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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

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.

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

Supplementary Figure from Proteogenomic Markers of Chemotherapy Resistance and Response in Triple-Negative Breast Cancer

Supplementary Table from Proteogenomic Markers of Chemotherapy Resistance and Response in Triple-Negative Breast Cancer

Supplementary Table S1 provides expression data of RNA, ribosome occupancy and protein used for multi-omics correlation analysis as well as complete lists of TE UP, TE DOWN and background genes.

Supplementary Figure S4 shows validation of TE UP and TE DOWN genes with polysome analysis.

Cooperativity between WNT and FGF signaling is well documented in embryonic development and cancer progression, but the molecular mechanisms underlying this cross-talk remain elusive. In this study, we interrogated the dynamics of RNA levels, ribosome occupancy, and protein expression as a function of inducible FGF signaling in mouse mammary glands with constitutive WNT hyperactivation. Multiomics correlation analysis revealed a substantial discrepancy between RNA and ribosome occupancy levels versus protein levels. However, this discrepancy decreased as cells became premalignant and dynamically responded to FGF signaling, implicating the importance of stringent gene regulation in nontransformed cells. Analysis of individual genes demonstrated that acute FGF hyperactivation increased translation of many stem cell self-renewal regulators, including WNT signaling components, and decreased translation of genes regulating cellular senescence. WNT pathway components translationally upregulated by FGF signaling had long and structured 5′ UTRs with a high frequency of polypurine sequences, several of which harbored (CGG)4 motifs that can fold into either stable G-quadruplexes or other stable secondary structures. The FGF-mediated increase in translation of WNT pathway components was compromised by silvestrol, an inhibitor of EIF4A that clamps EIF4A to polypurine sequences to block 43S scanning and inhibits its RNA-unwinding activity important for translation initiation. Moreover, silvestrol treatment significantly delayed FGF-WNT–driven tumorigenesis. Taken together, these results suggest that FGF signaling selectively enhances translation of structured mRNAs, particularly WNT signaling components, and highlight their vulnerability to inhibitors that target the RNA helicase EIF4A.Significance: The RNA helicase EIF4A may serve as a therapeutic target for breast cancers that require FGF and WNT signaling. Cancer Res; 78(15); 4229–40. ©2018 AACR.

Supplementary Methods and Figure Legends provide further details of the study methods and legends for supplementary figures and tables.

Shotgun proteomics is essential for protein identification and quantification in biomedical research, but protein isoform characterization is inhibited by 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 usingin silicoand experimental data, and then present SEPepQuant, a graph theory-based approach to enable 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 proteome 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.

Background: Molecularly targeted therapies are critical for improving cancer treatment. Since proteins are the targets of these therapies and functional effectors of genomic aberrations, proteogenomics data from the Clinical Proteomics Tumor Analysis Consortium (CPTAC) provides an unprecedented opportunity to characterize existing and future therapeutic targets for cancer treatment. Approach: CPTAC proteogenomics data from >1000 cancer patients spanning 10 cancer types was used to evaluate current and potential therapeutic targets curated from four databases. Cell line data from DepMap was further integrated to distinguish causations from associations. Computational pipelines were deployed to identify synthetic lethality for targeting tumor suppressor loss and to prioritize tumor associated antigens as immunotherapy targets. Results: We systematically collected 3050 druggable proteins and classified them into 5 tiers to facilitate different applications such as companion diagnostics, drug repurposing, and new therapy development. Many druggable proteins showed poor mRNA-protein correlation, including secreted proteins and proteins whose abundance was correlated with their interaction partners instead of cognate mRNA, highlighting the necessity of direct proteomic quantification of drug targets. 618 druggable proteins showed both overexpression in tumors compared to normal and significant dependency in CRISPR-Cas9 screens of cell lines of the same lineage. Notably, PAK1, a kinase targeted by investigational drugs, demonstrated both overexpression and dependency in all cancer types. A similar analysis of phosphoproteomics data focusing on known activating sites of druggable proteins further revealed targetable dependencies driven by protein hyperactivation. The phosphosite pS50 on PTPN1, a phosphatase targeted by experimental drugs, was increased in 7 cancer types and PTPN1 demonstrated dependency in related cancer cell lines. Based on tumor proteogenomic data and cell line CRISPR-Cas9 screen data, we identified synthetic lethality for difficult to target tumor suppressor losses, revealing TP53 mutations as a candidate biomarker to select breast cancer patients for CHEK1 inhibition, and endometrial cancer patients for treatment with doxorubicin. We identified 140 proteins whose expression was restricted in normal tissues but abnormal in tumors. Experimental analysis of peptides predicted to have high binding affinity to the most common allotype HLA-A02 for 7 prioritized proteins identified 21 peptides from 5 proteins with both strong binding affinity and immunogenicity which could be further investigated as immunotherapy targets. Conclusion: We generate a comprehensive resource of protein and peptide targets that covers multiple therapeutic modalities. This unique resource will pave the way for repurposing of currently available drugs and developing new drugs for cancer treatment. Citation Format: Jonathan T. Lei, Sara R. Savage, Xinpei Yi, Bo Wen, Hongwei Zhao, Lauren K. Somes, Paul W. Shafer, Yongchao Dou, Qiang Gao, Valentina Hoyos, Bing Zhang. Pan-cancer proteogenomics expands the landscape of therapeutic targets. [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 5726.

