Identifying and understanding drivers of chemoresistance in small cell lung cancer

摘要 小细胞肺癌(SCLC)是一种侵袭性和致死性的神经内分泌型肺癌。大多数患者最初 对化疗有反应，但在几个月内复发，以及导致化疗耐药的基因改变 人们对此了解甚少。除了MYC家族成员的扩增和SLFN11的表观遗传沉默之外， 菲尔德对促进小细胞肺癌耐药的基因了解极少。我们写了一本小说 我们从基因上改变小细胞肺癌高度化疗敏感性患者来源的异种移植(PDX)模型的系统 识别体内对顺铂/依托泊苷(CIS-ETO)耐药的干扰。慢病毒过表达 MYCN或MYCL的感染导致完全转为耐药(Grunblatt等人，2020年)。有系统地 确定SCLC耐药驱动因素，我们扩大了这种PDX慢病毒转导系统的使用，以执行 体内CRISPR失活筛选。我们发现了针对SAGA多个组件的sgRNAs(SPT- Ada-Gcn5乙酰转移酶)染色质修饰复合体作为屏幕点击并确认删除传奇 成员USP22，一个去泛素化酶，确实在两个SCLC PDX模型中赋予化疗耐药性，而返回 USP22到USP22缺失的SCLC PDX模型对化疗重新敏感。我们最重要的假设是 抑制USP22和SAGA复合体成员的表达可促进小细胞肺癌的化疗耐药 由SAGA抑制引起的转录变化是至关重要的。目标1，我们将询问基因是如何 在PDX模型中干扰多个佐贺复合体成员，包括USP22和TADA1，改变体内 对化疗的反应。Aim 2使用基因组和蛋白质组学方法开发深层分子 了解USP22调控的小细胞肺癌化疗反应相关基因和途径 并使用人类患者数据来确定功能研究的关键SAGA目标的优先顺序。几十年的学习 体外培养的小细胞肺癌细胞株的化疗反应几乎没有提供对化疗耐药性的了解 这表明，在组织培养条件下，这一过程的关键方面不能概括。我们的 新的系统优先使用体内方法进行化疗耐药性的研究，具有提供 帮助预防化疗耐药或使化疗耐药小细胞肺癌对化疗重新敏感的基础知识。

SUMMARY Small cell lung cancer (SCLC) is an aggressive and lethal neuroendocrine lung cancer type. Most patients initially respond to chemotherapy but relapse occurs within months and genetic alterations that drive chemoresistance are poorly understood. Beyond amplification of MYC family members and epigenetic silencing of SLFN11, the field has an extremely poor understanding of genes that promote SCLC chemoresistance. We developed a novel system in which we genetically alter highly chemosensitive patient derived xenograft (PDX) models of SCLC to identify perturbations that confer resistance to cisplatin/etoposide (CIS-ETO) in vivo. Lentiviral overexpression of either MYCN or MYCL caused complete switch to chemoresistance (Grunblatt et al, 2020). To systematically identify SCLC chemoresistance drivers, we expanded use of this PDX lentiviral transduction system to perform in vivo CRISPR inactivation screens. We identified sgRNAs targeting multiple components of the SAGA (Spt- Ada-Gcn5 acetyltransferase) chromatin modifying complex as screen hits and confirmed that deleting the SAGA member USP22, a deubiquitylase, indeed confers chemoresistance in two SCLC PDX models, while return of USP22 to a USP22-null SCLC PDX model re-sensitizes to chemotherapy. Our overarching hypothesis is that suppressing the expression of USP22 and SAGA complex members drives chemoresistance in SCLC, and that transcriptional changes caused by SAGA suppression are critical. Aim 1, we will interrogate how genetically perturbing multiple SAGA complex members, including USP22 and TADA1, in PDX models alters the in vivo response to chemotherapy. Aim 2 employs genomic and proteomic approaches to develop a deep molecular understanding of the USP22-regulated genes and pathways that contribute to chemotherapy response in SCLC and uses human patient data to prioritize key SAGA targets for functional study. Decades of studying chemotherapy response in SCLC cell lines grown in vitro have provided little insight into how chemoresistance emerges, suggesting that key aspects of this process are not recapitulated under tissue culture conditions. Our novel system prioritizes the study of chemoresistance using in vivo approaches with potential to provide foundational knowledge to help prevent chemoresistance or re-sensitize chemoresistant SCLC to chemotherapy.