Fast and broad-coverage lipidomics enabled by ion mobility-mass spectrometry.

Aberrant lipid metabolism has been widely recognized as a hallmark of various diseases. However, the comprehensive analysis of distinct lipids is challenging due to the complexity of lipid molecular structures, wide concentration ranges, and numerous isobaric and isomeric lipids. Usually, liquid chromatography-mass spectrometry (LC-MS)-based lipidomics requires a long time for chromatographic separation to achieve optimal separation and selectivity. Ion mobility (IM) adds a new separation dimension to LC-MS, significantly enhancing the coverage, sensitivity, and resolving power. We took advantage of the rapid separation provided by ion mobility and optimized a fast and broad-coverage lipidomics method using the LC-IM-MS technology. The method required only 8 minutes for separation and detected over 1000 lipid molecules in a single analysis of common biological samples. The high reproducibility and accurate quantification of this high-throughput lipidomics method were systematically characterized. We then applied the method to comprehensively measure dysregulated lipid metabolism in patients with colorectal cancer (CRC). Our results revealed 115 significantly changed lipid species between preoperative and postoperative plasma of patients with CRC and also disclosed associated differences in lipid classes such as phosphatidylcholines (PC), sphingomyelins (SM), and triglycerides (TG) regarding carbon number and double bond. Together, a fast and broad-coverage lipidomics method was developed using ion mobility-mass spectrometry. This method is feasible for large-scale clinical lipidomic studies, as it effectively balances the requirements of high-throughput and broad-coverage in clinical studies.

Encoding LC-MS-Based Untargeted Metabolomics Data into Images toward AI-Based Clinical Diagnosis.

Liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics provides comprehensive and quantitative profiling of metabolites in clinical investigations. The use of whole metabolome profiles is a promising strategy for disease diagnosis but technically challenging. Here, we developed an approach, namely MetImage, to encode LC-MS-based untargeted metabolomics data into multi-channel digital images. Then, the images that represent the comprehensive metabolome profiles can be employed for developing deep learning-based AI models toward clinical diagnosis. In this work, we demonstrated the application of MetImage for clinical screening of esophageal squamous cell carcinoma (ESCC) in a clinical cohort with 1104 participants. A convolutional neuronal network-based AI model was trained to distinguish ESCC screening positive and negative subjects using their serum metabolomics data. Superior performances such as sensitivity (85%), specificity (92%), and area under curve (0.95) were validated in an independent testing cohort (N = 442). Importantly, we demonstrated that our AI-based ESCC screening model is not a "black box". The encoded images reserved the characteristics of mass spectra from the raw LC-MS data; therefore, metabolite identifications in key image features were readily achieved. Altogether, MetImage is a unique approach that encodes raw LC-MS-based untargeted metabolomics data into images and facilitates the utilization of whole metabolome profiles for AI-based clinical applications with improved interpretability.

Serum metabolic traits reveal therapeutic toxicities and responses of neoadjuvant chemoradiotherapy in patients with rectal cancer.

Neoadjuvant chemoradiotherapy (nCRT) has become the standard treatment for patients with locally advanced rectal cancer (LARC). Therapeutic efficacy of nCRT is significantly affected by treatment-induced diarrhea and hematologic toxicities. Metabolic alternations in cancer therapy are key determinants to therapeutic toxicities and responses, but exploration in large-scale clinical studies remains limited. Here, we analyze 743 serum samples from 165 LARC patients recruited in a phase III clinical study using untargeted metabolomics and identify responsive metabolic traits over the course of nCRT. Pre-therapeutic serum metabolites successfully predict the chances of diarrhea and hematologic toxicities during nCRT. Particularly, levels of acyl carnitines are linked to sex disparity in nCRT-induced diarrhea. Finally, we show that differences in phenylalanine metabolism and essential amino acid metabolism may underlie distinct therapeutic responses of nCRT. This study illustrates the metabolic dynamics over the course of nCRT and provides potential to guide personalized nCRT treatment using responsive metabolic traits.

A two-step mechanism governing PARP1-DNA retention by PARP inhibitors.

PARP inhibitors (PARPi) have emerged as promising cancer therapeutics capable of targeting specific DNA repair pathways, but their mechanism of action with respect to PARP1-DNA retention remains unclear. Here, we developed single-molecule assays to directly monitor the retention of PARP1 on DNA lesions in real time. Our study reveals a two-step mechanism by which PARPi modulate the retention of PARP1 on DNA lesions, consisting of a primary step of catalytic inhibition via binding competition with NAD+ followed by an allosteric modulation of bound PARPi. While clinically relevant PARPi exhibit distinct allosteric modulation activities that can either increase retention of PARP1 on DNA or induce its release, their retention potencies are predominantly determined by their ability to outcompete NAD+ binding. These findings provide a mechanistic basis for improved PARPi selection according to their characteristic activities and enable further development of more potent inhibitors.

