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可溶性诱饵RTK亚型（sdRTK），产生的替代 剪接/内含子多聚腺苷酸化（IPA）机制，它可以作为异常RTK信号传导的有效天然抑制剂， 这是包括肺癌在内的大部分癌症的关键驱动因素。此外，我们开发了一种基于反义的方法， 以在体外和体内有效诱导抑制性可溶性诱饵RTK的表达。这些化合物代表了 一类新的药物，具有同时击倒病理目标的优势，同时引入 天然显性阴性同种型，从而大大增加功效。 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可溶性诱饵EGFR， EGFR依赖性肿瘤中的sdEGFR抑制性变体。sdEGFR变体，其通常在低水平表达， 水平，编码胞外配体结合结构域，但缺乏胞内激酶结构域。 我们假设sdEGFR诱导化合物通过三重机制抑制EGFR信号传导： 除去全长EGFR受体，B）螯合EGF和其它配体，和C）中和异源- 与残余受体（EGFR、HER 2/3等）的二聚化。我们进一步证实，随着所得产物的分泌， 它们同样影响靶细胞和旁观者细胞中的信号传导并干扰旁分泌和自分泌环， 从而发挥放大效应。由于其作用机制与目前的疗法不同， 在耐药机制仍然依赖于EGFR活性的所有难治性肿瘤中， 当这种活性由野生型EGFR扩增或EGFR激活突变驱动时。 为此，我们将努力实现以下具体目标： 1)天然抑制性sdEGFR剪接变体的表征。 2)靶向EGFR的剪接重定向化合物的优化。 3)治疗抗性肿瘤模型中内源性EGFR剪接和聚腺苷酸化的调节。 我们建议使用非小细胞肺癌（NSCLC）作为我们的初始癌症模型。NSCLC是最常见的原因 每年有超过130，000人死于癌症，其中四分之一与癌症相关，并依赖于癌症的异常。 EGFR信号传导。尽管第三代酪氨酸激酶抑制剂等靶向药物的初步疗效 奥希替尼对大多数活化EGFR的耐药性通常在2年内出现。因此， 治疗难治性EGFR依赖性癌症的另外的替代方法仍然是未满足的需要。 重要的是，虽然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 也可直接应用于EGFR发挥重要作用并经常突变或扩增的任何癌症， 如胶质母细胞瘤、结肠直肠癌等。此外，类似的化合物可以简单地针对其他化合物设计。 RTKs或其他负责各种耐药性机制和病理信号传导的靶标，从而增加了对耐药性的抑制作用。 一套强大的新工具来理解这种现象，并告知众多的治疗线索。

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.