Tumor-associated macrophages are pivotal constructors of the tumoral ECM structure and molecular composition. In particular, they orchestrate the buildup of the tumorigenic collagenous ECM niche.

Immunotherapy has revolutionized cancer treatment, yet most patients do not respond. Here, we investigated mechanisms of response by profiling the proteome of clinical samples from advanced stage melanoma patients undergoing either tumor infiltrating lymphocyte (TIL)-based or anti-programmed death 1 (PD1) immunotherapy. Using high-resolution mass spectrometry, we quantified over 10,300 proteins in total and $4,500 proteins across most samples in each dataset. Statistical analyses revealed higher oxidative phosphorylation and lipid metabolism in responders than in non-responders in both treatments. To elucidate the effects of the metabolic state on the immune response, we examined melanoma cells upon metabolic perturbations or CRISPR-Cas9 knockouts. These experiments indicated lipid metabolism as a regulatory mechanism that increases melanoma immunogenicity by elevating antigen presentation, thereby increasing sensitivity to T cell mediated killing both in vitro and in vivo. Altogether, our proteomic analyses revealed association between the melanoma metabolic state and the response to immunotherapy, which can be the basis for future improvement of therapeutic response.

Graphical Abstract Highlights d Homozygous UBQLN4 germline mutations lead to a genome instability syndrome d UBQLN4 removes ubiquitylated MRE11 from damaged chromatin to curtail DSB resection d UBQLN4 overexpression represses HRR and promotes the use of NHEJ for DSB repair d UBQLN4 overexpression in tumors promotes PARP1 inhibitor sensitivity SUMMARY Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4overexpressing tumors.of selected pairs defined in a contrast matrix using the R library multcomp. Error bars represent SD of the mean for 3 replicate wells analyzed in one experiment. Each experiment was carried out twice. *p < 0.05. (N) Quantification of the relative comet tail moment (n = 100) derived from the neutral comet assays at the indicated time points. Error bars represent SD of the mean of the relative comet tail moment analyzed in n = 3 experiments.

Studying the complex relationship between transcription, translation and protein degradation is essential to our understanding of biological processes in health and disease. The limited correlations observed between mRNA and protein abundance suggest pervasive regulation of post-transcriptional steps and support the importance of profiling mRNA levels in parallel to protein synthesis and degradation rates. In this work, we applied an integrative multi-omic approach to study gene expression along the mammalian cell cycle through side-by-side analysis of mRNA, translation and protein levels. Our analysis sheds new light on the significant contribution of both protein synthesis and degradation to the variance in protein expression. Furthermore, we find that translation regulation plays an important role at S-phase, while progression through mitosis is predominantly controlled by changes in either mRNA levels or protein stability. Specific molecular functions are found to be co-regulated and share similar patterns of mRNA, translation and protein expression along the cell cycle. Notably, these include genes and entire pathways not previously implicated in cell cycle progression, demonstrating the potential of this approach to identify novel regulatory mechanisms beyond those revealed by traditional expression profiling. Through this three-level analysis, we characterize different mechanisms of gene expression, discover new cycling gene products and highlight the importance and utility of combining datasets generated using different techniques that monitor distinct steps of gene expression.

Eukaryotic translation initiation factor 2B (eIF2B) is a master regulator of protein synthesis under normal and stress conditions. Mutations in any of the five genes encoding its subunits lead to vanishing white matter (VWM) disease, a recessive genetic deadly illness caused by progressive loss of white matter in the brain. In this study we used fibroblasts, which are not involved in the disease, to demonstrate the involvement of eIF2B in mitochondrial function and abundance. Mass spectrometry of total proteome of mouse embryonic fibroblasts (MEFs) isolated from Eif2b5 mice revealed unbalanced stoichiometry of proteins involved in oxidative phosphorylation and of mitochondrial translation machinery components, among others. Mutant MEFs exhibit 55% decrease in oxygen consumption rate per mtDNA content and 47% increase in mitochondrial abundance (p < 0.005), reflecting adaptation to energy requirements. A more robust eIF2B-associated oxidative respiration deficiency was found in mutant primary astrocytes, which exhibit > 3-fold lower ATP-linked respiration per cell despite a 2-fold increase in mtDNA content (p < 0.03). The 2-fold increase in basal and stimulated glycolysis in mutant astrocytes (p ≤ 0.03), but not in MEFs, demonstrates their higher energetic needs and further explicates their involvement in the disease. The data demonstrate the critical role of eIF2B in tight coordination of expression from nuclear and mitochondrial genomes and illuminates the importance of mitochondrial function in VWM pathology. Further dissection of the signaling network associated with eIF2B function will help generating therapeutic strategies for VWM disease and possibly other neurodegenerative disorders.

Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC–MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase.

Stable isotope labeling with amino acids in cell culture (SILAC) has risen as a powerful quantification technique in mass spectrometry (MS)-based proteomics in classical and modified forms. Previously, SILAC was limited to cultured cells because of the requirement of active protein synthesis; however, in recent years, it was expanded to model organisms and tissue samples. Specifically, the super-SILAC technique uses a mixture of SILAC-labeled cells as a spike-in standard for accurate quantification of unlabeled samples, thereby enabling quantification of human tissue samples. Here, we highlight the recent developments in super-SILAC and its application to the study of clinical samples, secretomes, post-translational modifications and organelle proteomes. Finally, we propose super-SILAC as a robust and accurate method that can be commercialized and applied to basic and clinical research.

Tumor relapse as a consequence of chemotherapy resistance is a major clinical challenge in advanced stage breast tumors. To identify processes associated with poor clinical outcome, we took a mass spectrometry‐based proteomic approach and analyzed a breast cancer cohort of 113 formalin‐fixed paraffin‐embedded samples. Proteomic profiling of matched tumors before and after chemotherapy, and tumor‐adjacent normal tissue, all from the same patients, allowed us to define eight patterns of protein level changes, two of which correlate to better chemotherapy response. Supervised analysis identified two proteins of proline biosynthesis pathway, PYCR1 and ALDH18A1, that were significantly associated with resistance to treatment based on pattern dominance. Weighted gene correlation network analysis of post‐treatment samples revealed that these proteins are associated with tumor relapse and affect patient survival. Functional analysis showed that knockdown of PYCR1 reduced invasion and migration capabilities of breast cancer cell lines. PYCR1 knockout significantly reduced tumor burden and increased drug sensitivity of orthotopically injected ER‐positive tumor in vivo, thus emphasizing the role of PYCR1 in resistance to chemotherapy.