Large-scale genetic perturbations reveal regulatory networks and an abundance of gene-specific repressors.

To understand regulatory systems, it would be useful to uniformly determine how different components contribute to the expression of all other genes. We therefore monitored mRNA expression genome-wide, for individual deletions of one-quarter of yeast genes, focusing on (putative) regulators. The resulting genetic perturbation signatures reflect many different properties. These include the architecture of protein complexes and pathways, identification of expression changes compatible with viability, and the varying responsiveness to genetic perturbation. The data are assembled into a genetic perturbation network that shows different connectivities for different classes of regulators. Four feed-forward loop (FFL) types are overrepresented, including incoherent type 2 FFLs that likely represent feedback. Systematic transcription factor classification shows a surprisingly high abundance of gene-specific repressors, suggesting that yeast chromatin is not as generally restrictive to transcription as is often assumed. The data set is useful for studying individual genes and for discovering properties of an entire regulatory system.

Truncated FGFR2 is a clinically actionable oncogene in multiple cancers.

Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2) occur in multiple types of cancer1. However, clinical responses to FGFR inhibitors have remained variable1-9, emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening10,11 and tumour modelling in mice12,13, and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1-E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 (FGFR2ΔE18). Functional in vitro and in vivo examination of a compendium of FGFR2ΔE18 and full-length variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies.

Ubiquitous atmospheric production of organic acids mediated by cloud droplets.

Atmospheric acidity is increasingly determined by carbon dioxide and organic acids1-3. Among the latter, formic acid facilitates the nucleation of cloud droplets4 and contributes to the acidity of clouds and rainwater1,5. At present, chemistry-climate models greatly underestimate the atmospheric burden of formic acid, because key processes related to its sources and sinks remain poorly understood2,6-9. Here we present atmospheric chamber experiments that show that formaldehyde is efficiently converted to gaseous formic acid via a multiphase pathway that involves its hydrated form, methanediol. In warm cloud droplets, methanediol undergoes fast outgassing but slow dehydration. Using a chemistry-climate model, we estimate that the gas-phase oxidation of methanediol produces up to four times more formic acid than all other known chemical sources combined. Our findings reconcile model predictions and measurements of formic acid abundance. The additional formic acid burden increases atmospheric acidity by reducing the pH of clouds and rainwater by up to 0.3. The diol mechanism presented here probably applies to other aldehydes and may help to explain the high atmospheric levels of other organic acids that affect aerosol growth and cloud evolution.

EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.

BACKGROUND: Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. METHODS: We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. RESULTS: Screening of 157 kinase inhibitors in wild-type (N=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (N=24 HCM and N=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (N=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. CONCLUSIONS: We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.

Insight in the (Phospho)proteome of Vascular Smooth Muscle Cells Derived From Patients With Abdominal Aortic Aneurysm Reveals Novel Disease Mechanisms.

BACKGROUND: Abdominal aortic aneurysm (AAA) is characterized by weakening and dilatation of the aortic wall in the abdomen. The aim of this study was to gain insight into cell-specific mechanisms involved in AAA pathophysiology by analyzing the (phospho)proteome of vascular smooth muscle cells derived from patients with AAA compared with those of healthy donors. METHODS: A (phospho)proteomics analysis based on tandem mass spectrometry was performed on vascular smooth muscle cells derived from patients with AAA (n=24) and healthy, control individuals (C-SMC, n=8). Following protein identification and quantification using MaxQuant, integrative inferred kinase activity analysis was used to calculate kinase activity scores. RESULTS: Expression differences between vascular smooth muscle cells derived from patients with AAA and healthy, control individuals were predominantly found in proteins involved in ECM (extracellular matrix) remodeling (THSD4 [thrombospondin type-1 domain-containing protein 4] and ADAMTS1 [A disintegrin and metalloproteinase with thrombospondin motifs 1]), energy metabolism (GYS1 [glycogen synthase 1] and PCK2 [phosphoenolpyruvate carboxykinase 2, mitochondrial]), and contractility (CACNA2D1 [calcium voltage-dependent channel subunit α-2/δ-1] and TPM1 [tropomyosin α-1 chain]). Phosphorylation patterns on proteins related to actin cytoskeleton organization dominated the phosphoproteome of vascular smooth muscle cells derived from patients with AAA . Besides, phosphorylation changes on proteins related to energy metabolism (GYS1), contractility (PARVA [α-parvin], PPP1R12A [protein phosphatase 1 regulatory subunit 12A], and CALD1 [caldesmon 1]), and intracellular communication (GJA1 [gap junction α-1 protein]) were seen. Kinase activity of NUAK1 (NUAK family SNF1-like kinase 1), FYN (tyrosine-protein kinase Fyn), MAPK7 (mitogen-activated protein kinase 7), and STK10 (serine/threonine kinase 10) was different in vascular smooth muscle cells derived from patients with AAA compared with those from healthy, control individuals. CONCLUSIONS: This study revealed changes in expression and phosphorylation levels of proteins involved in various processes responsible for AAA progression and development (eg, energy metabolism, ECM remodeling, actin cytoskeleton organization, contractility, intracellular communication, and cell adhesion). These newly identified proteins, phosphosites, and related kinases provide further insight into the underlying mechanism of vascular smooth muscle cell dysfunction within the aneurysmal wall. Our omics data thereby offer the opportunity to study the relevance, either as drug target or biomarker, of these proteins in AAA development.

