Modulating Cancer Stem Cell Signaling in Thoracic Malignancies

Division Of Basic Sciences - Nci

Investigators

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Abstract

Comprehensive efforts have been underway to explore the potential utility of induced pluripotent stem cells (iPSC) as models for characterizing epigenetic mediating stemness, treatment resistance and metastatic potential of thoracic malignancies, and identifying novel therapeutic targets in these neoplasms. Briefly, we reprogrammed normal human small airway epithelial cells (SAEC) to pluripotency using lentiviral transduction of Yamanaka factors. RNA-seq and DNA microarray experiments demonstrated that the Lu-iPSC transcriptome and DNA methylome signatures overlap much more with SCLC than 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 NFI family members as having the highest increase in digital footprinting and occupancy within open chromatin sites specifically in SCLC. Further studies demonstrated that NFIC is a novel mediator of metabolic reprogramming and growth of SCLC. A comprehensive manuscript pertaining to these studies has been submitted for peer review. Using our Lu-iPSC model system we have observed that 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 SCLC therapy. We have extended these published experiments and have observed that ASXL3 is tightly regulated in a cell cycle specific manner in normal cells and is a key mediator of replication timing and firing during early S phase, and that this regulation is highly perturbed by up-regulation of ASXL3 in the ASCL1 subtype of SCLC. After nearly three years of work, we have successfully cloned a full length flag-tagged ASXL3 locus into 293T cells, and are using pull-down assays to identify critical binding partners for ASXL3. We are waiting confirmation of successful knock-in of flag-tagged ASXL3 into SCLC to fully explore the mechanisms by which ASXL3 drives proliferation and stemness in these cells, and ascertain why the ASCL1 SCLC subtype is permissive for ASXL3 over-expression while all other cancer cells and all normal cells are not. Additional studies are in progress using conditional ASXL3 knock-out mouse fibroblasts and time course experiments to examine if ASXL3 expression is essential for establishing and/or maintaining pluripotency during reprogramming in normal cells. Such efforts could provide novel insights into the role of ASXL3 during the development of highly lethal SCLC and potential strategies to target stemness in these malignancies. 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 were reprogrammed to pluripotency using aforementioned techniques. ASXL3 was highly upregulated in the Eso-iPSC. We have compared transcriptomes and DNA methylomes of the Eso-iPSC with a panel of esophageal squamous cell cancer and adenocarcinoma lines as well as primary tumor specimens and several treatment-naive esophageal adenocarcinoma PDXs generated in our lab. This analysis identified approximately 8 novel genes that are related to stemness and appear to have prognostic significance in esophageal cancers. Using a novel PDX model we are not testing the impact of these genes in mediating metastasis of esophageal cancers. If successful, these efforts could lead to novel treatment strategies for these malignancies. 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. Predilections for organ specific metastases are associated with distinct transcriptome and DNA methylome signatures. We are now using this model to target specific epigenetic modulators for proof of concept experiments. A clone that metastasizes only to the lungs is being used for treatment experiments using aerosolized delivery of epigenetic agents. This clone as well as several others that exhibit broader metastatic potential are being used for preclinical immunotherapy experiments to establish proof of concept for the use of cancer stem cell vaccines as adjuvant therapy following resection of NSCLC. 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.