Pan-cancer multi-omics data produced from integrated genomic, epigenomic, transcriptomic, proteomic, and post-translational modification (PTM) profiling of a wide variety of cancer types holds great potential for understanding cancer biology and generating therapeutic hypotheses. To realize this potential and make analysis and visualization of these complex and interconnected data easily accessible to cancer biologists and clinicians, we have developed a web portal, LinkedOmicsKB. The web portal provides access to a harmonized proteogenomic dataset of over 1000 patient samples covering 10 cancer cohorts from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). We calculated associations between methylation, copy number variation (CNV), RNA, protein, and phosphosite data for each gene and further correlated the proteogenomics data with clinical and computed molecular phenotypes. All results are stored in a MongoDB database and visualizations are provided for exploring pan-cancer multi-omics relationships as well as individual statistics. We demonstrate the utility of LinkedOmicsKB to provide insights into clinical phenotypes, somatic mutations, and understudied genes. In the pan-cancer CPTAC data, overall survival was correlated with proteins involved in protein hydroxylation, including PLOD1 and PLOD2. Additionally, two phosphorylation sites on the tumor suppressor MIG-6 were associated with worse survival. These sites were also associated with hypoxia, MAPK, and EGFR pathway activity scores, suggesting a relationship between the signaling in these pathways and cancer prognosis. STK17B is an understudied kinase that regulates apoptosis. We found STK17B was upregulated in 6 cancer types at the protein level but only two at the RNA level. The protein abundance of STK17B was highly associated with immune scores and JAK/STAT signaling, supporting a role for STK17B in the immune response. We identified 3 phosphorylation sites on STK17B, which were associated with EGF pathway activity scores and immune-related scores. LinkedOmicsKB is a valuable tool that can be used to generate biological and clinical insights into any gene, phosphosite, mutation, or phenotype. Citation Format: Sara R. Savage, Yuxing Liao, Yongchao Dou, Zhiao Shi, Xinpei Yi, Wen Jiang, Jonathan T. Lei, Bing Zhang. LinkedOmicsKB: A web portal to explore pan-cancer molecular and phenotype associations [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 6575.

Background: Targeted therapy for HER2+ breast cancer has significantly improved outcomes for this aggressive subtype. However, a subset of patients do not achieve pathological complete response (pCR). In CALGB 40601, a randomized Phase III Trial for neoadjuvant treatment of HER2+ primary breast cancer with Paclitaxel (T: taxane) combined with HER2 antibody therapy (H: Herceptin/Trastuzumab), the small molecule inhibitor Lapatinib (L), or the antibody-inhibitor combination, pCR frequency was 56% for the combination (THL arm), 46% for Trastuzumab (TH arm), and 32% for Lapatinib (TL arm, closed early because of lower efficacy) (PMID: 26527775). While a recent publication reports relapse-free survival (RFS), overall survival (OS), and RNA-based gene expression signatures that can predict pCR (PMID: 33095682), understanding the proteogenomic landscape of treatment response should facilitate identification of alternative and therapeutically tractable protein targets for treatment-resistant tumors. Methods: Microscaled proteogenomic profiling (PMID: 31988290) was performed on treatment-naïve, flash-frozen core needle biopsies from the CALGB 40601 trial obtained from the Alliance for Clinical Trials in Oncology tissue bank. Multi-omics profiling included whole-exome sequencing (WES), RNA-sequencing, and mass spectroscopy-based proteomics and phosphoproteomics from one or two cores from each patient. Results: Eighty baseline core biopsies from 54 patients, including 22 patients from the THL arm, 24 from the TH arm, and 8 from the TL arm, from the CALGB 40601 tissue archive were of sufficient quality to yield genomics, transcriptomics, and/or proteomics profiling data. The frequency of pCR for profiled samples was representative of the overall trial cohort. Linear models were employed to identify baseline determinants of pCR for each arm and to assess differences in genes associated with response between the TH and THL arms. Pathways associated with RNA processing, translation, and the proteasome were elevated in pCR tumors in TH and THL arms, while cell cycle, DNA replication and repair pathways were higher in pCR only in the THL arm. While enrichment of similar pathways was observed in pCR in the transcriptome, the proteome specifically showed enrichment of pathways associated with extracellular matrix and EMT in non-pCR in the THL but not the TH arm. In particular, “EMT”, “ECM-receptor interaction”, and “extracellular structure organization” constituted the most enriched pathways and GO terms that were higher in non-pCR than in pCR tumors from the combination arm (THL) in the proteomics data despite showing no enrichment in the transcriptomics data. Driving this pathway enrichment were several collagens and matrix metalloproteinases that were significantly elevated in non-pCR tumors at the protein but not the RNA level. Finally, kinase target enrichment of differential phosphorylation sites suggested that the activity of PAK1, a regulator of cytoskeletal remodeling, is elevated in non-pCR tumors from the THL arm (p=0.006), but not the TH arm (p=0.69). Conclusion: Proteogenomic analysis of archival HER2+ breast cancer core biopsies provides opportunities for identifying proteins and phosphorylation sites in treatment-naive tumors that are associated with pCR to neoadjuvant Paclitaxel/anti-HER2 therapy. Notably, proteomic but not transcriptomic data showed that ECM and EMT pathways were elevated in non-pCR tumors; thus, signatures encompassing these pathways may serve as biomarkers for aggressive HER2+ breast cancer that is more likely to evade treatment. Non-pCR tumors in the THL arm were also marked by elevated levels of PAK1 target phosphorylation sites, suggesting that this kinase may be a potential therapeutic target in HER2+ breast cancer that is refractory to combination anti-HER2 therapy. Citation Format: Eric J. Jaehnig, Aranzazu Fernandez-Martinez, Tanmayi Vashist, Matthew V. Holt, LaTerrica Williams, Jonathan Lei, Beom-Jun Kim, Yongchao Dou, Viktoriya Korchina, Richard Gibbs, Donna Muzny, Harshavardhan Doddapaneni, Henry Rodriguez, Ana Robles, Tara Hiltke, DR Mani, Michael Gillette, Terry Hyslop, Yujia Wen, Linda McCart, George Miles, Steven Carr, Bing Zhang, Shankha Satpathy, Matthew Ellis, Meenakshi Anurag. Proteogenomic profiling of fresh frozen core biopsies from CALGB 40601 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-36.

