Enhancing Standard of Care Chemotherapy Efficacy Using DNA-Dependent Protein Kinase (DNA-PK) Inhibition in Preclinical Models of Ewing Sarcoma.

Disruption of DNA damage repair via impaired homologous recombination is characteristic of Ewing sarcoma (EWS) cells. We hypothesize that this disruption results in increased reliance on nonhomologous end joining to repair DNA damage. In this study, we investigated if pharmacologic inhibition of the enzyme responsible for nonhomologous end joining, the DNA-PK holoenzyme, alters the response of EWS cells to genotoxic standard of care chemotherapy. We used analyses of cell viability and proliferation to investigate the effects of clinical DNA-PK inhibitors (DNA-PKi) in combination with six therapeutic or experimental agents for EWS. We performed calculations of synergy using the Loewe additivity model. Immunoblotting evaluated treatment effects on DNA-PK, DNA damage, and apoptosis. Flow cytometric analyses evaluated effects on cell cycle and fate. We used orthotopic xenograft models to interrogate tolerability, drug mechanism, and efficacy in vivo. DNA-PKi demonstrated on-target activity, reducing phosphorylated DNA-PK levels in EWS cells. DNA-PKi sensitized EWS cell lines to agents that function as topoisomerase 2 (TOP2) poisons and enhanced the DNA damage induced by TOP2 poisons. Nanomolar concentrations of single-agent TOP2 poisons induced G2M arrest and little apoptotic response while adding DNA-PKi-mediated apoptosis. In vivo, the combination of AZD7648 and etoposide had limited tolerability but resulted in enhanced DNA damage, apoptosis, and EWS tumor shrinkage. The combination of DNA-PKi with standard of care TOP2 poisons in EWS models is synergistic, enhances DNA damage and cell death, and may form the basis of a promising future therapeutic strategy for EWS.

Endogenous EWSR1 Exists in Two Visual Modalities That Reflect Its Associations with Nucleic Acids and Concentration at Sites of Active Transcription.

EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

EWSR1's visual modalities are defined by its association with nucleic acids and RNA polymerase II.

We report systematic analysis of endogenous EWSR1's cellular organization. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Interestingly, EWSR1 and FUS, another FET protein, exhibit distinct spatial organizations. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

HNRNPH1 destabilizes the G-quadruplex structures formed by G-rich RNA sequences that regulate the alternative splicing of an oncogenic fusion transcript.

In the presence of physiological monovalent cations, thousands of RNA G-rich sequences can form parallel G-quadruplexes (G4s) unless RNA-binding proteins inhibit, destabilize, or resolve the formation of such secondary RNA structures. Here, we have used a disease-relevant model system to investigate the biophysical properties of the RNA-binding protein HNRNPH1's interaction with G-rich sequences. We demonstrate the importance of two EWSR1-exon 8 G-rich regions in mediating the exclusion of this exon from the oncogenic EWS-FLI1 transcripts expressed in a subset of Ewing sarcomas, using complementary analysis of tumor data, long-read sequencing, and minigene studies. We determined that HNRNPH1 binds the EWSR1-exon 8 G-rich sequences with low nM affinities irrespective of whether in a non-G4 or G4 state but exhibits different kinetics depending on RNA structure. Specifically, HNRNPH1 associates and dissociates from G4-folded RNA faster than the identical sequences in a non-G4 state. Importantly, we demonstrate using gel shift and spectroscopic assays that HNRNPH1, particularly the qRRM1-qRRM2 domains, destabilizes the G4s formed by the EWSR1-exon 8 G-rich sequences in a non-catalytic fashion. Our results indicate that HNRNPH1's binding of G-rich sequences favors the accumulation of RNA in a non-G4 state and that this contributes to its regulation of RNA processing.

Spatial Stable Isotopic Labeling by Amino Acids in Cell Culture: Pulse-Chase Labeling of Three-Dimensional Multicellular Spheroids for Global Proteome Analysis.