Functional overlap and regulatory links shape genetic interactions between signaling pathways.

To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy, and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multiprocess control.

Secretome processing for proteomics: A methods comparison.

The cancer cell secretome comprises a treasure-trove for biomarkers since it reflects cross-talk between tumor cells and their surrounding environment with high detectability in biofluids. In this study, we evaluated six secretome sample processing workflows coupled to single-shot mass spectrometry: (1) Protein concentration by ultrafiltration with a molecular weight cut-off (MWCO) filter and sample preparation through in-gel digestion (IGD); (2) Acetone protein precipitation coupled to IGD; (3) MWCO filter-based protein concentration followed by to in-solution digestion (ISD); (4) Acetone protein precipitation coupled to ISD; (5) Direct ISD; (6) Secretome lyophilization and ISD. To this end, we assessed workflow triplicates in terms of total number of protein identifications, unique identifications, reproducibility of protein identification and quantification and detectability of small proteins with important functions in cancer biology such as cytokines, chemokines, and growth factors. Our findings revealed that acetone protein precipitation coupled to ISD outperformed the other methods in terms of the number of identified proteins (2246) and method reproducibility (correlation coefficient between replicates (r = 0.94, CV = 19%). Overall, especially small proteins such as those from the classes mentioned above were better identified using ISD workflows. Concluding, herein we report that secretome protein precipitation coupled to ISD is the method of choice for high-throughput secretome proteomics via single shot nanoLC-MS/MS.

Donor natural killer cells trigger production of ß-2-microglobulin to enhance post-bone marrow transplant immunity.

Allogeneic hematopoietic transplantation is a powerful treatment for hematologic malignancies. Posttransplant immune incompetence exposes patients to disease relapse and infections. We previously demonstrated that donor alloreactive natural killer (NK) cells ablate recipient hematopoietic targets, including leukemia. Here, in murine models, we show that infusion of donor alloreactive NK cells triggers recipient dendritic cells (DCs) to synthesize ß-2-microglobulin (B2M) that elicits the release of c-KIT ligand and interleukin-7 that greatly accelerate posttransplant immune reconstitution. An identical chain of events was reproduced by infusing supernatants of alloreactive NK/DC cocultures. Similarly, human alloreactive NK cells triggered human DCs to synthesize B2M that induced interleukin-7 production by thymic epithelial cells and thereby supported thymocyte cellularity in vitro. Chromatography fractionation of murine and human alloreactive NK/DC coculture supernatants identified a protein with molecular weight and isoelectric point of B2M, and mass spectrometry identified amino acid sequences specific of B2M. Anti-B2M antibody depletion of NK/DC coculture supernatants abrogated their immune-rebuilding effect. B2M knock-out mice were unable to undergo accelerated immune reconstitution, but infusion of (wild-type) NK/DC coculture supernatants restored their ability to undergo accelerated immune reconstitution. Similarly, silencing the B2M gene in human DCs, before coculture with alloreactive NK cells, prevented the increase in thymocyte cellularity in vitro. Finally, human recombinant B2M increased thymocyte cellularity in a thymic epithelial cells/thymocyte culture system. Our studies uncover a novel therapeutic principle for treating posttransplant immune incompetence and suggest that, upon its translation to the clinic, patients may benefit from adoptive transfer of large numbers of cytokine-activated, ex vivo-expanded donor alloreactive NK cells.

