Biology subjects

Guha, U.

Publications and source records attributed to Guha, U..

9 recordsLinked to original sources

bioRxiv2026

Multifaceted regulations of HSV-1 ICP0 on the Anti-Viral Restrictions Imposed by the Host Hippo Kinases Reprogramming

The Hippo pathway is conserved across eukaryotes and controls key biological processes, including cell growth and organ development. Notably, humans with biallelic loss-of-function mutations in the Hippo kinase gene MST1 suffer from combined immunodeficiency, including recurrent infections of herpes simplex virus (HSV), implicating the pathway in host immune regulation. We investigated the role of MST1 and its homolog MST2 in HSV-1 infection. We found that human epithelial cells HEp-2 proteolytically converted full-length MST1/2 into smaller N-terminal fragments (MST1/2-NT) to enhance cell apoptosis in response to the HSV-1 infection. Moreover, while infection by mutants lacking ICP0 or US3 elevated the production of MST1/2-NT and apoptosis, the overexpression of MST1-NT significantly reduced HSV-1 replication, revealing anti-viral properties of MST1/2 cleavage and the viral counteractions by ICP0 and US3. Consistently, we discovered that MST1/2-NT production, which was high in {Delta}ICP0-infected HEp-2 cells, was completely diminished in cells permissive to the {Delta}ICP0 infection, linking the counteraction against host MST1/2 cleavage to ICP0 functions. In addition, host caspases cleaved MST1/2 with differential preferences toward MST1 or MST2 in different infection contexts, indicating multiple regulations on the MST1/2-NT production during HSV-1 infection. While double-knockouts of MST1/2 in HEp-2 cells had marginal effects on wild type HSV-1 replication, it substantially reduced the early intake of {Delta}ICP0 DNA, suggesting a role of full-length MST1/2 in early infection. Altogether, these results uncover the novel and distinct roles of full-length and cleaved Hippo kinases during HSV-1 infection and their multifaceted interactions with ICP0. ImportanceHSV poses serious threats to human health, ranging from cold sores to fatal brain infection. As a successful human pathogen, it deploys various viral proteins to counteract host defenses and subjugate host machinery, but mechanisms underlying these complex HSV-host interactions are not fully understood. For the first time, we report multifaceted interactions between the Hippo kinases and viral proteins during HSV-1 infection. We show that the Hippo kinases are converted to smaller fragments through protein cleavage in HSV-1 infected cells, and the cleaved Hippo fragments are accompanied by host cell death to execute their anti-viral activities. Moreover, multiple viral proteins contribute to counteracting this host defense, including ICP0, which executes a complex interplay with the Hippo kinases to promote infection. Understanding this new layer of virus-host interaction may pave the road to developing novel treatments for herpetic diseases.

microbiology↗

bioRxiv2024

Microenvironment Shapes Cell State, Plasticity, and Heterogeneity of Small Cell Lung Cancer

Small-cell lung cancer (SCLC) is the most fatal form of lung cancer. Intra-tumoral heterogeneity, marked by neuroendocrine (NE) and non-neuroendocrine (non-NE) cell states, defines SCLC, but the drivers of SCLC plasticity are poorly understood. To map the landscape of SCLC tumor microenvironment (TME), we apply spatially resolved transcriptomics and quantitative mass spectrometry-based proteomics to metastatic SCLC tumors obtained via rapid autopsy. The phenotype and overall composition of non-malignant cells in the tumor microenvironment (TME) exhibits substantial variability, closely mirroring the tumor phenotype, suggesting TME-driven reprogramming of NE cell states. We identify cancer-associated fibroblasts (CAF) as a crucial element of SCLC TME heterogeneity, contributing to immune exclusion, and predicting exceptionally poor prognosis. Together, our work provides a comprehensive map of SCLC tumor and TME ecosystems, emphasizing their pivotal role in SCLCs adaptable nature, opening possibilities for re-programming the intercellular communications that shape SCLC tumor states.

cancer biology↗

bioRxiv2021

NAD metabolism modulates mitochondrial function and inflammation and prevents progression of diabetic kidney disease

