Secretome of Cancer-Associated Fibroblasts (CAFs) Influences Drug Sensitivity in Cancer Cells.

Resistance is a major problem with effective cancer treatment and the stroma forms a significant portion of the tumor mass but traditional drug screens involve cancer cells alone. Cancer-associated fibroblasts (CAFs) are a major tumor stroma component and its secreted proteins may influence the function of cancer cells. The majority of secretome studies compare different cancer or CAF cell lines exclusively. Here, we present the direct characterization of the secreted protein profiles between CAFs and KRAS mutant-cancer cell lines from colorectal, lung, and pancreatic tissues using multiplexed mass spectrometry. 2573 secreted proteins were annotated, and differential analysis highlighted understudied CAF-enriched secreted proteins, including Wnt family member 5B (WNT5B), in addition to established CAF markers, such as collagens. The functional role of CAF secreted proteins was explored by assessing its effect on the response to 97 anticancer drugs since stromal cells may cause a differing cancer drug response, which may be missed on routine drug screening using cancer cells alone. CAF secreted proteins caused specific effects on each of the cancer cell lines, which highlights the complexity and challenges in cancer treatment and so the importance to consider stromal elements.

Structural basis for the modulation of MRP2 activity by phosphorylation and drugs.

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.

Degradation by Design: New Cyclin K Degraders from Old CDK Inhibitors.

Small molecules that induce protein degradation hold the potential to overcome several limitations of the currently available inhibitors. Monovalent or molecular glue degraders, in particular, enable the benefits of protein degradation without the disadvantages of high molecular weight and the resulting challenge in drug development that are associated with bivalent molecules like Proteolysis Targeting Chimeras. One key challenge in designing monovalent degraders is how to build in the degrader activity─how can we convert an inhibitor into a degrader? If degradation activity requires very specific molecular features, it will be difficult to find new degraders and challenging to optimize those degraders toward drugs. Herein, we demonstrate that an unexpectedly wide range of modifications to the degradation-inducing group of the cyclin K degrader CR8 are tolerated, including both aromatic and nonaromatic groups. We used these findings to convert the pan-CDK inhibitors dinaciclib and AT-7519 to Cyclin K degraders, leading to a novel dinaciclib-based compound with improved degradation activity compared to CR8 and confirm the mechanism of degradation. These results suggest that general design principles can be generated for the development and optimization of monovalent degraders.

Cancer-associated FBXW7 loss is synthetic lethal with pharmacological targeting of CDC7.

The F-box and WD repeat domain containing 7 (FBXW7) tumour suppressor gene encodes a substrate-recognition subunit of Skp, cullin, F-box (SCF)-containing complexes. The tumour-suppressive role of FBXW7 is ascribed to its ability to drive ubiquitination and degradation of oncoproteins. Despite this molecular understanding, therapeutic approaches that target defective FBXW7 have not been identified. Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens, focussed RNA-interference screens and whole and phospho-proteome mass spectrometry profiling in multiple FBXW7 wild-type and defective isogenic cell lines, we identified a number of FBXW7 synthetic lethal targets, including proteins involved in the response to replication fork stress and proteins involved in replication origin firing, such as cell division cycle 7-related protein kinase (CDC7) and its substrate, DNA replication complex GINS protein SLD5 (GINS4). The CDC7 synthetic lethal effect was confirmed using small-molecule inhibitors. Mechanistically, FBXW7/CDC7 synthetic lethality is dependent upon the replication factor telomere-associated protein RIF1 (RIF1), with RIF1 silencing reversing the FBXW7-selective effects of CDC7 inhibition. The delineation of FBXW7 synthetic lethal effects we describe here could serve as the starting point for subsequent drug discovery and/or development in this area.

Multifactorial Remodeling of the Cancer Immunopeptidome by IFNγ.

