Modulating Cancer Stem Cell Signaling in Thoracic Malignancies

关于诱导多能干细胞（iPSC）在研究介导胸部恶性肿瘤发生和进展的分子机制以及确定这些肿瘤的新治疗靶点方面的潜在效用，目前的信息有限。为了研究这个问题，我们使用慢病毒转导山中因子将正常的人小气道上皮细胞（SAEC）重新编程为多能性。肺iPSC （Lu-iPSC）在体外和体内都表现出多能性的特征，这与DNA甲基化的复杂改变、prc -2相关基因的显著上调以及15000个其他转录物的调节相吻合。为了探索Lu-iPSC模型的潜在临床相关性，我们比较了两种Lu-iPSC克隆、14种小细胞肺癌（SCLC）系和10种非小细胞肺癌（NSCLC）系与三种SAEC培养物的转录组特征。RNA-seq分析表明，Lu-iPSC转录组与SCLC的重叠比与NSCLC的重叠更多。Infinium array分析还表明，与NSCLC相比，Lu-iPSC中的DNA甲基化特征更接近于SCLC。然而，IPA并没有提供与SCLC和Lu-iPSC相关的特定致癌途径相关的有用信息。为了研究染色质景观对SCLC生物学的影响，我们将研究扩展到dna酶I超敏，然后进行深度测序（dna -seq），以确定相对于SAEC， Lu-iPSC和SCLC之间染色质配置状态所定义的调控元件。在Lu-iPSC或SCLC中发现超过20万个DNase 1超敏位点（DHS），而在SAEC中未发现。大约16%的DHS在Lu-iPSC和SCLC之间共享。大多数DHS是外部启动子，由内含子和基因间区包围，表明增强子是Lu-iPSC和SCLC基因组景观的主要贡献子。DHS和转录组数据的整合表明，不到5%的非启动子差异开放区域（DOR）映射到最近的邻居基因，表明基因受到远端调控元件的调控。DOR的一个子集是SCLC独有的。对所有样本的峰-基因链接和基因-峰链接的分析表明，95%的基因至少映射到一个开放的染色质区域，而每个峰平均映射到9个基因。许多预测的DOR-to-gene连接发生在簇中，其中多个附近的峰被预测连接到相同的基因，这表明这些簇作为调控单元或增强子的一部分起作用。基因组足迹（BaGFoot）的双变量分析发现NF1家族成员在开放染色质位点的数字足迹和占用率增加最高，特别是在SCLC中。与这些研究有关的一份全面的手稿正处于准备同行评议的最后阶段。额外的性梳样-3 （ASXL3）编码多梳去泛素酶复合体的一个组分，以前未被描述为在重编程细胞中上调，被确定为人类呼吸上皮多能性的重要介质和小细胞肺癌（SCLC）治疗的新表观遗传靶点。SCLC中ASXL3过表达与基因扩增相关。ASXL3基因的敲除减少了畸胎瘤的数量和大小，抑制了SCLC异种移植物的生长。这些研究结果发表在《癌症研究》杂志上。我们最近对5种不同组织来源的人类细胞系重编程获得的单细胞rna -seq数据进行分析，发现ASXL3上调与从正常细胞到完全重编程细胞的过渡状态一致。进一步的生物信息学分析表明，ASXL3仅在ASCL1 SCLC亚型中上调；敲除ASCL1或myc过表达，理论上会使SCLC从ASCL1亚型转变为NeuroD1或Yap亚型，但似乎不会影响SCLC中ASXL3的表达。目前，研究人员正在使用ASXL3基因的条件敲除以及在ASXL3编码区末端使用flag标记序列CRISPRed进行时间过程实验，以检查ASXL3基因的表达是否对正常细胞重编程过程中建立和/或维持多能性至关重要，阐明ASXL3基因在正常细胞和癌细胞中的表达调节机制，鉴定ASXL3结合伙伴。并明确ASXL3上调在SCLC中的分子和临床意义。进一步的生物信息学分析表明，ASXL3仅在ASCL1 SCLC亚型中上调；敲除ASCL1或myc过表达，理论上会使SCLC从ASCL1亚型转变为NeuroD1或Yap亚型，但似乎不会影响SCLC中ASXL3的表达。另外几个在Lu-iPSC中被发现以类似ASXL3的方式上调的基因，随后在SCLC中被发现上调，目前也在研究中。基于我们在肺癌建模方面的令人鼓舞的结果，我们已经扩展了我们的重编程工作，以表征和靶向食管癌细胞的表观基因组扰动。使用已建立的技术对正常食管鳞状上皮细胞（Heepic）和h-TERT永生化化巴雷特食管细胞（CP-A）进行重编程。主成分分析表明，Eso-iPSC完全重编程；而CP-A-iPSC则没有。ASXL3在CP-A-iPSC中的表达明显高于Eso-iPSC。与ASXL3促进干细胞的概念一致，Eso-iPSC在免疫缺陷小鼠中形成畸胎瘤，而CP-A-iPSC形成高级别癌。同样值得注意的是，尽管ASXL3在胸部肿瘤（SCLC除外）的2D培养中未被检测到，但当这些细胞过渡到非粘附条件时，ASXL3的表达上调。最后，ASXL3在干细胞样细胞中表达上调，我们现在可以从初级手术标本中分离出ASXL3，而不考虑组织组织学。在相关工作中，我们从Lewis肺癌细胞中分离出24个独立的干细胞样克隆，这些克隆稳定地转染了荧光素酶报告结构。这些克隆在体外表现出不同的干细胞和癌睾丸基因表达谱，以及独特的、高度可复制的器官特异性转移。ASXL3在许多克隆中表达非常高。因此，我们独特的模型现在使我们能够系统地剖析有助于胸部恶性肿瘤多能性和转移潜力的表观基因组机制，并评估针对这些肿瘤中CSC的新药理学和免疫方案。这些努力是我们目前研究工作的主要重点，将促进胸部癌症新疗法的临床开发，并可能广泛适用于治疗其他人类恶性肿瘤。

Limited information is available pertaining to the potential utility of induced pluripotent stem cells (iPSC) for investigating molecular mechanisms mediating initiation and progression of thoracic malignancies and identifying novel therapeutic targets in these neoplasms. To examine this issue, we reprogrammed normal human small airway epithelial cells (SAEC) to pluripotency using lentiviral transduction of Yamanaka factors. The lung iPSC (Lu-iPSC) exhibited hallmarks of pluripotency in-vitro and in-vivo which coincided with complex alterations in DNA methylation, marked up-regulation of PRC-2-related genes, and modulation of 15,000 other transcripts. To explore the potential clinical relevance of the Lu-iPSC model, we compared