Three-dimensional cell cultures, or spheroids, are important model systems for cancer research because they recapitulate chemical and phenotypic aspects of in vivo tumors. Spheroids develop radially symmetric chemical gradients, resulting in distinct cellular populations. Stable isotopic labeling by amino acids in cell culture (SILAC) is a well-established approach to quantify protein expression and has previously been used in a pulse-chase format to evaluate temporal changes. In this article, we demonstrate that distinct isotopic signatures can be introduced into discrete spatial cellular populations, effectively tracking proteins to original locations in the spheroid, using a platform that we refer to as spatial SILAC. Spheroid populations were grown with light, medium, and heavy isotopic media, and the concentric shells of cells were harvested by serial trypsinization. Proteins were quantitatively analyzed by ultraperformance liquid chromatography-tandem mass spectrometry. The isotopic signatures correlated with the spatial location and the isotope position do not significantly impact the proteome of each individual layer. Spatial SILAC can be used to examine the proteomic changes in the different layers of the spheroid and to identify protein biomarkers throughout the structure. We show that SILAC labels can be discretely pulsed to discrete positions, without altering the spheroid's proteome, promising future combined pharmacodynamic and pharmacokinetic studies.

Cancer biology functional genomics: From small RNAs to big dreams.

The year 2021 marks the 20th anniversary of the first publications reporting the discovery of the gene silencing mechanism, RNA interference (RNAi) in mammalian cells. Along with the many studies that delineated the proteins and substrates that form the RNAi pathway, this finding changed our understanding of the posttranscriptional regulation of mammalian gene expression. Furthermore, the development of methods that exploited the RNAi pathway began the technological revolution that eventually enabled the interrogation of mammalian gene function-from a single gene to the whole genome-in only a few days. The needs of the cancer research community have driven much of this progress. In this perspective, we highlight milestones in the development and application of RNAi-based methods to study carcinogenesis. We discuss how RNAi-based functional genetic analysis of exemplar tumor suppressors and oncogenes furthered our understanding of cancer initiation and progression and explore how such studies formed the basis of genome-wide scale efforts to identify cancer or cancer-type specific vulnerabilities, including studies conducted in vivo. Furthermore, we examine how RNAi technologies have revealed new cancer-relevant molecular targets and the implications for cancer of the first RNAi-based drugs. Finally, we discuss the future of functional genetic analysis, highlighting the increasing availability of complementary approaches to analyze cancer gene function.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Large-Scale Phosphoproteomics with the Production of over 11,000 Phosphopeptides from the Colon Carcinoma HCT116 Cell Line.

Phosphoproteomics requires better separation of phosphopeptides to boost the coverage of the phosphoproteome. We argue that an alternative separation method that produces orthogonal phosphopeptide separation to the widely used LC needs to be considered. Capillary zone electrophoresis (CZE) is one important alternative because CZE and LC are orthogonal for phosphopeptide separation and because the migration time of peptides in CZE can be accurately predicted. In this work, we coupled strong cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based platform identified 11,555 phosphopeptides. The phosphopeptide data set is at least 100% larger than that from previous CZE-MS/MS studies and will be a valuable resource for building a model for predicting the migration time of phosphopeptides in CZE. Phosphopeptides migrate significantly slower than corresponding unphosphopeptides under acidic conditions of CZE separations and in a normal polarity. According to our modeling data, phosphorylation decreases peptide's charge roughly by one charge unit, resulting in dramatic decrease in electrophoretic mobility. Preliminary investigations demonstrate that electrophoretic mobility of phosphopeptides containing one phosphoryl group can be predicted with the same accuracy as for nonmodified peptides ( R2 ≈ 0.99). The CZE-MS/MS and LC-MS/MS were complementary in large-scale phosphopeptide identifications and produced different phosphosite motifs from the HCT116 cell line. The data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary separation approach for not only improving the phosphoproteome coverage but also providing more insight into the phosphosite motifs.

Combined Short-Term Glucose Starvation and Chemotherapy in 3D Colorectal Cancer Cell Culture Decreases 14-3-3 Family Protein Expression and Phenotypic Response to Therapy.

