Selected Ion Monitoring for Orbitrap-Based Metabolomics.

Orbitrap mass spectrometry in full scan mode enables the simultaneous detection of hundreds of metabolites and their isotope-labeled forms. Yet, sensitivity remains limiting for many metabolites, including low-concentration species, poor ionizers, and low-fractional-abundance isotope-labeled forms in isotope-tracing studies. Here, we explore selected ion monitoring (SIM) as a means of sensitivity enhancement. The analytes of interest are enriched in the orbitrap analyzer by using the quadrupole as a mass filter to select particular ions. In tissue extracts, SIM significantly enhances the detection of ions of low intensity, as indicated by improved signal-to-noise (S/N) ratios and measurement precision. In addition, SIM improves the accuracy of isotope-ratio measurements. SIM, however, must be deployed with care, as excessive accumulation in the orbitrap of similar m/z ions can lead, via space-charge effects, to decreased performance (signal loss, mass shift, and ion coalescence). Ion accumulation can be controlled by adjusting settings including injection time and target ion quantity. Overall, we suggest using a full scan to ensure broad metabolic coverage, in tandem with SIM, for the accurate quantitation of targeted low-intensity ions, and provide methods deploying this approach to enhance metabolome coverage.

Glycine homeostasis requires reverse SHMT flux.

The folate-dependent enzyme serine hydroxymethyltransferase (SHMT) reversibly converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Such one-carbon unit production plays a critical role in development, the immune system, and cancer. Using rodent models, here we show that the whole-body SHMT flux acts to net consume rather than produce glycine. Pharmacological inhibition of whole-body SHMT1/2 and genetic knockout of liver SHMT2 elevated circulating glycine levels up to eight-fold. Stable-isotope tracing revealed that the liver converts glycine to serine, which is then converted by serine dehydratase into pyruvate and burned in the tricarboxylic acid cycle. In response to diets deficient in serine and glycine, de novo biosynthetic flux was unaltered, but SHMT2- and serine-dehydratase-mediated catabolic flux was lower. Thus, glucose-derived serine synthesis is largely insensitive to systemic demand. Instead, circulating serine and glycine homeostasis is maintained through variable consumption, with liver SHMT2 a major glycine-consuming enzyme.

Convergent and complementary selection shaped gains and losses of eusociality in sweat bees.

Sweat bees have repeatedly gained and lost eusociality, a transition from individual to group reproduction. Here we generate chromosome-length genome assemblies for 17 species and identify genomic signatures of evolutionary trade-offs associated with transitions between social and solitary living. Both young genes and regulatory regions show enrichment for these molecular patterns. We also identify loci that show evidence of complementary signals of positive and relaxed selection linked specifically to the convergent gains and losses of eusociality in sweat bees. This includes two pleiotropic proteins that bind and transport juvenile hormone (JH)-a key regulator of insect development and reproduction. We find that one of these proteins is primarily expressed in subperineurial glial cells that form the insect blood-brain barrier and that brain levels of JH vary by sociality. Our findings are consistent with a role of JH in modulating social behaviour and suggest that eusocial evolution was facilitated by alteration of the proteins that bind and transport JH, revealing how an ancestral developmental hormone may have been co-opted during one of life's major transitions. More broadly, our results highlight how evolutionary trade-offs have structured the molecular basis of eusociality in these bees and demonstrate how both directional selection and release from constraint can shape trait evolution.

Glycine homeostasis requires reverse SHMT flux.

The folate-dependent enzyme serine hydroxymethyltransferase (SHMT) reversibly converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Such one-carbon unit production plays a critical role in development, the immune system, and cancer. Here we show that the whole-body SHMT flux acts to net consume rather than produce glycine. Pharmacological inhibition of whole-body SHMT1/2 and genetic knockout of liver SHMT2 elevated circulating glycine levels up to eight-fold. Stable isotope tracing revealed that the liver converts glycine to serine, which is then converted by serine dehydratase into pyruvate and burned in the tricarboxylic acid cycle. In response to diets deficient in serine and glycine, de novo biosynthetic flux was unaltered but SHMT2- and serine dehydratase-mediated catabolic flux was lower. Thus, glucose-derived serine synthesis does not respond to systemic demand. Instead, circulating serine and glycine homeostasis is maintained through variable consumption, with liver SHMT2 as a major glycine-consuming enzyme.