Cancer proteogenomics combines genomics, transcriptomics and mass spectrometry-based proteomics to gain insights into cancer biology and treatment responsiveness. While proteogenomics analyses have already shown great potential to deepen our understanding of cancer tissue complexity and signaling, how a patient’s tumor changes upon treatment has largely been the province of genomics. This is due to technical difficulties associated with doing proteogenomic analysis on clinic-derived core-needle biopsies. To address this critical need, we have developed a “microscaled” proteogenomics approach for tumor-rich OCT-embedded core needle biopsies. Tissue-sparing specimen processing (“Biopsy Trifecta EXTraction”, BioTExt) and microscaled proteomics (MiProt) methodologies allowed generation of deep-scale proteogenomics datasets, with copy number and transcript information for >20,000 genes and mass spectrometry-based identification and quantification of nearly all expressed proteins in a tumor (>10,000 proteins) and more than >20,000 phosphosites starting with just 25 micrograms of protein per sample. In order to understand the capabilities and limitations our our approach relative to more conventional deepscale proteomics requiring >10X more starting material, we compared preclinical patient derived xenograft (PDX) models at conventional scale with data obtained by core-needle biopsy of the same tissues. Comparable depth and biological insights were obtained from the cores relative to surgically resected tumors. As a proof-of-concept for implementation in clinical trials, we applied microscaled proteogenomic methods to a small-scale clinical study where biopsies were accrued from patients with ERBB2+ locally advanced breast cancer before and 48 to 72 hours after the first dose of neoadjuvant Trastuzumab-based chemotherapy. Multi-omics comparisons were conducted between samples associated with residual disease versus samples associated with complete pathological response. Integrative, microscaled proteogenomic analyses efficiently diagnosed the molecular bases of diverse candidate treatment resistance mechanisms including: 1) absence of ERBB2 amplification (false-ERBB2+); 2) insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification (pseudo-ERBB2+); 3) resistance features in true-ERBB2+ cases including androgen receptor signaling, mucin expression and an inactive immune microenvironment; 4) lack of acute phospho-ERBB2 down-regulation in non-pCR cases. In summary, we have developed a robust proteogenomics pipeline well suited for large-scale cancer clinical studies to identify potential resistance mechanism in patients. We conclude that microscaled cancer proteogenomics could improve diagnostic precision in the clinical setting. Citation Format: Shankha Satpathy, Eric Jaehnig, Krug Karsten, Beom-Jun Kim, Alexander Saltzman, Doug Chan, Kimberly Holloway, Meenakshi Anurag, Chen Huang, Purba Singh, Ari Gao, Noel Namai, Yongchao Dou, Bo Wen, Suhas Vasaikar, David Mutch, Mark Watson, Cynthia Ma, Foluso Ademuyiwa, Mothaffar Rimawi, Jeremy Hoog, Samuel Jacobs, Anna Malovannaya, Terry Hyslop, D.R Mani, Charles Perou, George Miles, Bing Zhang, Michael Gillette, Steven Carr, Matthew Ellis. Microscaled proteogenomic methods for precision oncology [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-05.

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., Connecting genomics and proteomics allows the development of more efficient and specific treatments for cancer. Here, the authors develop proteogenomic methods to defining cancer signaling in-vivo starting from core needle biopsies and with application to a HER2 breast cancer focused clinical trial.

Summary 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.