IFNγ alters the immunopeptidome presented on HLA class I (HLA-I), and its activity on cancer cells is known to be important for effective immunotherapy responses. We performed proteomic analyses of untreated and IFNγ-treated colorectal cancer patient-derived organoids and combined this with transcriptomic and HLA-I immunopeptidomics data to dissect mechanisms that lead to remodeling of the immunopeptidome through IFNγ. IFNγ-induced changes in the abundance of source proteins, switching from the constitutive to the immunoproteasome, and differential upregulation of different HLA alleles explained some, but not all, observed peptide abundance changes. By selecting for peptides which increased or decreased the most in abundance, but originated from proteins with limited abundance changes, we discovered that the amino acid composition of presented peptides also influences whether a peptide is upregulated or downregulated on HLA-I through IFNγ. The presence of proline within the peptide core was most strongly associated with peptide downregulation. This was validated in an independent dataset. Proline substitution in relevant core positions did not influence the predicted HLA-I binding affinity or stability, indicating that proline effects on peptide processing may be most relevant. Understanding the multiple factors that influence the abundance of peptides presented on HLA-I in the absence or presence of IFNγ is important to identify the best targets for antigen-specific cancer immunotherapies such as vaccines or T-cell receptor engineered therapeutics. SIGNIFICANCE: IFNγ remodels the HLA-I-presented immunopeptidome. We showed that peptide-specific factors influence whether a peptide is upregulated or downregulated and identified a preferential loss or downregulation of those with proline near the peptide center. This will help selecting immunotherapy target antigens which are consistently presented by cancer cells.

SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.

A HUWE1 defect causes PARP inhibitor resistance by modulating the BRCA1-∆11q splice variant.

Although PARP inhibitors (PARPi) now form part of the standard-of-care for the treatment of homologous recombination defective cancers, de novo and acquired resistance limits their overall effectiveness. Previously, overexpression of the BRCA1-∆11q splice variant has been shown to cause PARPi resistance. How cancer cells achieve increased BRCA1-∆11q expression has remained unclear. Using isogenic cells with different BRCA1 mutations, we show that reduction in HUWE1 leads to increased levels of BRCA1-∆11q and PARPi resistance. This effect is specific to cells able to express BRCA1-∆11q (e.g. BRCA1 exon 11 mutant cells) and is not seen in BRCA1 mutants that cannot express BRCA1-∆11q, nor in BRCA2 mutant cells. As well as increasing levels of BRCA1-∆11q protein in exon 11 mutant cells, HUWE1 silencing also restores RAD51 nuclear foci and platinum salt resistance. HUWE1 catalytic domain mutations were also seen in a case of PARPi resistant, BRCA1 exon 11 mutant, high grade serous ovarian cancer. These results suggest how elevated levels of BRCA1-∆11q and PARPi resistance can be achieved, identify HUWE1 as a candidate biomarker of PARPi resistance for assessment in future clinical trials and illustrate how some PARPi resistance mechanisms may only operate in patients with particular BRCA1 mutations.

Unbiased differential proteomic profiling between cancer-associated fibroblasts and cancer cell lines.

Cancer-associated fibroblasts (CAFs) are a key component of tumors. We aimed to profile the proteome of cancer cell lines representing three common cancer types (lung, colorectal and pancreatic) and a representative CAF cell line from each tumor type to gain insight into CAF function and novel CAF biomarkers. We used isobaric labeling, liquid chromatography and mass spectrometry to evaluate the proteome of 9 cancer and 3 CAF cell lines. Of the 9460 proteins evaluated, functional enrichment analysis revealed an upregulation of N-glycan biosynthesis and extracellular matrix proteins in CAFs. 85 proteins had 16-fold higher expression in CAFs compared to cancer cells, including previously known CAF markers like fibroblast activation protein (FAP). Novel overexpressed CAF biomarkers included heat shock protein ß-6 (HSPB6/HSP20) and cyclooxygenase 1 (PTGS1/COX1). SiRNA knockdown of the genes encoding these proteins did not reduce contractility in lung CAFs, suggesting they were not crucial to this function. Immunohistochemical analysis of 30 tumor samples (10 lung, 10 colorectal and 10 pancreatic) showed restricted HSPB6 and PTGS1 expression in the stroma. Therefore, we describe an unbiased differential proteome analysis of CAFs compared to cancer cells, which revealed higher expression of HSPB6 and PTGS1 in CAFs. Data are available via ProteomeXchange (PXD040360). SIGNIFICANCE: Cancer-associated fibroblasts (CAFs) are highly abundant stromal cells present in tumors. CAFs are known to influence tumor progression and drug resistance. Characterizing the proteome of CAFs could give potential insights into new stromal drug targets and biomarkers. Mass spectrometry-based analysis comparing proteomic profiles of CAFs and cancers characterized 9460 proteins of which 85 proteins had 16-fold higher expression in CAFs compared to cancer cells. Further interrogation of this rich resource could provide insight into the function of CAFs and could reveal putative stromal targets. We describe for the first time that heat shock protein ß-6 (HSPB6/HSP20) and cyclooxygenase 1 (PTGS1/COX1) are overexpressed in CAFs compared to cancer cells.

