Bungum, Aaron O.

Abstract RATIONALE: Alveolar Type 2 (AT2) cells play a critical role in maintaining alveolar function and facilitating repair processes in the lung. Despite recent advancement on transcriptomic mapping of cell identities and AT fate selection, the mechanisms governing AT2 fate plasticity remain elusive. Notably, in AT2-derived pulmonary diseases such as COPD and IPF, disruptions in AT2 differentiation are observed, suggesting alterations may exist in the regulation of fate plasticity. Therefore, a refined model describing the regulation of AT2 fate plasticity is needed, along with an assessment of the model's conservation across murine and human AT2s. METHODS: We performed scRNA-sequencing, ATAC-seq, and bioinformatic analysis on samples across murine lung development and validating findings by immunostaining of embryonic lungs. We conducted organotypic cultures as well as genetically engineered mouse model experiments to assess changes in AT2 fate plasticity in. Further, we established a Mayo Clinic patient lung cell biorepository from patient tissue derived AT2s. In parallel, we performed long read scRNA-sequencing for isoform discovery, organotypic culture and immunostaining in human AT2 organoids, to characterize the presence of similar regulatory mechanisms. RESULTS: Throughout the late embryonic and early perinatal period, we found a window of fate plasticity exists in AT2s whereby they retain the capacity to give rise to AT1s, and that its being closed over time by the bZIP transcription factor C/EBPα, as its genetic deletion in AT2s results in regaining fate plasticity. By ATAC-seq and ChIP-seq, we found C/EBPα binds closely to the Dlk1 locus, a Notch signaling regulator implicated in AT2-to-AT1 conversion that is upregulated following Cebpa deletion. Following adult lung injury, we found C/EBPα downregulation is required for re-accessing AT2 fate plasticity and is likely mediated by the dominant negative C/EBP family member CHOP. Finally, we observe Cebpa loss also activates a “defender” AT2 state enriched in interferon stimulated antipathogenic gene expression that is distinct from its reparative state and propose AT2s can toggle between these states following infection to robustly protect and repair alveoli.Further, we validated the presence of C/EBPα and absence of Dlk1 in human AT2s isolated from healthy adult patients, which persists following in vitro expansion. We conclude that AT2 cells window of fate plasticity is dictated by a molecular circuit involving C/EBPα suppressing and Dlk1. Future works aim to remove C/EBPα in human AT2s to reopen their fate plasticity for cell therapy.

Abstract RATIONALE: Alveoli form late in development and, for first breath, alveolar type 1 (AT1) as well as AT2 epithelial cell types must be functional. While prior studies have mapped the cellular and transcriptional programs underlying this process and identified AT fate inductive cues, questions remain as to the timing and regulation of their emergence. Thus, we sought to identify when, where, and how AT2 cells first arise and motivated by recent work in embryonic stem cells, we investigated whether the biophysical parameter cell membrane tension (CMT) plays a role. METHODS: Single-cell RNA-sequencing (scRNAseq) datasets sampling mouse lung development were processed using cluster correlation, velocity, and gene ontology analysis. Marker expression and AT2 morphology was assessed by immunostaining, and their migration confirmed by time-lapse confocal microscopy on embryonic precision-cut lung slices (PCLS). CMT was measured via optical tweezers (OT) on isolated cells as well as fluorescence lifetime (FLIM) on organoids and PCLS. Progenitors (E15.5) and AT2s (E16.5- >PN50) were isolated from distal region of the mouse lung, as well as human AT2s recently isolated, expanded, and banked in Mayo Clinic's recently established patient lung cell biorepository. CMT was raised using methyl-β-cyclodextran (MBCD) or polyethylene glycol (PEG). Small molecules CHIR and ICG-001 were used in culture to raise and lower β-catenin levels, respectively. Fluorescently labeled mesenchymal cells were isolated, expanded, and added to organoids in suspension for co-association experiments. RESULTS: No evidence of distal progenitors selecting either AT fate was found until E16.5, at which time we identified a distinct nascent AT2 (nAT2) state that ontology analysis detects upregulation of single-cell migration genes. Resistin-like molecule alpha (RELM-α), a type 2 inflammation regulator, labels nAT2s – which first individually emerged around E16.5 (although instances exist at E15.5) at intermediate zones between the branch tips and stalks of the distal epithelium. Following molecular emergence, we observed nAT2s extruded basally and can subsequently interact with nearby but anatomically distinct lumens, a behavior we provisionally name interlumenal junctioning and whose dynamics we validated by timelapse microscopy. Regarding CMT, we found its reduction in distal progenitors occurs prior to, and is required for, differentiation. We observed CMT reduction is sensitive to the manner of organoid culture, with on top culture resulting in more reduction and differentiation. Finally, we observed CMT is regulated cell-intrinsically via the adaptor protein β-catenin, as well as -extrinsically through osmotic (via PEG) or mesenchymal cell interactions, both of which reduce membrane fluidity.