Introduction: There are no robust molecular predictors for response of TNBC to chemotherapy. Microscaled proteogenomics (PMC6985126) was therefore applied to biopsies from TNBC patients undergoing neoadjuvant carboplatin/docetaxel. Approach: Sufficient tumor-rich tumor core biopsies were accrued from 59 patients. A second biopsy was collected from 16 patients on day 3. Study endpoints were pathological complete response (pCR) and residual cancer burden (RCB). Analyses included exome-based mutational signatures, RNA-based TNBC subtyping, immune cell infiltrate scores, and multi-gene proliferation scores, protein and phosphoprotein-based stimulatory and inhibitory immunomodulators. Single-sample Gene Set Enrichment Analysis (ssGSEA) was used for gene set and pathway scoring. Non-parametric tests and outlier analyses were applied to identify differential genes, proteins and pathways. Results: Most cases were PAM50 basal-like and all Lehmann TNBC subtypes were represented. BRCA1/2 and PALB2 homologous recombination (HRD) mutations were observed in 7 cases. Neither subtype, HRD mutation nor HRD mutation signature associated with pCR, but higher mismatch repair defect signature associated with higher RCB. Comparison of baseline pCR vs non-pCR cases showed higher ssGSEA scores for metabolic pathways including oxidative phosphorylation, fatty-acid metabolism, and glycolysis in the non-pCR cases at the protein but not at mRNA level. Non-pCR cases were associated with chromosomal deletions in chemokine receptors, JAK2, and PD-L1, lower PD-L1 protein levels, and lower immune activation. Consistently, ssGSEA scores for interferon alpha and gamma response pathways were higher in pCR cases. Phospho-PD-L1 levels were anti-correlated with developmental pathways. Matched comparisons of baseline and on-treatment samples revealed increase in proteins involved in cell cycle, DNA replication, and mismatch repair following treatment. Metabolic proteins were also upregulated following treatment, while complement activation, immune, and cell adhesion-related proteins were downregulated. Conclusion: Microscaled proteogenomic analysis revealed a wealth of biological features associated with chemotherapy resistance beyond immune response markers, including metabolic features that are only present at the protein level. These data suggests that the development of a microscaled proteogenomic chemotherapy response predictor is a feasible objective for future studies. Citation Format: Meenakshi Anurag, Eric Jaehnig, Shankha Satpathy, Karsten krug, Jonathan T. Lei, Yongchao Dou, Beom-Jun Kim, Cathy M. Sullivan, D. R. Mani, Erik J. Bergstrom, Gloria V. Echeverria, Ian S. Hagemann, Kristen Otte, Henry Rodriguez, Ana I. Robles, Michael T. Lewis, Michael Gillette, Bing Zhang, Mothaffar F. Rimawi, Steven Carr, Foluso O. Ademuyiwa, Matthew J. Ellis. Molecular dissection of chemotherapy response in triple negative breast cancer (TNBC) using microscaled proteogenomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 18.

Introduction: Cytotoxic chemotherapy for sporadic Triple Negative Breast Cancer (TNBC) remains the standard of care and the recent approval for adjuvant PD1 therapy is not biomarker guided. Pathological complete response (pCR) is often not achieved and portends poor survival. Predictive markers for individual drugs have proven elusive. Approach: Microscaled proteogenomics (MPG) was applied to snap-frozen TNBC clinical trial core needle biopsies obtained before treatment with carboplatin and docetaxel (WashU: NCT201404107 and BCM: NCT02544987). Clinical endpoints for discovery analysis were pathological complete response (pCR) and residual cancer burden (RCB). Standard non-parametric statistical tests were employed to identify proteogenomic features associated with these endpoints. Results: Copy number aberrations (CNA) are a recurrent feature of TNBC and a potential driver of chemotherapy sensitivity. We therefore sought CNA with concordant changes at the mRNA and protein levels that also associate with pCR status. Genes located within a recurrent interstitial deletion at chromosomal location 19q13.3 were the most significantly down-regulated at mRNA and protein level in chemotherapy resistant cases. 19q13.3 encodes multiple DNA damage response (DDR) genes; however, only LIG1, a DNA ligase required for lagging strand synthesis and DNA repair, showed concordant changes at both the mRNA and protein level. In multiple independent TNBC data sets, LIG1 deletion was associated lack of pCR and poor metastasis-free survival. Additionally in the BrighTNess TNBC trial lower LIG1 mRNA levels were associated with increased chemotherapy resistance in the carboplatin containing arms (no pCR and residual cancer burden I-III; p=0.0008 and 0.003 respectively). In PDX-derived short-term cultures and PDXs treated with docetaxel or carboplatin, a specific association of carboplatin resistance with LIG1 deletion was observed. LIG1 depleted-tumors did not harbor elevated scores for homologous recombination defect signature, suggesting LIG1 loss is an orthogonal pathway for TNBC pathogenesis The high chromosomal instability index in LIG1 deletion tumors in our TNBC study was robustly reproduced in multiple datasets (including TCGA-BRCA ; Metastatic breast cancer project). LIG1 copy number deletion was also associated with poor progression free survival, and high chromosomal instability in multiple other cancers (including TCGA-​UCEC HR=2.23, TCGA-HNSC HR=1.46, TCGA-PRAD HR=2.07, TCGA- COAD HR=1.75 and TCGA-KIRP HR=4.00). Conclusion: Deletion of LIG1 is associated with chromosomal instability in TNBC and occurs in tumors without genomic evidence for defects in homologous recombination. Other clinical features of LIG1 deleted TNBC and how LIG1 loss may cause chromosomal instability and tumorigenesis will be discussed. Citation Format: Meenakshi Anurag, Eric Jaehnig, Jonathan Lei, Beom-Jun Kim, Anh Minh Tran Huynh, Yongchao Dou, Tanmayi Vashist, Erik Bergstrom, Xuxu Gou, Viktoriya Korchina, Donna Marie Muzny, Kristen Otte, Harshavardhan Doddapaneni, Lacey Dobrolecki, Gloria Vittone Echeverria, Bora Lim, Mothaffar Rimawi, Karsten Krug, Ian Hageman, Henry Rodriguez, Ana I. Robles, Tara Hiltke, Kent Osborne, Michael Gillette, George Miles, Steven Carr, Michael T Lewis, Bing Zhang, Foluso Ademuyiwa, Shankha Satpathy, Matthew J. Ellis. LIG1 deletion predicts chemotherapy resistance, chromosomal instability, and poor prognosis in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1010.

