Multiomics profiling of urothelial carcinoma in situ reveals CIS-specific gene signature and immune characteristics.

Urothelial carcinoma in situ (CIS) is an aggressive phenotype of non-muscle-invasive bladder cancer. Molecular features unique to CIS compared to high-grade papillary tumors are underexplored. RNA sequencing of CIS, papillary tumors, and normal urothelium showed lower immune marker expression in CIS compared to papillary tumors. We identified a 46-gene expression signature in CIS samples including selectively upregulated known druggable targets MTOR, TYK2, AXIN1, CPT1B, GAK, and PIEZO1 and selectively downregulated BRD2 and NDUFB2. High expression of selected genes was significantly associated with CIS in an independent dataset. Mutation analysis of matched CIS and papillary tumors revealed shared mutations between samples across time points and mutational heterogeneity. CCDC138 was the most frequently mutated gene in CIS. The immunological landscape showed higher levels of PD-1-positive cells in CIS lesions compared to papillary tumors. We identified CIS lesions to have distinct characteristics compared to papillary tumors potentially contributing to the aggressive phenotype.

Loss of ZNRF3/RNF43 Unleashes EGFR in Cancer.

ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the most negatively correlated protein with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo, whereas knockout of ZNRF3/RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptor, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.

Frozen tissue coring and layered histological analysis improves cell type-specific proteogenomic characterization of pancreatic adenocarcinoma.

BACKGROUND: Omics characterization of pancreatic adenocarcinoma tissue is complicated by the highly heterogeneous and mixed populations of cells. We evaluate the feasibility and potential benefit of using a coring method to enrich specific regions from bulk tissue and then perform proteogenomic analyses. METHODS: We used the Biopsy Trifecta Extraction (BioTExt) technique to isolate cores of epithelial-enriched and stroma-enriched tissue from pancreatic tumor and adjacent tissue blocks. Histology was assessed at multiple depths throughout each core. DNA sequencing, RNA sequencing, and proteomics were performed on the cored and bulk tissue samples. Supervised and unsupervised analyses were performed based on integrated molecular and histology data. RESULTS: Tissue cores had mixed cell composition at varying depths throughout. Average cell type percentages assessed by histology throughout the core were better associated with KRAS variant allele frequencies than standard histology assessment of the cut surface. Clustering based on serial histology data separated the cores into three groups with enrichment of neoplastic epithelium, stroma, and acinar cells, respectively. Using this classification, tumor overexpressed proteins identified in bulk tissue analysis were assigned into epithelial- or stroma-specific categories, which revealed novel epithelial-specific tumor overexpressed proteins. CONCLUSIONS: Our study demonstrates the feasibility of multi-omics data generation from tissue cores, the necessity of interval H&E stains in serial histology sections, and the utility of coring to improve analysis over bulk tissue data.

SEPepQuant enhances the detection of possible isoform regulations in shotgun proteomics.

Shotgun proteomics is essential for protein identification and quantification in biomedical research, but protein isoform characterization is challenging due to the extensive number of peptides shared across proteins, hindering our understanding of protein isoform regulation and their roles in normal and disease biology. We systematically assess the challenge and opportunities of shotgun proteomics-based protein isoform characterization using in silico and experimental data, and then present SEPepQuant, a graph theory-based approach to maximize isoform characterization. Using published data from one induced pluripotent stem cell study and two human hepatocellular carcinoma studies, we demonstrate the ability of SEPepQuant in addressing the key limitations of existing methods, providing more comprehensive isoform-level characterization, identifying hundreds of isoform-level regulation events, and facilitating streamlined cross-study comparisons. Our analysis provides solid evidence to support a widespread role of protein isoform regulation in normal and disease processes, and SEPepQuant has broad applications to biological and translational research.

Proteogenomic data and resources for pan-cancer analysis.