Short-term glucose starvation prior to chemotherapy has the potential to preferentially weaken cancer cells, making them more likely to succumb to treatment, while protecting normal cells. In this study, we used 3D cell cultures of colorectal cancer and assessed the effects of short-term glucose starvation and chemotherapy compared to treatment of either individually. We evaluated both phenotypic changes and protein expression levels. Our findings indicate that the combined treatment results in more significant phenotypic responses, including decreased cell viability and clonogenicity. These phenotypic responses can be explained by the decreased expression of LDHA and 14-3-3 family proteins, which were found only in the combined treatment groups. This study indicates that short-term glucose starvation has the potential to increase the efficacy of current cancer treatment regimes. Graphical Abstract ᅟ.

Calcitriol Supplementation Causes Decreases in Tumorigenic Proteins and Different Proteomic and Metabolomic Signatures in Right versus Left-Sided Colon Cancer.

Vitamin D deficiency is a common problem worldwide. In particular, it is an issue in the Northern Hemisphere where UVB radiation does not penetrate the atmosphere as readily. There is a correlation between vitamin D deficiency and colorectal cancer incidence and mortality. Furthermore, there is strong evidence that cancer of the ascending (right side) colon is different from cancer of the descending (left side) colon in terms of prognosis, tumor differentiation, and polyp type, as well as at the molecular level. Right-side tumors have elevated Wnt signaling and are more likely to relapse, whereas left-side tumors have reduced expression of tumor suppressor genes. This study seeks to understand both the proteomic and metabolomic changes resulting from treatment of the active metabolite of vitamin D, calcitriol, in right-sided and left-sided colon cancer. Our results show that left-sided colon cancer treated with calcitriol has a substantially greater number of changes in both the proteome and the metabolome than right-sided colon cancer. We found that calcitriol treatment in both right-sided and left-sided colon cancer causes a downregulation of ribosomal protein L37 and protein S100A10. Both of these proteins are heavily involved in tumorigenesis, suggesting a possible mechanism for the correlation between low vitamin D levels and colon cancer.

iTRAQ Quantitative Proteomic Profiling and MALDI-MSI of Colon Cancer Spheroids Treated with Combination Chemotherapies in a 3D Printed Fluidic Device.

For a patient with metastatic colorectal cancer there are limited clinical options aside from chemotherapy. Unfortunately, the development of new chemotherapeutics is a long and costly process. New methods are needed to identify promising drug candidates earlier in the drug development process. Most chemotherapies are administered to patients in combinations. Here, an in vitro platform is used to assess the penetration and metabolism of combination chemotherapies in three-dimensional colon cancer cell cultures, or spheroids. Colon carcinoma HCT 116 cells were cultured and grown into three-dimensional cell culture spheroids. These spheroids were then dosed with a common combination chemotherapy, FOLFIRI (folinic acid, 5-fluorouracil, and irinotecan) in a 3D printed fluidic device. This fluidic device allows for the dynamic treatment of spheroids across a semipermeable membrane. Following dosing, the spheroids were harvested for quantitative proteomic profiling to examine the effects of the combination chemotherapy on the colon cancer cells. Spheroids were also imaged to assess the spatial distribution of administered chemotherapeutics and metabolites with MALDI-imaging mass spectrometry. Following treatment, we observed penetration of folinic acid to the core of spheroids and metabolism of the drug in the outer proliferating region of the spheroid. Proteomic changes identified included an enrichment of several cancer-associated pathways. This innovative dosing device, along with the proteomic evaluation with iTRAQ-MS/MS, provides a robust platform that could have a transformative impact on the preclinical evaluation of drug candidates. This system is a high-throughput and cost-effective approach to examine novel drugs and drug combinations prior to animal testing.

Glucose Restriction Combined with Autophagy Inhibition and Chemotherapy in HCT 116 Spheroids Decreases Cell Clonogenicity and Viability Regulated by Tumor Suppressor Genes.