Paired Box 8 (PAX8) is a transcription factor clinically utilized as a histologic biomarker for cancers arising in tissues derived from the embryonic Mullerian tract. Although dysregulated PAX8 expression has recently been shown to promote the growth of ovarian and renal cancers, its role in uterine cancer remains poorly understood. Here, we hypothesized that overexpression of PAX8 promotes uterine cancer growth and metastasis. When 108 uterine cancer specimens were evaluated by mass spectroscopy, the highest levels of PAX8 were found in TP53-mutated, copy number (CN)-high specimens. In vitro, we found that targeting PAX8 expression resulted in decreased rates of both proliferation and apoptosis in multiple representative cell lines (ARK2 and ARK4). PAX8 knockdown also negatively impacted rates of in vitro migration and invasion assessed using commercially available Boyden chamber assays. Using whole-exome and transcriptomic profiling performed by Next Generation Sequencing, we found that PAX8 knockdown resulted in increased expression of 268 gene products by >1.5-fold, and decreased expression of 179 gene products (adj p Citation Format: Lauren Karnolt, Maja Okuka, Susan Read, Yongchao Dou, Bing Zhang, Thomas J. Rutherford, Matthew L. Anderson. The role of PAX8 in copy number-high uterine cancers: Inflammatory cytokine modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1317.

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. Wang et al. perform integrated proteogenomic analysis of adult glioblastoma (GBM), including metabolomics, lipidomics, and single nuclei RNA-Seq, revealing insights into the immune landscape of GBM, cell-specific nature of EMT signatures, histone acetylation in classical GBM, and the existence of signaling hubs which could provide therapeutic vulnerabilities.

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.

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.

Summary 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., Graphical abstract, Highlights • Extensive collection of 43 immunopeptidomic datasets with 1018 samples • Standardized and rigorous identification of HLA-bound peptides, including PTM peptides • Comprehensive annotation of CT antigens and cancer-associated antigens • User-friendly data dissemination through the caAtlas web portal, Immunology; Proteomics; Cancer

At the core of immunotherapy is the T cell recognition of tumor antigens. Thus, comprehensive characterization of tumor antigens is essential for the design of effective and safe cancer immunotherapy strategies. Mass spectrometry (MS)-based immunopeptidomics allows high-throughput, direct identification of major histocompatibility complex (MHC)-bound peptides in vivo. Here, we collected 42 published human immunopeptidomic datasets, which include 327 cancer samples covering nine cancer types and 216 non-cancerous samples. A systematic, quality-controlled analysis of all 51.6 million MS/MS spectra using an open search algorithm identified 364,283 peptides, including 11,647 modified peptides associated with 80 types of modifications. Unmodified peptides showed high levels of predicted binding affinities by NetMHCpan; however, the predicted binding affinities for the modified peptides were much lower, suggesting a deficiency of existing computational tools in making predictions for modified peptides. Analysis of the 416 phosphorylated and 1,239 acetylated HLA class I peptides revealed distinct sequence motifs, and the pattern for the phosphopeptides was consistent with previous reports. Further interrogation of all identified peptides revealed evidence for tumor specific-presentation of hundreds of known and novel cancer-testis antigens as well as tumor associated antigens. We also found non-tumor-specific presentation of 44 previously annotated cancer-testis antigens. Applying NeoQuery, a newly developed computational pipeline, to our dataset identified immunopeptidomic evidence for more than 1,000 somatic mutation-derived neoantigens, and a subset of them were further validated through a traditional MS/MS peptide identification pipeline. We make all these data together with annotated MS/MS spectra supporting each individual antigen identification in a publicly accessible, easily browsable web portal named cancer antigen atlas (caAtlas, http://www.zhang-lab.org/caatlas/). caAtlas provides a fundamental resource for the selection and prioritization of MHC-bound peptides for immunogenicity testing and cancer immunotherapy development. Citation Format: Xinpei Yi, Yuxing Liao, Bo Wen, Kai Li, Yongchao Dou, Sara R. Savage, Bing Zhang. caAtlas: An immunopeptidome atlas of human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1895.

Schizaphis graminum (green bug; GB) and Sipha flava (yellow sugarcane aphid; YSA) are two cereal aphid species with broad host ranges capable of establishing on sorghum (Sorghum bicolor) and several switchgrass (Panicum virgatum) cultivars. Switchgrass and sorghum are staple renewable bioenergy crops that are vulnerable to damage by aphids, therefore, identifying novel targets to control aphids has the potential to drastically improve yields and reduce losses in these bioenergy crops. Despite the wealth of genomic and transcriptomic information available from a closely related model aphid species, the pea aphid (Acyrthosiphon pisum), similar genomic information, including the identification of small RNAs, is still limited for GB and YSA. Deep sequencing of miRNAs expressed in GB and YSA was conducted and 72 and 56 miRNA candidates (including 14 and eight novel) were identified, respectively. Of the identified miRNAs, 45 were commonly expressed in both aphid species. Further, plant derived miRNAs were also detected in both aphid samples, including 13 (eight known and five novel) sorghum miRNAs and three (novel) barley miRNAs. In addition, potential aphid gene targets for the host plant-derived miRNAs were predicted. The establishment of miRNA repertoires in these two aphid species and the detection of plant-derived miRNA in aphids will ultimately lead to a better understanding of the role of miRNAs in regulating gene expression networks in these two aphids and the potential roles of plant miRNAs in mediating plant-insect interactions.