Local production of lactate, ribose phosphate, and amino acids within human triple-negative breast cancer.

BACKGROUND: Upregulated glucose metabolism is a common feature of tumors. Glucose can be broken down by either glycolysis or the oxidative pentose phosphate pathway (oxPPP). The relative usage within tumors of these catabolic pathways remains unclear. Similarly, the extent to which tumors make biomass precursors from glucose, versus take them up from the circulation, is incompletely defined. METHODS: We explore human triple negative breast cancer (TNBC) metabolism by isotope tracing with [1,2-13C]glucose, a tracer that differentiates glycolytic versus oxPPP catabolism and reveals glucose-driven anabolism. Patients enrolled in clinical trial NCT03457779 and received IV infusion of [1,2-13C]glucose during core biopsy of their primary TNBC. Tumor samples were analyzed for metabolite labeling by liquid chromatography-mass spectrometry (LC-MS). Genomic and proteomic analyses were performed and related to observed metabolic fluxes. FINDINGS: TNBC ferments glucose to lactate, with glycolysis dominant over the oxPPP. Most ribose phosphate is nevertheless produced by oxPPP. Glucose also feeds amino acid synthesis, including of serine, glycine, aspartate, glutamate, proline and glutamine (but not asparagine). Downstream in glycolysis, tumor pyruvate and lactate labeling exceeds that found in serum, indicating that lactate exchange via monocarboxylic transporters is less prevalent in human TNBC compared with most normal tissues or non-small cell lung cancer. CONCLUSIONS: Glucose directly feeds ribose phosphate, amino acid synthesis, lactate, and the TCA cycle locally within human breast tumors.

Opposing immune and genetic mechanisms shape oncogenic programs in synovial sarcoma.

Synovial sarcoma (SyS) is an aggressive neoplasm driven by the SS18-SSX fusion, and is characterized by low T cell infiltration. Here, we studied the cancer-immune interplay in SyS using an integrative approach that combines single-cell RNA sequencing (scRNA-seq), spatial profiling and genetic and pharmacological perturbations. scRNA-seq of 16,872 cells from 12 human SyS tumors uncovered a malignant subpopulation that marks immune-deprived niches in situ and is predictive of poor clinical outcomes in two independent cohorts. Functional analyses revealed that this malignant cell state is controlled by the SS18-SSX fusion, is repressed by cytokines secreted by macrophages and T cells, and can be synergistically targeted with a combination of HDAC and CDK4/CDK6 inhibitors. This drug combination enhanced malignant-cell immunogenicity in SyS models, leading to induced T cell reactivity and T cell-mediated killing. Our study provides a blueprint for investigating heterogeneity in fusion-driven malignancies and demonstrates an interplay between immune evasion and oncogenic processes that can be co-targeted in SyS and potentially in other malignancies.

The nucleosome acidic patch and H2A ubiquitination underlie mSWI/SNF recruitment in synovial sarcoma.

Interactions between chromatin-associated proteins and the histone landscape play major roles in dictating genome topology and gene expression. Cancer-specific fusion oncoproteins, which display unique chromatin localization patterns, often lack classical DNA-binding domains, presenting challenges in identifying mechanisms governing their site-specific chromatin targeting and function. Here we identify a minimal region of the human SS18-SSX fusion oncoprotein (the hallmark driver of synovial sarcoma) that mediates a direct interaction between the mSWI/SNF complex and the nucleosome acidic patch. This binding results in altered mSWI/SNF composition and nucleosome engagement, driving cancer-specific mSWI/SNF complex targeting and gene expression. Furthermore, the C-terminal region of SSX confers preferential affinity to repressed, H2AK119Ub-marked nucleosomes, underlying the selective targeting to polycomb-marked genomic regions and synovial sarcoma-specific dependency on PRC1 function. Together, our results describe a functional interplay between a key nucleosome binding hub and a histone modification that underlies the disease-specific recruitment of a major chromatin remodeling complex.

A Novel SS18-SSX Fusion-specific Antibody for the Diagnosis of Synovial Sarcoma.

