Role of ULK3 in Sensitive and Refractory Multiple Myeloma

总结 多发性骨髓瘤（MM）是一种无法治愈的致命性疾病。试剂如蛋白酶体抑制剂， 硼替佐米显著延长了总生存期，但耐药性可能迅速出现。为了产生治疗方法 可以提供持久的反应，特别是在难治性疾病的背景下， 需要驱动疾病的遗传学和治疗抗性的进化。为此，我们的团队 对来自疾病谱患者的CD 138 + MM细胞进行RNASeq分析（n=815） 并确定Unc-51样激酶-3（ULK 3）在新诊断和难治性MM中高度表达。 新兴的研究表明ULK 3在调节MM中的自噬中具有新的作用，这是维持MM的关键程序。 细胞生存的时间压力，并已牵连作为一个主要的机制，蛋白酶体抑制剂 阻力值得注意的是，已知MM高度依赖于自噬。目前，特异性ULK 3抑制剂是 缺乏作为克服对单一药物耐药性的策略，我们的团队专注于开发 新型抑制剂，如SG 3，靶向多种激酶，包括ULK 3（EC 50 90 nM）以及布罗莫结构域 蛋白4（BRD 4）（3）。BRD 4是MYC的已知驱动因素，对我们的患者RNASeq数据的分析再次揭示了 BRD 4抑制剂JQ 1有效地削弱了难治性MM中BRD 4的表达。 MM的潜力，但也注意到该试剂的耐药性。我们的代理SG 3抑制BRD 4活性， nM范围（27 nM）与JQ 1（20 nM）相似，并与我们ULK 3研究一致，SG 3处理迅速 抑制MM细胞中的自噬。在体内，我们发现SG 3显著抑制MM进展，并诱导骨形成， 疾病令人兴奋的是，我们的团队已经开发出第二代SG 3衍生产品MA 9， 在体外和体内与SG 3一样有效，但具有优越的上级稳定性。我们还发现MA 9可以使蛋白酶体再敏感 对硼替佐米具有抑制剂抗性的MM细胞。该提案的中心假设是ULK 3是一个关键监管机构 自噬在MM中的作用，代表了治疗难治性疾病的新靶点， 有三个目标。目标1将采取遗传方法，删除ULK 3，以发挥其在MM中调节自噬的作用 以及使用临床相关的动物模型在体内研究疾病的进展。目标二号将审问 我们的双重ULK 3/BRD 4化合物MA 9在体内MM进展和总生存期中的影响， JQ 1和自噬抑制剂氯喹（CQ）。目的3将检查MA 9用于治疗以下病症的功效： 使用开发的新型离体高通量平台从初治和难治性患者中分离的CD 138 + MM 在莫菲特在这里，我们还将使用免疫组织化学方法来确定ULK 3在介导蛋白酶体抑制剂抗性中的作用。 硼替佐米耐药MM细胞系。重要的是，在每个目标中，我们还将确定ULK 3在MM中的作用。 诱发骨质破坏-这是该疾病的临床重要方面。我们预计我们的预期结果将 为将我们的新型双重ULK 3/BRD 4抑制剂转化为临床提供了强有力的理由。

SUMMARY Multiple myeloma (MM) is an incurable and fatal disease. Reagents such as the proteasome inhibitor, bortezomib, have significantly extended overall survival but resistance can rapidly arise. To generate therapies that can provide durable responses, especially in the context of refractory disease, a deeper understanding of the genetics driving the disease and the evolution of therapy resistance is required. To this end, our team has performed RNASeq analysis of CD138+ MM cells derived from patients across the disease spectrum (n=815) and identified that Unc-51 Like Kinase-3 (ULK3) is highly expressed in newly diagnosed and refractory MM. Our emerging studies demonstrate a novel role for ULK3 in regulating autophagy in MM, a key program that sustains cell survival under times of stress and has been implicated as a major mechanism of proteasome inhibitor resistance. Of note, MM is known to be highly dependent on autophagy. Currently, specific ULK3 inhibitors are lacking. As a strategy to overcome resistance to single agents, our team has focused on the development of novel inhibitors such as SG3, that target multiple kinases including ULK3 (EC50 90nM) as well bromodomain protein 4 (BRD4)(3). BRD4 is a known driver of MYC and analysis of our patient RNASeq data again reveals increased BRD4 expression in refractory MM. The BRD4 inhibitor, JQ1, effectively impairs the tumorigenic potential of MM but resistance has also been noted with this reagent. Our agent SG3 inhibits BRD4 activity in the nM range (27 nM) similar to that of JQ1 (20 nM), and consistent with our ULK3 studies, SG3 treatment rapidly inhibits autophagy in MM cells. In vivo, we found SG3 significantly inhibits MM progression and induced bone disease. Excitingly, our team has already developed a 2nd generation SG3 derivative coined MA9 that is just as effective as SG3 in vitro and in vivo but has superior stability. We also show that MA9 can resensitize proteosome inhibitor resistant MM cells to bortezomib. The central hypothesis of this proposal is that ULK3 is a key regulator of autophagy in MM and represents a novel target for treatment of refractory disease and it will be tested with three Aims. Aim 1 will take a genetic approach and delete ULK3 to its role in regulating autophagy in MM as well in the progression of the disease in vivo using clinically relevant animal models. Aim 2 will interrogate the impact of our dual ULK3/BRD4 compound, MA9, in MM progression and overall survival in vivo compared to JQ1 and the autophagy inhibitor, chloroquine (CQ). Aim 3 will examine the efficacy of MA9 for the treatment of CD138+ MM isolated from naïve and refractory patients using a novel ex vivo high throughput platform developed at Moffitt. Here, we will also define the contribution of ULK3 in mediating proteasome inhibitor resistance using bortezomib resistant MM cell lines. Importantly, in each Aim, we will also determine the role of ULK3 in MM induced bone destruction - a clinically significant aspect of this disease. We expect our anticipated results will provide strong rationale for the translation of our novel dual ULK3/BRD4 inhibitors to the clinic.