BMP4-Induced Suppression of Breast Cancer Metastasis Is Associated with Inhibition of Cholesterol Biosynthesis.

We reported previously that in preclinical models, BMP4 is a potent inhibitor of breast cancer metastasis and that high BMP4 protein levels predict favourable patient outcomes. Here, we analysed a breast cancer xenograft with or without enforced expression of BMP4 to gain insight into the mechanisms by which BMP4 suppresses metastasis. Transcriptomic analysis of cancer cells recovered from primary tumours and phosphoproteomic analyses of cancer cells exposed to recombinant BMP4 revealed that BMP4 inhibits cholesterol biosynthesis, with many genes in this biosynthetic pathway being downregulated by BMP4. The treatment of mice bearing low-BMP4 xenografts with a cholesterol-lowering statin partially mimicked the anti-metastatic activity of BMP4. Analysis of a cohort of primary breast cancers revealed a reduced relapse rate for patients on statin therapy if their tumours exhibited low BMP4 levels. These findings indicate that BMP4 may represent a predictive biomarker for the benefit of additional statin therapy in breast cancer patients.

Identification of biological pathways and processes regulated by NEK5 in breast epithelial cells via an integrated proteomic approach.

Specific members of the Nima-Related Kinase (NEK) family have been linked to cancer development and progression, and a role for NEK5, one of the least studied members, in breast cancer has recently been proposed. However, while NEK5 is known to regulate centrosome separation and mitotic spindle assembly, NEK5 signalling mechanisms and function in this malignancy require further characterization. To this end, we established a model system featuring overexpression of NEK5 in the immortalized breast epithelial cell line MCF-10A. MCF-10A cells overexpressing NEK5 exhibited an increase in clonogenicity under monolayer conditions and enhanced acinar size and abnormal morphology in 3D Matrigel culture. Interestingly, they also exhibited a marked reduction in Src activation and downstream signalling. To interrogate NEK5 signalling and function in an unbiased manner, we applied a variety of MS-based proteomic approaches. Determination of the NEK5 interactome by Bio-ID identified a variety of protein classes including the kinesins KIF2C and KIF22, the mitochondrial proteins TFAM, TFB2M and MFN2, RhoH effectors and the negative regulator of Src, CSK. Characterization of proteins and phosphosites modulated upon NEK5 overexpression by global MS-based (phospho)proteomic profiling revealed impact on the cell cycle, DNA synthesis and repair, Rho GTPase signalling, the microtubule cytoskeleton and hemidesmosome assembly. Overall, the study indicates that NEK5 impacts diverse pathways and processes in breast epithelial cells, and likely plays a multifaceted role in breast cancer development and progression. Video Abstract.

Proteomics-based interrogation of the kinome and its implications for precision oncology.

The identification of specific protein kinases as oncogenic drivers in a variety of cancer types, coupled with the clinical success of particular kinase-directed targeted therapies, has cemented the human kinome as an attractive source of "actionable" targets for cancer therapy. However, "mining" of the human kinome for precision oncology applications has yet to yield its full potential. This reflects a variety of issues, including oncogenic kinase dysregulation at levels not detectable by genomic sequencing and the uncharacterized nature of a considerable fraction of the kinome. In addition, selective therapeutic targeting of specific kinases requires efficient mapping of total kinome space impacted by candidate small molecule drugs. Fortunately, recent developments in proteomics techniques, particularly in mass spectrometry-based phosphoproteomics and kinomics, provide the necessary technology platforms to address these impediments. Moreover, initiatives such as the Clinical Proteomic Tumour Analysis Consortium have enabled the generation, deposition and integration of genomic, transcriptomic and (phospho)proteomic data for many cancer types, providing unprecedented insights into oncogenic kinases and cancer cell signalling generally. These multi-omic data are identifying novel therapeutic targets, highlighting opportunities for drug re-purposing, and helping assign optimal therapies to specific tumour subtypes, heralding a new era of "enhanced" precision oncology.

Evaluation of FGFR targeting in breast cancer through interrogation of patient-derived models.