transcriptome signatures in two Lu-iPSC clones, 14 small cell lung cancer (SCLC) lines, and 10 non-small cell lung cancer (NSCLC) lines relative to three SAEC cultures. RNA-seq analysis demonstrated that the Lu-iPSC transcriptomes overlap much more with SCLC than NSCLC. Infinium array analysis also indicated that DNA methylation signatures in the Lu-iPSC more closely aligned with SCLC compared to NSCLC. However, IPA did not provide useful information related to specific oncogenic pathways related to SCLC vs Lu-iPSC. To investigate how chromatin landscape contributes to SCLC biology, we expanded our study to perform DNase I hypersensitivity followed by deep sequencing (DNase-seq) to identify regulatory elements defined by the state of chromatin configuration among Lu-iPSC and SCLC relative to SAEC. More than 200,000 DNase 1 Hypersensitivity Sites (DHS) were identified in Lu-iPSC or SCLC but not in SAEC. Approximately 16% of these DHS were shared between Lu-iPSC and SCLC. The majority of DHS were outside promoters, encompassed by introns and intergenic regions, indicating that enhancers were the major contributors of genomic landscape in Lu-iPSC and SCLC. Integration of DHS and transcriptome data indicated that less than 5% of non-promoter differentially open regions (DOR) mapped to the nearest neighbor gene, indicating gene regulation by distant regulatory elements. A subset of DOR was unique to SCLC. Analysis of peak-to-gene links and gene-to-peak links across all samples showed that 95% of genes mapped to at least one open chromatin region, whereas each peak mapped to a mean number of 9 genes. Many of the predicted DOR-to-gene links occurred in clusters where multiple nearby peaks are predicted to be linked to the same gene, suggesting that these clusters function as part of a regulatory unit or enhancer. Bivariate analysis of Genomic Footprint (BaGFoot) identified NF1 family members as having the highest increase in digital footprinting and occupancy within open chromatin sites specifically in SCLC. A comprehensive manuscript pertaining to these studies is in final stages of preparation for peer review. Additional Sex Combs Like-3 (ASXL3), encoding a component of the polycomb deubiquitinase complex not previously described to be up-regulated in reprogrammed cells was identified as an essential mediator of pluripotency in human respiratory epithelia and a novel epigenetic target for small cell lung cancer (SCLC) therapy. ASXL3 over-expression correlated with gene amplification in SCLC. Knock-down of ASXL3 decreased the number and size of teratomas and inhibited growth of SCLC xenografts. Results of these studies were published in Cancer Research. Our recent analysis of single cell RNAs-seq data obtained from reprogramming of 5 human cell lines of different tissue origins revealed that ASXL3 up-regulation coincides with a transition state from normal to fully reprogrammed cells. Additional bioinformatics analysis indicates that ASXL3 is only up-regulated in the ASCL1 SCLC subtype; knock-out