The proteomic landscape of soft tissue sarcomas.

Soft tissue sarcomas (STS) are rare and diverse mesenchymal cancers with limited treatment options. Here we undertake comprehensive proteomic profiling of tumour specimens from 321 STS patients representing 11 histological subtypes. Within leiomyosarcomas, we identify three proteomic subtypes with distinct myogenesis and immune features, anatomical site distribution and survival outcomes. Characterisation of undifferentiated pleomorphic sarcomas and dedifferentiated liposarcomas with low infiltrating CD3 + T-lymphocyte levels nominates the complement cascade as a candidate immunotherapeutic target. Comparative analysis of proteomic and transcriptomic profiles highlights the proteomic-specific features for optimal risk stratification in angiosarcomas. Finally, we define functional signatures termed Sarcoma Proteomic Modules which transcend histological subtype classification and show that a vesicle transport protein signature is an independent prognostic factor for distant metastasis. Our study highlights the utility of proteomics for identifying molecular subgroups with implications for risk stratification and therapy selection and provides a rich resource for future sarcoma research.

Mechanism of assembly, activation and lysine selection by the SIN3B histone deacetylase complex.

Lysine acetylation in histone tails is a key post-translational modification that controls transcription activation. Histone deacetylase complexes remove histone acetylation, thereby repressing transcription and regulating the transcriptional output of each gene. Although these complexes are drug targets and crucial regulators of organismal physiology, their structure and mechanisms of action are largely unclear. Here, we present the structure of a complete human SIN3B histone deacetylase holo-complex with and without a substrate mimic. Remarkably, SIN3B encircles the deacetylase and contacts its allosteric basic patch thereby stimulating catalysis. A SIN3B loop inserts into the catalytic tunnel, rearranges to accommodate the acetyl-lysine moiety, and stabilises the substrate for specific deacetylation, which is guided by a substrate receptor subunit. Our findings provide a model of specificity for a main transcriptional regulator conserved from yeast to human and a resource of protein-protein interactions for future drug designs.

Regulators of male and female sexual development are critical for the transmission of a malaria parasite.

Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite.

Identifying individuals using proteomics: are we there yet?

Multi-omics approaches including proteomics analyses are becoming an integral component of precision medicine. As clinical proteomics studies gain momentum and their sensitivity increases, research on identifying individuals based on their proteomics data is here examined for risks and ethics-related issues. A great deal of work has already been done on this topic for DNA/RNA sequencing data, but it has yet to be widely studied in other omics fields. The current state-of-the-art for the identification of individuals based solely on proteomics data is explained. Protein sequence variation analysis approaches are covered in more detail, including the available analysis workflows and their limitations. We also outline some previous forensic and omics proteomics studies that are relevant for the identification of individuals. Following that, we discuss the risks of patient reidentification using other proteomics data types such as protein expression abundance and post-translational modification (PTM) profiles. In light of the potential identification of individuals through proteomics data, possible legal and ethical implications are becoming increasingly important in the field.

HSP90-CDC37-PP5 forms a structural platform for kinase dephosphorylation.

Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, the kinase-specific co-chaperone CDC37, and the kinase client itself. Removal of regulatory phosphorylation from client kinases and their release from the HSP90-CDC37 system depends on the Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here, we present the cryoEM structure of the oncogenic protein kinase client BRAFV600E bound to HSP90-CDC37, showing how the V600E mutation favours BRAF association with HSP90-CDC37. Structures of HSP90-CDC37-BRAFV600E complexes with PP5 in autoinhibited and activated conformations, together with proteomic analysis of its phosphatase activity on BRAFV600E and CRAF, reveal how PP5 is activated by recruitment to HSP90 complexes. PP5 comprehensively dephosphorylates client proteins, removing interaction sites for regulatory partners such as 14-3-3 proteins and thus performing a 'factory reset' of the kinase prior to release.