LaSap vaccine: Immunotherapy and immunochemotherapy associated with allopurinol in dogs naturally infected with Leishmania infantum.

Canine visceral leishmaniasis is a parasitic zoonosis that has a profound impact on public health in countries where it is endemic. Chemotherapeutic treatments cannot keep dogs stable for long periods, and the risk of generating parasitic resistance must be considered. Forty-four symptomatic and naturally infected dogs with Leishmania infantum were tested with two treatment protocols (i) immunotherapy with LaSap vaccine and (ii) immunochemotherapy with LaSap vaccine plus allopurinol. At 90 days after the end of the treatment, it was verified that, although both protocols had generated significant clinical improvements with a greater production of IFN-γ/IL-10, in relation to the parasite load, mainly in the skin, the dogs treated only with immunotherapy maintained the same profile. These results indicate that LaSap is a good strategy to control dog parasitism.

Phosphoproteomic Analysis of FLCN Inactivation Highlights Differential Kinase Pathways and Regulatory TFEB Phosphoserines.

In Birt-Hogg-Dubé (BHD) syndrome, germline loss-of-function mutations in the Folliculin (FLCN) gene lead to an increased risk of renal cancer. To address how FLCN inactivation affects cellular kinase signaling pathways, we analyzed comprehensive phosphoproteomic profiles of FLCNPOS and FLCNNEG human renal tubular epithelial cells (RPTEC/TERT1). In total, 15,744 phosphorylated peptides were identified from 4329 phosphorylated proteins. INKA analysis revealed that FLCN loss alters the activity of numerous kinases, including tyrosine kinases EGFR, MET, and the Ephrin receptor subfamily (EPHA2 and EPHB1), as well their downstream targets MAPK1/3. Validation experiments in the BHD renal tumor cell line UOK257 confirmed that FLCN loss contributes to enhanced MAPK1/3 and downstream RPS6K1/3 signaling. The clinically available MAPK inhibitor Ulixertinib showed enhanced toxicity in FLCNNEG cells. Interestingly, FLCN inactivation induced the phosphorylation of PIK3CD (Tyr524) without altering the phosphorylation of canonical Akt1/Akt2/mTOR/EIF4EBP1 phosphosites. Also, we identified that FLCN inactivation resulted in dephosphorylation of TFEB Ser109, Ser114, and Ser122, which may be linked to increased oxidative stress levels in FLCNNEG cells. Together, our study highlights differential phosphorylation of specific kinases and substrates in FLCNNEG renal cells. This provides insight into BHD-associated renal tumorigenesis and may point to several novel candidates for targeted therapies.

Optic Nerve Amyloid Deposition Disguised as Optic Nerve Sheath Meningioma.

Localized orbital amyloidosis is a rare clinical entity. Periocular and orbital amyloid deposits are mainly located at the lacrimal apparatus, eyelid, conjunctiva, ocular adnexa, extraocular muscles, and levator palpebrae muscle. In this article, we report an unusual case of optic nerve amyloid deposition in an 82-year-old African American woman who presented with vertical diplopia. MRI revealed an enhancing mass from the optic nerve sheath, and CT showed foci of calcifications suggestive of optic nerve meningioma. However, an incisional biopsy demonstrated lymphoproliferative disease with focal optic nerve sheath amyloid deposition confirmed by histologic Congo red staining and immunohistochemistry.

Apoptotic Marker Expression of Resected Lacrimal Gland Adenoid Cystic Carcinoma Tumor Margins After Intra-arterial Chemotherapy and Globe-Sparing Excision.