Abstract Rationale Global Lung Function Initiative (GLI) Global spirometry reference equations were recently derived to offer a “race-neutral” interpretation option. The impact of transitioning from the race-specific GLI-2012 to the GLI Global reference equations is unknown. Objectives Describe the direction and magnitude of changes in predicted lung function measurements in a population of diverse race and ethnicity using GLI Global in place of GLI-2012 reference equations. Methods In this multicenter cross-sectional study using a large pulmonary function laboratory database, 109,447 spirometry tests were reanalyzed using GLI Global reference equations and compared with the existing GLI-2012 standard, stratified by self-reported race and ethnicity. Measurements and Main Results Mean FEV1 and FVC percent predicted increased in the White and Northeast Asian groups and decreased in the Black, Southeast Asian, and mixed/other race groups. The prevalence of obstruction increased by 9.7% in the White group, and prevalences of possible restriction increased by 51.1% and 37.1% in the Black and Southeast Asian groups, respectively. Using GLI Global in a population with equal representation of all five race and ethnicity groups altered the interpretation category for 10.2% of spirometry tests. Subjects who self-identified as Black were the only group with a relative increase in the frequency of abnormal spirometry test results (32.9%). Conclusions The use of GLI Global reference equations will significantly impact spirometry interpretation. Although GLI Global offers an innovative approach to transition from race-specific reference equations, it is important to recognize the continued need to place these data within an appropriate clinical context.

Abstract Background: Non-small cell lung cancer (NSCLC) tissue is a valuable resource for diagnosis, treatment planning, and drug development. Current 2D histopathological techniques introduce under-sampling error (i.e., a single 5 um section represents 0.5% of a 1 mm thick biopsy), interobserver variability, and fail to capture the biology contained within the entire tissue sample. We have developed a suite of technologies to stain, chemically clarify, image, visualize, and analyze entire intact NSCLC tissue samples. Methods: Human NSCLC tissue, stored frozen in OCT, was fixed in 4% paraformaldehyde, stained with nuclear (TOPRO-3) and general protein (Eosin) fluorescent dyes, and optically cleared using a modified iDISCO protocol with ethyl cinnamate as the refractive index matching solution. Whole, intact tissue samples, roughly 1-5mm3 in volume, were imaged at 2 microns/pixel resolution with an open-top light-sheet microscope (3Di, Alpenglow Biosciences). Smaller regions of interest (ROIs) with key pathologic features were reimaged at higher resolution, 0.17 microns/pixel, to reveal subnuclear features and for cell typing. Visualization was performed using Aivia software. Results: NSCLC tissue samples were successfully imaged in 3D. Low resolution images (2 microns/pixel) were obtained within 4-31 minutes, depending on the tissue volume. The 3D distribution of cancer cells, immune cells, vessels, and fibrosis varied substantially throughout the volume of the tissue. Recognizable histologic features, including nests of tumor cells surrounded by vasculature and immune cells, were readily visualized. Squamous and adenocarcinoma with its subtypes (solid, acinar, lepidic, and micropapillary) morphologies were recognizable in 2D optical sections of the 3D datasets. Imaging quality degraded in tissue deeper than 1 mm due to light scattering. Conclusion: We assessed intact NSCLC tissue samples measuring up to 5 mm3 using our custom light-sheet microscope and tissue clearing techniques. This novel method enables us to visualize key features of NSCLC such as the tumor interfaces, tertiary lymphoid structures, vessels and fibrosis in the entire tissue sample, preventing under sampling error, and potentially enabling new biologic insights. Next steps include segmentation and quantification of key tissue structures such as tumor volume, immune cell distribution, and fibrosis/immune cell exclusion. This proof-of-concept study provides motivation for further investigation into the significance of 3D tissue features in NSCLC tissue samples. Citation Format: Alexandra Alvarsson, Carl Storey, Brandy Olin Pope, Caleb Stoltzfus, Robert Vierkant, Jessica Tufariello, Aaron Bungum, Julia Naso, Cheuk Ki Chan, Eric Edell, Christopher Hartley, Janani Reisenauer, Nicholas Reder. 3D assessment of the lung cancer microenvironment using multi-resolution open-top light-sheet microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6624.

