Development of selective HDAC6 inhibitors to improve cancer immunotherapy

项目摘要 转移性黑色素瘤的中位生存期约为8-16个月，很少有治疗能提供有效的治疗。 总体生存率显著改善。然而，消除免疫原性的免疫策略， 检查点或改善免疫监视已显示出希望，特别是在黑色素瘤中。 我们已经发现HDAC 6的遗传废除和药理学抑制都导致HDAC 6基因的缺失。 减少促肿瘤肿瘤相关巨噬细胞的浸润和下调 免疫抑制介质。这些效应转化为体内黑色素瘤肿瘤的显著延迟 生长，这是，至少部分地，依赖于完整的免疫，如证明了肿瘤的恢复 CD 4+和CD 8+耗竭后的生长。我们的研究结果表明， HDAC 6在黑色素瘤中的作用，为使用选择性HDAC 6 is改善抗肿瘤免疫提供了理论基础。 我们最感兴趣的是鉴定能够最好地减少HDAC 6的促肿瘤表型的HDAC 6。 肿瘤相关的巨噬细胞，并减少免疫抑制表面分子的表达， 与PD-L1和PD-L2一样，但其本身几乎没有细胞毒性。 因此，我们的目标是在体外和体内设计、合成和测试新的HDAC 6 i，以用于 黑色素瘤和其他恶性肿瘤的治疗。该补助金拟实现的目标如下： 1.使用分子建模，设计和合成每年约50个新的HDAC 6，前三年， 测试这些HDAC同工酶的选择性;最后两年的赠款将集中在化合物的规模扩大， 临床前转化研究和先进的ADMET测试。 2.使用体外试验评估IC 50值<50 nM且HDAC 6选择性至少为400倍的化合物 黑色素瘤模型，以测量乙酰化微管蛋白、pSTAT 3、PD-L1水平和细胞毒性。化合物的 发现降低pSTAT 3和PD-L1的水平，同时增加乙酰化微管蛋白，进行早期 使用CRO进行ADMET研究（2 - 3/年），如果需要，使用药物原则修改化合物 基于化学和分子建模的方法来增加化合物的药物样特征。 3.最后，测试最佳化合物在同基因小鼠黑色素瘤中延迟肿瘤生长的能力 模型，作为独立的和与PD-1阻断抗体组合。此外，我们将评估 我们最好的候选人使用人源化PDX模型和作为细胞疗法预处理巨噬细胞，以避免 它们转变为M2样的前肿瘤表型。那些表现出最佳体内疗效的候选药物将被发送到 用于晚期ADMET，包括大鼠慢性毒性研究、心脏活动和肺活动。 我们提出的工作是重要的，因为它有助于填补这一关键的知识空白，从而推动表观遗传- 将免疫疗法提升到一个新的水平。待验证的概念是新颖的，因为它显著地 偏离了HDAC抑制剂作为抗癌剂的经典细胞毒性作用。

Project Summary The median survival for metastatic melanoma is approximately 8–16 months, and few therapies offer a significant improvement in overall survival. However, immunotherapeutic strategies that abrogate immunologic checkpoints or improve immunosurveillance have shown promise, especially in melanoma. We have found that both genetic abrogation and pharmacologic inhibition of HDAC6 leads to a decreased infiltration of pro-tumoral tumor-associated macrophages and downregulation of immunosuppressive mediators. These effects translated into a pronounced delay of in vivo melanoma tumor growth, which is, at least in part, dependent on intact immunity, as evidenced by the restoration of tumor growth after CD4+ and CD8+ depletion. Our findings demonstrate a significant immunoregulatory role of HDAC6 in melanoma, providing a rationale for the use of selective HDAC6is to improve antitumor immunity. We are most interested in identifying HDAC6is that are best able to reduce the pro-tumoral phenotype of tumor-associated macrophages and decrease the expression of immunosuppressive surface molecules such as PD-L1 and PD-L2 while showing little cytotoxicity on their own. Our goal is thus to design, synthesize, and test new HDAC6i both in vitro and in vivo for use in the treatment of melanoma and other malignancies. The aims to be accomplished under this grant are as follows: 1. Using molecular modeling, design, and synthesize ~50 new HDAC6is per year, for the first three years and test these for HDAC isozyme selectivity; the last two years of the grant will focus on compound scale-up, preclinical translational studies, and advanced ADMET testing. 2. Evaluate compounds that have IC50 values of <50nM and at least 400-fold HDAC6 selectivity using in vitro melanoma models to measure acetylated tubulin, pSTAT3, PD-L1 levels, and cytotoxicity. For compounds found to decrease levels of pSTAT3 and PD-L1, while increasing acetylated tubulin, conduct early-stage ADMET studies (2 – 3/year) using a CRO, and if needed, modify the compounds using principles of medicinal chemistry and molecular modeling based methods to increase the compound’s drug-like character. 3. Lastly, test the best compounds for their ability to delay tumor growth in syngeneic murine melanoma models, both as stand-alone and in combination with PD-1 blocking antibodies. Additionally, we will evaluate our best candidates using humanized PDX models and as a cell therapy pre-treating macrophages to avoid their switch to M2-like protumoral phenotype. Those candidates showing the best in vivo efficacy will be sent for advanced ADMET, including chronic toxicity studies, cardiac activity, and pulmonary activity in rats. Our proposed work is significant as it helps to fill this critical knowledge gap and thus moves epigenetic- based combination immunotherapies to a new level. The concept to be validated is novel as it significantly deviates from the classical cytotoxic role of HDAC inhibitors as anti-cancer agents.