Pandey, Akhilesh

Abstract BRCA2 mutations contribute to the pathogenesis and treatment sensitivity of a subset of ovarian, breast, prostate, and pancreatic cancers. When these cancers become therapy resistant, secondary mutations that restore the BRCA2 open reading frame are found in half the cases, but other causes of resistance remain incompletely understood. Here, we identified translational readthrough of a premature termination codon (PTC) as a cause of resistance to poly(ADP-ribose) polymerase inhibitors (PARPis) and cisplatin in cells derived from the BRCA2-mutated ovarian cancer line PEO1 by PARPi selection. Despite persistence of the signature 4965C > G (p.Y1655X) BRCA2 mutation, low-level expression of full-length BRCA2 protein was detectable in these cells by immunoblotting and tandem mass spectrometry. Either BRCA2 knockdown or gene interruption 5′ or 3′ to the PTC restored treatment sensitivity, implicating BRCA2 in the resistance. Reporter assays demonstrated UAG-selective readthrough in the resistant clones but not parental cells. Moreover, custom searching of global proteomic data indicated readthrough of stop codons, particularly UAGs, in additional proteins in the resistant clones. Finally, multi-omic analysis identified multiple changes in the nonsense-mediated decay and termination machineries that favor readthrough. Accordingly, the present results identify PTC readthrough as a potential mechanism of drug resistance in cells with BRCA2 nonsense mutations.

Patient-derived organoids (PDOs) have emerged as powerful tools in personalized medicine applicable to both non-malignant conditions and to cancer, where they are increasingly used for personalized drug screening and precision treatment strategies in part due to their ability to replicate tumor heterogeneity. They also serve as study model systems to understand disease mechanisms, pathways, and the impact of ex vivo exposures. We present a detailed step-by-step protocol for generating organoids from normal crypts, polyps, and tumors, including methods for tissue processing, crypt isolation, culture establishment, and the transition from basolateral to apical-out polarity for co-culture and exposure-based studies. The protocol also includes immunofluorescence staining procedures for cellular characterization and quality control measures. Our standardized approach successfully generates organoids from diverse colorectal tissues with high efficiency and reproducibility. This comprehensive guide addresses common technical challenges and provides troubleshooting strategies to improve success rates across different sample types. We believe that this resource will enhance reproducibility in organoid research and expand their utility in translational applications, particularly for personalized medicine approaches in colorectal cancer.

Key Points Increased protein expression of tissue inhibitor of metalloproteinase-3, glypican 6, synuclein γ , apolipoprotein A-4, and Ecto-5′-nucleotidase was observed in old adults compared with young adults. In old adults, overexpressed proteins were enriched in mitochondrial processes while under expressed proteins enriched in RNA metabolism. Validated differentially expressed proteins were not significantly correlated with kidney pathology after adjusting for age. Background Kidney aging is characterized by a loss of glomeruli, predominantly in the superficial cortex, with a resultant decline in GFR and an increased risk of various kidney-related diseases. The early molecular alterations in glomeruli associated with the aging process are not well studied. Methods We combined laser capture microdissection and mass spectrometry–based unbiased proteomic analysis of nonsclerosed, nonischemic glomeruli in the superficial cortex from young and old adults who underwent a radical nephrectomy for a tumor to understand the age-related molecular changes in glomeruli. Twenty-four young and 30 old adults were used for the discovery dataset, and the significant differentially expressed proteins were further validated using an independent set comprising six young and eight old adults. Results Kidneys from old adults had lower eGFR, less kidney parenchyma on computed tomography imaging, and more glomerulosclerosis and arteriosclerosis on histology. Quantitative proteomic analysis of nonsclerosed, nonischemic glomeruli identified increased expression of tissue inhibitor of metalloproteinase-3, glypican 6, synuclein γ , apolipoprotein A-4, and Ecto-5′-nucleotidase in old adults that were further validated in an independent set. Pathway analysis indicated that proteins with increased expression in old adults were enriched in mitochondrial translational processes, aerobic respiration, and TCA cycle, whereas proteins with decreased expression in old adults were enriched in mRNA splicing, mRNA processing, and nonsense mediated decay. Furthermore, validated differentially expressed proteins did not show any significant correlation with the kidney pathology independent of age group. Conclusions Overall, this study identified proteins that are specifically associated with the aging process in otherwise normal-appearing glomeruli.

