Pandey, Akhilesh

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.

Abstract Congenital disorders of glycosylation (CDG) are a group of neurogenetic conditions resulting from disruptions in the cellular glycosylation machinery. The majority of CDG patients have compound heterozygous pathogenic variants in the phosphomannomutase 2 ( PMM2) gene. Individuals with PMM2-CDG exhibit multi-systemic symptoms, prominently featuring neurological deficits with nearly all patients exhibiting cerebellar hypoplasia and ataxia. To overcome embryonic lethality caused by whole body knock-out of Pmm2 and mimic patient-related compound heterozygous pathogenic variants, we paired a Pmm2 flox allele ( Pmm2 fl ) with a catalytically inactive knock-in allele ( Pmm2 R137H ), commonly present in PMM2-CDG patients. Mice with post-mitotic loss of PMM2 from neurons or astrocytes are indistinguishable from unaffected littermates, including in a broad battery of neurological assessments. In contrast, removal of PMM2 from embryonic neural precursor cells leads to cerebellar hypoplasia, ataxia, seizures, and early lethality. Comprehensive multi-omics profiling, including metabolomics, glycomics, single-cell transcriptomics, proteomics, and glycoproteomics, reveal widespread molecular disturbances throughout the brain, with the cerebellum showing the most pronounced disruption. These findings highlight the heightened dependency of the developing cerebellum on intact N-glycosylation, aligning with clinical observations in PMM2-CDG patients. Importantly, glycoproteomic alterations identified in our mouse model are corroborated in PMM2-CDG patient post-mortem cerebellar tissue, underscoring the translational relevance of our findings and implicating impaired synaptic transmission as a key pathogenic mechanism.

Circulating glucagon concentrations differ between individuals with no diabetes (ND) and those with type 1 diabetes (T1D). We combined an isotope dilution technique using stable tracers [6,22-13C9,15N1]glucagon and [6,14,19,22-13C9,15N1]glucagon with splanchnic and leg catheterization in participants with ND (n = 8; age 23.1 ± 2.9 years, BMI 26.6 ± 3.5 kg/m2, HbA1c 5.0 ± 0.2% [31 ± 2 mmol/mol]) and T1D (n = 6; 29.0 ± 8.8 years, BMI 26.3 ± 5.0 kg/m2, HbA1c 7.9 ± 0.8% [63 ± 8 mmol/mol]) in the overnight fasted state. After baseline period, exogenous glucagon was infused at rates designed to achieve plasma glucagon concentrations spanning the physiological ranges, to determine the effects of rising glucagon concentrations on splanchnic and leg glucagon balance. At baseline, splanchnic glucagon extraction (SGE) was similar (30.7 ± 2.7 vs. 29.1 ± 2.9%) but leg glucagon extraction (LGE) was lower (27.0 ± 4.2 vs. 40.6 ± 3.1%) in participants with T1D versus those with ND. However, with increasing plasma glucagon concentrations, while SGE remained unchanged within and between groups, LGE fell in participants with ND (41 vs. 31 vs. 24%) but did not change in those with T1D. Despite a numerically lower net splanchnic glucagon production in participants with T1D than in those with ND, no changes were observed with increasing glucagon concentrations within the physiological range in both groups. This is the first human study applying novel glucagon isotopes that describes regional glucagon metabolism in participants with ND and T1D. Our observations provide translational relevance for dual hormone closed-loop systems and provide tools for probing the effects of GLP-1, dual, and triple receptor agonists on pancreatic α-cell functions. Article Highlights This study was conducted to assess splanchnic and leg glucagon metabolism in humans using stable glucagon isotopes. We wanted to evaluate whether splanchnic and leg glucagon metabolism differed between participants with no diabetes (ND) and those with type 1 diabetes (T1D) at glucagon concentrations spanning the physiological range. Whereas splanchnic glucagon extraction did not differ between participants with ND and those with T1D, leg glucagon extraction fell in those with ND but did not change in those with T1D as glucagon concentrations increased. Net splanchnic glucagon production did not change with exogenous glucagon infusion. Our study has implications for dual hormone closed-loop control in T1D where glucagon is infused for prevention of hypoglycemia and for investigating the effects of emerging GLP-1, glucose-dependent insulinotropic polypeptide, and glucagon receptor agonists on endogenous glucagon secretion and clearance.

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.

