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Table S5: The protein expression matrix used for input in the Principle Component Analysis. Table S6: The list of gene ontology terms in each cluster observed in the SAFE network. Table S7: The regression coefficients and constants of each protein in each proteomics subtype in the Partial Least Squares-Discriminant Analysis. Table S8: The gene ontology analysis of Protein Cluster 1 from ClueGO. Table S9: The gene ontology analysis of Protein Cluster 2 from ClueGO. Table S10: The gene ontology analysis of Protein Cluster 3 from ClueGO. Table S11: The Reactome pathway enrichment analysis on the average protein expression of the 4 proteomics subtypes. Table S12: The tertile assignments of each of the 916 proteins used for the analysis in each 56 samples. Table S13: The comparative analysis of the gemcitabine-treated proteome.

Purpose:Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease that can be separated into distinct subtypes based on molecular signatures. Identifying PDAC subtype-specific therapeutic vulnerabilities is necessary to develop precision medicine approaches to treat PDAC.Experimental Design:A total of 56 PDAC liver metastases were obtained from the UNMC Rapid Autopsy Program and analyzed with quantitative proteomics. PDAC subtypes were identified by principal component analysis based on protein expression profiling. Proteomic subtypes were further characterized by the associated clinical information, including but not limited to survival analysis, drug treatment response, and smoking and drinking status.Results:Over 3,960 proteins were identified and used to delineate four distinct PDAC microenvironment subtypes: (i) metabolic; (ii) progenitor-like; (iii) proliferative; and (iv) inflammatory. PDAC risk factors of alcohol and tobacco consumption correlate with subtype classifications. Enhanced survival is observed in FOLFIRINOX treated metabolic and progenitor-like subtypes compared with the proliferative and inflammatory subtypes. In addition, TYMP, PDCD6IP, ERAP1, and STMN showed significant association with patient survival in a subtype-specific manner. Gemcitabine-induced alterations in the proteome identify proteins, such as serine hydroxymethyltransferase 1, associated with drug resistance.Conclusions:These data demonstrate that proteomic analysis of clinical PDAC liver metastases can identify molecular signatures unique to disease subtypes and point to opportunities for therapeutic development to improve the treatment of PDAC.

Figure S6: Protein expression and gene ontology analysis related to PDAC subtypes. Figure S7: The characterization of Protein Cluster 1. Figure S8: The characterization of Protein Cluster 2. Figure S9: The characterization of Protein Cluster 3. Figure S10: The Kaplan-Meier curves of patients separated based on individual PDAC liver metastasis subtypes.

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Table S4: The total list of peptides identified.

Tumor necrosis factor (TNF) α-induced nuclear translocation of the NF-κB subunit RELA has been implicated in several pathological conditions. Here we report the discovery of a spirocyclic dimer (SpiD7) that covalently modifies RELA to inhibit TNFα-induced nuclear translocation. This is a previously unexplored strategy to inhibit TNFα-induced NF-κB activation., Discovery of a spirocyclic dimer (SpiD7) that covalently modifies RELA to generate stable high molecular weight complexes. SpiD7 inhibits TNFα-induced nuclear translocation of RELA resulting in the blockade of NF-kB gene transcription, through a previously unexplored modality.

BRCA1 C-terminal domains are found in a specialized group of 23 proteins that function in the DNA damage response to protect genomic integrity. C-terminal domain phosphatase 1 (CTDP1) is the only phosphatase with a BRCA1 C-terminal domain in the human proteome, yet direct participation in the DNA damage response has not been reported. Examination of the CTDP1 BRCA1 C-terminal domain-specific protein interaction network revealed 103 high confidence interactions enriched in DNA damage response proteins, including FANCA and FANCI that are central to the Fanconi anemia DNA repair pathway necessary for the resolution of DNA interstrand crosslink damage. CTDP1 expression promotes DNA damage-induced FANCA and FANCD2 foci formation and enhances homologous recombination repair efficiency. CTDP1 was found to regulate multiple aspects of FANCI activity, including chromatin localization, interaction with γ-H2AX, and SQ motif phosphorylations. Knockdown of CTDP1 increases MCF-10A sensitivity to DNA interstrand crosslinks and double-strand breaks, but not ultraviolet radiation. In addition, CTDP1 knockdown impairs in vitro and in vivo growth of breast cancer cell lines. These results elucidate the molecular functions of CTDP1 in Fanconi anemia interstrand crosslink repair and identify this protein as a potential target for breast cancer therapy.

