Faculty Bibliography

Introduction: Gene rearrangements play a critical role in the development of brain tumors. RNA next-generation sequencing (NGS) panels cover a limited number of genes, are rarely successful in FFPE samples > 5 years old, and cannot detect rearrangements between genes and non-coding regulatory regions. We evaluated whole genome Hi-C NGS for detection of gene fusions and cryptic rearrangements.
Method(s): DNA was extracted from FFPE scrolls of 55 glial and non-glial brain tumors and processed using Arima-HiC+ FFPE Sample protocol, consisting of chromatin fragmentation, labeling, and re-ligation, followed by DNA purification and library preparation for paired-end Illumina sequencing with an average of 10X genome coverage (100M PE reads per sample). Data were analyzed using the Arima-SV pipeline using Juicer and HiCUP, SV detection using HiC-Breakfinder, loop calling using Juicer Tools, and integrative data visualization using Juicebox. Overexpression of putative driver genes was confirmed by immunohistochemistry.
Result(s): Hi-C libraries were prepared and sequenced from FFPE tissues including samples that failed RNA NGS. Hi-C successfully detected gene-gene fusions including actionable EML4-NTRK3, ETV6-NTRK3, fusions. We detected rearrangements missed by RNA NGS (i.e., complex MYBL1 rearrangement) or between non-coding regions and known cancer genes (i.e. PDL1, PAX5, NRAS, TERT, KAT6A, GATA6, and ARID1B). Since Hi-C data captures 3D genome structural features such as chromatin loops and topological domains, datasets were of high quality and capable of detecting up to 13,000 chromatin loops per tumor.
Conclusion(s): Genome-wide Hi-C NGS is successful in detecting gene fusions and cryptic rearrangements between coding and non-coding regions in archival FFPE tissue including degraded samples. Because Hi-C data captures 3D genome structures, these datasets simultaneously inform gene regulatory mechanisms that may play a role in oncogenesis or tumor progression. Whole-genome Hi-C NGS expands our ability to detect actionable and novel drivers, and potentially new therapeutic targets in a single NGS workflow

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of mutated pre-leukemic cells. Individuals with CH are at an increased risk of developing hematopoietic malignancies. Here, we describe a novel animal model carrying a recurrent TET2 missense mutation, frequently found in CH and leukemic patients. In a fashion similar to CH, animals show signs of disease late in life when they develop a wide range of myeloid neoplasms, including acute myeloid leukemia (AML). Using single cell transcriptomic profiling of the bone marrow, we show that disease progression in aged animals correlates with an enhanced inflammatory response and the emergence of an aberrant inflammatory monocytic cell population. The gene signature characteristic of this inflammatory population is associated to poor prognosis in AML patients. Our study illustrates an example of collaboration between a genetic lesion found in CH and inflammation, leading to transformation and the establishment of blood neoplasms.

PURPOSE/OBJECTIVE:Lung adenocarcinoma (LUAD) is a clinically heterogenous disease, which is highlighted by the unpredictable recurrence in low-stage tumors and highly variable responses observed in patients treated with immunotherapies, which cannot be explained by mutational profiles. DNA methylation-based classification and understanding of microenviromental heterogeneity may allow stratification into clinically relevant molecular subtypes of LUADs. EXPERIMENTAL DESIGN/METHODS:We characterize the genome-wide DNA methylation landscape of 88 resected LUAD tumors. Exome sequencing focusing on a panel of cancer-related genes was used to genotype these adenocarcinoma samples. Bioinformatic and statistical tools, the immune cell composition, DNA methylation age (DNAm age), and DNA methylation clustering were used to identify clinically relevant subgroups. RESULTS:Deconvolution of DNA methylation data identified immunologically hot and cold subsets of lung adenocarcinomas. Additionally, concurrent factors were analyzed that could affect the immune microenvironment, such as smoking history, ethnicity, or presence of KRAS or TP53 mutations. When the DNAm age was calculated, a lower DNAm age was correlated with the presence of a set of oncogenic drivers, poor overall survival, and specific immune cell populations. Unsupervised DNA methylation clustering identified 6 molecular subgroups of LUAD tumors with distinct clinical and microenvironmental characteristics. CONCLUSIONS:Our results demonstrate that DNA methylation signatures can stratify lung adenocarcinoma into clinically relevant subtypes, and thus such classification of LUAD at the time of resection may lead to better methods in predicting tumor recurrence and therapy responses.

Mitofusins reside on the outer mitochondrial membrane and regulate mitochondrial fusion, a physiological process that impacts diverse cellular processes. Mitofusins are activated by conformational changes and subsequently oligomerize to enable mitochondrial fusion. Here, we identify small molecules that directly increase or inhibit mitofusins activity by modulating mitofusin conformations and oligomerization. We use these small molecules to better understand the role of mitofusins activity in mitochondrial fusion, function, and signaling. We find that mitofusin activation increases, whereas mitofusin inhibition decreases mitochondrial fusion and functionality. Remarkably, mitofusin inhibition also induces minority mitochondrial outer membrane permeabilization followed by sub-lethal caspase-3/7 activation, which in turn induces DNA damage and upregulates DNA damage response genes. In this context, apoptotic death induced by a second mitochondria-derived activator of caspases (SMAC) mimetic is potentiated by mitofusin inhibition. These data provide mechanistic insights into the function and regulation of mitofusins as well as small molecules to pharmacologically target mitofusins.