The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) investigates tumors from a proteogenomic perspective, creating rich multi-omics datasets connecting genomic aberrations to cancer phenotypes. To facilitate pan-cancer investigations, we have generated harmonized genomic, transcriptomic, proteomic, and clinical data for >1000 tumors in 10 cohorts to create a cohesive and powerful dataset for scientific discovery. We outline efforts by the CPTAC pan-cancer working group in data harmonization, data dissemination, and computational resources for aiding biological discoveries. We also discuss challenges for multi-omics data integration and analysis, specifically the unique challenges of working with both nucleotide sequencing and mass spectrometry proteomics data.

A proteogenomics data-driven knowledge base of human cancer.

By combining mass-spectrometry-based proteomics and phosphoproteomics with genomics, epi-genomics, and transcriptomics, proteogenomics provides comprehensive molecular characterization of cancer. Using this approach, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) has characterized over 1,000 primary tumors spanning 10 cancer types, many with matched normal tissues. Here, we present LinkedOmicsKB, a proteogenomics data-driven knowledge base that makes consistently processed and systematically precomputed CPTAC pan-cancer proteogenomics data available to the public through ∼40,000 gene-, protein-, mutation-, and phenotype-centric web pages. Visualization techniques facilitate efficient exploration and reasoning of complex, interconnected data. Using three case studies, we illustrate the practical utility of LinkedOmicsKB in providing new insights into genes, phosphorylation sites, somatic mutations, and cancer phenotypes. With precomputed results of 19,701 coding genes, 125,969 phosphosites, and 256 genotypes and phenotypes, LinkedOmicsKB provides a comprehensive resource to accelerate proteogenomics data-driven discoveries to improve our understanding and treatment of human cancer. A record of this paper's transparent peer review process is included in the supplemental information.

Proteogenomic insights suggest druggable pathways in endometrial carcinoma.

We characterized a prospective endometrial carcinoma (EC) cohort containing 138 tumors and 20 enriched normal tissues using 10 different omics platforms. Targeted quantitation of two peptides can predict antigen processing and presentation machinery activity, and may inform patient selection for immunotherapy. Association analysis between MYC activity and metformin treatment in both patients and cell lines suggests a potential role for metformin treatment in non-diabetic patients with elevated MYC activity. PIK3R1 in-frame indels are associated with elevated AKT phosphorylation and increased sensitivity to AKT inhibitors. CTNNB1 hotspot mutations are concentrated near phosphorylation sites mediating pS45-induced degradation of ß-catenin, which may render Wnt-FZD antagonists ineffective. Deep learning accurately predicts EC subtypes and mutations from histopathology images, which may be useful for rapid diagnosis. Overall, this study identified molecular and imaging markers that can be further investigated to guide patient stratification for more precise treatment of EC.

Proteogenomic Approaches for the Identification of NF1/Neurofibromin-depleted Estrogen Receptor-positive Breast Cancers for Targeted Treatment.

NF1 is a key tumor suppressor that represses both RAS and estrogen receptor-α (ER) signaling in breast cancer. Blocking both pathways by fulvestrant (F), a selective ER degrader, together with binimetinib (B), a MEK inhibitor, promotes tumor regression in NF1-depleted ER+ models. We aimed to establish approaches to determine how NF1 protein levels impact B+F treatment response to improve our ability to identify B+F sensitive tumors. We examined a panel of ER+ patient-derived xenograft (PDX) models by DNA and mRNA sequencing and found that more than half of these models carried an NF1 shallow deletion and generally have low mRNA levels. Consistent with RAS and ER activation, RET and MEK levels in NF1-depleted tumors were elevated when profiled by mass spectrometry (MS) after kinase inhibitor bead pulldown. MS showed that NF1 can also directly and selectively bind to palbociclib-conjugated beads, aiding quantification. An IHC assay was also established to measure NF1, but the MS-based approach was more quantitative. Combined IHC and MS analysis defined a threshold of NF1 protein loss in ER+ breast PDX, below which tumors regressed upon treatment with B+F. These results suggest that we now have a MS-verified NF1 IHC assay that can be used for patient selection as a complement to somatic genomic analysis. Significance: A major challenge for targeting the consequence of tumor suppressor disruption is the accurate assessment of protein functional inactivation. NF1 can repress both RAS and ER signaling, and a ComboMATCH trial is underway to treat the patients with binimetinib and fulvestrant. Herein we report a MS-verified NF1 IHC assay that can determine a threshold for NF1 loss to predict treatment response. These approaches may be used to identify and expand the eligible patient population.