of ASCL1 or over-expression of myc which theoretically would drive SCLC from ASCL1 to either NeuroD1 or Yap subtypes does not appear to impact ASXL3 expression in SCLC. Studies are in progress using conditional ASXL3 knock-out as well as time course experiments using a FLAG-tagged sequence CRISPRed into the end of the ASXL3 coding region to examine if ASXL3 expression is essential for establishing and/or maintaining pluripotency during reprogramming in normal cells, elucidate the mechanisms by which ASXL3 expression is regulated in normal cells and cancer cells, identify ASXL3 binding partners, and define molecular and clinical implications of ASXL3 up-regulation in SCLC. Additional bioinformatics analysis indicates that ASXL3 is only up-regulated in the ASCL1 SCLC subtype; knock-out of ASCL1 or over-expression of myc which theoretically would drive SCLC from ASCL1 to either NeuroD1 or Yap subtypes does not appear to impact ASXL3 expression in SCLC. Several additional genes which were noted to be upregulated in a manner similar to ASXL3 in Lu-iPSC and subsequently found to be elevated in SCLC are also under investigation at this time. Based on our encouraging results pertaining to modeling of lung cancers, we have extended our reprogramming efforts to characterize and target epigenomic perturbations in esophageal cancer cells. Normal esophageal squamous epithelial cells (Heepic) as well as h-TERT immortalized metaplastic Barrett's esophageal cells (CP-A) were reprogrammed using established techniques. PCA demonstrated that the Eso-iPSC were fully reprogrammed; however, CP-A-iPSC were not. ASXL3 expression was much higher in the CP-A-iPSC compared to Eso-iPSC. Consistent with the notion that ASXL3 facilitates stemness, Eso-iPSC form teratomas in immunodeficient mice whereas CP-A-iPSC form high grade carcinomas. Also of note, whereas ASXL3 is not detected in 2D cultures of thoracic cancers (except SCLC), ASXL3 expression is upregulated when these cells are transitioned to non-adherent conditions. Lastly, ASXL3 is up-regulated in stem-like cells that we can now reproducibly isolate from primary surgical specimens irrespective of tissue histology. In related efforts, we isolated 24 separate stem-like clones from Lewis lung cancer cells stably transfected with a luciferase reporter construct. These clones exhibit distinct stem cell and cancer-testis gene expression profiles in-vitro, and unique, highly reproducible organ-specific metastases. ASXL3 expression is very high in many of the clones. As such, our unique models now enable us to systematically dissect epigenomic mechanisms contributing to pluripotency and metastatic potential in thoracic malignancies and to evaluate novel pharmacologic and immunologic regimens targeting CSC in these neoplasms. These efforts, which are a major focus of our current investigative work, will facilitate clinical development of novel therapies for thoracic cancers with potentially broad applicability for the treatment of other human malignancies.