BACKGROUND: Particular breast cancer subtypes pose a clinical challenge due to limited targeted therapeutic options and/or poor responses to the existing targeted therapies. While cell lines provide useful pre-clinical models, patient-derived xenografts (PDX) and organoids (PDO) provide significant advantages, including maintenance of genetic and phenotypic heterogeneity, 3D architecture and for PDX, tumor-stroma interactions. In this study, we applied an integrated multi-omic approach across panels of breast cancer PDXs and PDOs in order to identify candidate therapeutic targets, with a major focus on specific FGFRs. METHODS: MS-based phosphoproteomics, RNAseq, WES and Western blotting were used to characterize aberrantly activated protein kinases and effects of specific FGFR inhibitors. PDX and PDO were treated with the selective tyrosine kinase inhibitors AZD4547 (FGFR1-3) and BLU9931 (FGFR4). FGFR4 expression in cancer tissue samples and PDOs was assessed by immunohistochemistry. METABRIC and TCGA datasets were interrogated to identify specific FGFR alterations and their association with breast cancer subtype and patient survival. RESULTS: Phosphoproteomic profiling across 18 triple-negative breast cancers (TNBC) and 1 luminal B PDX revealed considerable heterogeneity in kinase activation, but 1/3 of PDX exhibited enhanced phosphorylation of FGFR1, FGFR2 or FGFR4. One TNBC PDX with high FGFR2 activation was exquisitely sensitive to AZD4547. Integrated 'omic analysis revealed a novel FGFR2-SKI fusion that comprised the majority of FGFR2 joined to the C-terminal region of SKI containing the coiled-coil domains. High FGFR4 phosphorylation characterized a luminal B PDX model and treatment with BLU9931 significantly decreased tumor growth. Phosphoproteomic and transcriptomic analyses confirmed on-target action of the two anti-FGFR drugs and also revealed novel effects on the spliceosome, metabolism and extracellular matrix (AZD4547) and RIG-I-like and NOD-like receptor signaling (BLU9931). Interrogation of public datasets revealed FGFR2 amplification, fusion or mutation in TNBC and other breast cancer subtypes, while FGFR4 overexpression and amplification occurred in all breast cancer subtypes and were associated with poor prognosis. Characterization of a PDO panel identified a luminal A PDO with high FGFR4 expression that was sensitive to BLU9931 treatment, further highlighting FGFR4 as a potential therapeutic target. CONCLUSIONS: This work highlights how patient-derived models of human breast cancer provide powerful platforms for therapeutic target identification and analysis of drug action, and also the potential of specific FGFRs, including FGFR4, as targets for precision treatment.

Allelic association with ankylosing spondylitis fails to correlate with human leukocyte antigen B27 homodimer formation.

Expression of human leukocyte antigen (HLA)-B27 is strongly associated with predisposition toward ankylosing spondylitis (AS) and other spondyloarthropathies. However, the exact involvement of HLA-B27 in disease initiation and progression remains unclear. The homodimer theory, which proposes that HLA-B27 heavy chains aberrantly form homodimers, is a central hypothesis that attempts to explain the role of HLA-B27 in disease pathogenesis. Here, we examined the ability of the eight most prevalent HLA-B27 allotypes (HLA-B*27:02 to HLA-B*27:09) to form homodimers. We observed that HLA-B*27:03, a disease-associated HLA-B27 subtype, showed a significantly reduced ability to form homodimers compared with all other allotypes, including the non-disease-associated/protective allotypes HLA-B*27:06 and HLA-B*27:09. We used X-ray crystallography and site-directed mutagenesis to unravel the molecular and structural mechanisms in HLA-B*27:03 that are responsible for its compromised ability to form homodimers. We show that polymorphism at position 59, which differentiates HLA-B*27:03 from all other allotypes, is responsible for its compromised ability to form homodimers. Indeed, histidine 59 in HLA-B*27:03 leads to a series of local conformational changes that act in concert to reduce the accessibility of the nearby cysteine 67, an essential amino acid residue for the formation of HLA-B27 homodimers. Considered together, the ability of both protective and disease-associated HLA-B27 allotypes to form homodimers and the failure of HLA-B*27:03 to form homodimers challenge the role of HLA-B27 homodimers in AS pathoetiology. Rather, this work implicates other features, such as peptide binding and antigen presentation, as pivotal mechanisms for disease pathogenesis.

