Reprogramming myogenic regulatory factors in RMS to promote differentiation and halt growth

总结 横纹肌肉瘤（RMS）占所有儿科癌症的3-4%，总5年生存率不到30 诊断为转移性RMS的儿童的发病率。肉瘤患者的发病率也较高， 死亡率高于其他癌症患者，这在儿童中尤为明显。由于他们的治疗，42% 的儿童癌症幸存者经历严重、致残或危及生命的疾病，包括继发性 肿瘤的因此，显然需要为儿科肿瘤开发新的，更有针对性的治疗策略 例如RMS;抑制肿瘤进展但产生有限副作用的治疗。在许多癌症中， 胚胎程序，包括获得干细胞/祖细胞状态，被制定为有助于肿瘤的发生。 进展这种恢复或保留发展计划可能是一个关键的驱动因素， 胚胎性横纹肌肉瘤（ERMS，通常为融合阴性，也称为FN-RMS）。 在RMS中，观察到肌源性谱系转录因子（TF）MYOD 1和MYOG的高表达。 然而，尽管这些TF的高表达，RMS细胞不能分化。相反，这些TF驱动RMS 恶性表型，因为这些MRF的敲除导致RMS细胞的致死性。因此，一个未回答的关键 该领域的一个问题是为什么以及如何生肌调节因子（MRF）偏离了它们的典型作用， 肌肉分化的驱动因素，而不是维持RMS细胞在未分化状态。在本提案中，我们 正在研究SIX 1，一种调节肌肉发育的关键TF，是否负责全球改变 MRF的功能。我们的数据表明SIX 1表达的升高促进FN-RMS的进展和生长 通过促进RMS祖细胞样状态。有趣的是，SIX 1敲低诱导肌肉分化， 标记，伴随着关键MRF基因组范围的重新定位。这些数据表明，在FN-RMS中，SIX 1 过表达改变MRF功能，促进RMS祖细胞样表型并增强肿瘤生长 和进步。因此，在本提案中，我们将测试以下假设：SIX 1及其专性辅因子 EYA 2/3通过改变基因组格局，协同驱动FN-RMS的进展，导致MRF 以利于生长而不是分化。由于SIX 1在分化组织中的表达较低，因此靶向它可以 因此是一种抑制FN-RMS的手段，对未转化的细胞具有有限的副作用，其中SIX 1是 - 是的具体目的如下：1）确定SIX 1作为一种蛋白质的分子机制， 肌肉祖细胞与发育和RMS中分化状态的主要调节因子。2)识别 关键的SIX 1辅因子（重点是EYA蛋白），当靶向时，可以诱导分化并抑制 RMS增长。这项工作将利用正常的发育调节机制来抑制肿瘤， 并且由于SIX/EYA在成体组织中表达的缺乏，因此可能具有有限的毒性。我们将联合收割机 斑马鱼和人类模型将最大限度地发挥每个模型系统的优势， 确定抑制RMS生长和进展的方法。了解RMS细胞的机制 因此，被困在早期发育状态可能会导致针对该疾病的新方法。

Summary Rhabdomyosarcoma (RMS) accounts for 3-4% of all pediatric cancers, with less than a 30% overall 5-year survival rate for children diagnosed with metastatic RMS. Sarcoma patients also experience higher rates of morbidity and mortality than other cancer patients, and this is particularly evident in children. As a result of their therapies, 42% of childhood cancer survivors experience severe, disabling, or life threatening conditions, including secondary tumors. Thus, there is clearly a need to develop new, more targeted treatment strategies for pediatric tumors such as RMS; treatments that inhibit tumor progression yet confer limited side effects. In many cancers, embryonic programs, including the acquisition of stem/progenitor states, are instituted to contribute to tumor progression. Such reinstatement or retention of developmental programs may be a key driving factor in Embryonal Rhabdomyosarcoma (ERMS, which are generally fusion negative and also referred to as FN-RMS). In RMS, high expression of myogenic-lineage transcription factors (TF), MYOD1 and MYOG, is observed. However, despite high expression of these TFs, RMS cells fail to differentiate. Instead, these TFs drive the RMS malignant phenotype, since knockout of these MRFs results in lethality to the RMS cell. Thus, a key unanswered question in the field is why and how the myogenic regulatory factors (MRFs) depart from their canonical roles as drivers of muscle differentiation to instead maintain RMS cells in an undifferentiated state. In this proposal, we are examining whether SIX1, a critical TF that regulates muscle development, is responsible for globally altering the function of MRFs. Our data show that elevated SIX1 expression promotes FN-RMS progression and growth by promoting an RMS progenitor-like state. Intriguingly, SIX1 knockdown induces a muscle differentiation signature, concomitant with re-localization of key MRFs genome-wide. These data suggest that in FN-RMS, SIX1 overexpression alters MRF function, promoting an RMS progenitor-like phenotype and enhancing tumor growth and progression. Thus, in this proposal we will test the following hypothesis: SIX1 and its obligate cofactors EYA2/3 cooperatively drive the progression of FN-RMS by altering the genomic landscape, causing MRFs to favor growth over differentiation. Since SIX1 expression in differentiated tissues is low, targeting it may therefore be a means to inhibit FN-RMS with limited side effects to untransformed cells where SIX1 is dispensable. Specific aims are as follows: 1) To determine the molecular mechanism by which SIX1 serves as a master regulator of the muscle progenitor vs differentiated state in development and in RMS. 2) To identify the critical SIX1 cofactors (with a focus on EYA proteins) that, when targeted, can induce differentiation and inhibit RMS growth. This work will take advantage of normal developmental regulatory mechanisms to inhibit the tumor, and thus may have limited toxicity due to the paucity of SIX/EYA expression in adult tissue. Our ability to combine zebrafish and human models will maximize the benefits of each model system to rapidly and inexpensively identify means to inhibit RMS growth and progression. Understanding the mechanisms by which RMS cells are trapped in an early developmental state may therefore lead to novel means to target the disease.