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，并且有新的治疗方法 战略是迫切需要的。MAP激酶(MAPK)和PI3K/AKT通路是这两种疾病的中心 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 将受益于更高的目标特异性和选择性。AKT丝氨酸/苏氨酸激酶家族 由三种高度同源且功能截然不同的异构体(AKT1、AKT2和AKT3)组成 在黑色素瘤中的独特作用，但还没有被利用来进行异构体特异性靶向。Akt2是一款有吸引力的 由于AKT2突变和AKT2基因扩增已知，因此是治疗干预的候选对象 适应性耐受当前靶向治疗的机制，如BRAF抑制剂(BRAFi) 维莫拉非尼。我们假设AKT2参与了黑色素瘤的侵袭和转移，并作为一种 布拉菲反应的调解人。我们进一步建议，测试AKT2亚型特异性靶向 与BRAF抑制剂联合使用是一种可避免严重剂量限制毒性的抗黑色素瘤策略 通常与PAN-AKT抑制有关。 这项工作将确定AKT2在BRAF突变型黑色素瘤进展和 转移，使用人类黑色素瘤细胞系和小鼠模型。在Aim1中，我们将调查 Akt2基因敲除对人黑色素瘤细胞迁移和侵袭的影响 布拉菲·维穆拉菲尼。此外，我们将确定影响BRAFi反应的AKT2特定底物 通过免疫沉淀在细胞中与AKT磷酸底物特异性抗体反应的磷蛋白 用Vemurafenib治疗，有或没有AKT2特异性基因敲除。免疫沉淀物将通过 质谱仪，使用AKT2野生型和敲除细胞进行优先排序和验证，以确定 任何底物的操作都会改变细胞对BRAF抑制的敏感性。在目标2中，我们将调查 AKT2基因缺失在BRAF突变黑色素瘤易感小鼠模型中的作用，并进行转移 心脏内注射AKT2零细胞系的种子实验以及同种异体移植的药物研究 肿瘤。我们将研究联合靶向BRAF和AKT2与特定抑制剂的效果，作为- 未来研究的原则。将AKT抑制剂用于黑色素瘤的临床试验，我们的发现可能会产生新的结果 治疗策略和提高现有疗法的效率，以改善治疗选择和 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.