USP1-trapping lesions as a source of DNA replication stress and genomic instability.

The deubiquitinase USP1 is a critical regulator of genome integrity through the deubiquitylation of Fanconi Anemia proteins and the DNA replication processivity factor, proliferating cell nuclear antigen (PCNA). Uniquely, following UV irradiation, USP1 self-inactivates through autocleavage, which enables its own degradation and in turn, upregulates PCNA monoubiquitylation. However, the functional role for this autocleavage event during physiological conditions remains elusive. Herein, we discover that cells harboring an autocleavage-defective USP1 mutant, while still able to robustly deubiquitylate PCNA, experience more replication fork-stalling and premature fork termination events. Using super-resolution microscopy and live-cell single-molecule tracking, we show that these defects are related to the inability of this USP1 mutant to be properly recycled from sites of active DNA synthesis, resulting in replication-associated lesions. Furthermore, we find that the removal of USP1 molecules from DNA is facilitated by the DNA-dependent metalloprotease Spartan to counteract the cytotoxicity caused by "USP1-trapping". We propose a utility of USP1 inhibitors in cancer therapy based on their ability to induce USP1-trapping lesions and consequent replication stress and genomic instability in cancer cells, similar to how non-covalent DNA-protein crosslinks cause cytotoxicity by imposing steric hindrances upon proteins involved in DNA transactions.

A basal-level activity of ATR links replication fork surveillance and stress response.

Mammalian cells use diverse pathways to prevent deleterious consequences during DNA replication, yet the mechanism by which cells survey individual replisomes to detect spontaneous replication impediments at the basal level, and their accumulation during replication stress, remain undefined. Here, we used single-molecule localization microscopy coupled with high-order-correlation image-mining algorithms to quantify the composition of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a basal-level activity of ATR that monitors and regulates the amounts of RPA at forks during normal replication. Replication-stress amplifies the basal activity through the increased volume of ATR-RPA interaction and diffusion-driven enrichment of ATR at forks. This localized crowding of ATR enhances its collision probability, stimulating the activation of its replication-stress response. Finally, we provide a computational model describing how the basal activity of ATR is amplified to produce its canonical replication stress response.

Super-resolution visualization of distinct stalled and broken replication fork structures.

Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). Super-resolution microscopy has made it possible to directly observe replication stress and DNA damage inside cells, however new approaches to sample preparation and analysis are required. Here we develop and apply multicolor single molecule microscopy to visualize individual replication forks under mild stress from the trapping of Topoisomerase I cleavage complexes, a damage induction which closely mimics endogenous replicative stress. We observe RAD51 and RAD52, alongside RECQ1, as the first responder proteins to stalled but unbroken forks, whereas Ku and MRE11 are initially recruited to seDSBs. By implementing novel super-resolution imaging assays, we are thus able to discern closely related replication fork stress motifs and their repair pathways.

CDK7 Inhibition Potentiates Genome Instability Triggering Anti-tumor Immunity in Small Cell Lung Cancer.

Cyclin-dependent kinase 7 (CDK7) is a central regulator of the cell cycle and gene transcription. However, little is known about its impact on genomic instability and cancer immunity. Using a selective CDK7 inhibitor, YKL-5-124, we demonstrated that CDK7 inhibition predominately disrupts cell-cycle progression and induces DNA replication stress and genome instability in small cell lung cancer (SCLC) while simultaneously triggering immune-response signaling. These tumor-intrinsic events provoke a robust immune surveillance program elicited by T cells, which is further enhanced by the addition of immune-checkpoint blockade. Combining YKL-5-124 with anti-PD-1 offers significant survival benefit in multiple highly aggressive murine models of SCLC, providing a rationale for new combination regimens consisting of CDK7 inhibitors and immunotherapies.

Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors.

Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II are necessary for DNA-damage-response (DDR) focus formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNA polymerase II preinitiation complex, MED1 and CDK9. Absence or inactivation of these factors causes a reduction in DDR foci both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins, such as 53BP1, into foci that exhibit liquid-liquid phase-separation condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci.

Disruption of Ca2+i Homeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice.

BACKGROUND: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+]i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. CONCLUSIONS: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Ultrafast data mining of molecular assemblies in multiplexed high-density super-resolution images.