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.

Purpose: Assess the suitability of PAX8 as a therapeutic target in copy number (CNV)-high uterine cancers. Methods: Patterns of gene expression and somatic mutations were evaluated across 97 specimens of uterine cancers characterized by the National Institute of Health’s Clinical Proteomics Tumor Analysis Consortium. Three established USC cell lines (ARK1, ARK2, ARK4) were transfected with small interfering RNAs (siRNAs) targeting PAX8 or a non-targeting siRNA control. Proliferation and apoptosis were evaluated using MTS and Caspase 3/7 assays, respectively. Migration and invasion were evaluated using Boyden chamber assays. PAX8 expression was evaluated using semi-quantitative rtPCR and Western blot. Statistical significance was evaluated using two-tailed t-tests. Results: Our analyses revealed that PAX8 is highly expressed in a subset of uterine cancers and correlates with advanced clinical stage, serous histology and inactivating TP53 mutations. In vitro, knockdown of PAX8 expression resulted in significantly decreased rates of proliferation and apoptosis when compared to cells transfected with non-targeting controls. Decreased proliferation was seen most profoundly at the 72- and 96-hour time points. Enforced reductions in PAX8 expression also decreased rates of tumor migration and invasion. Invasion was significantly decreased in ARK1 (p = 0.05). Migration was significantly decreased in ARK2 (p = 0.002), and the decrease in invasion approached statistical significance (p=0.07). In ARK4, both migration (p = 0.02) and invasion (p = 0.02) were significantly decreased. Knockdown of PAX8 was confirmed using rtPCR for all in vitro experiments. Conclusion: High levels of PAX8 expression are a key feature of a CNV-high uterine cancers that drive tumor proliferation, invasion and migration. These results indicate that PAX8 overexpression contributes to the aggressive clinical behavior of these cancers. They also establish that agents targeting PAX8 activity or expression could ultimately prove to be effective therapeutically. Citation Format: Susan H. Read, Matthew L. Anderson, Thomas J. Rutherford, Venkata A. Jonnalagadda, Yongchao Dou, Bing Zhang. PAX8 represents a potential novel therapeutic target in copy number high uterine serous carcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO018.

SUMMARY 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., In Brief Breast cancer is a highly heterogeneous disease with variable outcomes and subtype-driven treatment approaches, making precision medicine a considerable challenge. Proteogenomic analyses of 122 primary breast cancers provide insights into clinically relevant biology, including cell cycle dysregulation, tumor immunogenicity, aberrant metabolism, and heterogeneity in therapeutic target expression., Graphical Abstract

Patients with head and neck squamous cell carcinomas (HNSCCs) are treated with surgery, radiation, chemotherapy, and limited targeted therapies. Compared to human papillomavirus (HPV)-positive HNSCCs, HPV-negative cases have worse treatment response and prognosis and represent an unmet clinical need. We performed comprehensive proteogenomic characterization of tumor specimens, matched normal adjacent tissues (NATs), and blood samples from 109 HPV-negative HNSCC patients. This cohort is dominated by tumors from oral cavity (45, 41%) and larynx (49, 45%). Somatic mutation and somatic copy number analyses validated previously reported genomic aberrations in HPV-negative HNSCC. Proteomics analysis linked p53 loss of heterozygosity to increased expression of EPCAM, a stemness marker. Additionally, FAT1 truncation mutations were associated with increased expression of proteins involved in keratinization, a key feature of SCC differentiation. Deletions of 3p and 9p led to the loss of genes encoding p16, chemokine receptors, and interferon/JAK/STAT signaling pathway proteins, whereas amplifications of 3q and 11q led to overexpression of proteins involved in cell proliferation and anti-apoptosis pathways. Comparative analysis of tumor and NAT proteomes and phosphoproteomes identified putative diagnostic biomarkers and druggable targets, and proteogenomic integration further identified putative neoantigens. Tumor site-specific characterization associated epigenetic silencing of neurofilaments with laryngeal but not oral cavity SCC. Protein targets of FDA approved or investigational drugs for HNSCC treatment showed high inter-tumor heterogeneity in their protein abundances. DNA copy number and RNA expression level were good surrogates of protein abundance for some targets, such as EGFR and PD-L1, but they failed to reflect protein levels or kinase activities for other targets, such as MMP9 and MTOR. Thus, there is a critical need for protein biomarker-driven treatment stratification. Deconvolution of bulk tumor gene expression data revealed an immune-hot subgroup and an immune-cold subgroup. Immune-hot tumors broadly overexpressed multiple immune checkpoints including PD-L1, IDO1, and CTLA4, underscoring the necessity of combination immune checkpoint inhibition to improve treatment efficacy. Immune-cold tumors were characterized by smoking, chromosomal instability, and activation of the CDK4/6-pRb axis, suggesting they could be targeted by CDK4/6 inhibitors. We also noted that EGFR-amplified tumors frequently harbor copy number aberrations of downstream signaling components of the EGFR pathway. This may explain the low response rate of EGFR-amplified tumors to EGFR inhibitors, and targeting multiple pathway components, including EGFR, PIK3CA and STAT3, may be required for these tumors. In summary, our integrative proteogenomic characterization revealed multiple novel insights into the pathogenesis and treatment of HPV-negative HNSCCs. Citation Format: Chen Huang, Lijun Chen, Yize Li, Sara Savage, Michael Schnaubelt, Felipe V. Leprevost, Marcin Cieslik, Yongchao Dou, Bo Wen, Jonathan T. Lei, Kai Li, Eric Jaehnig, Zhiao Shi, Meenakshi Anurag, Jianbo Pan, Yingwei Hu, Rodrigo V. Eguez, David J. Clark, Matthew Wyczalkowski, Saravana M. Dhanasekaran, Chandan Kumar, Antonio Colaprico, Karsten Krug, Michael Gillette, D. R. Mani, Seungyeul Yoo, Jiayi Ji, Xiaoyu Song, Weiping Ma, Xi Steven Chen, Alex Pico, Nathan J. Edwards, Scott D. Jewell, Mathangi Thiagarajan, Emily S. Boja, Henry Rodriguez, Andrew Sikora, Pei Wang, Matthew Ellis, Gilbert S. Omenn, Li Ding, Alexey I. Nesvizhskii, Adel K. EI-Naggar, Daniel W. Chan, Hui Zhang, Bing Zhang, Clinical Proteomic Tumor Analysis Consortium. Proteogenomics characterization of HPV-negative head and neck squamous cell carcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5118.

