Serial multi-omic analysis of proteome, phosphoproteome, and acetylome provides insights into changes in protein expression, cell signaling, cross-talk and epigenetic pathways involved in disease pathology and treatment. However, ubiquitylome and HLA peptidome data collection used to understand protein degradation and antigen presentation have not together been serialized, and instead require separate samples for parallel processing using distinct protocols. Here we present MONTE, a highly sensitive multi-omic native tissue enrichment workflow, that enables serial, deep-scale analysis of HLA-I and HLA-II immunopeptidome, ubiquitylome, proteome, phosphoproteome, and acetylome from the same tissue sample. We demonstrate that the depth of coverage and quantitative precision of each ‘ome is not compromised by serialization, and the addition of HLA immunopeptidomics enables the identification of peptides derived from cancer/testis antigens and patient specific neoantigens. We evaluate the technical feasibility of the MONTE workflow using a small cohort of patient lung adenocarcinoma tumors.

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

Abstract 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

Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early-phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR/Cas9 screens implicated decreased translation initiation as protective following GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to the effects of GSPT1 degradation. We defined 2 Crbn amino acids that prevent Gspt1 degradation in mice, generated a knockin mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant acute myeloid leukemia.

Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites is necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-ε-GG peptides are enriched with anti-K-ε-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-ε-GG antibody (mK-ε-GG) and a magnetic particle processor. We report the identification of ∼20,000 ubiquitylation sites from a TMT10-plex with 500 μg input per sample processed in ∼2 hours. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility and significantly reduced processing time. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of this method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples.

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.

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.

Serial multiomic analyses of proteome, phosphoproteome and acetylome provides functional insights into disease pathology and drug effects while conserving precious human material. To date, ubiquitylome and HLA peptidome analyses have required separate samples for parallel processing each using distinct protocols. Here we present MONTE, a highly-sensitive m ulti- o mic n ative t issue e nrichment workflow that enables serial, deepscale analysis of HLA-I and HLA-II immunopeptidome, ubiquitylome, proteome, phosphoproteome and acetylome from the same tissue samples. We demonstrate the capabilities of MONTE in a proof-of-concept study of primary patient lung adenocarcinoma(LUAD) tumors. Depth of coverage and quantitative precision at each of the ‘omes is not compromised by serialization, and the addition of HLA immunopeptidomics enables identification of putative immunotherapeutic targets such as cancer/testis antigens and neoantigens. MONTE can provide insights into disease-specific changes in antigen presentation, protein expression, protein degradation, cell signaling, cross-talk and epigenetic pathways involved in disease pathology and treatment.