Faculty Bibliography

The relative contributions of genetic and environmental factors to variation in immune responses are still poorly understood. Here, we performed a deep phenotypic analysis of immunological parameters of laboratory mice released into an outdoor enclosure, carrying susceptibility genes (Nod2 and Atg16l1) implicated in the development of inflammatory bowel diseases. Variations of immune cell populations were largely driven by environment, whereas cytokine production in response to stimulation was affected more by genetic mutations. Multi-omic models identified transcriptional signatures associated with differences in T cell populations. Subnetworks associated with responses against Clostridium perfringens, Candida albicans and Bacteroides vulgatus were also coupled with rewilding. Hence, exposing laboratory mice carrying different genetic mutations to a natural environment uncovered important contributors to immune variation

Multiple sclerosis (MS) is a neuroinflammatory disorder resulting from infiltration of T cells into the central nervous system (CNS) and demyelination of neurons. Since T follicular helper (TFH) cells are associated with MS relapse, their selective inhibition could be an ideal therapeutic. TFH cells require transcription factor BCL6 and active mTORC1/2 for development. When mTORC is active, cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) initiates translation of selective mRNAs. However, the requirement of eIF4E for translation of mRNAs necessary for TFH cell development has not been investigated. Disruption of eIF4E binding to the 5' mRNA cap with drug 4EGI-1 inhibits TFH and germinal center (GC) B cell development while having no effect on differentiation and effector function of TH1, TH2, TH17, or Tregs. Silencing of eIF4E in only CD4 T cells is sufficient to inhibit their formation of TFH cells. We used polysome profiling to determine which mRNAs are selectively translated by eIF4E and identified salient programs regulated by transcription (BCL6, NFAT) and costimulation (CD28, SLAM). eIF4E is required for translation of BCL6 in human lymph node TFH and GC B cells. Administration of 4EGI-1 during experimental autoimmune encephalitis (EAE) results in significantly decreased infiltration of CD4 T cells in the CNS, demyelination, and clinical score. Further, 4EGI-1 treatment following initiation of symptoms results in rapid improvement of symptoms and partial remission earlier than vehicle-treated animals. Thus, eIF4E is required for differentiation of TFH cells and pathogenesis of autoimmune encephalitis, and 4EGI-1 represents a potential therapeutic.

We undertook a comprehensive proteogenomic characterization of 95 prospectively collected endometrial carcinomas, comprising 83 endometrioid and 12 serous tumors. This analysis revealed possible new consequences of perturbations to the p53 and Wnt/β-catenin pathways, identified a potential role for circRNAs in the epithelial-mesenchymal transition, and provided new information about proteomic markers of clinical and genomic tumor subgroups, including relationships to known druggable pathways. An extensive genome-wide acetylation survey yielded insights into regulatory mechanisms linking Wnt signaling and histone acetylation. We also characterized aspects of the tumor immune landscape, including immunogenic alterations, neoantigens, common cancer/testis antigens, and the immune microenvironment, all of which can inform immunotherapy decisions. Collectively, our multi-omic analyses provide a valuable resource for researchers and clinicians, identify new molecular associations of potential mechanistic significance in the development of endometrial cancers, and suggest novel approaches for identifying potential therapeutic targets.

Melanoma brain metastasis is the largest cause of melanoma morbidity and mortality, and melanoma has the highest rate of brain metastasis of any cancer. The mechanisms that mediate melanoma brain metastasis remain poorly understood. We characterized patient-derived Short-Term Cultures (STCs) as a novel model system for the study of melanoma brain metastasis. Unbiased proteomics analysis of STCs revealed striking alterations in brain metastasis vs non-brain metastasis derived STCs in proteins related to neurodegeneration. Through in-vivo assays, we show that loss of Amyloid Precursor Protein (APP) in melanoma cells dramatically inhibits melanoma brain metastasis formation without affecting metastasis to other organs and that amyloid beta is the form of APP critically required for melanoma brain metastasis. Additionally, we demonstrate that APP is required for late growth and survival of melanoma cells in the brain parenchyma. Furthermore, we demonstrate that melanoma-derived amyloid beta polarizes astrocytes to an anti-inflammatory secretory phenotype that inhibits microglial phagocytosis of melanoma cells. Finally, we show that treatment of mice with a beta secretase inhibitor (LY2886721), which prevents amyloid beta production, decreases brain metastatic burden. Our results demonstrate a critical role for amyloid beta in melanoma brain metastasis, establish a novel connection between brain metastasis and neurodegenerative pathologies, and show that amyloid beta is a promising therapeutic target for brain metastasis treatment. Studies to further characterize how amyloid beta acts in the melanoma brain metastasis microenvironment are currently underway

Advances in mass spectrometry-based methods have enabled deeper biological insights using proteomics and metabolomics approaches. This has also resulted in improvements in our ability to compare and correlate these data with other 'omics methods such as genomics and transcriptomics-thus creating the new field of multi-omics. In this session, experts will present their work in the area of multi-omics research. Robert Hettich (Oak Ridge National Laboratory) will present his experiences with integrating advanced mass spectrometry technology and meta-omics analysis for characterizing complex microbiomes. Kelly Ruggles (NYU Langone Health) will discuss ways to integrate the results from diverse experiments and develop complementary statistical analysis methods for large comprehensive proteogenomics datasets. Ewy Mathe (Ohio State University) will cover analytical methods in genomics, epigenomics, and metabolomics to characterize disease biomarkers and therapeutic targets. Lastly, Tejaswini Mis-hra (Stanford University) will tell of her work in multi-omics profiling. The presentations will be followed by a panel discussion that will address challenges and solutions for adopting multi-omics methods in core facility settings

To elucidate the deregulated functional modules that drive clear cell renal cell carcinoma (ccRCC), we performed comprehensive genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic characterization of treatment-naive ccRCC and paired normal adjacent tissue samples. Genomic analyses identified a distinct molecular subgroup associated with genomic instability. Integration of proteogenomic measurements uniquely identified protein dysregulation of cellular mechanisms impacted by genomic alterations, including oxidative phosphorylation-related metabolism, protein translation processes, and phospho-signaling modules. To assess the degree of immune infiltration in individual tumors, we identified microenvironment cell signatures that delineated four immune-based ccRCC subtypes characterized by distinct cellular pathways. This study reports a large-scale proteogenomic analysis of ccRCC to discern the functional impact of genomic alterations and provides evidence for rational treatment selection stemming from ccRCC pathobiology.

Aberrant phospho-signaling is a hallmark of cancer. We investigated kinase-substrate regulation of 33,239 phosphorylation sites (phosphosites) in 77 breast tumors and 24 breast cancer xenografts. Our search discovered 2,134 quantitatively-correlated kinase-phosphosite pairs, enriching for and extending experimental or binding-motif predictions. Among the 91 kinases with auto-phosphorylation, elevated EGFR, ERBB2, PRKG1, and WNK1 phosphosignaling were enriched in basal, HER2-E, Luminal A, and Luminal B breast cancers, respectively, revealing subtype-specific regulation. CDKs, MAPKs, and ataxia-telangiectasia proteins were dominant, master regulators of substrate-phosphorylation, whose activities are not captured by genomic evidence. We unveiled phospho-signaling and druggable targets from 113 kinase-substrate pairs and cascades downstream of kinases, including AKT1, BRAF and EGFR. We further identified kinase-substrate-pairs associated with clinical or immune signatures and experimentally validated activated phosphosites of ERBB2, EIF4EBP1, and EGFR. Overall, kinase-substrate regulation revealed by the largest unbiased global phosphorylation data to date connects driver events to their signaling effects.