A role for AKT2 in BRAFV600E melanoma initiation, progression, and response to therapy

项目摘要 尽管最近取得了进展，黑色素瘤仍然是最致命的皮肤癌形式， 迫切需要战略。MAP激酶（MAPK）和PI 3 K/AKT通路是这两种疾病的核心 黑色素瘤的进展和化疗耐药性，双靶向方法产生了有希望的结果， 将受益于增加的目标特异性和选择性。丝氨酸/苏氨酸激酶的AKT家族 包括三种高度同源且功能不同的同种型（AKT 1、AKT 2和AKT 3）， 在黑色素瘤中具有独特的作用，但尚未用于同种型特异性靶向。AKT 2是一个有吸引力的 考虑到已知AKT 2突变和AKT 2基因扩增， 对当前靶向治疗（如BRAF抑制剂（BRAFi））的适应性耐药机制 维罗非尼我们假设AKT 2参与黑色素瘤的侵袭和转移，并作为一种免疫调节剂。 BRAFi反应的中介。我们进一步建议，测试AKT 2同种型特异性靶向， 联合BRAF抑制剂是一种抗黑色素瘤策略，可避免严重的剂量限制性毒性 通常与泛AKT抑制相关。 这项工作将确定AKT 2对BRAF突变型黑色素瘤进展的贡献， 转移，使用人黑素瘤细胞系和小鼠模型。在目标1中，我们将研究以下因素的影响： AKT 2敲低对人黑色素瘤细胞迁移和侵袭的影响，以及这是否在存在AKT 2的情况下有所不同。 维罗非尼此外，我们将确定影响BRAFi反应的AKT 2特异性底物， 通过免疫沉淀与细胞中AKT磷酸底物特异性抗体反应的磷蛋白 用具有或不具有AKT 2特异性敲低的维罗非尼处理。免疫沉淀物将通过以下方法进行分析： 使用AKT 2野生型和敲低细胞进行优先级排序和验证，以确定 任何底物的操作改变了细胞对BRAF抑制的敏感性。在目标2中，我们将研究 在BRAF突变型黑色素瘤易感小鼠模型中基因AKT 2缺失的贡献，并进行转移 通过心内注射AKT 2无效细胞系的接种实验，以及同种异体移植中的药物研究 肿瘤的我们将研究用特异性抑制剂共靶向BRAF和AKT 2的效果，作为对 未来研究的原则。随着AKT抑制剂在黑色素瘤临床试验中的应用，我们的发现可能会产生新的 治疗策略，提高现有疗法的效率，以改善治疗方案， 这种毁灭性疾病的后果。

Project Summary Despite recent advances, melanoma remains the deadliest form of skin cancer, and new therapeutic strategies are urgently needed. The MAP kinase (MAPK) and PI3K/AKT pathways are central to both disease progression and chemoresistance in melanoma, with dual targeting approaches yielding promising results that would benefit from increased target specificity and selectivity. The AKT family of serine/threonine kinases comprises three highly homologous and functionally distinct isoforms (AKT1, AKT2, and AKT3) that play unique roles in melanoma but have not yet been leveraged for isoform-specific targeting. AKT2 is an attractive candidate for therapeutic intervention, given that AKT2 mutations and AKT2 gene amplification are known mechanisms of adaptive resistance to current targeted therapies such as the BRAF inhibitor (BRAFi) Vemurafenib. We hypothesize that AKT2 is involved in melanoma invasion and metastasis, and acts as a mediator of the BRAFi response. We further suggest that testing AKT2 isoform-specific targeting in combination with BRAF inhibitors is an anti-melanoma strategy that may avoid serious dose limiting toxicities commonly associated with pan-AKT inhibition. This work will establish the contribution of AKT2 to BRAF-mutant melanoma progression and metastasis, using both human melanoma cell lines and mouse models. In Aim1, we will investigate the effect of AKT2 knockdown on human melanoma cell migration and invasion, and whether this differs in the presence of the BRAFi Vemurafenib. Further, we will identify AKT2-specific substrates that influence the BRAFi response by immunoprecipitating phosphoproteins reacting with AKT phospho-substrate-specific antibodies in cells treated with Vemurafenib with or without AKT2-specific knockdown. Immunoprecipitates will be analyzed by mass spectrometry, prioritized and validated using AKT2 wild-type and knockdown cells to determine if manipulation of any substrate alters cell sensitivity to BRAF inhibition. In Aim 2, we will investigate the contribution of genetic AKT2 loss in a BRAF-mutant melanoma prone mouse model, and perform metastasis seeding experiments by intra-cardiac injection of AKT2 null cell lines, as well as drug studies in allograft tumors. We will investigate the effect of co-targeting BRAF and AKT2 with specific inhibitors, as a proof-of- principle for future studies. With AKT inhibitors in clinical trials for melanoma, our findings could yield novel therapeutic strategies and increase the efficiency of existing therapies to improve treatment options and outcome for this devastating disease.