Endometrial cancer (EC) is the sixth most common cancer in women globally and is one of the few cancers for which mortality is increasing; to address the need to develop new methods for the diagnosis and treatment of this disease, we performed a comprehensive proteogenomic characterization of 95 endometrial carcinomas. We prospectively collected 83 endometrioid and 12 serous tumors, along with 49 normal tissue samples, for multi-omic characterization. Each sample underwent whole exome sequencing, whole genome sequencing, total RNA and miRNA sequencing, and DNA methylation array analyses. In addition, we evaluated all samples for protein, phosphorylation, and acetylation levels using an integrated, isobaric tandem mass tag labeling–based quantitative proteomics workflow. This dataset, publicly available through the Genomic Data Commons, CPTAC data portal, and other sources, provides a valuable resource for researchers and clinicians. Our analyses of these tumors revealed, among other things, a potential role for circRNAs in the epithelial-mesenchymal transition and new information about proteomic markers of clinical and genomic tumor subgroups, including relationships to known druggable pathways. We found possible new consequences of perturbations to the Wnt/β-catenin pathway, particularly CTNNB1 hotspot mutations; moreover, from the genome-wide acetylation survey, we found potential regulatory mechanisms linking Wnt signaling and histone acetylation. Phosphorylation data analysis suggests an elevated DNA damage response in serous tumors, which we hypothesize may be targetable by certain FDA-approved drugs. We found that TP53 hotspot mutations play unique trans-acting roles as compared to the same mutations in colon and ovarian cancers. 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; of particular translational interest, we identified a subset of patients who, despite having high amounts of microsatellite instability, exhibit biomarkers which suggest that immunotherapy would be an ineffective treatment for their disease. By integrating global measurements of protein abundance, phosphorylation, and acetylation with next-generation genomics and transcriptomics, we have provided novel insights into biological processes underlying the development of endometrial cancers and generated strong hypotheses for new approaches to personalized treatment of individuals with endometrial cancer. Citation Format: Emily A. Kawaler, Yongchao Dou, Daniel Cui Zhou, Marina A. Gritsenko, Karin D. Rodland, Li Ding, Bing Zhang, Tao Liu, David Fenyö, Clinical Proteomic Tumor Analysis Consortium. Proteogenomic characterization of endometrial carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6580.

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.

The steady-state levels of miRNAs are under sophisticated control to ensure their proper functions such as development and responses to environmental stimuli. Nevertheless, enzymes responsible for the degradation of various forms of unmethylated miRNAs remain enigmatic, which largely impedes our understanding of miRNA homeostasis and active turnover. Here we report a 3′ to 5′ exoribonuclease Atrimmer 2 that may degrade unmethylated miRNAs in their miRNA/miRNA* duplex status, at places distinct from their production sites (i.e., Dicing bodies). Our study not only increases the complexity of miRNA surveillance, but also provides clues into how nascent miRNA/miRNA* duplexes undergo methylation and RNA-induced silencing complex loading, which is a big challenge in the plant small RNA field.