Background and Aims: Cholangiocarcinoma (CCA) is one of the most lethal cancers, characterized by molecular heterogeneity and treatment resistance. To uncover new biological signals and therapeutic opportunities, we employed multiomic characterization combined with machine learning. Approach and Results: We profiled all anatomical CCA subtypes using whole exome sequencing, mRNA sequencing, and proteome/phosphoproteome analysis. Integrative dimensional reduction revealed RNA, protein, and phosphoprotein features driving tumor heterogeneity, enabling clustering. Machine learning algorithms identified molecular features for each cluster and mapped external datasets and patient-derived xenograft (PDX) models onto these clusters. Kinase enrichment analysis highlighted targetable kinases active in each cluster. In vivo validation was performed in cluster-specific PDX models using the selective TNK1 inhibitor, TP-5801. We identified 3 molecular clusters with distinct pathway characterization: immunomodulatory (cluster 1), metabolic (cluster 2), and gene regulation/cellular fate (cluster 3). Cluster assignment was independent of anatomic subtype but correlated with overall survival following curative-intent resection. We also identified multiomic features and pathways linked to overall survival and lymph node metastases, crucial for patient treatment selection. Kinase enrichment analysis pinpointed TNK1 as a highly active kinase in the metabolic cluster. Treatment with TP-5801 significantly reduced tumor growth in a metabolic PDX model, but not in models representing the other clusters. Combining internal data with publicly available datasets, we identified the immunomodulatory cluster as most responsive to gemcitabine/cisplatin therapy, confirmed in vivo using cluster-specific PDX models. Conclusions: Integrated multiomic characterization provides translational insights by defining unique molecular subtypes associated both with therapeutic response and overall clinical outcomes. This approach identified TNK1 as a previously unrecognized therapeutic target in a defined subset of CCA tumors.

ABSTRACT T‐cell receptor (TCR) signaling plays a crucial role in various biological processes and is usually studied using global mass spectrometry‐based phosphoproteomic studies. Despite advancements in targeted mass spectrometry‐based assays for protein quantification, their application in studying signaling processes, for example, reproducible measurements of post‐translational modifications (PTMs) such as phosphorylation, remains limited. Tyrosine phosphorylation is critical for many signaling pathways but presents challenges due to the low abundance of phosphotyrosine‐containing peptides. Conventional untargeted methods often encounter data gaps when analyzing large sample sets, particularly for low‐abundance peptides. To address this issue, a targeted proteomics method called “SureQuant” was employed, which relies on triggered data acquisition with heavy isotope‐labeled peptides. This method has been shown to provide sensitive and reproducible quantification of low‐abundance peptides. Here we describe the development of a SureQuant‐based method to quantify phosphotyrosine peptides that are involved in the TCR signaling pathway. To monitor the change in phosphotyrosine signals upon activation, the T‐cells were stimulated with anti‐CD3/CD28 antibodies. We successfully quantified changes in important phosphotyrosine peptides in primary T‐cells upon stimulation with anti‐CD3/CD28 antibodies. This study showcases the ability of the SureQuant approach to accurately quantify low‐abundance phosphotyrosine peptides, highlighting its broader potential to study a diverse set of PTMs in physiological or clinical settings. Summary T‐cell receptor (TCR) signaling plays a fundamental role in immune responses, regulating T‐cell activation, differentiation, and function. While tyrosine phosphorylation is a key regulatory mechanism in this pathway, the low abundance of phosphotyrosine peptides presents a major challenge for their detection and quantification in complex biological samples. By employing the SureQuant targeted mass spectrometry approach, we achieved highly sensitive and reproducible quantification of key phosphotyrosine sites involved in T‐cell activation. This study provides a systematic view of TCR signaling dynamics, revealing distinct phosphorylation patterns across different activation timepoints. Our findings demonstrate the effectiveness of SureQuant in quantifying low‐abundance, post‐translationally modified peptides, offering a valuable tool for studying signaling pathways with greater precision. Additionally, this methodological framework can be extended to investigate other signaling networks, immune cell functions, and disease‐associated phosphotyrosine modifications.