Abstract Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Consistent with this carcinogenic exposure, cytogenetic analyses have identified multiple recurrent structural chromosomal abnormalities in this malignancy, but more recent high-throughput sequencing evaluations of point mutations suggest that there is a low mutational burden in mesothelioma. Since tumor mutational burden has been correlated with responses to treatment with immune checkpoint inhibitors such as nivolumab, it was not consistent that patients with mesothelioma and low mutation burdens would have similar response rates in clinical trials with immune checkpoint inhibitors as patients with non-small cell lung cancer which is associated with a high mutation burden. In order to reconcile these differences, and given the potential for an improved understanding of the molecular pathogenesis of mesothelioma to improve therapeutic options, we used mate-pair sequencing (MPseq) and RNA sequencing (RNAseq) to understand how structural variants affect the transcriptome. MPseq differs from standard next generation sequencing approaches by tiling the whole genome with larger fragments (2-5kb) to reliably detect structural variants such as insertions, deletions and rearrangements. Amongst 22 mesothelioma specimens there were 1535 chromosomal rearrangements (median 41, range 3-298 per specimen), that resulted in junctions or novel fusions of non-coding DNA or genes. Six-hundred thirty-seven of these rearrangements (median 22, range 5-103 range per specimen) resulted in novel fusions of genes. Many of these inter- or intra-chromosomal rearrangements were consistent with a pattern of chromoanagesis such as chromoplexy or chromothripsis. Chromosomal rearrangements detected by MPseq were used to guide analysis of RNAseq data and revealed that these chromosomal junctions resulted in the expression of 179 novel amino acid sequences (median 5, 0-51 range per specimen). To determine whether transcription of chromosomal rearrangement-related junctions have neoantigenic potential, we used in silico tools to determine whether any of the expressed junctions contained peptides that could be presented by patient-specific HLA molecules. The top candidate rearrangement-related peptides with neoantigenic potential bound patient-specific HLA molecules nearly as well or as well as a positive control in competitive binding assays. Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel therapeutic strategies, the selection of patients to receive immunotherapy, and blood-based treatment monitoring strategies. Citation Format: Aaron S. Mansfield, Tobias Peikert, James B. Smadbeck, Julia B. Udell, Farhad Kosari, Stephen J. Murphy, Hongzheng Ren, Vishnu V. Serla, Janet L. Schaefer Klein, Giannoula Karagouga, Faye R. Harris, Carlos Sosa, Sarah H. Johnson, Wendy Nevala, Svetomir N. Markovic, Aaron O. Bungum, Eric S. Edell, Haidong Dong, John C. Cheville, Marie Christine Aubry, Jin Jen, George Vasmatzis. Rearrangement-related peptides with neoantigenic potential in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5726.

Abstract Lung adenocarcinoma is the most common type of primary lung cancer. Previous studies have shown that gene expression signatures may predict clinical outcome. In this study, we used a systems biology approach, weighted gene co-expression network analysis (WGCNA), to identify survival related networks and gene signatures. We first analyzed whole genome gene expression profiles in 82 lung adenocarcinoma tumor tissues from never smokers (smoked <100 cigarettes lifetime) and found a significant correlation of an expression module with tumor grade of differentiation (r=0.38, p=4×10−4) and overall survival (OS, p =0.027, HR, 1.71; 95% CI, 1.06-2.74) after adjusting for potential confounders (age and tumor grade). When this module signature was incorporated and compared to the conventional model consisting of clinical variables only, the survival prediction accuracy was increased from 0.70 to 0.75. Gene Ontology (GO) enrichment analysis indicated this module was significantly enriched in biological process GO term “cell cycle” (p = 2.1×10−53). To explore the effect of this network module at germline level, we performed the WGCNA on normal lung tissues derived from 78 of the 82 patients and observed significant association of an expression module with OS (adjusted p=3×10−4, HR=1.98; 95% CI, 1.36-2.87). Interestingly, this module was also significantly enriched in cell cycle-related genes (p=4.1×10−41). To further validate this result, we analyzed an independent microarray gene expression dataset including 442 lung adenocarcinoma patients from NCI Director's Challenge Consortium. We found the same association: the cell cycle-enriched module was correlated with tumor grade of cell differentiation (r=0.55, p-value=5×10−37) and OS (adjusted p=7.4×10−3; HR, 1.20; 95% CI, 1.05-1.37). We further identified 30 genes in the OS-related module that overlapped between the discovery and validation data sets. Furthermore, all these 30 genes were found to be up-regulated in adenocarcinoma compared to adjacent normal lung tissues in the discovery data set. Of the 30 genes, three key genes, UBE2C, TPX2 and MELK, were identified with highest connectivity in cell cycle-enriched module; their expression levels were significantly associated with OS in both discovery and validation sets. UBE2C and TPX2 have been reported as gene signatures for human lung cancer prognosis in vitro lung carcinogenesis system; MELK was identified as a key regulator of the proliferation of malignant brain tumors and malignant grade in human astrocytomas, and was also associated with breast cancer prognosis. Our results suggest that genes involved in cell-cycle are likely related with tumor aggressiveness and therefore can predict survival in lung adenocarcinoma. Further studies are needed to validate the key genes that could improve survival prediction and provide potential new targets to intervene progression of lung adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5060. doi:10.1158/1538-7445.AM2011-5060

Abstract Background: Patients with lung cancer with mutations in EGF receptor (EGFR) tyrosine kinase have improved prognosis when treated with EGFR inhibitors. We hypothesized that EGFR mutations may be related to residential radon or passive tobacco smoke. Methods: This hypothesis was investigated by analyzing EGFR mutations in 70 lung tumors from a population of never and long-term former female smokers from Missouri with detailed exposure assessments. The relationship with passive smoking was also examined in never-smoking female lung cancer cases from the Mayo clinic. Results: Overall, the frequency of EGFR mutation was 41% [95% confidence interval (CI), 32%–49%]. Neither radon nor passive-smoking exposure was consistently associated with EGFR mutations in lung tumors. Conclusions: The results suggest that EGFR mutations are common in female, never-smoking lung cancer cases from the United States, and EGFR mutations are unlikely due to exposure to radon or passive smoking. Cancer Epidemiol Biomarkers Prev; 21(6); 988–92. ©2012 AACR.