Human Leukocyte Antigen (HLA) B27 Allotype-Specific Binding and Candidate Arthritogenic Peptides Revealed through Heuristic Clustering of Data-independent Acquisition Mass Spectrometry (DIA-MS) Data.

Expression of HLA-B27 is strongly associated with ankylosing spondylitis (AS) and other spondyloarthropathies. While this is true for the majority of HLA-B27 allotypes, HLA-B*27:06 and HLA-B*27:09 are not associated with AS. These two subtypes contain polymorphisms that are ideally positioned to influence the bound peptide repertoire. The existence of disease-inducing peptides (so-called arthritogenic peptides) has therefore been proposed that are exclusively presented by disease-associated HLA-B27 allotypes. However, we have recently demonstrated that this segregation of allotype-bound peptides is not the case and that many peptides that display sequence features predicted to favor binding to disease-associated subtypes are also capable of being presented naturally by protective alleles. To further probe more subtle quantitative changes in peptide presentation, we have used a combination of data-independent acquisition (DIA) and multiple reaction monitoring (MRM) mass spectrometry to quantify the abundance of 1646 HLA-B27 restricted peptides across the eight most frequent HLA-B27 allotypes (HLA-B*27:02-HLA-B*27:09). We utilized K means cluster analysis to group peptides with similar allelic binding preferences across the eight HLA-B27 allotypes, which enabled us to identify the most-stringent binding characteristics for each HLA-B27 allotype and further refined their existing consensus-binding motifs. Moreover, a thorough analysis of this quantitative dataset led to the identification of 26 peptides, which are presented in lower abundance by HLA-B*27:06 and HLA-B*27:09 compared with disease-associated HLA-B27 subtypes. Although these differences were observed to be very subtle, these 26 peptides might encompass the sought-after arthritogenic peptide(s).

A Semiautomated Proteomics and Phosphoproteomics Protocol for the Identification of Novel Therapeutic Targets and Predictive Biomarkers in In Vivo Xenograft Models of Pediatric Cancers.

Genomic profiling has identified therapeutic targets for precision treatment of certain cancers, but many patients lack actionable mutations. Additional omics approaches, like proteomics and phosphoproteomics, are essential for comprehensive mapping of cancer-associated molecular phenotypes. In vivo models, such as cell line and patient-derived xenografts (PDX), offer valuable insights into cancer biology and treatment strategies.This chapter presents a semiautomated high-throughput workflow for integrated proteomics and phosphoproteomics analysis on the Kingfish platform coupled with MagReSyn® Zr-IMAC HP. It enhances protein extraction from in vivo xenograft samples and provides better insights into cancers with poor prognosis. The approach successfully identified over 11,000 unique phosphosites and ~6000 proteins in SJSA-1 pediatric osteosarcoma xenografts, demonstrating its efficacy. This workflow is a valuable tool for studying tumor biology and developing precision oncology strategies.

PRMT1 acts as a suppressor of MHC-I and anti-tumor immunity.

Cancer immunotherapies have demonstrated remarkable success; however, the majority of patients do not respond or develop resistance. Here, we conduct epigenetic gene-targeted CRISPR-Cas9 screens to identify epigenomic factors that limit CD8+ T cell-mediated anti-tumor immunity. We identify that PRMT1 suppresses interferon gamma (Ifnγ)-induced MHC-I expression, thus dampening CD8+ T cell-mediated killing. Indeed, PRMT1 knockout or pharmacological targeting of type I PRMT with the clinical inhibitor GSK3368715 enhances Ifnγ-induced MHC-I expression through elevated STAT1 expression and activation, while re-introduction of PRMT1 in PRMT1-deficient cells reverses this effect. Importantly, loss of PRMT1 enhances the efficacy of anti-PD-1 immunotherapy, and The Cancer Genome Atlas analysis reveals that PRMT1 expression in human melanoma is inversely correlated with expression of human leukocyte antigen molecules, infiltration of CD8+ T cells, and overall survival. Taken together, we identify PRMT1 as a negative regulator of anti-tumor immunity, unveiling clinical type I PRMT inhibitors as immunotherapeutic agents or as adjuncts to existing immunotherapies.

Phosphoproteomics-directed manipulation reveals SEC22B as a hepatocellular signaling node governing metabolic actions of glucagon.

