Targeting oncogenic Myb fusions in salivary gland cancer with the elongation inhibitor SVC112

概括。唾液腺癌（SGC）是一种孤儿疾病，目前尚未批准靶向治疗。 SGC 分为组织型，最常见的是腺样囊性癌 (ACC) 和粘液表皮样癌 癌（MEC）。我们已经生成了报道最大的 SGC PDX 库之一，包括主要组织型 例如 ACC 和首次报道的 MEC PDX 模型。我们的 SGC PDX 库包含来自同一国家的多个案例 患者，在随后的复发后收集，这使得我们能够探索致癌基因的获取 基因事件。与其他报告类似，我们的 SGC 模型通过全外显子组测序具有较低的突变负担， RNA-seq 分析鉴定了 ACC 中已知的 (MYB-NFIB)、新型 (NFIB-MTFR2) 甚至双基因融合 产生致癌融合蛋白产物的病例。 SGC 中的其他遗传事件聚集在 PI3K 中 和 mTOR 途径，影响蛋白质合成。 MEC 和 ACC 均过度表达关键转录 Myc 或 SOX2 等因素决定了 SGC 的致瘤性和生长。总的来说，我们建议蛋白质 合成是 SGC 中尚未探索的目标。 SVC112是一种小分子，可抑制蛋白质合成 通过抑制 eEF2 实现延伸步骤。在我们首次报道 SVC112 的开创性研究中，蛋白质合成 和生长抑制相关； SVC112 对癌症细胞的影响大于对非癌细胞的影响； SVC112 耗尽 SOX2、Myc 和 Cyclin D1，并抑制体内生长。在 ACC 和 MEC 细胞系中，我们发现 SVC112 抑制天然 Myb 以及 MYB 融合蛋白和关键蛋白（如 Myc）的蛋白产物。 SVC112 在 ACC 和 MEC 体外测定中抑制增殖和球体形成，并且具有显着的单药效果 2 个 ACC PDX 中的活性（包括实际肿瘤缩小），其中一个具有 MYB 融合，另一个显示非 融合介导的 Myb 上调。我们建议研究致癌融合在SGC中的作用，其机制 SVC112 在 SGC 中的作用，以及 SVC112 在复杂 SGC 模型中的功效。首先，我们将编目并 优先考虑来自大型患者和 PDX 队列的 MYB 融合。然后，为了理解关键的遗传事件，我们将插入 在非癌细胞系中进行融合，并在融合和非融合的 SGC 中部署 CRISPR 和慢病毒载体 细胞系，分别；我们还将测试 SVC112 抑制，以添加额外一层的影响测试 蛋白质调节。 SVC112 的选择性作用是由于关键蛋白质的选择性耗尽所致的假设 将通过核糖体分析（以识别 mRNA 目标）和蛋白质组学分析（以识别蛋白质）进行测试 目标）。最后，我们将使用带有 MYB 和 MYB 的天然 SGC PDX 模型测试 SVC112 的体内功效。 其他融合和野生型，以及 CRISPR 耗尽的 MYB 融合菌株的体内模型。总的来说我们的 目标是检查融合事件与其他分子机制对蛋白质过度表达的依赖性 SVC112 敏感性。该项目将推动SVC112的转化，SVC112是2017年发现的SGC新药。 科罗拉多州，通过剖析其作用和治疗窗的基础，并确定其在先进技术中的功效 SGC 模型的计划能够识别哪一部分 SGC 患者可能会获得更多益处。

SUMMARY. Salivary gland cancer (SGC) is an orphan disease for which no targeted therapies are approved. SGCs are divided into histotypes, the most common being adenoid cystic carcinoma (ACC) and mucoepidermoid carcinoma (MEC). We have generated one of the largest reported SGC PDX banks including major histotypes like ACC and the first reported PDX models of MEC. Our SGC PDX bank includes several cases from the same patient, collected after subsequent relapses, which has allowed an exploration of the acquisition of oncogenic gene events. Similar to other reports, our SGC models had low mutation burden by whole exome sequencing, and RNA-seq analysis identified known (MYB-NFIB), novel (NFIB-MTFR2), and even dual gene fusions in ACC cases that gave rise to oncogenic fusion protein products. Other genetic events in SGC clustered in the PI3K and mTOR pathways, which impinge upon protein synthesis. Both MEC and ACC overexpress key transcription factors such as Myc or SOX2 that dictated tumorigenicity and growth in SGC. Overall, we propose that protein synthesis is an unexplored target in SGC. SVC112 is a small molecule that inhibits protein synthesis at the elongation step by inhibiting eEF2. In our seminal studies where SVC112 was first reported, protein synthesis and growth inhibition were associated; SVC112 had greater effect on cancer over non-cancer cells; SVC112 depleted SOX2, Myc, and Cyclin D1, and arrested growth in vivo. In both ACC and MEC cell lines we found that SVC112 inhibits both native Myb and the protein products of MYB fusions and key proteins like Myc. SVC112 inhibited proliferation and sphere formation in both ACC and MEC in vitro assays, and had notable single agent activity (including actual tumor shrinkage) in 2 ACC PDX, one with a MYB fusion and another one showing non- fusion mediated Myb upregulation. We propose to study the role of oncogenic fusions in SGC, the mechanism of action of SVC112 in SGC, and the efficacy of SVC112 in complex SGC models. First, we will catalogue and prioritize MYB fusions from large patient and PDX cohorts. Then, to understand key genetic events we will insert fusions in non-cancer cell lines, and deploy CRISPR and lentiviral vectors in fusion-and non-fusion-bearing SGC cell lines, respectively; we will also test SVC112 inhibition to add an additional layer of testing of the impact of protein modulation. The hypothesis that SVC112’s selective effect is due to selective depletion of key proteins will be tested by ribosome profiling (to identify mRNA targets) and proteomics analysis (to identify proteins targets). Lastly, we will test the in vivo efficacy of SVC112 using both native SGC PDX models bearing MYB and other fusions and wild-tpy, as well as in vivo models with the CRISPR-depleted MYB fusions strains. Overall our goal is to examine dependence on fusion events vs protein overexpression by other molecular mechanisms for SVC112 susceptibility. This project will propel the translation of SVC112, a new drug for SGC discovered in Colorado, by dissecting the basis for its effect and therapeutic window, and identifying its efficacy in advanced SGC models with a plan that enables identification of which subset of SGC patients may derive more benefit.