Targeting BMP signaling to treat advanced melanoma and suppress therapeutic resistance

项目摘要 黑色素瘤是美国皮肤癌死亡的主要原因，5年生存率为20% 对于晚期疾病患者1，2.尽管在治疗方面有所改进，但许多患者的生存率很低 受益，并往往产生耐药性的标准治疗3 -8。此外，存在患者群体 没有合适的突变或肿瘤特征，不适合接受新的靶向治疗， 免疫疗法为了给产生耐药性或耐药的患者提供适当的治疗选择， 如果这些患者不适合当前的尖端疗法，则必须确定新的治疗靶点。我们的实验室发现 一种新的黑色素瘤癌基因，生长分化因子6（GDF 6），一种分泌的骨形态发生蛋白 (BMP)通过调节特定神经嵴因子的表达促进黑色素瘤的配体，其具有 防止分化和抑制分化的双重作用9.除了这些具体因素，我们发现 GDF 6更广泛地促进模仿胚胎神经嵴的基因表达特征。神经 先前已将嵴同一性确定为涉及黑色素瘤发生、进展和 治疗耐药性5，6，10，11.我们的研究表明，GDF 6的敲低抑制了许多这些基因的表达。 神经嵴基因总之，这些数据表明GDF 6是黑色素瘤治疗的最佳靶标。作为 分泌的细胞外蛋白GDF 6易于被抗体靶向。我们生产了一组单克隆抗体， 靶向GDF 6的C-末端结合区的抗体，并开发了多种体外和体内测定， 评估对GDF 6具有最有效作用的候选抗体，并评估其有效性， 潜在的黑色素瘤治疗剂。我假设一个抗体子集将有效地阻断GDF 6活性， 导致体外和体内黑素瘤细胞的分化和细胞死亡增加。我进一步假设， 阻断GDF 6将抑制黑色素瘤细胞中的神经嵴特性，导致侵袭性较低的肿瘤细胞 特征和使先前耐药的细胞对标准护理疗法敏感。我将评估一个预- 筛选了一组42种单克隆抗体在黑素瘤细胞中对GDF 6的体外和体内活性， 确定具有最有效活性的最佳候选物，同时表征药代动力学和动力学 抗体在体内的特性。我将进一步表征这些抗体对GDF 6抑制的作用 神经嵴表达谱和晚期黑色素瘤的关键特征，如治疗抗性， 入侵性和锚定独立生长。此外，我将分析潜在的组合疗法， 以评估已知治疗抗性机制的途径活性的变化。结果 本研究的第一部分将确定用于首次人体（FIH）研究的主要候选抗GDF 6抗体，并提供 用于提交FIH申请的适当临床前安全性数据。此外，这些数据将提供 广泛了解与神经嵴身份有关的致瘤特征和逆转 神经嵴的特征。

PROJECT SUMMARY Melanoma is the leading cause of skin cancer death in the United States, with the 5-year survival rate of 20% for patients with advanced disease1,2. Despite improvements in therapy, many patients receive minimal survival benefit and often develop resistance to standard-of-care therapies3–8. Furthermore, a population of patients exist who do not have the appropriate mutations or tumor characteristics to be eligible for new targeted and immunotherapies. In order to provide adequate treatment options for patients who develop resistance or are ineligible for current cutting-edge therapies, new therapeutic targets must be identified. Our lab has discovered a novel melanoma oncogene, growth differentiation factor 6 (GDF6), a secreted bone morphogenetic protein (BMP) ligand that promotes melanoma by regulating expression of specific neural crest factors, which has the dual effect of preventing differentiation and suppressing apoptosis9. In addition to these specific factors, we found GDF6 more broadly promotes a gene expression signature that mimics that of the embryonic neural crest. Neural crest identity has previously been identified as a key feature involved in melanoma initiation, progression, and therapeutic resistance5,6,10,11. Our studies show knockdown of GDF6 suppresses expression of many of these neural crest genes. Taken together, these data indicate GDF6 is an optimal target for melanoma therapy. As a secreted extracellular protein, GDF6 is amenable to targeting by antibodies. We produced a panel of monoclonal antibodies to target the C-terminal binding region of GDF6 and developed multiple in vitro and in vivo assays to assess candidate antibodies with the most potent action against GDF6 and evaluate their effectiveness as potential melanoma therapeutics. I hypothesize that a subset of antibodies will effectively block GDF6 activity leading to increase differentiation and cell death of melanoma cells in vitro and in vivo. I further hypothesize that blocking GDF6 will suppress neural crest identity in melanoma cells, leading to less aggressive tumor cell characteristics and sensitizing previously resistant cells to standard-of-care therapy. I will evaluate a pre- screened panel of 42 monoclonal antibodies for in vitro and in vivo activity against GDF6 in melanoma cells to identify top candidates with the most potent activity, in parallel with characterizing pharmacokinetic and dynamic properties of the antibodies in vivo. I will further characterize the effects of GDF6 inhibition by these antibodies on neural crest expression profiles and key features of advanced melanoma such as therapeutic resistance, invasiveness, and anchorage independent growth. Additionally, I will analyze potential combinatorial therapies in vitro and in vivo to assess changes in pathway activity for known therapeutic resistance mechanisms. Results of this study will identify lead candidate anti-GDF6 antibodies for first-in-human (FIH) studies and provide appropriate pre-clinical safety data for submission of an FIH application. Furthermore, these data will provide broad insight into the tumorigenic features that are connected to neural crest identity and the result of reversing neural crest characteristics in established melanomas.