Metabolic modulation of alkylating agent efficacy in MEK inhibitor resistant thyroid cancers.

项目总结 间变性甲状腺癌(ATC)是一种致命性实体，其特征是BRAF癌基因频繁突变。 直到最近，还没有显著延长ATC患者生存期的治疗选择。与BRAFV600E不同- BRAFV600E突变的甲状腺癌由黑色素瘤驱动，对靶向激酶的反应不明显 抑制剂单一疗法，强调分子中显著的上下文相关差异 补偿致癌信号阻断的机制。最近批准的双BRAF+MEK 抑制是对抗晚期ATC的一个关键补充，结果显示肿瘤控制有所改善。 尽管靶向治疗最初取得了成功，但获得耐药性和复发的情况经常发生 严格限制完全的应对和治疗。这种治疗诱导的适应机制包括重新连接 然而，对于保护BRAF途径抑制的代谢途径，尚不清楚是否代谢 在获得性耐药的演变过程中，ATCs会发生变化，如果它们具有任何潜在的临床实用价值。 在初步实验中，我们发现MEK抑制剂诱导ATC细胞脂解和脂滴(LD)丢失。 LD是动态细胞器，参与重要的细胞功能，包括新陈代谢、蛋白质运输、 膜合成和信号转导。我们建立了对MEK抑制剂获得性耐药性的体外模型 重新建立了LD丰度并分析了它们的基因表达模式以确定候选漏洞 这可以通过临床批准的疗法进行药理学靶向治疗。我们发现DNA的表达 修复酶MGMT在LDHigh而不是LDlow MEK抑制剂抗性(MEKiR)克隆中表达下调。 在这里，我们建议严格评估LD丰度和MGMT表达可以满足的假设 在临床前Meki耐药甲状腺模型中作为烷化剂敏感性的生物标志物 癌症。在目标1中，我们将通过揭示MGMT在MEKiR中表达的代谢决定因素 控制MGMT表达和对烷化剂敏感性的营养需求和表观遗传标记。 我们将设计具有限速甘油三酯脂肪酶干扰表达的等基因细胞系 ATGL/PNPLA2询问LD积累对于MGMT沉默是必要的还是充分的，并将评估蛋白质- 蛋白质相互作用调节MEKiR中ATGL的活性。在目标2中，我们将量化烷基化试剂在 异种移植MEKiR模型肿瘤。此外，我们将产生新的Meki体内临床前模型 曲美替尼间歇治疗对小鼠肿瘤的耐药性，并将验证LD丰度作为生物标志物 通过确定肿瘤LD周转率、MGMT表达和烷化反应来确定烷化剂的疗效 这些肿瘤的药物治疗。

PROJECT SUMMARY Anaplastic thyroid cancer (ATC) is a lethal entity characterized by frequent mutations in the BRAF oncogene. Until recently there were no treatment options to substantially prolong survival in ATC patients. Unlike BRAFV600E- driven melanomas, thyroid cancers with BRAFV600E mutations are only modestly responsive to targeted kinase inhibitor monotherapy, which emphasizes the significant context dependent differences in the molecular mechanisms that compensate for oncogenic signaling blockade. The recent approval of dual BRAF + MEK inhibition is a critical addition to the arsenal against advanced ATC, with results showing improved tumor control. Despite the initial success of targeted therapies, acquisition of resistance and relapse occur frequently and severely limit complete responses and cures. Such therapy-induced adaptive mechanisms include the rewiring of metabolic pathways that protect from BRAF pathway inhibition, however, it is not known if metabolic alterations occur in ATCs during the evolution of acquired resistance and if they have any potential clinical utility. In preliminary experiments we found that MEK inhibitors induce lipolysis and lipid droplet (LD) loss in ATC cells. LD are dynamic organelles participating in crucial cellular functions including metabolism, protein trafficking, membrane synthesis and signaling. We generated in vitro models of acquired resistance to MEK inhibitors with re-established LD abundance and analyzed their gene expression patterns to identify candidate vulnerabilities that can be pharmacologically targeted with clinically approved therapies. We found that expression of the DNA repair enzyme MGMT was downregulated in LDhigh but not LDlow MEK inhibitor resistant (MEKiR) clones. Here we propose to rigorously evaluate the hypothesis that LD abundance and MGMT expression can serve as biomarkers indicating alkylating agent sensitivity in preclinical models of MEKi resistant thyroid cancers. In aim 1 we will identify metabolic determinants of MGMT expression in MEKiR by uncovering nutritional requirements and epigenetic marks that control MGMT expression and sensitivity to alkylating agents. We will engineer isogenic cell lines with perturbed expression of the rate limiting triglyceride lipase ATGL/PNPLA2 to ask if LD accumulation is necessary or sufficient for MGMT silencing, and will assess if protein- protein interactions regulate ATGL activity in MEKiR. In aim 2 we will quantify the efficacy of alkylating agents in xenotransplanted MEKiR model tumors. Further, we will generate novel in vivo preclinical models of MEKi resistance by intermittent trametinib treatment of tumors in mice, and will validate LD abundance as a biomarker for alkylating agent efficacy by determining tumoral LD turnover, MGMT expression and response to alkylating agent therapy in these tumors.