Structure of a nucleosome-bound MuvB transcription factor complex reveals DNA remodelling.

Genes encoding the core cell cycle machinery are transcriptionally regulated by the MuvB family of protein complexes in a cell cycle-specific manner. Complexes of MuvB with the transcription factors B-MYB and FOXM1 activate mitotic genes during cell proliferation. The mechanisms of transcriptional regulation by these complexes are still poorly characterised. Here, we combine biochemical analysis and in vitro reconstitution, with structural analysis by cryo-electron microscopy and cross-linking mass spectrometry, to functionally examine these complexes. We find that the MuvB:B-MYB complex binds and remodels nucleosomes, thereby exposing nucleosomal DNA. This remodelling activity is supported by B-MYB which directly binds the remodelled DNA. Given the remodelling activity on the nucleosome, we propose that the MuvB:B-MYB complex functions as a pioneer transcription factor complex. In this work, we rationalise prior biochemical and cellular studies and provide a molecular framework of interactions on a protein complex that is key for cell cycle regulation.

SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics.

Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.

PBAF loss leads to DNA damage-induced inflammatory signaling through defective G2/M checkpoint maintenance.

The PBRM1 subunit of the PBAF (SWI/SNF) chromatin remodeling complex is mutated in ∼40% of clear cell renal cancers. PBRM1 loss has been implicated in responses to immunotherapy in renal cancer, but the mechanism is unclear. DNA damage-induced inflammatory signaling is an important factor determining immunotherapy response. This response is kept in check by the G2/M checkpoint, which prevents progression through mitosis with unrepaired damage. We found that in the absence of PBRM1, p53-dependent p21 up-regulation is delayed after DNA damage, leading to defective transcriptional repression by the DREAM complex and premature entry into mitosis. Consequently, DNA damage-induced inflammatory signaling pathways are activated by cytosolic DNA. Notably, p53 is infrequently mutated in renal cancer, so PBRM1 mutational status is critical to G2/M checkpoint maintenance. Moreover, we found that the ability of PBRM1 deficiency to predict response to immunotherapy correlates with expression of the cytosolic DNA-sensing pathway in clinical samples. These findings have implications for therapeutic responses in renal cancer.

Proteomic characterisation of triple negative breast cancer cells following CDK4/6 inhibition.

When used in combination with hormone treatment, Palbociclib prolongs progression-free survival of patients with hormone receptor positive breast cancer. Mechanistically, Palbociclib inhibits CDK4/6 activity but the basis for differing sensitivity of cancer to Palbociclib is poorly understood. A common observation in a subset of Triple Negative Breast Cancers (TNBCs) is that prolonged CDK4/6 inhibition can engage a senescence-like state where cells exit the cell cycle, whilst, remaining metabolically active. To better understand the senescence-like cell state which arises after Palbociclib treatment we used mass spectrometry to quantify the proteome, phosphoproteome, and secretome of Palbociclib-treated MDA-MB-231 TNBC cells. We observed altered levels of cell cycle regulators, immune response, and key senescence markers upon Palbociclib treatment. These datasets provide a starting point for the derivation of biomarkers which could inform the future use CDK4/6 inhibitors in TNBC subtypes and guide the development of potential combination therapies.

Aneuploidy tolerance caused by BRG1 loss allows chromosome gains and recovery of fitness.

Aneuploidy results in decreased cellular fitness in many species and model systems. However, aneuploidy is commonly found in cancer cells and often correlates with aggressive growth, suggesting that the impact of aneuploidy on cellular fitness is context dependent. The BRG1 (SMARCA4) subunit of the SWI/SNF chromatin remodelling complex is frequently lost in cancer. Here, we use a chromosomally stable cell line to test the effect of BRG1 loss on the evolution of aneuploidy. BRG1 deletion leads to an initial loss of fitness in this cell line that improves over time. Notably, we find increased tolerance to aneuploidy immediately upon loss of BRG1, and the fitness recovery over time correlates with chromosome gain. These data show that BRG1 loss creates an environment where karyotype changes can be explored without a fitness penalty. At least in some genetic backgrounds, therefore, BRG1 loss can affect the progression of tumourigenesis through tolerance of aneuploidy.