PURPOSE: Lacrimal gland adenoid cystic carcinoma (LGACC) is a rare orbital malignancy with devastating lethality. Neoadjuvant intra-arterial chemotherapy (IACC) has demonstrated cytoreductive effects on LGACC macroscopically, but limited studies have examined cellular and molecular determinants of the cytoreductive effect. This post hoc study assessed apoptotic marker expression on excised tumor specimens after neoadjuvant IACC and globe-sparing resection, emphasizing the examination of tumor margins. METHODS: This retrospective study identified LGACC specimens resected in a globe-sparing technique after neoadjuvant IACC by reviewing the Florida Lions Ocular Pathology database at Bascom Palmer Eye Institute. Histopathology slides of the specimens were re-examined to confirm the diagnosis and identify the tumor margin. Immunofluorescent staining was performed for apoptotic markers, including P53, cleaved caspase-3, cleaved PARP-1, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Positive expression was determined by comparison to the negative control. RESULTS: Tumor specimens from 5 patients met inclusion criteria. All 5 cases were positive at the center and the margin for TUNEL, p53, and cleaved caspase-3. One case did not show positive expression of cleaved PARP-1 at the margin but was positive for the other apoptotic markers. CONCLUSIONS: This post hoc study demonstrated positive staining for multiple apoptotic markers in post-IACC tumor specimens at the tumor center and margin. Apoptotic marker expression along the margins of post-treatment specimens is important, as it may offer surrogate information to speculate on the state of residual cancer cells adjacent to the excision margin inadvertently remaining in the orbit.

VRK1 Regulates Sensitivity to Oxidative Stress by Altering Histone Epigenetic Modifications and the Nuclear Phosphoproteome in Tumor Cells.

The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.

A transformer architecture for retention time prediction in liquid chromatography mass spectrometry-based proteomics.

Accurate retention time (RT) prediction is important for spectral library-based analysis in data-independent acquisition mass spectrometry-based proteomics. The deep learning approach has demonstrated superior performance over traditional machine learning methods for this purpose. The transformer architecture is a recent development in deep learning that delivers state-of-the-art performance in many fields such as natural language processing, computer vision, and biology. We assess the performance of the transformer architecture for RT prediction using datasets from five deep learning models Prosit, DeepDIA, AutoRT, DeepPhospho, and AlphaPeptDeep. The experimental results on holdout datasets and independent datasets exhibit state-of-the-art performance of the transformer architecture. The software and evaluation datasets are publicly available for future development in the field.

Interaction between KDELR2 and HSP47 as a Key Determinant in Osteogenesis Imperfecta Caused by Bi-allelic Variants in KDELR2.

Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.

Inferring kinase activity from phosphoproteomic data: Tool comparison and recent applications.

Aberrant cellular signaling pathways are a hallmark of cancer and other diseases. One of the most important signaling mechanisms involves protein phosphorylation/dephosphorylation. Protein phosphorylation is catalyzed by protein kinases, and over 530 protein kinases have been identified in the human genome. Aberrant kinase activity is one of the drivers of tumorigenesis and cancer progression and results in altered phosphorylation abundance of downstream substrates. Upstream kinase activity can be inferred from the global collection of phosphorylated substrates. Mass spectrometry-based phosphoproteomic experiments nowadays routinely allow identification and quantitation of >10k phosphosites per biological sample. This substrate phosphorylation footprint can be used to infer upstream kinase activities using tools like Kinase Substrate Enrichment Analysis (KSEA), Posttranslational Modification Substrate Enrichment Analysis (PTM-SEA), and Integrative Inferred Kinase Activity Analysis (INKA). Since the topic of kinase activity inference is very active with many new approaches reported in the past 3 years, we would like to give an overview of the field. In this review, an inventory of kinase activity inference tools, their underlying algorithms, statistical frameworks, kinase-substrate databases, and user-friendliness is presented. The most widely-used tools are compared in-depth. Subsequently, recent applications of the tools are described focusing on clinical tissues and hematological samples. Two main application areas for kinase activity inference tools can be discerned. (1) Maximal biological insights can be obtained from large data sets with group comparisons using multiple complementary tools (e.g., PTM-SEA and KSEA or INKA). (2) In the oncology context where personalized treatment requires analysis of single samples, INKA for example, has emerged as tool that can prioritize actionable kinases for targeted inhibition.

Candidate biomarkers for treatment benefit from sunitinib in patients with advanced renal cell carcinoma using mass spectrometry-based (phospho)proteomics.