Synovial sarcoma (SS), an aggressive soft tissue sarcoma with a predilection for the extremities of young adults, harbors the pathognomonic t(X;18)(p11;q11) translocation, resulting in SS18-SSX rearrangements. SS includes monophasic, biphasic, and poorly differentiated variants, which show considerable histologic overlap with a range of other tumor types, making the diagnosis challenging on limited biopsies. Immunohistochemistry (IHC) is routinely used in the differential diagnosis; however, presently available markers lack specificity. Thus, cytogenetic or molecular genetic techniques are often employed to confirm the diagnosis. Here, we report the development and characterization of 2 novel antibodies: an SS18-SSX fusion-specific antibody (E9X9V, designed to the breakpoint) as well as an SSX-specific antibody (E5A2C, designed to the SSX C-terminus). We validated the selectivity and specificity of the antibodies using immunoblotting, immunoprecipitation, and chromatin immunoprecipitation followed by next-generation sequencing in SS cell lines and demonstrated that both antibodies capture SS18-SSX on chromatin at established target sites (eg, TLE1 and BCL2) genome-wide. Using IHC in whole sections from 400 tumors including 100 genetically confirmed cases of SS and 300 histologic mimics, the SS18-SSX fusion-specific antibody revealed strong diffuse nuclear staining in 95 of 100 (95%) SS cases, whereas none of the 300 control tumors showed any staining. The SSX antibody showed strong diffuse nuclear staining in all 100 (100%) SS cases; 13 (4%) of the 300 other tumors were also positive, 5 of which displayed >50% nuclear staining. In summary, a novel SS18-SSX fusion-specific antibody is highly sensitive (95%) and specific (100%) for SS, and an antibody to the SSX C-terminus is also highly sensitive (100%), but slightly less specific (96%). IHC using the SS18-SSX antibody could replace molecular genetic or cytogenetic testing in most cases, and these reagents together will also provide the research community with valuable tools for further biochemical and genomic interrogation of the SS18-SSX fusion protein.

Recent Changes in the Scaffold Diversity of Organic Chemistry As Seen in the CAS Registry.

A large set of organic compounds extracted from the CAS Registry is analyzed to study recent changes in structural diversity. The diversity is characterized using the framework content of the compounds; the framework of a molecule is the scaffold consisting of all its ring systems and all the chain fragments connecting them. The compounds are partitioned based on their year of first report in the literature, which allows framework occurrence frequencies to be compared across a 10-year interval. The results are consistent with a process in which frameworks with the greatest frequency of use in the past are the most likely to be used again, but it is also found that the frequency ordering changes over time. These fluctuations in ordering are attributed to stochastic factors, scientific and economic, that can affect how chemical space is explored. Framework diversity is found to have increased over time despite the extensive reuse of a relatively small number of frameworks; this increase is due to the large number of new frameworks. The long tail of the framework distribution, composed of frameworks that occur in few compounds or only one compound, is found to be a large and growing part of framework space.

A non-canonical SWI/SNF complex is a synthetic lethal target in cancers driven by BAF complex perturbation.

Mammalian SWI/SNF chromatin remodelling complexes exist in three distinct, final-form assemblies: canonical BAF (cBAF), PBAF and a newly characterized non-canonical complex (ncBAF). However, their complex-specific targeting on chromatin, functions and roles in disease remain largely undefined. Here, we comprehensively mapped complex assemblies on chromatin and found that ncBAF complexes uniquely localize to CTCF sites and promoters. We identified ncBAF subunits as synthetic lethal targets specific to synovial sarcoma and malignant rhabdoid tumours, which both exhibit cBAF complex (SMARCB1 subunit) perturbation. Chemical and biological depletion of the ncBAF subunit, BRD9, rapidly attenuates synovial sarcoma and malignant rhabdoid tumour cell proliferation. Importantly, in cBAF-perturbed cancers, ncBAF complexes maintain gene expression at retained CTCF-promoter sites and function in a manner distinct from fusion oncoprotein-bound complexes. Together, these findings unmask the unique targeting and functional roles of ncBAF complexes and present new cancer-specific therapeutic targets.

Binding of TMPRSS2-ERG to BAF Chromatin Remodeling Complexes Mediates Prostate Oncogenesis.

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.

