Understanding tumor addiction to TRK fusions and sensitivity to TRK inhibition

NTRK融合导致具有组成型激活激酶的致癌嵌合TRK蛋白的表达， 类似于ALK和ROS 1融合。最重要的是，NTRK融合在临床上是高度可行的。首先- 产生TRK激酶抑制（larotrectinib）导致快速和持久的组织学不可知的反应， 在多种实体瘤中的客观反应率为76%（Drilon等，NEJM，暂时 接受）。尽管有这些令人印象深刻的结果，但对NTRK融合的生物学和耐药性知之甚少。 到第一代疗法的发展。虽然第二代抑制剂解决了靶向耐药性， 对这些药物的获得性耐药性的发展也是一个挑战。 该提案的主要目标是阐明介导激活的信号通路， TRK融合蛋白的转化能力，并鉴定内在或获得性抗性的机制 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)我们先前在识别和表征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， 活动，我们将使用患者来源的模型进行无偏倚的全球蛋白质组学/转录组学筛选 用第一代或第二代TRK抑制剂治疗。参与TRK介导的细胞凋亡的候选蛋白/途径 肿瘤发生将在遗传学和生物学上得到验证。 确定对第1代（larotrectinib和entrectinib）和第2代的耐药机制 （LOXO-195和TPX-0005）抑制剂在临床上的应用，我们将对配对的 来自在我们机构治疗的NTRK重排实体瘤患者的治疗前和治疗后活检。 肿瘤将通过基于靶向捕获的外显子组测序（MSK-IMPACT）、锚定的外显子组测序（MSK-IMPACT）和测序来表征。 多重PCR（MSK Archer实体瘤组）和泛TRK IHC;此外，系列血浆分析（ddPCR 和杂合捕获）将在接受TRK抑制剂治疗的患者中进行。根据我们先前确定的 收敛性、靶向耐药（溶剂前沿突变），我们确定了由 MAPK通路再激活（NRAS/BRAF/GNAS突变），可能适合联合治疗。 对于第二代抑制剂耐药性，我们已经确定了一种新的化合物NTRK突变。 这些努力将通过常规创建NTRK重排的患者来源的细胞系来补充， 异种移植物和工程化模型（转导的原代细胞系/CRISPR修饰的）。除了探索 如前所述，将在体外和体内探索治疗策略， 靶向耐药（第二代TKI转换）和脱靶耐药（联合治疗，例如， TRK和MEK抑制）。在可行的情况下，候选策略将在诊所进行探索。

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.