The tyrosine kinase inhibitor sunitinib is an effective first-line treatment for patients with advanced renal cell carcinoma (RCC). Hypothesizing that a functional read-out by mass spectrometry-based (phospho, p-)proteomics will identify predictive biomarkers for treatment outcome of sunitinib, tumor tissues of 26 RCC patients were analyzed. Eight patients had primary resistant (RES) and 18 sensitive (SENS) RCC. A 78 phosphosite signature (p < 0.05, fold-change > 2) was identified; 22 p-sites were upregulated in RES (unique in RES: BCAR3, NOP58, EIF4A2, GDI1) and 56 in SENS (35 unique). EIF4A1/EIF4A2 were differentially expressed in RES at the (p-)proteome and, in an independent cohort, transcriptome level. Inferred kinase activity of MAPK3 (p = 0.026) and EGFR (p = 0.045) as determined by INKA was higher in SENS. Posttranslational modifications signature enrichment analysis showed that different p-site-centric signatures were enriched (p < 0.05), of which FGF1 and prolactin pathways in RES and, in SENS, vanadate and thrombin treatment pathways, were most significant. In conclusion, the RCC (phospho)proteome revealed differential p-sites and kinase activities associated with sunitinib resistance and sensitivity. Independent validation is warranted to develop an assay for upfront identification of patients who are intrinsically resistant to sunitinib.

The potential of PARP as a therapeutic target across pediatric solid malignancies.

BACKGROUND: Pediatric cancer is the leading cause of disease-related death in children and the need for better therapeutic options remains urgent. Due to the limited number of patients, target and drug development for pediatrics is often supplemented by data from studies focused on adult cancers. Recent evidence shows that pediatric cancers possess different vulnerabilities that should be explored independently from adult cancers. METHODS: Using the publicly available Genomics of Drug Sensitivity in Cancer database, we explore therapeutic targets and biomarkers specific to the pediatric solid malignancies Ewing sarcoma, medulloblastoma, neuroblastoma, osteosarcoma, and rhabdomyosarcoma. Results are validated using cell viability assays and high-throughput drug screens are used to identify synergistic combinations. RESULTS: Using published drug screening data, PARP is identified as a drug target of interest across multiple different pediatric malignancies. We validate these findings, and we show that efficacy can be improved when combined with conventional chemotherapeutics, namely topoisomerase inhibitors. Additionally, using gene set enrichment analysis, we identify ribosome biogenesis as a potential biomarker for PARP inhibition in pediatric cancer cell lines. CONCLUSION: Collectively, our results provide evidence to support the further development of PARP inhibition and the combination with TOP1 inhibition as a therapeutic approach in solid pediatric malignancies. Additionally, we propose ribosome biogenesis as a component to PARP inhibitor sensitivity that should be further investigated to help maximize the potential utility of PARP inhibition and combinations across pediatric solid malignancies.

Feasibility of Phosphoproteomics on Leftover Samples After RNA Extraction With Guanidinium Thiocyanate.

In daily practice, different types of biomolecules are usually extracted for large-scale "omics" analysis with tailored protocols. However, when sample material is limited, an all-in-one strategy is preferable. Although lysis of cells and tissues with urea is widely used for phosphoproteomic applications, DNA, RNA, and proteins can be simultaneously extracted from small samples using acid guanidinium thiocyanate-phenol-chloroform (AGPC). Use of AGPC for mass spectrometry-based phosphoproteomics was reported but has not yet been thoroughly evaluated against a classical phosphoproteomic protocol. Here we compared urea- with AGPC-based protein extraction, profiling phosphorylations in the DNA damage response pathway after ionizing irradiation of U2OS cells as proof of principle. On average we identified circa 9000 phosphosites per sample with both extraction methods. Moreover, we observed high similarity of phosphosite characteristics (e.g., 94% shared class 1 identifications) and deduced kinase activities (e.g., ATM, ATR, CHEK1/2, PRKDC). We furthermore extended our comparison to murine and human tissue samples yielding similar and highly correlated results for both extraction protocols. AGPC-based sample extraction can thus replace common cell lysates for phosphoproteomic workflows and may thus be an attractive way to obtain input material for multiple omics workflows, yielding several data types from a single sample.