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，很少有治疗方法能提供 总体存活率显著提高。然而，废除免疫学的免疫治疗策略 检查站或改善免疫监控已经显示出希望，特别是在黑色素瘤方面。 我们发现，HDAC6的基因去除和药物抑制都会导致 促肿瘤相关巨噬细胞的侵袭减少和下调 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，这至少部分依赖于完整的免疫，肿瘤的恢复证明了这一点 CD_4~+和CD_8~+耗尽后生长。我们的发现显示出显著的免疫调节作用。 HDAC6在黑色素瘤中的作用，为使用选择性HDAC6提高抗肿瘤免疫力提供了理论依据。 我们最感兴趣的是确定HDAC6是最能减少肿瘤前表型的 肿瘤相关巨噬细胞和减少免疫抑制表面分子的表达 作为PD-L1和PD-L2，其本身显示的细胞毒性很小。 因此，我们的目标是在体外和体内设计、合成和测试新的HDAC6i，用于 治疗黑色素瘤和其他恶性肿瘤。根据这项拨款，我们将会达到的目标如下： 1.使用分子建模，设计和合成每年约50个新的HDAC6，在前三年和 测试这些HDAC同工酶的选择性；拨款的最后两年将专注于化合物的放大， 临床前转化性研究和高级ADMET测试。 2.使用体外实验评估IC50值为50 nm且HDAC6选择性至少为400倍的化合物 测量乙酰化微管蛋白、pSTAT3、PD-L1水平和细胞毒性的黑色素瘤模型。对于化合物 发现降低pSTAT3和PD-L1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(每年2-3次)，如果需要，使用药物原理修改化合物 基于化学和分子模拟的方法来增加化合物的类药物特性。 3.最后，测试最好的化合物对同基因小鼠黑色素瘤肿瘤生长的延缓作用 模型，既是单独的，也是与PD-1阻断抗体联合使用的。此外，我们还将评估 我们最好的候选者使用人源化的PDX模型，并将其作为细胞治疗的前处理巨噬细胞以避免 他们转变为M2样癌前表型。那些在体内表现最好的候选人将被送往 用于晚期ADMET，包括对大鼠的慢性毒性研究、心脏活动和肺活动。 我们提出的工作意义重大，因为它有助于填补这一关键的知识缺口，从而推动表观遗传学-- 基础联合免疫疗法达到一个新的水平。要验证的概念是新的，因为它意义重大 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.