Drug resistance is a prevalent phenomenon that decreases the efficacy of cancer treatments and contributes to cancer progression and metastasis. Weakening drug-resistant cancer cells prior to chemotherapy is a potential strategy to combat chemoresistance. One approach to damage resistant cancer cells is modulation of nutritional intake. The combination of nutrient restriction with targeted compound treatment results in pronounced molecular changes. This study provides valuable information about augmenting existing chemotherapeutic regimes with simultaneous glucose restriction and autophagy inhibition in colorectal cancer cells. In this study, we explore the chemical pathways that drive the cellular response to nutrient restriction, autophagy inhibition, and the chemotherapy irinotecan using global quantitative proteomics and imaging mass spectrometry. We determined that significant pathways were altered including autophagy and metabolism via glycolysis, gluconeogenesis, and sucrose degradation. We also found that period circadian clock 2 (PER2), a tumor suppressor protein, was significantly up-regulated only when glucose was restricted with autophagy inhibition and chemotherapy. The upstream regulators of these differentially regulated pathways were determined to have implications in cancer, showing an increase in tumor suppressor proteins and a decrease in nuclear protein 1 (NUPR1) an important protein in chemoresistance. We also evaluated the phenotypic response of these cells and discovered autophagy inhibition and chemotherapy treatment increased apoptosis and decreased cell clonogenicity and viability. When glucose restriction was combined with autophagy inhibition and chemotherapy, all of the phenotypic results were intensified. In sum, our results indicate that glucose metabolism is of great importance in the ability of cancer cells to survive chemotherapy. By weakening cancer cells with glucose restriction and autophagy inhibition prior to chemotherapy, cancer cells become more sensitive to therapy.

Chemical cytometry of thiols using capillary zone electrophoresis-laser induced fluorescence and TMPAB-o-M, an improved fluorogenic reagent.

Low molecular weight thiol compounds play crucial roles in many physiological processes. Most methods for determination of thiol compounds are population-averaged; few methods for quantification of thiol compounds in single cells have been reported. We report an ultrasensitive method for determination of thiol compounds in single cells by use of 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M), a fluorogenic probe with useful spectral properties, coupled with capillary zone electrophoresis and laser induced fluorescence detection using a post-column sheath flow cuvette. TMPAB-o-M provides low background, high sensitivity, and excellent reactivity. After optimization of the separation method, we achieved baseline separation of labeled glutathione (GSH), cysteine (Cys), homocysteine, and γ-glutamylcysteine within 11 min, and produced concentration limits of detection from 10 to 20 pM and mass LODs of 65 to 100 zmol. The method was applied for analysis of thiol containing compounds in both cell homogenates and in single HCT-29 and MCF-10A cells. GSH was the main thiol, and Cys was also detected in both cell types. Cells were treated with N-ethylmaleimide, which significantly attenuated thiol levels.

Over 2300 phosphorylated peptide identifications with single-shot capillary zone electrophoresis-tandem mass spectrometry in a 100 min separation.

Ultraperformance liquid chromatography (UPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) is typically employed for phosphoproteome analysis. Alternatively, capillary zone electrophoresis (CZE)-ESI-MS/MS has great potential for phosphoproteome analysis due to the significantly different migration times of phosphorylated and unphosphorylated forms of peptides. In this work, we systematically compared UPLC-MS/MS and CZE-MS/MS for phosphorylated peptide identifications (IDs) using an enriched phosphoproteome from the MCF-10A cell line. When the sample loading amount of UPLC was 10 times higher than that of CZE (2 µg vs 200 ng), UPLC generated more phosphorylated peptide IDs than CZE (3313 vs 1783). However, when the same sample loading amounts were used for CZE and UPLC (2-200 ng), CZE-MS/MS consistently and significantly outperformed UPLC-MS/MS in terms of phosphorylated peptide and total peptide IDs. This superior performance is most likely due to the higher peptide intensity generated by CZE-MS/MS. More importantly, compared with UPLC data from a 2 µg sample, CZE-MS/MS can identify over 500 unique phosphorylated peptides from a 200 ng sample, suggesting that CZE and UPLC are complementary for phosphorylated peptide IDs. With further improved loading capacity via a dynamic pH junction method, 2313 phosphorylated peptides were identified with single-shot CZE-MS/MS in a 100 min analysis. This number of phosphorylated peptide IDs is over 1 order of magnitude higher than the number of phosphorylated peptide IDs previously reported by single-shot CZE-MS/MS.