写了一份方案。然而，由于在动物模型中缺乏疗效，我们将不会进行这项临床试验。目前的工作旨在确定与rigosertib协同作用的药物，以便我们可以提高该药物在儿童实体瘤中的疗效。最近，其他研究人员表明rigosertib对高风险NB模型有效，并与vincristine协同作用。除了这些临床前研究，我们也在评估微管蛋白翻译后修饰在横纹肌肉瘤形成中的作用。使用质谱法，我们已经能够证明rigosertib共价修饰微管蛋白。此外，在与Sackett博士的合作中，我们已经表明一些FP和FN RMS细胞系增加了微管蛋白的牛磺酸化。

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. Recently, other investigators have shown rigosertib to be effective in high-risk NB models and synergistic with vincristine. In addition to these preclinical studies, we are also evaluating the role of post-translational modifications of tubulin in rhabdomyosarcomagenesis. Using mass spectrometry, we have been able to show that rigosertib covalently modifies tubulin. In addition, in collaboration with Dr. Sackett, we have shown that some FP and FN RMS cell lines have increased taurination of tubulin.