Vulnerability of SCLC based on bi-allelic genetic inactivation of RB1 and TP53

目前迫切需要找到治疗小细胞肺癌（SCLC）的新的有效药物， 其特征在于侵袭性生长、早期转移和小于2%的5年存活率。虽然 最初化疗敏感，对顺铂加依托泊苷一线治疗的反应率为70%，大多数患者 耐药肿瘤初始治疗后复发。数十种新药已被测试的活动， SCLC在过去的几十年里，包括超过40个药物，已在III期试验失败。没有一个目标 用于非小细胞肺癌或其他实体瘤的药物对SCLC有效。 SCLC的一个突出特征是RB 1和TP 53的几乎一致的双等位基因遗传失活。 然而，由于基因失活的RB 1和TP 53不能被重新激活， 尽管将它们重新引入SCLC细胞，但这种定义性的遗传特征并没有导致SCLC的治疗策略。在 在这次拨款中，我们将测试抑制Rb 1下游靶点可以重建肿瘤的假设 抑制和促凋亡作用，当Rb 1失活时失去。 在初步研究中，我们发现Rb 1的下游靶点E3泛素的调节 连接酶SCFSkp 2/Cks 1（Skp 2），可以显著阻断Rb 1丢失的促肿瘤发生后果， 诱导小细胞肺癌中Rb 1-Skp 2合成致死性凋亡。这是由于Rb 1和Skp 2在细胞中的作用。 调节p27（CDKN 1B），从而Rb 1的缺失导致Skp 2介导的泛素化， p27的降解和随后的细胞周期调控的丧失。最重要的是，我们已经确定了活跃的， 小分子Skp 2抑制剂专门针对这种脆弱性。 具体目的是：1）确定Cks 1-Skp 2相互作用在Rb 1和p53缺失中的作用- 诱发SCLC。如果发现这种作用是必要的，我们将确定另一个靶点来抑制Skp 2介导的 p27泛素化。由于一些具有p53错义突变的人SCLC可能获得了新的致癌基因， 除了经典p53功能的丧失外，我们还将确定这些p53 GOF突变是否 影响对Skp 2抑制的敏感性。2)为了确定Skp 2失活的影响， 药理学方法。四种不同的小分子Skp 2抑制剂，具有不同的分子靶点， 用于几种新的体外和体内模型。特别关注SCLC肝转移，以及 化疗初治和化疗耐药SCLC细胞之间的比较。3)为了确定抗肿瘤 Skp 2抑制剂在使用大量SCLC PDX的更具临床相关性的小鼠肿瘤模型中的作用 （患者来源的异种移植物）。将在来自未经化疗的PDX和来自未接受化疗的PDX之间进行比较。 化疗耐药SCLC肿瘤，包括来自同一患者的系列标本的“同基因”PDX 化疗前和化疗后。完成后，本提案中的研究将使大多数SCLC受益 因为RB 1和TP 53的双等位基因失活在SCLC中几乎是一致的。

There is an urgent need to identify new and effective drugs for small cell lung cancer (SCLC), a tumor characterized by aggressive growth, early metastases, and a 5-year survival rate of less than 2%. While it is initially chemosensitive, with 70% response rates to first-line therapy of cisplatin plus etoposide, most patients relapse after initial therapy with drug-resistant tumors. Dozens of new drugs have been tested for activity in SCLC over the decades, including more than 40 agents that have failed in phase III trials. None of the targeted drugs used in non-small cell lung cancer or other solid tumors are effective in SCLC. An outstanding feature of SCLC is the near uniform bi-allelic genetic inactivation of RB1 and TP53. However, since genetically inactivated RB1 and TP53 cannot be reactivated nor is it clinically feasible to reintroduce them into SCLC cells, this defining genetic feature has not led to treatment strategies for SCLC. In this grant, we will test the hypothesis that inhibiting a downstream target of Rb1 can re-establish the tumor suppressor and pro-apoptotic actions that were lost when Rb1 was inactivated. In preliminary studies, we found that modulation of only one downstream target of Rb1, the E3 ubiquitin ligase SCFSkp2/Cks1 (Skp2), can dramatically block the pro-tumorigenic consequences of the loss of Rb1, and induce Rb1-Skp2 synthetic lethal apoptosis in SCLC. This is due to the role Rb1 and Skp2 play in the regulation of p27 (CDKN1B), whereby the loss of Rb1 leads to the Skp2-mediated ubiquitination and degradation of p27 and the subsequent loss of cell cycle regulation. Most critically, we have identified active, small molecule Skp2 inhibitors to specifically target this vulnerability. The specific aims are: 1) To determine the role of the Cks1-Skp2 interaction in Rb1 and p53 deletion- induced SCLC. If the role is found to be essential, we will have identified another target to inhibit Skp2-medited p27 ubiquitination. Since some human SCLCs with p53 missense mutations may have gained new oncogenic functions (GOF), in addition to the loss of classic p53 functions, we will determine if these p53 GOF mutations affects the sensitivity to Skp2 inhibition. 2) To determine effects of Skp2 inactivation using genetic and pharmacologic approaches. Four different small molecule Skp2 inhibitors, with distinct molecular targets, will be used in several novel in vitro and in vivo models. Particular focus will be on SCLC liver metastasis, and on comparisons between chemotherapy-naïve and chemo-resistant SCLC cells. 3) To determine the antitumor effects of Skp2 inhibitors in a more clinically relevant mouse tumor model using a large panel of SCLC PDXs (patient-derived xenografts). Comparisons will be made between PDXs derived from chemotherapy-naïve and chemo-resistant SCLC tumors, including “isogenic” PDXs derived from serial specimens from the same patient pre- and post-chemotherapy. When completed, research in this proposal will potentially benefit most SCLC patients since bi-allelic inactivation of RB1 and TP53 is nearly uniform in SCLC.