Understanding tumor addiction to TRK fusions and sensitivity to TRK inhibition

NTRK融合导致癌基因嵌合TRK蛋白与成分激活的激酶的表达， 类似于ALK和ROS1融合。最重要的是，NTRK融合在临床上具有很高的可操作性。首先- 生成TRK激酶抑制(Larotrectinib)可导致快速和持久的组织学不可知性反应， 在各种实体肿瘤中的客观有效率为76%(Drilon等人，《新英格兰医学杂志》，暂定 接受)。尽管有这些令人印象深刻的结果，但人们对NTRK融合和耐药性的生物学知之甚少 到第一代疗法最终发展起来。虽然第二代抑制剂解决了靶向耐药性，但 对这些药物产生获得性耐药性同样是一项挑战。 这一建议的主要目的是阐明介导激活和 TRK融合蛋白的转化能力，并鉴定固有或获得性耐药的机制 到TRK抑制剂。对于这个项目，我们计划利用(1)我们在正在进行的TRK抑制剂临床试验中的领导地位， (2)我们先前在识别和描述靶向治疗耐药机制方面的经验，以及(3) 我们创建了一个由科学家、病理学家和临床医生组成的多学科“TRK团队”来研究TRK生物学。 为了阐明介导TRK融合激酶的信号通路和/或基因表达模式 我们将使用患者衍生的模型进行无偏倚的全球蛋白质组/转录组筛选 使用第一代或第二代TRK抑制剂治疗。TRK介导的候选蛋白质/通路 肿瘤的发生将从遗传学和药理学两个方面进行验证。 确定对第一代(larotrectinib和entrectinib)和第二代的抗性机制 (Loxo-195和TPX-0005)抑制剂在临床上，我们将对配对进行全面的表征 在我们机构治疗的NTRK重排实体瘤患者的治疗前后的活检。 肿瘤的特征将是基于靶向捕获的外显子组测序(MSK-IMPACT)，锚定 多重聚合酶链式反应(MSK Archer Solid Tumor Panel)和PAN-TRK IHC；此外，系列血浆分析(DdPCR) 和杂交捕获)将在接受TRK抑制剂治疗的患者中进行。建立在我们先前确定的 会聚的、靶标上的抗性(溶剂前沿突变)，我们鉴定了由 MAPK通路重新激活(NRAS/BRAF/GNAS突变)，可能适用于联合治疗。 对于第二代抑制剂的耐药性，我们已经鉴定出一种新的复合NTRK突变。 这些努力的补充将是常规创建NTRK重组患者来源的细胞系和 异种移植和工程模型(转导原代细胞系/CRISPR修饰)。除了探索 下游信号转导如前所述，治疗策略将在体外和体内探索 靶上阻力(第二代TKI切换)和靶外阻力(联合治疗，例如 TrK和MEK抑制)。如果可行，随后将在临床上探索候选策略。

NTRK fusions lead to the expression of oncogenic chimeric TRK proteins with constitutively activated kinases, similar to ALK and ROS1 fusions. Most importantly, NTRK fusions are highly actionable in the clinic. First- generation TRK kinase inhibition (larotrectinib) results in rapid and durable histology-agnostic responses, with an objective response rate of 76% across a wide variety of solid tumors (Drilon et al., NEJM, provisionally accepted). Despite these impressive results, little is known about the biology of NTRK fusions, and resistance to 1st-generation therapy ultimately develops. While 2nd-generation inhibitors address on-target resistance, the development of acquired resistance to these drugs likewise represents a challenge. The main objectives of this proposal are to elucidate signaling pathways that mediate the activation and transformative capacity of TRK fusion proteins, and to identify mechanisms of intrinsic or acquired resistance to TRK inhibitors. For this project, we plan to leverage (1) our leadership in ongoing TRK inhibitor clinical trials, (2) our prior experience in identifying and characterizing resistance mechanisms to targeted therapy, and (3) our creation of a multidisciplinary “TRK team” of scientists, pathologists, and clinicians to study TRK biology. In order to shed light on signaling pathways and/or gene expression patterns that mediate TRK fusion kinase activity, we will perform an unbiased global proteomic/transcriptomic screening using patient-derived models treated with 1st- or 2nd-generation TRK inhibitors. Candidate proteins/pathways involved in TRK-mediated tumorigenesis will be validated both genetically and pharmacologically. To determine mechanisms of resistance to 1st-generation (larotrectinib and entrectinib) and 2nd-generation (LOXO-195 and TPX-0005) inhibitors in the clinic, we will perform comprehensive characterization of paired pre- and post-treatment biopsies from patients with NTRK-rearranged solid tumors treated at our institution. Tumors will be characterized by targeted capture-based exome sequencing (MSK-IMPACT), anchored multiplex PCR (MSK Archer Solid Tumor Panel), and pan-TRK IHC; in addition, serial plasma profiling (ddPCR and hybrid capture) will be performed in patients on TRK inhibitor therapy. Building on our prior identification of convergent, on-target resistance (solvent-front mutations), we identified off-target resistance mediated by MAPK pathway reactivation (NRAS/BRAF/GNAS mutations) that may be amenable to combination therapy. For 2nd-generation inhibitor resistance, we have already identified a novel compound NTRK mutation. These efforts will be complemented by the routine creation of NTRK-rearranged patient-derived cell lines and xenografts, and engineered models (transduced primary cell lines/CRISPR-modified). In addition to exploring downstream signaling as previously described, therapeutic strategies will be explored in vitro and in vivo for both on-target resistance (2nd-generation TKI switching), and off-target resistance (combination therapy, e.g. TRK and MEK inhibition). When feasible, candidate strategies will then be explored in the clinic.