Efficient cancer modeling through CRISPR-Cas9/HDR-based somatic precision gene editing in mice.

CRISPR-Cas9 has been used successfully to introduce indels in somatic cells of rodents; however, precise editing of single nucleotides has been hampered by limitations of flexibility and efficiency. Here, we report technological modifications to the CRISPR-Cas9 vector system that now allows homology-directed repair-mediated precise editing of any proto-oncogene in murine somatic tissues to generate tumor models with high flexibility and efficiency. Somatic editing of either Kras or Pik3ca in both normal and hyperplastic mammary glands led to swift tumorigenesis. The resulting tumors shared some histological, transcriptome, and proteome features with tumors induced by lentivirus-mediated expression of the respective oncogenes, but they also exhibited some distinct characteristics, particularly showing less intertumor variation, thus potentially offering more consistent models for cancer studies and therapeutic development. Therefore, this technological advance fills a critical gap between the power of CRISPR technology and high-fidelity mouse models for studying human tumor evolution and preclinical drug testing.

Proteogenomic Markers of Chemotherapy Resistance and Response in Triple-Negative Breast Cancer.

Microscaled proteogenomics was deployed to probe the molecular basis for differential response to neoadjuvant carboplatin and docetaxel combination chemotherapy for triple-negative breast cancer (TNBC). Proteomic analyses of pretreatment patient biopsies uniquely revealed metabolic pathways, including oxidative phosphorylation, adipogenesis, and fatty acid metabolism, that were associated with resistance. Both proteomics and transcriptomics revealed that sensitivity was marked by elevation of DNA repair, E2F targets, G2-M checkpoint, interferon-gamma signaling, and immune-checkpoint components. Proteogenomic analyses of somatic copy-number aberrations identified a resistance-associated 19q13.31-33 deletion where LIG1, POLD1, and XRCC1 are located. In orthogonal datasets, LIG1 (DNA ligase I) gene deletion and/or low mRNA expression levels were associated with lack of pathologic complete response, higher chromosomal instability index (CIN), and poor prognosis in TNBC, as well as carboplatin-selective resistance in TNBC preclinical models. Hemizygous loss of LIG1 was also associated with higher CIN and poor prognosis in other cancer types, demonstrating broader clinical implications. SIGNIFICANCE: Proteogenomic analysis of triple-negative breast tumors revealed a complex landscape of chemotherapy response associations, including a 19q13.31-33 somatic deletion encoding genes serving lagging-strand DNA synthesis (LIG1, POLD1, and XRCC1), that correlate with lack of pathologic response, carboplatin-selective resistance, and, in pan-cancer studies, poor prognosis and CIN. This article is highlighted in the In This Issue feature, p. 2483.

caAtlas: An immunopeptidome atlas of human cancer.

Comprehensive characterization of tumor antigens is essential for the design of cancer immunotherapies, and mass spectrometry (MS)-based immunopeptidomics enables high-throughput identification of major histocompatibility complex (MHC)-bound peptide antigens in vivo. Here we construct an immunopeptidome atlas of human cancer through an extensive collection of 43 published immunopeptidomic datasets and standardized analysis of 81.6 million MS/MS spectra using an open search engine. Our analysis greatly expands the current knowledge of MHC-bound antigens, including an unprecedented characterization of post-translationally modified antigens and their cancer-association. We also perform systematic analysis of cancer-testis antigens, cancer-associated antigens, and neoantigens. We make all these data together with annotated MS/MS spectra supporting identification of each antigen in an easily browsable web portal named cancer antigen atlas (caAtlas). caAtlas provides a central resource for the selection and prioritization of MHC-bound peptides for in vitro HLA binding assay and immunogenicity testing, which will pave the way to eventual development of cancer immunotherapies.

