Elucidating the therapeutic utility of targeting metabolic dependencies in osteosarcoma

项目摘要/摘要 骨肉瘤(OS)是人类和狗最常见的恶性骨肿瘤，它具有以下几个特征 两种包括临床表现和分子改变。尽管做出了许多努力，但还是有 在过去的30年里，这两个物种的疾病结果都没有改善：30%的人和90%的人 狗仍然死于转移。虽然一些方法在微观疾病的背景下显示出了希望， 在这两个物种中，宏观转移表现出对多种药物(激酶抑制剂， 化疗、免疫治疗等)。我最近描述了犬类OS基因组的特征，发现 与人类OS一样，体细胞突变负荷很低，拷贝数异常/结构变异占主导地位， 而且没有明显的分子驱动因素。这些数据，以及临床试验失败的历史表明 当代治疗进展的方法，如激酶抑制剂和免疫检查点 封锁可能会对临床产生有限的影响，需要开发创新的治疗方法 战略。癌细胞的一个显著特征是它们有能力进行有氧糖酵解，使它们能够 在各种微环境中茁壮成长。单羧酸转运体(MCT)是这一过程的关键促进者， 乳酸穿过质膜，对糖酵解性肿瘤的生长和转移至关重要，例如 操作系统。在以前的工作中，我发现OS细胞中MCT1或MCT4功能的丧失会降低基础和代偿性 糖酵解、细胞增殖和侵袭能力。我还证明了MCT4是一个直接转录的靶点。 STAT3和FOXM1，两者都在操作系统中表现出结构性激活，支持 MCT4/STAT3/FOXM1和有氧糖酵解。在这些数据的基础上，我将通过利用比较交叉- 物种方法首次定义支持MCT1/MCT4介导的有氧糖酵解的调节回路 OS，然后识别和验证与MCT控制细胞代谢相关的治疗漏洞。我 假设在OS细胞中，持续的MCT1/MCT4表达是由结构型STAT3/FOXM1驱动的 激活和增加MYC拷贝数，从而促进有氧糖酵解。我进一步预测， 通过基因操作或靶向抑制物失去MCT1/MCT4将损害体内肿瘤的生长， 而这可以通过合理的药物组合来最大化。要做到这一点，我将首先 用芯片测序相结合的方法研究MCT1/MCT4在OS中的转录调控， 亚硫酸氢盐测序和4C/CHIA-PET分析。对小鼠的异种移植研究将补充体外分析 评估MCT1/4功能缺失如何影响OS代谢谱、肿瘤表型和肿瘤生长。最后，我 将使用斑马鱼模型来筛选具有MCT阻断的合成致命性选定的制剂，然后验证发现 在老鼠身上。塔夫茨大学及其合作伙伴提供的丰富研究环境确保了资源的获取 以及完成拟议工作所需的专业知识。我在兽医肿瘤学、临床试验方面的培训 和遗传学，以及我的指导团队在基因组学、生物信息学、动物方面的专业知识 模型和翻译肿瘤学使我为成功过渡到独立做好了准备。

PROJECT SUMMARY/ABSTRACT Osteosarcoma (OS), the most common malignant bone tumor in humans and dogs, shares several features in both species including clinical presentation and molecular alterations. Despite numerous efforts there have been no improvements in disease outcome for either species in the past three decades: 30% of people and 90% of dogs still die of metastasis. While some approaches have shown promise in the setting of microscopic disease, in both species, macroscopic metastasis exhibits inherent resistance to multiple agents (kinase inhibitors, chemotherapy, immunotherapy, among others). I recently characterized the canine OS genome and found that as with human OS, the somatic mutational load is low, copy number aberrations/structural variants predominate, and no clear molecular drivers are evident. These data, along with a history of failed clinical trial efforts suggest that contemporary approaches to therapeutic advancement such as kinase inhibitors and immune checkpoint blockade will likely have limited clinical impact, necessitating the development of innovative therapeutic strategies. A distinguishing feature of cancer cells is their ability to undergo aerobic glycolysis, allowing them to thrive in a variety of microenvironments. Monocarboxylate transporters (MCTs) are key facilitators of this, moving lactic acid across the plasma membrane, and are critical for growth and metastasis of glycolytic tumors, such as OS. In previous work, I found that loss of MCT1 or MCT4 function in OS cells decreases basal and compensatory glycolysis, cellular proliferation and invasive capacity. I also showed that MCT4 is a direct transcriptional target of STAT3 and FOXM1, both of which exhibit constitutive activation in OS, supporting a link between MCT4/STAT3/FOXM1 and aerobic glycolysis. Building on these data, I will by leverage a comparative cross- species approach to first define regulatory circuits that support aerobic glycolysis mediated by MCT1/MCT4 in OS and then identify and validate therapeutic vulnerabilities related to MCT control of cellular metabolism. I hypothesize that in OS cells, sustained MCT1/MCT4 expression is driven by constitutive STAT3/FOXM1 activation and increased MYC copy number, thereby promoting aerobic glycolysis. I further predict that loss of MCT1/MCT4 through genetic manipulation or targeted inhibitors will impair tumor growth in vivo, and that this can be maximized through rational drug combination. To accomplish this, I will first characterize the transcriptional regulation of MCT1/MCT4 in OS using a combination of ChIP-sequencing, bisulfite sequencing and 4C/ChiA-PET analysis. Xenograft studies in mice will complement in vitro analyses to assess how loss of MCT1/4 function affects OS metabolic profile, tumor phenotype and tumor growth. Lastly, I will use a zebrafish model to screen select agents for synthetic lethality with MCT blockade, then validate findings in mice. The rich research environment afforded by Tufts University and its partners ensures access to resources and expertise necessary for completion of the proposed work. My training in veterinary oncology, clinical trials and genetics along with guidance from my mentoring team with expertise in genomics, bioinformatics, animal models and translational oncology make me well positioned for successful transition to independence.