In addition to having broad applicability as cellular therapies in regenerative medicine, transplantation, and oncology, induced pluripotent stem cells (iPSC) may be useful models for studying a variety of benign and malignant diseases. Limited information is available pertaining to the potential utility of iPSC for investigating molecular mechanisms mediating initiation and progression of thoracic cancers and identifying novel therapeutic targets in these malignancies. To examine these issues, we recently generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) by lentiviral transduction of Yamanaka factors. The lung iPSC (Lu-iPSC) exhibited complex alterations in DNA methylation, with marked up-regulation of PRC-2-related genes, and modulation of 15,000 additional genes. Additional Sex Combs Like-3 (ASXL3), an epigenetic modifier not previously described in reprogrammed cells or any human malignancy, was up-regulated 400-fold in Lu-iPSC and was markedly over-expressed in SCLC lines and primary tumors. Knock-down of ASXL3 inhibited proliferation and teratoma formation by Lu-iPSC, and significantly diminished growth of SCLC cells in-vitro and in-vivo. These initial studies were published in Cancer Research. Ongoing studies are focused on the mechanisms and clinical implications of ASXL3 up-regulation in SCLC. These studies have progressed a bit slower than anticipated due to the number of alternative transcripts, difficulty in constitutively over-expressing ASXL3 and lack of high-titer ASXL3-specific antibodies. Several additional genes which were noted to be upregulated in Lu-iPSC and subsequently found to be elevated in primary lung cancers are under investigation at this time. To further examine the potential of Lu-iPSC to model lung cancer stem cells, RNA- seq experiments were used to examine gene expression profiles in 2 Lu-iPSC clones, 10 SCLC lines, and 10 NSCLC lines relative to SAEC. 6318, 12051, and 6504 genes were differentially expressed in Lu-iPSC, SCLC, and NSCLC lines, respectively. 2804 differentially expressed genes (44%) in Lu-iPSC were unique to these cells. Approximately 1/3rd of genes differentially expressed in Lu-iPSC were modulated in a histology specific manner. 1515 genes representing 24%, 12%, and 23% of differentially expressed genes in Lu-iPSC, SCLC, and NSCLC lines, respectively, were commonly regulated across Lu-iPSC, SCLC and NSCLC. Top canonical pathways included cell cycle control of chromosomal replication, mitotic roles of Polo-like kinases, and role of cytokines in mediating communication between immune cells. 3499 genes were commonly regulated in SCLC and NSCLC; metabolic pathways were enriched in this gene subset. 1937 genes were commonly modulated in SCLC and Lu-iPSC; top canonical pathways included GABA receptor signaling, TREM1 signaling, and IL-15 production. In contrast, only 102 genes were commonly modulated in Lu-iPSC and NSCLC; top canonical pathways included tight junction signaling, hepatic fibrosis/hepatic stellate cell activation, and PTEN signaling. Pluripotency pathways modulated in Lu-iPSC were not enriched in SCLC or NSCLC, possibly due to limited numbers of pluripotent cells in these lines. RNA-seq is being performed on side population (SP) fractions of SCLC and NSCLC lines to identify novel genes/pathways mediating stemness that could be potential targets for lung cancer therapy. Collectively, these findings suggest that Lu-iPSC models may prove useful for examining mechanisms of pulmonary carcinogenesis, and developing novel regimens targeting pluripotency for lung cancer therapy. In collaboration with Dr. Gordon Hager's team, we have recently performed DNAse hypersensitivity sequencing (DHS) experiments to examine epigenomic perturbations in lung cancer cells in a genome-wide manner. 14 SCLC, 10 NSCLC and Lu-iPSC were compared to SAEC. Re-analysis of RNA-seq data confirmed that the Lu-iPSC transcriptome overlaps much more with SCLC than NSCLC. Infinium array analysis also indicated that DNA methylation signatures in iPSC more closely aligned with SCLC compared to NSCLC. 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 LuiPS 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. Further analysis demonstrated that the vast 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), a computational approach that combines genomic footprinting and genome-wide chromatin accessibility, identified NF1 family members as having the highest increase in digital footprinting and occupancy within open chromatin sites specifically in SCLC. Interestingly, NF1 has been recently linked to metastatic progression in S a mouse model of SCLC. A comprehensive manuscript pertaining to these studies is in final stages of preparation for peer review. In related efforts, we have developed reliable methods for isolation and propagation of cancer cells which exhibit stem-like properties from primary lung cancers. These putative cancer stem cells (CSC)/cancer progenitor cells (CPC) grow as spheroids and exhibit high level expression of pluripotency-associated genes when cultured under non-adherent conditions, and can be reproducibly isolated from subsequently established adherent lung cancer lines. As such, our unique models now enable us to systematically dissect epigenomic mechanisms contributing to pluripotency in pulmonary carcinomas and to evaluate novel pharmacologic and immunologic regimens targeting CSC in these malignancies. These efforts, which are a major focus of our investigative work, will facilitate clinical development of novel therapies for lung cancers with potentially broad applicability for the treatment of other solid tumors.