The 3' end methylation catalyzed by HUA Enhancer 1 (HEN1) is a crucial step of small RNA stabilization in plants, yet how unmethylated small RNAs undergo degradation remains largely unknown. Using a reverse genetic approach, we here show that Atrimmer 2 (ATRM2), a DEDDy-type 3' to 5' exoribonuclease, acts in the degradation of unmethylated miRNAs and miRNA*s in Arabidopsis Loss-of-function mutations in ATRM2 partially suppress the morphological defects caused by HEN1 malfunction, with restored levels of a subset of miRNAs and receded expression of corresponding miRNA targets. Dysfunction of ATRM2 has negligible effect on miRNA trimming, and further increase the fertility of hen1 heso1 urt1, a mutant with an almost complete abolishment of miRNA uridylation, indicating that ATRM2 may neither be involved in 3' to 5' trimming nor be the enzyme that specifically degrades uridylated miRNAs. Notably, the fold changes of miRNAs and their corresponding miRNA*s were significantly correlated in hen1 atrm2 versus hen1 Unexpectedly, we observed a marked increase of 3' to 5' trimming of several miRNA*s but not miRNAs in ATRM2 compromised backgrounds. These data suggest an action of ATRM2 on miRNA/miRNA* duplexes, and the existence of an unknown exoribonuclease for specific trimming of miRNA*. This asymmetric effect on miRNA/miRNA* is likely related to Argonaute (AGO) proteins, which can distinguish miRNAs from miRNA*s. Finally, we show that ATRM2 colocalizes and physically interacts with Argonaute 1 (AGO1). Taken together, our results suggest that ATRM2 may be involved in the surveillance of unmethylated miRNA/miRNA* duplexes during the initiation step of RNA-induced silencing complex assembly.

DAWDLE (DDL) is a conserved forkhead-associated (FHA) domain-containing protein with essential roles in plant development and immunity. It acts in the biogenesis of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs), which regulate gene expression at the transcriptional and/or posttranscriptional levels. However, the functional mechanism of DDL and its impact on exogenous siRNAs remain elusive. Here, we report that DDL is required for the biogenesis of siRNAs derived from sense transgenes and inverted-repeat transgenes. Furthermore, we show that a mutation in the FHA domain of DDL disrupts the interaction of DDL with DICER-LIKE1 (DCL1), which is the enzyme that catalyzes miRNA maturation from primary miRNA transcripts (pri-miRNAs), resulting in impaired pri-miRNA processing. Moreover, we demonstrate that DDL interacts with DCL3, which is a DCL1 homolog responsible for siRNA production, and this interaction is crucial for optimal DCL3 activity. These results reveal that the interaction of DDL with DCLs is required for the biogenesis of miRNAs and siRNAs in Arabidopsis (Arabidopsis thaliana).

Cooperativity between WNT and FGF signaling is well documented in embryonic development and cancer progression, but the molecular mechanisms underlying this cross-talk remain elusive. In this study, we interrogated the dynamics of RNA levels, ribosome occupancy, and protein expression as a function of inducible FGF signaling in mouse mammary glands with constitutive WNT hyperactivation. Multiomics correlation analysis revealed a substantial discrepancy between RNA and ribosome occupancy levels versus protein levels. However, this discrepancy decreased as cells became premalignant and dynamically responded to FGF signaling, implicating the importance of stringent gene regulation in nontransformed cells. Analysis of individual genes demonstrated that acute FGF hyperactivation increased translation of many stem cell self-renewal regulators, including WNT signaling components, and decreased translation of genes regulating cellular senescence. WNT pathway components translationally upregulated by FGF signaling had long and structured 5′ UTRs with a high frequency of polypurine sequences, several of which harbored (CGG)4 motifs that can fold into either stable G-quadruplexes or other stable secondary structures. The FGF-mediated increase in translation of WNT pathway components was compromised by silvestrol, an inhibitor of EIF4A that clamps EIF4A to polypurine sequences to block 43S scanning and inhibits its RNA-unwinding activity important for translation initiation. Moreover, silvestrol treatment significantly delayed FGF-WNT–driven tumorigenesis. Taken together, these results suggest that FGF signaling selectively enhances translation of structured mRNAs, particularly WNT signaling components, and highlight their vulnerability to inhibitors that target the RNA helicase EIF4A. Significance: The RNA helicase EIF4A may serve as a therapeutic target for breast cancers that require FGF and WNT signaling. Cancer Res; 78(15); 4229–40. ©2018 AACR.

To improve the applicability of RNA-seq technology, a large number of RNA-seq data analysis methods and correction algorithms have been developed. Although these new methods and algorithms have steadily improved transcriptome analysis, greater prediction accuracy is needed to better guide experimental designs with computational results. In this study, a new tool for the identification of differentially expressed genes with RNA-seq data, named GExposer, was developed. This tool introduces a local normalization algorithm to reduce the bias of nonrandomly positioned read depth. The naive Bayes classifier is employed to integrate fold change, transcript length, and GC content to identify differentially expressed genes. Results on several independent tests show that GExposer has better performance than other methods. The combination of the local normalization algorithm and naive Bayes classifier with three attributes can achieve better results; both false positive rates and false negative rates are reduced. However, only a small portion of genes is affected by the local normalization and GC content correction.

Background Recently, identification and functional studies of circular RNAs, a type of non-coding RNAs arising from a ligation of 3' and 5' ends of a linear RNA molecule, were conducted in mammalian cells with the development of RNA-seq technology. Method Since compared with animals, studies on circular RNAs in plants are less thorough, a genome-wide identification of circular RNA candidates in Arabidopsis was conducted with our own developed bioinformatics tool to several existing RNA-seq datasets specifically for non-coding RNAs. Results A total of 164 circular RNA candidates were identified from RNA-seq data, and 4 circular RNA transcripts, including both exonic and intronic circular RNAs, were experimentally validated. Interestingly, our results show that circular RNA transcripts are enriched in the photosynthesis system for the leaf tissue and correlated to the higher expression levels of their parent genes. Sixteen out of all 40 genes that have circular RNA candidates are related to the photosynthesis system, and out of the total 146 exonic circular RNA candidates, 63 are found in chloroplast.