Abstract The lack of a molecular understanding of mood disorders has impeded progress in diagnosis and treatment. Glycosylation may provide insights into the complex mechanisms underlying these conditions. We conducted N-glycoproteomic analysis on dorsolateral prefrontal cortex samples from individuals with major depressive disorder (MDD) and bipolar disorder (BD), in depressive or manic states at death. Additionally, we examined depression prevalence in congenital disorders of glycosylation (CDG) through a literature review and assessment of 110 CDG patients. Glycoproteomic analysis revealed a significant increase in protein glycosylation in individuals with MDD relative to both controls and individuals with BD. Depression prevalence was lower in our pediatric and adult cohort of individuals with CDG. These results suggest brain glycosylation changes may play a role in mood disorder pathology and highlight the distinct biology of unipolar and bipolar depression. Our findings propose that impaired glycosylation may confer resilience to depression, offering potential therapeutic insights.

The quinoxaline core is found in several biologically active compounds, with Erdafitinib being the first FDA-approved quinoxaline derivative that targets a kinase and exhibits anti-cancer properties. We previously reported a quinoxaline analog (84) that displayed anti-cancer effects by inhibiting IKKβ, a key kinase in the NFκB pathway. Here, we present the synthesis of a regioisomer (51-106) and its characterization as a selective MAP3K1 inhibitor with improved metabolic stability and oral bioavailability. We used the small molecule MAP3K1 inhibitor in a proteomics study that identified NPM1 as a member of the MAP3K1 network.

Increased infiltration of CD3 + and CD8 + T cells into ovarian cancer (OC) is linked to better prognosis, but the specific antigens involved are unclear. Recent reports suggest that HLA class I can present peptides from noncoding genomic regions, known as noncanonical or cryptic peptides, but their immunogenicity is underexplored. To address this, we used immunopeptidomic analysis and RNA sequencing on five metastatic OC samples, which identified 311 cryptic peptides (40 to 83 per patient). Despite comprising less than 1% of total peptides, cryptic peptides from noncoding transcripts emerged as the predominant antigen class when compared to the other major classes of known tumor-specific and tumor-associated antigens in OC samples. Notably, nearly 70% of the prioritized cryptic peptides elicited T cell activation, as evidenced by increased 4-1BB and IFN-γ expression in autologous CD8 + T cells. This study reveals noncoding cryptic peptides as an important class of immunogenic antigens in OC.

Abstract DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematologic neoplasms. Although individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Here, we make use of a natural inflammatory response occurring during coronavirus disease 2019 (COVID-19), to understand the association of these mutations with inflammatory morbidity (acute respiratory distress syndrome [ARDS]) and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant (DNMT3Amt) CH on COVID-19–related ARDS and mortality. Using single-cell proteogenomics we show that DNMT3A mutations involve myeloid and lymphoid lineage cells. Using single-cell multiomics sequencing, we identify cell-specific gene expression changes associated with DNMT3A mutations, along with significant epigenomic deregulation affecting enhancer accessibility, resulting in overexpression of interleukin-32 (IL-32), a proinflammatory cytokine that can result in inflammasome activation in monocytes and macrophages. Finally, we show with single-cell resolution that the loss of function of DNMT3A is directly associated with increased chromatin accessibility in mutant cells. Hence, we demonstrate the negative prognostic impact of DNMT3Amt CH on COVID-19–related ARDS and mortality. DNMT3Amt CH in the context of COVID-19, was associated with inflammatory transcriptional priming, resulting in overexpression of IL32. This overexpression was secondary to increased chromatic accessibility, specific to DNMT3Amt CH cells. DNMT3Amt CH can thus serve as a potential biomarker for adverse outcomes in COVID-19.