Targeting Refractory EGFR-Driven Tumors By Induction Of Dominant-Negative EGFR Splicing Variants

摘要 我们最近发现了一些新的分泌型可溶性诱骗RTK亚型(SdRTK)，它是由一种替代的 剪接/内含子多聚腺苷酸化(IPA)机制，可以作为异常RTK信号的有效天然抑制物，a 很大一部分癌症的关键驱动因素，包括肺癌。此外，我们还开发了一种基于反义的方法 目的：在体内外有效诱导抑制性可溶性诱骗RTKs的表达。这些化合物代表一种 一种新型药物，其优点是在引入的同时击倒病理靶点 自然显性-负性异构体，从而大大提高疗效。 我们建议应用这种基于反义的剪接重定向策略来诱导内源性的、可溶的诱骗EGFR EGFR依赖肿瘤中的抑制变异体(SdEGFR)。SdEGFR变异体，通常在低水平表达 水平，编码细胞外配体结合域，但缺乏细胞内激酶域。 我们假设sdEGFR诱导的化合物通过三种机制抑制EGFR信号转导：a) 去除全长EGFR受体，B)隔离EGF和其他配体，以及C)中和异源受体。 与残留受体(EGFR、HER2/3等)的二聚化。我们进一步假设，由于所产生的产品是秘密的， 它们影响靶细胞和旁观者细胞中的信号，并干扰旁分泌和自分泌环路， 从而产生放大的效果。由于其作用机制与目前的疗法不同，这一策略 对于耐药机制仍然依赖于EGFR活性的所有难治性肿瘤应该有效，两者 当这种活性是由野生型EGFR扩增或EGFR激活突变驱动时。 为此，我们将追求以下具体目标： 1)天然抑制sdEGFR剪接变异体的特征。 2)针对EGFR的剪接重定向化合物的优化。 3)耐药肿瘤模型中内源性EGFR剪接和多聚腺苷酸化的调控。 我们建议使用非小细胞肺癌(NSCLC)作为我们的初始癌症模型。非小细胞肺癌是最常见的原因 每年有超过13万人死于癌症，其中四分之一与异常有关并依赖于 EGFR信号转导。尽管靶向药物的初步疗效，如第三代酪氨酸激酶抑制剂 Osimertinib对大多数激活的EGFR的耐药性通常在2年内出现。因此，发展 此外，治疗难治性EGFR依赖型癌症的替代方法仍然是一个未得到满足的需求。 重要的是，尽管我们建议使用非小细胞肺癌作为体外和体内的主要模型系统，但我们的发现将 也直接适用于任何EGFR发挥重要作用并经常发生突变或扩增的癌症，如 如胶质母细胞瘤、结直肠癌等。此外，类似的化合物可以简单地针对其他化合物进行设计 RTK或其他负责各种耐药机制和病理信号转导的靶点，从而增加了一个 一套强大的新工具来理解这种现象，并为大量的治疗线索提供信息。

ABSTRACT We recently identified a number of novel secreted soluble decoy RTK isoforms (sdRTKs), generated by an alternative splicing/intronic polyadenylation (IPA) mechanism, which can act as potent natural inhibitors of aberrant RTK signaling, a key driving aspect of a large fraction of cancers, including lung cancer. Further, we developed an antisense-based method to effectively induce expression of the inhibitory soluble decoy RTKs in vitro and in vivo. These compounds represent a new class of drugs that carry the advantage of simultaneously knocking down the pathological targets while introducing natural dominant-negative isoforms, thus greatly increasing efficacy. We propose to apply this antisense-based splicing redirection strategy to induce endogenous, soluble decoy EGFR inhibitory variants (sdEGFR) in EGFR-dependent tumors. sdEGFR variants, which are normally expressed at low levels, encode the extracellular ligand-binding domain but lack the intracellular kinase domain. We hypothesize that sdEGFR-inducing compounds inhibit EGFR signaling by a three-fold mechanism: A) removal of full-length EGFR receptor, B) sequestration of EGF and other ligands, and C) neutralizing hetero- dimerization with residual receptors (EGFR, HER2/3, etc.). We further posit that, as the resulting products are secreted, they affect signaling in both targeted and bystander cells alike and interfere with paracrine and autocrine loops, thus exerting an amplified effect. Since its mechanism of action is different from that of current therapies, this strategy should be effective in all refractory tumors where the mechanisms of resistance still relies on EGFR activity, both when such activity is being driven by wild-type EGFR amplification or EGFR-activating mutations. To this end, we will pursue the following specific aims: 1) Characterization of natural inhibitory sdEGFR splicing variants. 2) Optimization of splicing redirection compounds targeted to EGFR. 3) Modulation of endogenous EGFR splicing and polyadenylation in treatment-resistant tumor models. We propose to use non-small cell lung cancer (NSCLC) as our initial cancer model. NSCLC is the most frequent cause of cancer fatalities, with over 130,000 deaths per year, a quarter of which are associated to, and depend on, aberrant EGFR signaling. Notwithstanding the initial efficacy of targeted drugs, such as third-generation tyrosine kinase inhibitor Osimertinib, against most activated EGFR, resistance typically emerges within 2 years. Thus, the development of additional, alternative approaches to treat refractory EGFR-dependent cancers remains an unfulfilled need. Importantly, whereas we propose to use NSCLC as our main model system both in vitro and in vivo, our findings will also directly apply to any cancer where EGFR plays an important role and is frequently mutated or amplified, such as glioblastoma, colorectal cancer and others. Furthermore, analogous compounds can be simply designed against other RTKs or other targets responsible for various mechanisms of drug-resistance and pathological signaling, thus adding a powerful new set of tools to understand such phenomena, and to inform a multitude of therapeutic leads.