Abstract Background: Cholangiocarcinoma (CCA) is a diverse type of cancer. For several years, platinum-based chemotherapy (Gemcitabine and Cisplatin) has been the standard treatment and serves as the foundation for current immunotherapy combinations. The typical response rates are generally between 20% and 30%. In this study, we present the results of preclinical efficacy trials conducted on CCA patient-derived xenograft (PDX) models, correlating them with the baseline multiomics profiling of these models to identify molecular characteristics linked to treatment response. Methods: Tumor samples from twenty-eight CCA PDX were used to isolate RNA and protein to be included in multiomics analysis (Phosphoproteomics, Proteomics, and Transcriptomics). A subset of PDX models were treated with various doses of Gemcitabine and Cisplatin. The Median Efficacy Index (MEI) was calculated as the ratio of the difference in median of delta in tumor volume at the last day of the study between the treatment and the control arms divided by the median of delta in tumor volume of the control at the last day of the treatment (positive= growth, negative=inhibition, and 0=no efficacy). A Hierarchical All-Against-All algorithm was utilized to compare multiomics features to the MEI and pathway analysis completed for identified molecular features. Pathway enrichment was calculated using the KEGG database, and random-forest variable selection chose the pathways most influential in predicting sensitivity or resistance. Results: After conducting preclinical efficacy trials, the 11 tested CCA PDX models were classified into sensitive, moderately sensitive, and resistant models based on the MEI on each treatment arm. The top up-regulated genes were identified for Gemcitabine response (SRPK2, GLK2, and RIOK2) and Cisplatin response (NTRK1, PRPF4B, and SRPK2). The top up-regulated genes related to Gemcitabine resistance were NTRK1, PIP5K1C, and CSNK2A2, while those related to Cisplatin resistance were SRPK1, PRPF4B, and CSNK2A1. Downregulation of the anti-folate resistance pathway is a strong predictor of gemcitabine sensitivity, and the lysine degradation pathway is a strong predictor of cisplatin sensitivity. Conclusion: Multiomics signatures of cholangiocarcinoma can forecast responses to standard chemotherapy in preclinical models. New markers of resistance to Gemcitabine and Cisplatin were discovered, linked to NTRK1, SRPK1, and SRPK2. These resistance profiles will be tested using suitable targeted therapies on the resistant CCA patient-derived xenograft (PDX) models. Citation Format: Amro M. Abdelrahman, Danielle M. Carlson, Ellen L. Larson, Erik Jessen, Dong-Gi Mun, Isaac Lynch, Alessandro Fogliati, Stella Konadu Adjei Antwi, Aushinie Abeynayake, Akhilesh Pandey, Mark J. Truty, Gregory J. Gores, Rory L. Smoot. Baseline multiomics and chemotherapy efficacy in preclinical trials using patient-derived xenograft models of cholangiocarcinoma can reveal distinct signatures of chemotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6762.

Abstract Introduction: Cholangiocarcinoma (CCA) is an aggressive biliary malignancy with poor survival outcomes and multiple mechanisms of resistance to targeted therapies. We previously validated NXP800, a novel GCN2 small-molecule activator, as an effective anti-cancer drug in six CCA cell lines. In vitro testing identified upregulation of phosphorylated eIF2a, as well as upregulation of ATF4, in CCA cells treated with of NXP800 compared to vehicle, suggesting that treated cells led to stimulation of stress-induced genes and subsequently, apoptosis. Furthermore, patient derived xenografts (PDX) models treated with NXP800 were found to have decreased tumor size, particularly in models containing ARID1A, FGFR1, and ATM mutations. Here, we aimed to predict response to NXP800 using sensitivity and resistance signatures from our profiled PDX models. Methods: GCN2 activation in multiple previously profiled PDX models was evaluated. Whole Exome Sequencing (WES) was done on multiple PDX models to characterize the mutational signature and activity in CCA. Two additional CCA xenografts were expanded into the flank of NOD/SCID mice. Tumor bearing mice were treated with NXP800 (35 mg/kg) or vehicle via oral gavage for 28 days once tumors averaged 120 mm3. Tumor volume and animal weight were collected twice weekly. Median Efficacy Index (MEI) scores were generated as drug response rates based on tumor growth. Predicted sensitivity and resistance kinase signatures to predict drug response were produced using multi-omics. Results: Treatment with NXP800 was associated with a statistically significant decrease in tumor size in two additional PDX models, which included ARID1A and BRAF mutants. Preliminary multi-omics on the PDX models revealed that sensitivity mechanisms were upregulated most in EGFR signaling, while resistance signatures were focused around CSNK2A1 signaling. Conclusion: Multi-omics analysis revealed a sensitivity mechanism structured around EGFR signaling, providing insights into which patients should be selected for treatment with NXP800. A Phase 1b clinical trial investigating the effects of NXP800 use in patients with advanced CCA is open and currently accepting patients. Citation Format: Danielle Marie Carlson, Hendrien Kuipers, Amro Abdelrahman, Erik Jessen, Joshua Roldan Kalill, Jennifer Tomlinson, Jack Sample, Akhilesh Pandey, Mark Truty, Rory Smoot. Modeling response prediction to a novel GCN2 kinase activator (NXP800) in patient-derived xenograft cholangiocarcinoma models using a multi-omics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5592.