Hyperinsulinemia affects 72% of Fanconi anemia (FA) patients and an additional 25% experience lowered glucose tolerance or frank diabetes. The underlying molecular mechanisms contributing to the dysfunction of FA pancreas β cells is unknown. Therefore, we sought to evaluate the functional role of FANCA, the most commonly mutated gene in FA, in glucose-stimulated insulin secretion (GSIS). This study reveals that FANCA or FANCB knockdown impairs GSIS in human pancreas β cell line EndoC-βH3. To identify potential pathways by which FANCA might regulate GSIS, we employed a proteomics approach to identify FANCA protein interactions in EndoC-βH3 differentially regulated in response to elevated glucose levels. Glucose-dependent changes in the FANCA interaction network were observed, including increased association with other FA family proteins, suggesting an activation of the DNA damage response in response to elevated glucose levels. Reactive oxygen species increase in response to glucose stimulation and are necessary for GSIS in EndoC-βH3 cells. Glucose-induced activation of the DNA damage response was also observed as an increase in the DNA damage foci marker γ-H2AX and dependent upon the presence of reactive oxygen species. These results illuminate the role of FANCA in GSIS and its protein interactions regulated by glucose stimulation that may explain the prevalence of β cell-specific endocrinopathies in FA patients.

Little is known on the possible association between impaired glucose/insulin metabolism, the pattern of IGFBP-1 phosphorylation and the complex formation with other serum proteins. In this study, the concentration, isoform, multimer and complex pattern of IGFBP-1 was compared in healthy persons and patients with type 2 diabetes mellitus or with hypoglycemia. Concentrations of insulin and IGFBP-1 were determined by radioimmunoassay. Metal affinity and immunoaffinity chromatography were used for the separation of molecular forms of IGFBP-1, which were detected by immunoblotting and SELDI. The counter directional change in insulin and IGFBP-1 concentrations, expressed as a factor that takes into consideration the rate of insulin increase and IGFBP-1 decrease after glucose intake was approximately twice more pronounced in patients with diabetes than in healthy and hypoglycemic persons. The alteration in the phosphorylation pattern of IGFBP-1 due to diabetes or hypoglycemia was not observed. IGFBP-1 multimers found in the circulation of patients with diabetes type 2 differed from those detected in the circulation of others: there were 3 molecular forms between 90 and 100 kDa (compared to one in patients with hypoglycemia or 2 in healthy persons), 2 of which were α (2)M-reactive and one not. These results suggest a possible greater involvement of IGF system in glucose regulation in patients with diabetes type 2.

Structural and ligand-binding properties of the insulin-like growth factor-binding protein (IGFBP)-3 in patients with poorly controlled diabetes mellitus type 2 were investigated using boronic acid- and lectin-affinity chromatography. IGFBP-3 species separated by chromatography were analyzed by immunoblotting and surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Increased IGFBP-3 binding to boronic acid in patients was shown to be accompanied by the increased ligand-binding. Increased binding of IGFBP-3 forms to lectins from Sambucus nigra (SNA) and Canavalia ensiformis (ConA) in patients, on the other hand, was either not accompanied by altered ligand-binding (in the case of ConA) or it was reduced (in the case of SNA). Strong and opposite effects of glycation and additional sialylation on ligand binding qualify them as factors that may be involved in the regulation of the amount of free, physiologically active IGFs, and modulation of processes that accompany development and progression of diabetes. SELDI-TOF MS analysis revealed a fragment of 13.9 kDa as representative for the non-glycosylated form of IGFBP-3, whereas a fragment of 28.0 kDa profiled as typical for the glycosylated/glycated IGFBP-3 species. The same fragmentation pattern found in healthy persons and in patients indicates that the same degradation process predominantly occurs in both groups of individuals.

Physical activity is accompanied by the changes in Insulin-like Growth Factor I (IGF-I)/IGF-Binding Protein 1 (IGFBP-1) axis. Inconsistent results concerning IGF-I and IGFBP-1 levels were reported. In this study we have raised some questions on the events that occur at the molecular level of the exercise-related IGFBP-1 changes. We have examined the fragmentation pattern of IGFBP-1, IGFBP-1 protease activity, interaction between IGFBP-1 and alpha2-macroglobulin (α2M), and possible existence of minor structural changes of IGFBP-1 in professional soccer players. Athletes had significantly greater amounts of fragmented IGFBP- 1, whereas no difference was found in the amount of intact IGFBP-1 compared with controls. An increased activity of matrix metalloprotease-9 (MMP-9) was detected in athletes, causing IGFBP-1 degradation down to the fragment of 9 kDa as the major one. The amount of α2M, which protects IGFBP-1 from proteolysis, or the amount of IGFBP-1/α2M complexes was unaltered. Finally, we have examined whether IGFBP-1 isolated from soccer players exhibited altered reactivity with several chemical surfaces used in surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Different reactivity was detected with anion and cation exchangers, suggesting existence of at least one sequence within IGFBP-1, whose ionization pattern was not equal in athletes and controls. Differences in spectra obtained with ion exchanges may reflect differences in IGFBP-1 phosphorylation. Physiological implications of the events described in this study on the IGF-I availability are, at this time, unknown. It can be hypothesized that IGFBP-1 proteolysis leads to altered distribution of IGF-I among IGFBPs, which may affect the final IGF-associated response.