Identifying and understanding drivers of chemoresistance in small cell lung cancer

总结 小细胞肺癌（SCLC）是一种侵袭性和致死性的神经内分泌肺癌类型。大多数患者最初 对化疗有反应，但在几个月内复发，遗传改变导致化疗耐药性 我们对此知之甚少。除了MYC家族成员的扩增和SLFN 11的表观遗传沉默之外， field对促进小细胞肺癌耐药性的基因的理解非常贫乏。我们开发了一种新的 在该系统中，我们遗传改变SCLC的高度化学敏感性患者来源的异种移植物（PDX）模型， 鉴定在体内赋予顺铂/依托泊苷（CIS-ETO）抗性的扰动。慢病毒过表达 MYCN或MYCL引起完全转变为化疗耐药性（Grunblatt et al，2020）。系统地 为了鉴定SCLC化学抗性驱动因子，我们扩大了这种PDX慢病毒转导系统的使用， 体内CRISPR失活筛选。我们鉴定了靶向佐贺的多个组分的sgRNA（Spt. Ada-Gcn 5乙酰转移酶）染色质修饰复合物作为筛选命中物，并证实缺失佐贺 成员USP 22，一种去泛素化酶，确实在两种SCLC PDX模型中赋予化学抗性，而 USP 22至USP 22无效SCLC PDX模型对化疗重新敏感。我们的首要假设是 抑制USP 22和佐贺复合体成员的表达可驱动SCLC的化学抗性， 由佐贺抑制引起的转录变化是关键的。目标1，我们将询问基因如何 在PDX模型中干扰多个佐贺复合物成员，包括USP 22和TADA 1，改变了体内 对化疗的反应。目的2采用基因组学和蛋白质组学方法， 了解USP 22调节的基因和途径，有助于SCLC的化疗反应 并使用人类患者数据来优先考虑功能研究的关键佐贺靶标。几十年的研究 体外培养的SCLC细胞系的化疗反应几乎没有提供关于化疗耐药性如何 出现，这表明这一过程的关键方面没有重演组织培养条件下。我们 一种新的系统优先考虑使用体内方法研究化学抗性， 基础知识，以帮助预防化疗耐药性或重新敏感化疗耐药SCLC化疗。

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.