The peptide hormone glucagon is a fundamental metabolic regulator that is also being considered as a pharmacotherapeutic option for obesity and type 2 diabetes. Despite this, we know very little regarding how glucagon exerts its pleiotropic metabolic actions. Given that the liver is a chief site of action, we performed in situ time-resolved liver phosphoproteomics to reveal glucagon signaling nodes. Through pathway analysis of the thousands of phosphopeptides identified, we reveal "membrane trafficking" as a dominant signature with the vesicle trafficking protein SEC22 Homolog B (SEC22B) S137 phosphorylation being a top hit. Hepatocyte-specific loss- and gain-of-function experiments reveal that SEC22B was a key regulator of glycogen, lipid and amino acid metabolism, with SEC22B-S137 phosphorylation playing a major role in glucagon action. Mechanistically, we identify several protein binding partners of SEC22B affected by glucagon, some of which were differentially enriched with SEC22B-S137 phosphorylation. In summary, we demonstrate that phosphorylation of SEC22B is a hepatocellular signaling node mediating the metabolic actions of glucagon and provide a rich resource for future investigations on the biology of glucagon action.

Cell-Type-Specific Signalling Networks Impacted by Prostate Epithelial-Stromal Intercellular Communication.

Prostate cancer is the second most common cause of cancer death in males. A greater understanding of cell signalling events that occur within the prostate cancer tumour microenvironment (TME), for example, between cancer-associated fibroblasts (CAFs) and prostate epithelial or cancer cells, may identify novel biomarkers and more effective therapeutic strategies for this disease. To address this, we used cell-type-specific labelling with amino acid precursors (CTAP) to define cell-type-specific (phospho)proteomic changes that occur when prostate epithelial cells are co-cultured with normal patient-derived prostate fibroblasts (NPFs) versus matched CAFs. We report significant differences in the response of BPH-1 benign prostate epithelial cells to CAF versus NPF co-culture. Pathway analysis of proteomic changes identified significant upregulation of focal adhesion and cytoskeleton networks, and downregulation of metabolism pathways, in BPH-1 cells cultured with CAFs. In addition, co-cultured CAFs exhibited alterations in stress, DNA damage, and cytoskeletal networks. Functional validation of one of the top differentially-regulated proteins in BPH-1 cells upon CAF co-culture, transglutaminase-2 (TGM2), demonstrated that knockdown of this protein significantly reduced the proliferation and migration of prostate epithelial cells. Overall, this study provides novel insights into intercellular communication in the prostate cancer TME that may be exploited to improve patient management.

SAPrIm, a semi-automated protocol for mid-throughput immunopeptidomics.

Human leukocyte antigen (HLA) molecules play a crucial role in directing adaptive immune responses based on the nature of their peptide ligands, collectively coined the immunopeptidome. As such, the study of HLA molecules has been of major interest in the development of cancer immunotherapies such as vaccines and T-cell therapies. Hence, a comprehensive understanding and profiling of the immunopeptidome is required to foster the growth of these personalised solutions. We herein describe SAPrIm, an Immunopeptidomics tool for the Mid-Throughput era. This is a semi-automated workflow involving the KingFisher platform to isolate immunopeptidomes using anti-HLA antibodies coupled to a hyper-porous magnetic protein A microbead, a variable window data independent acquisition (DIA) method and the ability to run up to 12 samples in parallel. Using this workflow, we were able to concordantly identify and quantify ~400 - 13000 unique peptides from 5e5 - 5e7 cells, respectively. Overall, we propose that the application of this workflow will be crucial for the future of immunopeptidome profiling, especially for mid-size cohorts and comparative immunopeptidomics studies.

Engineering Cell Lines for Specific Human Leukocyte Antigen Presentation.

Epitope-specific immunotherapies have enabled the targeted treatment of a variety of diseases, ranging from cancer, infection, and autoimmune disorders. For CD8+ T cell-based therapies, the precise identification of immunogenic peptides presented by human leukocyte antigen (HLA) class I is essential which can be achieved by immunopeptidomics. Here, using lentivirus-mediated transduction and cell sorting approaches, we present a method to engineer a cell line that does not express its native HLA but instead expresses an HLA of interest (in this instance HLA-A*02:01). This technique can be used to elucidate the immunopeptidome of cell lines expressing different HLAs.

