To further examine epigenetic mechanisms regulating stemness in lung cancers and identify novel therapeutic targets in these neoplasms, we recently generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) by lentiviral transduction of OSKM (Yamanaka) factors. SAEC were derived from a 57 year old Hispanic female non-smoker. Numerous lung iPSC (Lu-iPSC) clones were generated, several of which were randomly selected for further analysis. These Lu-iPSC demonstrated hallmarks of pluripotency including morphology, cytoplasmic alkaline phosphatase expression, cell surface expression of SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, up-regulation of stem cell genes including Nanog, Oct4, and SALL4, with decreased expression of cytokeratins, as well as in-vitro proliferation and teratoma formation. Spectral karyotyping and qRT-PCR analyses demonstrated no chromosomal aberrations and complete silencing of reprogramming transgenes in Lu-iPSCs. Chromatin immunoprecipitation (ChIP)-seq, RNA-seq, qRT-PCR, and immunoblot experiments demonstrated complex alterations in DNA methylation with a shift toward a more hypermethylated state; this unexpected finding was also observed following analysis of A549 and Calu-6 lung cancer cells as well as publically available data sets for other iPSC as well as embryonic stem cells (ESC). RNAseq analysis demonstrated that 15,000 genes were commonly altered Lu-iPSC relative to SAEC. A marked up-regulation of PRC-2-related genes was observed. Notably, ASXL3, an epigenetic modifier not previously described in reprogrammed cells or thoracic malignancies, was up-regulated 400-fold in Lu-iPSC. No significant up-regulation of ASXL1 or ASXL2 was observed in Lu-iPSC relative to SAEC. Subsequent qRT-PCR and immunoblot experiments demonstrated over-expression of ASXL3 in SCLC lines relative to NSCLC lines or normal or immortalized respiratory epithelial cells. ASXL3 expression was particularly high in a subset of SCLC lines and correlated with increased gene copy number. Immunohistochemistry experiments confirmed significant up-regulation of ASXL3 in primary SCLC. Knock-down of ASXL3 inhibited proliferation and teratoma formation by Lu-iPSC, and significantly diminished growth of SCLC cells in-vitro and in-vivo. Collectively these experiments are the first description of reprogramming of human airway epithelial cells, and the first demonstration that up-regulation of ASXL3 contributes to human cancer. A comprehensive manuscript pertaining to these findings has been tentatively accepted for publication in Cancer Research pending minor revisions. Our findings highlight the potential utility of this Lu-iPSC model for elucidation of epigenomic mechanisms of pulmonary carcinogenesis and identification of other novel epigenetic targets for lung cancer therapy. In additional studies, microarray analysis of cultured lung cancer cells and xenografts demonstrated that mithramycin decreased expression of musashi-2 (Msi-2), a novel RNA binding protein which mediates self-renewal in normal stem cells and aggressive phenotype of several human cancers. qRT-PCR and immunoblot experiments confirmed that mithramycin depletes Msi-2 in lung cancer cells in a time and dose-dependent manner. Expression levels of Msi-2 were significantly elevated in non-small cell as well as small-cell lung cancer lines relative to normal/immortalized human respiratory epithelial cells (p 0.001). Consistent with these findings, Msi-2 mRNA levels in primary lung cancers were significantly higher than those detected in adjacent paired normal lung parenchyma (p 0.0003). Msi-2 expression was enriched in side populations of cultured lung cancer cells, and was significantly increased in Lu-iPSC. si-RNA-mediated knock-down of Msi-2 decreased expression of Oct4, Nanog and Myc, and transiently inhibited proliferation of lung cancer cells. Attempts to permanently knockdown Msi-2 by shRNA techniques were unsuccessful, suggesting strong selection pressure to maintain Msi-2 expression in these cells. Experiments are underway using tet-inducible lentiviral vectors targeting MSI-2 to definitively characterize mechanisms and effects of MSI-2 depletion in lung cancer cells in-vitro and in-vivo. Collectively, these data suggest that pharmacologic depletion of MSI-2 may be a novel strategy for lung cancer therapy. Results of these studies were presented at a Keystone Epigenetics conference in January 2017, and a manuscript pertaining to these studies will be submitted for publication once the knock-down and gene expression profiling experiments have been completed.