Investigation of sub-lineages in pulmonary neuroendocrine cells and identification of the cells of origin of small cell lung cancer

项目总结 小细胞肺癌(SCLC)由于其频率、致命性和致命性，仍然是公共卫生中的一大挑战。 缺乏方便的模型来探讨其发病机制和潜在的治疗策略。肺 神经内分泌细胞(PNECs)被认为是小细胞肺癌的前体。然而，越来越多的证据表明， PNEC包含位置、细胞大小和生理功能不同的亚系。 在我之前的研究中，我开发了一种新的实验方法来研究PNECs的生物学-并且 小细胞肺癌的启动--通过将人类胚胎干细胞(HESCs)分化为肺系，并随后 扰乱在小细胞肺癌中经常改变的三个肿瘤抑制基因。通过干扰缺口信号，肺 前体细胞可以分化为PNECs，进一步进行致癌转化，形成小细胞肺癌样瘤 在小鼠中，当Rb和P53表达减少时。单细胞RNA(ScRNA)图谱显示出 人和小鼠肺内hESC来源的PNECs和天然PNECs。特别重要的是，scRNA分析 进一步揭示了hESC来源的PNEC的亚系。其中，有一份档案显示出显著的相似性。 到早期人类小细胞肺癌肿瘤和小细胞肺癌细胞系的RNA图谱。上述发现和最近的研究由 其他的，使我进一步假设PNEC亚系具有不同的致癌潜力，其中，一个 特定人群是小细胞肺癌的主要起源细胞。 我建议将该模型与其他方法一起使用，如scRNA转录、基因工程 小鼠模型，以检验这一假说，并从几个方面研究小细胞肺癌的起源。首先，我将确定PNEC的分支- 正常人和小鼠肺组织中的谱系与hESC来源的PNEC中的谱系相似。或者， 小鼠肺中PNECs的亚系将通过scRNA图谱和新的细胞命运标记来表征 将外推小鼠PNEC亚系，以进一步描述人类PNEC中的异质性群体。 接下来，我将纯化hESC来源的PNEC亚群，并测试它们在培养和 小细胞肺癌中已知致癌事件导致的免疫受损小鼠。或者，小鼠肺中的PNEC亚系可以 使用有条件的Rb1/Trp53基因敲除小鼠来测试它们形成肿瘤的潜力。通过这些研究，我希望 为了确定对SCLC突变最敏感并能够转化的特定PNEC亚系， 表明它们是小细胞肺癌的起源细胞。 在R00阶段，我建议扩大研究范围，探索PNEC血统结构的驱动机制 以及它们的不同致癌潜能。这些措施包括研究PNEC亚族之间的分化， Notch、SOX2等单通路对PNEC命运决定的影响及CRISPR筛查探讨 维持PNEC层级模式及其特异性致癌调控的新分子事件 容量。这些研究不仅有望促进我们对PNECs致癌机制的理解，而且还将为我们提供 为诊断、分类、治疗甚至可能更有效地预防这种疾病提供了新的机会。

PROJECT SUMMARY Small cell lung cancer (SCLC) remains a major challenge in public health because of its frequency, its lethality, and the paucity of convenient models for exploring its pathogenesis and potential therapeutic strategies. Pulmonary neuroendocrine cells (PNECs) are believed to be the putative precursor of SCLC. However, increasing evidence shows that PNECs contain sub-lineages varying in location, cell size, and physiological functions. In my previous research, I developed a novel experimental approach for studying the biology of PNECs - and the initiation of SCLC - by differentiating human embryonic stem cells (hESCs) into the lung lineage, and subsequently perturbing three tumor suppressor genes that are frequently altered in SCLC. By perturbing NOTCH signaling, the lung progenitor cells can be differentiated into PNECs that further undergo oncogenic transformation and form SCLC-like tumors in mice, when RB and P53 expression are reduced. Single cell RNA (scRNA) profiles demonstrated great similarity between the hESC-derived PNECs and the native PNECs in human and mouse lung. Of particular significance, scRNA analysis further revealed sub-lineages within the hESC-derived PNECs. Among them, one profile demonstrated significant similarity to the RNA profiles of early stage human SCLC tumors and SCLC cell lines. The above findings and recent studies by others, led me to further hypothesize that the PNEC sub-lineages have different oncogenic potential, and among them, one specific population serves as the dominant cell of origin of SCLC. I propose to use this model together with other methods such as scRNA transcriptomics, genetically engineering mouse models, to test this hypothesis and to study the origins of SCLC in several ways. First, I will identify the PNEC sub- lineages in normal human and mouse lung tissues that are similar to the ones in the hESC-derived PNECs. Alternatively, sub-lineages of PNECs in mouse lung will be characterized by scRNA profiling and new cell-fate markers identified from the mouse PNEC sub-lineages will be extrapolated to further delineate the heterogeneous populations in human PNECs. Next, I will purify the sub-populations of hESC-derived PNECs and test their transformative potential in culture and in immunocompromised mice by known oncogenic events in SCLC. Alternatively, the PNEC sub-lineages in mouse lung can be tested for their potential to form tumors by using conditional Rb1/Trp53 knockout mice. Through these studies, I expect to identify a specific sub-lineage of PNECs that are most sensitive to SCLC mutations and capable of transformation, which would implicate them as the cell of origin in SCLC. In the R00 phase, I propose to expand the research to explore mechanisms driving the lineage hierarchies of PNECs and their variant oncogenic potentials. These include studying inter-differentiation among the PNEC sub-lineages, the effects of NOTCH, SOX2 and other single pathways on PNEC fate determination, and using CRISPR screening to explore new molecular events important in maintenance of PNEC hierarchical patterns and the regulation of their specific oncogenic capacity. These studies are expected to not only advance our understanding of carcinogenesis of PNECs, but also provide new opportunities to diagnose, categorize, treat, and possibly even prevent this disease more effectively.