The pseudokinase NRBP1 activates Rac1/Cdc42 via P-Rex1 to drive oncogenic signalling in triple-negative breast cancer.

We have determined that expression of the pseudokinase NRBP1 positively associates with poor prognosis in triple negative breast cancer (TNBC) and is required for efficient migration, invasion and proliferation of TNBC cells in culture as well as growth of TNBC orthotopic xenografts and experimental metastasis. Application of BioID/MS profiling identified P-Rex1, a known guanine nucleotide exchange factor for Rac1, as a NRBP1 binding partner. Importantly, NRBP1 overexpression enhanced levels of GTP-bound Rac1 and Cdc42 in a P-Rex1-dependent manner, while NRBP1 knockdown reduced their activation. In addition, NRBP1 associated with P-Rex1, Rac1 and Cdc42, suggesting a scaffolding function for this pseudokinase. NRBP1-mediated promotion of cell migration and invasion was P-Rex1-dependent, while constitutively-active Rac1 rescued the effect of NRBP1 knockdown on cell proliferation and invasion. Generation of reactive oxygen species via a NRBP1/P-Rex1 pathway was implicated in these oncogenic roles of NRBP1. Overall, these findings define a new function for NRBP1 and a novel oncogenic signalling pathway in TNBC that may be amenable to therapeutic intervention.

In-depth mining of the immunopeptidome of an acute myeloid leukemia cell line using complementary ligand enrichment and data acquisition strategies.

The identification of T cell epitopes derived from tumour specific antigens remains a significant challenge for the development of peptide-based vaccines and immunotherapies. The use of mass spectrometry-based approaches (immunopeptidomics) can provide powerful new avenues for the identification of such epitopes. In this study we report the use of complementary peptide antigen enrichment methods and a comprehensive mass spectrometric acquisition strategy to provide in-depth immunopeptidome data for the THP-1 cell line, a cell line used widely as a model of human leukaemia. To accomplish this, we combined robust experimental workflows that incorporated ultrafiltration or off-line reversed phase chromatography to enrich peptide ligand as well as a multifaceted data acquisition strategy using an Orbitrap Fusion LC-MS instrument. Using the combined datasets from the two ligand enrichment methods we gained significant depth in immunopeptidome coverage by identifying a total of 41,816 HLA class I peptides from THP-1 cells, including a significant number of peptides derived from different oncogenes or over expressed proteins associated with cancer. The physicochemical properties of the HLA-bound peptides dictated their recovery using the two ligand enrichment approaches and their distribution across the different precursor charge states considered in the data acquisition strategy. The data highlight the complementarity of the two enrichment procedures, and in cases where sample is not limiting, suggest that the combination of both approaches will yield the most comprehensive immunopeptidome information.

Spliced Peptides and Cytokine-Driven Changes in the Immunopeptidome of Melanoma.

Antigen recognition by CD8+ T cells is governed by the pool of peptide antigens presented on the cell surface in the context of HLA class I complexes. Studies have shown not only a high degree of plasticity in the immunopeptidome, but also that a considerable fraction of all presented peptides is generated through proteasome-mediated splicing of noncontiguous regions of proteins to form novel peptide antigens. Here, we used high-resolution mass spectrometry combined with new bioinformatic approaches to characterize the immunopeptidome of melanoma cells in the presence or absence of IFNγ. In total, we identified more than 60,000 peptides from a single patient-derived cell line (LM-MEL-44) and demonstrated that IFNγ induced changes in the peptidome, with an overlap of only approximately 50% between basal and treated cells. Around 6% to 8% of the peptides were identified as cis-spliced peptides, and 2,213 peptides (1,827 linear and 386 cis-spliced peptides) were derived from known melanoma-associated antigens. These peptide antigens were equally distributed between the constitutive- and IFNγ-induced peptidome. We next examined additional HLA-matched patient-derived cell lines to investigate how frequently these peptides were identified and found that a high proportion of both linear and spliced peptides was conserved between individual patient tumors, drawing on data amassing to more than 100,000 peptide sequences. Several of these peptides showed in vitro immunogenicity across multiple patients with melanoma. These observations highlight the breadth and complexity of the repertoire of immunogenic peptides that can be exploited therapeutically and suggest that spliced peptides are a major class of tumor antigens.