The SS18-SSX Fusion Oncoprotein Hijacks BAF Complex Targeting and Function to Drive Synovial Sarcoma.

Synovial sarcoma (SS) is defined by the hallmark SS18-SSX fusion oncoprotein, which renders BAF complexes aberrant in two manners: gain of SSX to the SS18 subunit and concomitant loss of BAF47 subunit assembly. Here we demonstrate that SS18-SSX globally hijacks BAF complexes on chromatin to activate an SS transcriptional signature that we define using primary tumors and cell lines. Specifically, SS18-SSX retargets BAF complexes from enhancers to broad polycomb domains to oppose PRC2-mediated repression and activate bivalent genes. Upon suppression of SS18-SSX, reassembly of BAF47 restores enhancer activation, but is not required for proliferative arrest. These results establish a global hijacking mechanism for SS18-SSX on chromatin, and define the distinct contributions of two concurrent BAF complex perturbations.

Disruption of mammalian SWI/SNF and polycomb complexes in human sarcomas: mechanisms and therapeutic opportunities.

Soft-tissue sarcomas are increasingly characterized and subclassified by genetic abnormalities that represent underlying drivers of their pathology. Hallmark tumor suppressor gene mutations and pathognomonic gene fusions collectively account for approximately one-third of all sarcomas. These genetic abnormalities most often result in global transcriptional misregulation via disruption of protein regulatory complexes which govern chromatin architecture. Specifically, alterations to mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes and polycomb repressive complexes cause disease-specific changes in chromatin architecture and gene expression across a number of sarcoma subtypes. Understanding the functions of chromatin regulatory complexes and the mechanisms underpinning their roles in oncogenesis will be required for the design and development of new therapeutic strategies in sarcomas. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

SMARCB1 is required for widespread BAF complex-mediated activation of enhancers and bivalent promoters.

Perturbations to mammalian SWI/SNF (mSWI/SNF or BAF) complexes contribute to more than 20% of human cancers, with driving roles first identified in malignant rhabdoid tumor, an aggressive pediatric cancer characterized by biallelic inactivation of the core BAF complex subunit SMARCB1 (BAF47). However, the mechanism by which this alteration contributes to tumorigenesis remains poorly understood. We find that BAF47 loss destabilizes BAF complexes on chromatin, absent significant changes in complex assembly or integrity. Rescue of BAF47 in BAF47-deficient sarcoma cell lines results in increased genome-wide BAF complex occupancy, facilitating widespread enhancer activation and opposition of Polycomb-mediated repression at bivalent promoters. We demonstrate differential regulation by two distinct mSWI/SNF assemblies, BAF and PBAF complexes, enhancers and promoters, respectively, suggesting that each complex has distinct functions that are perturbed upon BAF47 loss. Our results demonstrate collaborative mechanisms of mSWI/SNF-mediated gene activation, identifying functions that are co-opted or abated to drive human cancers and developmental disorders.

Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain.

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

Determination of Abraham model solute descriptors for the monomeric and dimeric forms of trans-cinnamic acid using measured solubilities from the Open Notebook Science Challenge.

BACKGROUND: Calculating Abraham descriptors from solubility values requires that the solute have the same form when dissolved in all solvents. However, carboxylic acids can form dimers when dissolved in non-polar solvents. For such compounds Abraham descriptors can be calculated for both the monomeric and dimeric forms by treating the polar and non-polar systems separately. We illustrate the method of how this can be done by calculating the Abraham descriptors for both the monomeric and dimeric forms of trans-cinnamic acid, the first time that descriptors for a carboxylic acid dimer have been obtained. RESULTS: Abraham descriptors were calculated for the monomeric form of trans-cinnamic acid using experimental solubility measurements in polar solvents from the Open Notebook Science Challenge together with a number of water-solvent partition coefficients from the literature. Similarly, experimental solubility measurements in non-polar solvents were used to determine Abraham descriptors for the trans-cinnamic acid dimer. CONCLUSION: Abraham descriptors were calculated for both the monomeric and dimeric forms of trans-cinnamic acid. This allows for the prediction of further solubilities of trans-cinnamic acid in both polar and non-polar solvents with an error of about 0.10 log units. Graphical abstractMolar concentration of trans-cinnamic acid in various polar and non-polar solvents.