Multicolor single-molecule localization super-resolution microscopy has enabled visualization of ultrafine spatial organizations of molecular assemblies within cells. Despite many efforts, current approaches for distinguishing and quantifying such organizations remain limited, especially when these are contained within densely distributed super-resolution data. In theory, higher-order correlation such as the Triple-Correlation function is capable of obtaining the spatial configuration of individual molecular assemblies masked within seemingly discorded dense distributions. However, due to their enormous computational cost such analyses are impractical, even for high-end computers. Here, we developed a fast algorithm for Triple-Correlation analyses of high-content multiplexed super-resolution data. This algorithm computes the probability density of all geometric configurations formed by every triple-wise single-molecule localization from three different channels, circumventing impractical 4D Fourier Transforms of the entire megapixel image. This algorithm achieves 102-folds enhancement in computational speed, allowing for high-throughput Triple-Correlation analyses and robust quantification of molecular complexes in multiplexed super-resolution microscopy.

Spatiotemporal dynamics of homologous recombination repair at single collapsed replication forks.

Homologous recombination (HR) is a crucial pathway for the repair of DNA double-strand breaks. BRCA1/2 breast cancer proteins are key players in HR via their mediation of RAD51 nucleofilament formation and function; however, their individual roles and crosstalk in vivo are unknown. Here we use super-resolution (SR) imaging to map the spatiotemporal kinetics of HR proteins, revealing the interdependent relationships that govern the dynamic interplay and progression of repair events. We show that initial single-stranded DNA/RAD51 nucleofilament formation is mediated by RAD52 or, in the absence of RAD52, by BRCA2. In contrast, only BRCA2 can orchestrate later RAD51 recombinase activity during homology search and resolution. Furthermore, we establish that upstream BRCA1 activity is critical for BRCA2 function. Our analyses reveal the underlying epistatic landscape of RAD51 functional dependence on RAD52, BRCA1, and BRCA2 during HR and explain the phenotypic similarity of diseases associated with mutations in these proteins.

SWATHtoMRM: Development of High-Coverage Targeted Metabolomics Method Using SWATH Technology for Biomarker Discovery.

The complexity of metabolome presents a great analytical challenge for quantitative metabolite profiling, and restricts the application of metabolomics in biomarker discovery. Targeted metabolomics using multiple-reaction monitoring (MRM) technique has excellent capability for quantitative analysis, but suffers from the limited metabolite coverage. To address this challenge, we developed a new strategy, namely, SWATHtoMRM, which utilizes the broad coverage of SWATH-MS technology to develop high-coverage targeted metabolomics method. Specifically, SWATH-MS technique was first utilized to untargeted profile one pooled biological sample and to acquire the MS2 spectra for all metabolites. Then, SWATHtoMRM was used to extract the large-scale MRM transitions for targeted analysis with coverage as high as 1000-2000 metabolites. Then, we demonstrated the advantages of SWATHtoMRM method in quantitative analysis such as coverage, reproducibility, sensitivity, and dynamic range. Finally, we applied our SWATHtoMRM approach to discover potential metabolite biomarkers for colorectal cancer (CRC) diagnosis. A high-coverage targeted metabolomics method with 1303 metabolites in one injection was developed to profile colorectal cancer tissues from CRC patients. A total of 20 potential metabolite biomarkers were discovered and validated for CRC diagnosis. In plasma samples from CRC patients, 17 out of 20 potential biomarkers were further validated to be associated with tumor resection, which may have a great potential in assessing the prognosis of CRC patients after tumor resection. Together, the SWATHtoMRM strategy provides a new way to develop high-coverage targeted metabolomics method, and facilitates the application of targeted metabolomics in disease biomarker discovery. The SWATHtoMRM program is freely available on the Internet ( http://www.zhulab.cn/software.php ).

ATR-mediated phosphorylation of FANCI regulates dormant origin firing in response to replication stress.

Excess dormant origins bound by the minichromosome maintenance (MCM) replicative helicase complex play a critical role in preventing replication stress, chromosome instability, and tumorigenesis. In response to DNA damage, replicating cells must coordinate DNA repair and dormant origin firing to ensure complete and timely replication of the genome; how cells regulate this process remains elusive. Herein, we identify a member of the Fanconi anemia (FA) DNA repair pathway, FANCI, as a key effector of dormant origin firing in response to replication stress. Cells lacking FANCI have reduced number of origins, increased inter-origin distances, and slowed proliferation rates. Intriguingly, ATR-mediated FANCI phosphorylation inhibits dormant origin firing while promoting replication fork restart/DNA repair. Using super-resolution microscopy, we show that FANCI co-localizes with MCM-bound chromatin in response to replication stress. These data reveal a unique role for FANCI as a modulator of dormant origin firing and link timely genome replication to DNA repair.