Molecular classification has improved diagnosis and treatment for patients with malignant gliomas. However, classification has relied on individual assays that are both costly and slow, leading to frequent delays in treatment. Here, we propose the use of DNA methylation, as an emerging clinical diagnostic platform, to classify gliomas based on major genomic alterations and provide insight into subtype characteristics. We show that using machine learning models, DNA methylation signatures can accurately predict somatic alterations and show improvement over existing classifiers. The established Unified Diagnostic Pipeline (UniD) we develop is rapid and cost-effective for genomic alterations and gene expression subtypes diagnostic at early clinical phase and improves over individual assays currently in clinical use. The significant relationship between genetic alteration and epigenetic signature indicates broad applicability of our approach to other malignancies.

Mammalian cells autonomously activate hypoxia-inducible transcription factors (HIFs) to ensure survival in low-oxygen environments. We report here that injury-induced hypoxia is insufficient to trigger HIF1α in damaged epithelium. Instead, multimodal single-cell and spatial transcriptomics analyses and functional studies reveal that retinoic acid-related orphan receptor γt⁺ (RORγt⁺) γδ T cell-derived interleukin-17A (IL-17A) is necessary and sufficient to activate HIF1α. Protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling proximal of IL-17 receptor C (IL-17RC) activates mammalian target of rapamycin (mTOR) and consequently HIF1α. The IL-17A-HIF1α axis drives glycolysis in wound front epithelia. Epithelial-specific loss of IL-17RC, HIF1α, or blockade of glycolysis derails repair. Our findings underscore the coupling of inflammatory, metabolic, and migratory programs to expedite epithelial healing and illuminate the immune cell-derived inputs in cellular adaptation to hypoxic stress during repair.

The APOE gene is diversified by three alleles ε2, ε3, and ε4 encoding corresponding apolipoprotein (apo) E isoforms. Possession of the ε4 allele is signified by increased risks of age-related cognitive decline, Alzheimer's disease (AD), and the rate of AD dementia progression. ApoE is secreted by astrocytes as high-density lipoprotein-like particles and these are internalized by neurons upon binding to neuron-expressed apoE receptors. ApoE isoforms differentially engage neuronal plasticity through poorly understood mechanisms. We examined here the effects of native apoE lipoproteins produced by immortalized astrocytes homozygous for ε2, ε3, and ε4 alleles on the maturation and the transcriptomic profile of primary hippocampal neurons. Control neurons were grown in the presence of conditioned media from Apoe ^-/- astrocytes. ApoE2 and apoE3 significantly increase the dendritic arbor branching, the combined neurite length, and the total arbor surface of the hippocampal neurons, while apoE4 fails to produce similar effects and even significantly reduces the combined neurite length compared to the control. ApoE lipoproteins show no systemic effect on dendritic spine density, yet apoE2 and apoE3 increase the mature spines fraction, while apoE4 increases the immature spine fraction. This is associated with opposing effects of apoE2 or apoE3 and apoE4 on the expression of NR1 NMDA receptor subunit and PSD95. There are 1,062 genes differentially expressed across neurons cultured in the presence of apoE lipoproteins compared to the control. KEGG enrichment and gene ontology analyses show apoE2 and apoE3 commonly activate expression of genes involved in neurite branching, and synaptic signaling. In contrast, apoE4 cultured neurons show upregulation of genes related to the glycolipid metabolism, which are involved in dendritic spine turnover, and those which are usually silent in neurons and are related to cell cycle and DNA repair. In conclusion, our work reveals that lipoprotein particles comprised of various apoE isoforms differentially regulate various neuronal arbor characteristics through interaction with neuronal transcriptome. ApoE4 produces a functionally distinct transcriptomic profile, which is associated with attenuated neuronal development. Differential regulation of neuronal transcriptome by apoE isoforms is a newly identified biological mechanism, which has both implication in the development and aging of the CNS.

Embryos are diagnosed as mosaic if their chromosomal copy number falls between euploid and aneuploid. The purpose of this study was to investigate the impact of mosaicism on global gene expression. This study included 42 blastocysts that underwent preimplantation genetic testing for aneuploidy (PGT-A) and were donated for IRB approved research. Fourteen blastocysts were diagnosed as mosaic with Next-generation Sequencing (NGS). Three NGS diagnosed euploid embryos, and 25 aneuploid embryos (9 NGS, 14 array Comparative Genomic Hybridization, 2 Single Nucleotide Polymorphism array) were used as comparisons. RNA-sequencing was performed on all of the blastocysts. Differentially expressed genes (DEGs) were calculated using DESeq2/3.5 (R Bioconductor Package) with p < 0.05 considered significantly differentially expressed. Pathway analysis was performed on mosaic embryos using EnrichR with p < 0.05 considered significant. With euploid embryo gene expression used as a control, 12 of 14 mosaic embryos had fewer DEGs compared to aneuploid embryos involving the same chromosome. On principal component analysis (PCA), mosaic embryos mapped separately from aneuploid embryos. Pathways involving cell proliferation, differentiation, and apoptosis were the most disrupted within mosaic embryos. Mosaic embryos have decreased disruption of global gene expression compared to aneuploid embryos. This study was limited by the small sample size, lack of replicate samples for each mosaic abnormality, and use of multiple different PGT-A platforms for the diagnosis of aneuploid embryos.