Proteogenomic characterization of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.

A proteogenomic portrait of lung squamous cell carcinoma.

Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic options. We characterized the proteogenomic landscape of LSCC, providing a deeper exposition of LSCC biology with potential therapeutic implications. We identify NSD3 as an alternative driver in FGFR1-amplified tumors and low-p63 tumors overexpressing the therapeutic target survivin. SOX2 is considered undruggable, but our analyses provide rationale for exploring chromatin modifiers such as LSD1 and EZH2 to target SOX2-overexpressing tumors. Our data support complex regulation of metabolic pathways by crosstalk between post-translational modifications including ubiquitylation. Numerous immune-related proteogenomic observations suggest directions for further investigation. Proteogenomic dissection of CDKN2A mutations argue for more nuanced assessment of RB1 protein expression and phosphorylation before declaring CDK4/6 inhibition unsuccessful. Finally, triangulation between LSCC, LUAD, and HNSCC identified both unique and common therapeutic vulnerabilities. These observations and proteogenomics data resources may guide research into the biology and treatment of LSCC.

Proteogenomic and metabolomic characterization of human glioblastoma.

Glioblastoma (GBM) is the most aggressive nervous system cancer. Understanding its molecular pathogenesis is crucial to improving diagnosis and treatment. Integrated analysis of genomic, proteomic, post-translational modification and metabolomic data on 99 treatment-naive GBMs provides insights to GBM biology. We identify key phosphorylation events (e.g., phosphorylated PTPN11 and PLCG1) as potential switches mediating oncogenic pathway activation, as well as potential targets for EGFR-, TP53-, and RB1-altered tumors. Immune subtypes with distinct immune cell types are discovered using bulk omics methodologies, validated by snRNA-seq, and correlated with specific expression and histone acetylation patterns. Histone H2B acetylation in classical-like and immune-low GBM is driven largely by BRDs, CREBBP, and EP300. Integrated metabolomic and proteomic data identify specific lipid distributions across subtypes and distinct global metabolic changes in IDH-mutated tumors. This work highlights biological relationships that could contribute to stratification of GBM patients for more effective treatment.

Proteogenomic insights into the biology and treatment of HPV-negative head and neck squamous cell carcinoma.

We present a proteogenomic study of 108 human papilloma virus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs). Proteomic analysis systematically catalogs HNSCC-associated proteins and phosphosites, prioritizes copy number drivers, and highlights an oncogenic role for RNA processing genes. Proteomic investigation of mutual exclusivity between FAT1 truncating mutations and 11q13.3 amplifications reveals dysregulated actin dynamics as a common functional consequence. Phosphoproteomics characterizes two modes of EGFR activation, suggesting a new strategy to stratify HNSCCs based on EGFR ligand abundance for effective treatment with inhibitory EGFR monoclonal antibodies. Widespread deletion of immune modulatory genes accounts for low immune infiltration in immune-cold tumors, whereas concordant upregulation of multiple immune checkpoint proteins may underlie resistance to anti-programmed cell death protein 1 monotherapy in immune-hot tumors. Multi-omic analysis identifies three molecular subtypes with high potential for treatment with CDK inhibitors, anti-EGFR antibody therapy, and immunotherapy, respectively. Altogether, proteogenomics provides a systematic framework to inform HNSCC biology and treatment.

MLK4-mediated phosphorylation of histone H3T3 promotes flowering by transcriptional silencing of FLC/MAF in Arabidopsis thaliana.