AbstractBackgroundTandem mass spectrometry (MS/MS) has become a common clinical laboratory testing modality has demonstrated success in distinguishing between small protein variations in transthyretin amyloidosis. Since many common clinically significant RBC antigens are also small protein variations, this study aimed to determine if MS/MS could correctly detect common RBC antigens within the Rh, Kell, Duffy, MNS, Kidd, Diego, and Lutheran blood group systems.Study Design and MethodsResidual samples from serotyped/genotyped blood donors at a hospital‐based blood donation center from February to August 2021 were analyzed. RBC membrane protein preparations underwent protease digestion prior to MS/MS analysis for RhD, RhCE, Kell, atypical chemokine receptor 1 (Duffy), glycophorin A (MNS), glycophorin B (MNS), urea transporter 1 (Kidd), band 3 anion transport protein (Diego), and basal cell adhesion molecule (Lutheran). Untargeted liquid chromatography (LC)‐MS/MS detected protein‐specific peptides, while targeted LC‐selected reaction monitoring (LC‐SRM) detected RBC antigen‐containing peptides.ResultsThrough the use of multiple proteases, untargeted LC–MS/MS detected protein‐specific peptides in all but glycophorin B, with band 3 anion transport protein, basal cell adhesion molecule, Kell, and glycophorin A having the greatest protein sequence coverage. Targeted LC‐SRM detected antigen‐specific peptides for Cw, Dia/Dib, Kpb, and Wra/Wrb, which agreed with the donors' previous typing results.DiscussionMS/MS can successfully detect peptides from several blood group systems but only detected a subset of their common RBC antigens. Further sample enrichment and MS/MS detection improvements will need to occur before MS/MS could be considered a clinical RBC phenotyping modality.

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.

Introduction: Extracranial carotid artery pathology accounts for 15-20% of ischemic strokes. Advancements in magnetic resonance angiography (MRA) with vessel wall imaging (VWI) have enabled the identification of vulnerable plaques, aiding in risk stratification for neurovascular events. This pilot study aimed to identify proteins in plaques with and without vulnerable features on MRA with VWI. Hypothesis: There is a differential proteomic profile of vulnerable and non-vulnerable, thus, these can serve as potential biomarkers for disease progression, stroke prevention, and therapeutic targets. Methods: Consecutive patients undergoing carotid endarterectomy (CEA) were included in the study cohort with pre-operative MRA with VWI. A retrospective chart review was conducted to extract pertinent clinical data including cardiovascular risk factors and medications. The proteomic analysis involved Tandem Mass Tag (TMTpro) labeling of peptides, basic pH HPLC fractionation, and NanoLC-tandem mass spectrometry. Results: A total of sixteen plaques were retrieved from fifteen patients. There were 73.3% male patients. The most common comorbidities were hyperlipidemia (93.3%) and hypertension (80%). 31.2% of patients had symptomatic disease. Eleven plaques were vulnerable in preoperative MRI with VWI. From those, intraplaque hemorrhage was the most common feature (81.1%) . Proteomic analysis revealed 23 relevant proteins significantly elevated in vulnerable plaques, including Proteinase 3 (PRTN3), Phospholipid Transfer Protein (PLTP), and S100 Calcium-Binding Protein A12 (S100A12), with increased abundance exceeding two-fold changes or above (P<0.001). Conversely, three proteins exhibited reduced abundance in vulnerable plaques including Dynamin-3 (DNM3), Transmembrane Protein 181 (TMEM181), and Adducin-3 (ADD3) (P<0.05). All results are summarized in Figure 1A-C and Table I and II. Conclusion: This study contributes to the understanding of protein biomarkers associated with carotid plaque vulnerability, offering insights into disease progression, stroke prevention, and identification of potential therapeutic targets (Figure 1D) . Furthermore, these patients could benefit from early surgical intervention if these biomarkers were to be detected. We present a comprehensive study of multiple proteins with significant bioavailability in explanted plaque specimens that can serve as a foundation for identifying blood biomarkers for future validation studies.