Casein kinase I (CK1), a ubiquitous Ser/Thr protein kinase in eukaryotes, plays a critical role in higher plant flowering. Arabidopsis CK1 family member MUT9-LIKE KINASEs, such as MLK1 and MLK3, have been shown to phosphorylate histone H3 at threonine 3 (H3T3), an evolutionarily conserved residue, and the modification is associated with the transcriptional repression of euchromatic and heterochromatic loci. This study demonstrates that mlk4-3, a T-DNA insertion mutant of MLK4, flowered late, and that overexpression of MLK4 caused early flowering. The nuclear protein MLK4 phosphorylated histone H3T3 both in vitro and in vivo, and this catalytic activity required the conserved lysine residue K175. mutation of MLK4 at K175 failed to restore the level of phosphorylated H3T3 (H3T3ph) or to complement the phenotypic defects of mlk4-3. The FLC/MAF-clade genes, including FLC, MAF4 and MAF5, were significantly upregulated in mlk4-3. The double mutant mlk4-3 flc-3 flowered earlier than mlk4-3, suggesting that functional FLC is crucial for flowering repression in mlk4-3. Chromatin immunoprecipitation assays showed that MLK4 bound to FLC/MAF chromatin and that H3T3ph occupancy at the promoter of FLC/MAF was negatively associated with its transcriptional level. In accordance, H3T3ph accumulated at FLC/MAF in 35S::MLK4/mlk4-3 but diminished in 35S::MLK4(K175R)/mlk4-3 plants. Moreover, the amount of RNA Pol II deposited at FLC/MAF was clearly enriched in mlk4-3 relative to the wild type. Therefore, MLK4-dependent phosphorylation of H3T3 contributes to accelerating flowering by repressing the transcription of negative flowering regulator FLC/MAF. This study sheds light on the delicate control of flowering by the plant-specific CK1, MLK4, via post-translational modification of histone H3.

Proteogenomic Landscape of Breast Cancer Tumorigenesis and Targeted Therapy.

The integration of mass spectrometry-based proteomics with next-generation DNA and RNA sequencing profiles tumors more comprehensively. Here this "proteogenomics" approach was applied to 122 treatment-naive primary breast cancers accrued to preserve post-translational modifications, including protein phosphorylation and acetylation. Proteogenomics challenged standard breast cancer diagnoses, provided detailed analysis of the ERBB2 amplicon, defined tumor subsets that could benefit from immune checkpoint therapy, and allowed more accurate assessment of Rb status for prediction of CDK4/6 inhibitor responsiveness. Phosphoproteomics profiles uncovered novel associations between tumor suppressor loss and targetable kinases. Acetylproteome analysis highlighted acetylation on key nuclear proteins involved in the DNA damage response and revealed cross-talk between cytoplasmic and mitochondrial acetylation and metabolism. Our results underscore the potential of proteogenomics for clinical investigation of breast cancer through more accurate annotation of targetable pathways and biological features of this remarkably heterogeneous malignancy.

Proteogenomic Characterization of Endometrial Carcinoma.

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/ß-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.

Microscaled proteogenomic methods for precision oncology.

Cancer proteogenomics promises new insights into cancer biology and treatment efficacy by integrating genomics, transcriptomics and protein profiling including modifications by mass spectrometry (MS). A critical limitation is sample input requirements that exceed many sources of clinically important material. Here we report a proteogenomics approach for core biopsies using tissue-sparing specimen processing and microscaled proteomics. As a demonstration, we analyze core needle biopsies from ERBB2 positive breast cancers before and 48-72 h after initiating neoadjuvant trastuzumab-based chemotherapy. We show greater suppression of ERBB2 protein and both ERBB2 and mTOR target phosphosite levels in cases associated with pathological complete response, and identify potential causes of treatment resistance including the absence of ERBB2 amplification, insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification, and candidate resistance mechanisms including androgen receptor signaling, mucin overexpression and an inactive immune microenvironment. The clinical utility and discovery potential of proteogenomics at biopsy-scale warrants further investigation.