Diabetes mellitus is the leading cause of cardiovascular and renal disease in the United States. In spite of the beneficial interventions available for patients with diabetes, there remains a need for additional therapeutic targets and therapies in diabetic kidney disease (DKD). Inflammation and oxidative stress are increasingly recognized as important causes of renal diseases. Inflammation is closely associated with mitochondrial damage. The molecular connection between inflammation and mitochondrial metabolism remains to be elucidated. Recently, nicotinamide adenine nucleotide (NAD+) metabolism has been found to regulate immune function and inflammation. In the present studies we tested the hypothesis that enhancing NAD metabolism could prevent inflammation in and progression of DKD. We found that treatment of db/db mice with type 2 diabetes with nicotinamide riboside (NR) prevented several manifestations of kidney dysfunction (i.e., albuminuria, increased urinary kidney injury marker-1 (KIM1) excretion and pathologic changes). These effects were associated with decreased inflammation, at least in part via inhibiting the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway. An antagonist of the serum stimulator of interferon genes (STING) and whole-body STING deletion in diabetic mice showed similar renoprotection. Further analysis found that NR increased SIRT3 activity and improved mitochondrial function, which led to decreased mitochondrial DNA damage, a trigger for mitochondrial DNA leakage which activates the cGAS-STING pathway. Overall, these data show that NR supplementation boosted NAD metabolism to augment mitochondrial function, reducing inflammation and thereby preventing progression of diabetic kidney disease.

pathology↗

bioRxiv2021

A novel small molecule that induces cytotoxicity in lung cancer cells inhibits disulfide reductases GSR and TXNRD1

High-throughput phenotype-based screening of large libraries of novel compounds without known targets can identify small molecules that elicit a desired cellular response, but additional approaches are required to find and characterize their targets and mechanisms of action. Here we show that a compound termed lung cancer screen 3 (LCS3), previously selected for its ability to impair the growth of human lung adenocarcinoma (LUAD) cell lines, but not normal lung cells, induces oxidative stress and activates the NRF2 signaling pathway by generating reactive oxygen species (ROS) in sensitive LUAD cell lines. To identify the target that mediates this effect, we applied thermal proteome profiling (TPP) and uncovered the disulfide reductases GSR and TXNRD1 as LCS3 targets. Through enzymatic assays using purified protein, we confirmed that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are correspondingly sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identified the loss of NQO1 as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential utility of inhibiting disulfide reductases as a therapeutic strategy for LUAD.

cancer biology↗

bioRxiv2020

Proteogenomic analysis unveils the HLA Class I presented immunopeptidome in melanoma and EGFR mutant lung adenocarcinoma

Immune checkpoint inhibitor and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential for cancers with high tumor mutation burden (TMB). Here, we employed mass spectrometry (MS)-based proteogenomic large-scale profiling to identify potential immunogenic human leukocyte antigen (HLA) Class I-presented peptides in both melanoma, a "hot tumor" with high TMB, and EGFR mutant lung adenocarcinoma, a "cold tumor" with low TMB. We identified several classes of neopeptides, including mutated neoantigens and more than 1000 post-translationally modified peptides representing 58 different PTMs. We constructed a cancer germline (CG) antigen database with 285 antigens and identified 42 Class I-presented CG antigens. Finally, we developed a non-canonical peptide discovery pipeline to identify 44 lncRNA-derived peptides and validated Class I binding for select neopeptides. We provide direct evidence of HLA Class I presentation of a large number of neopeptides for potential vaccine or adoptive cell therapy in melanoma and mutant EGFR lung cancer.

immunology↗

bioRxiv2020

Global proteome and phosphoproteome alterations in third generation EGFR TKI resistance reveal drug targets to circumvent resistance

Lung cancer is the leading cause of cancer mortality worldwide. The treatment of lung cancer patients harboring mutant EGFR with orally administered EGFR TKIs has been a paradigm shift. Osimertinib and rociletinib are 3rd generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here, we have characterized the proteome and phosphoproteome of a series of isogenic EGFR mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs. To our knowledge, this is the most comprehensive proteomic dataset resource to date to investigate 3rd generation EGFR TKI resistance in lung adenocarcinoma. We have interrogated this unbiased global quantitative mass spectrometry dataset to uncover alterations in signaling pathways, reveal a proteomic signature of epithelial mesenchymal transition (EMT) and identify kinases and phosphatases with altered expression and phosphorylation in TKI resistant cells. Decreased tyrosine phosphorylation of key sites in the phosphatase SHP2 suggests its inhibition resulting in inhibition of RAS/MAPK and activation of PI3K/AKT pathways. Furthermore, we performed anticorrelation analyses of this phosphoproteomic dataset with the published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures (LINCS) program to predict drugs with the potential to overcome EGFR TKI resistance. We identified dactolisib, a PI3K/mTOR inhibitor, which in combination with osimertinib overcomes resistance both in vitro and in vivo.One Sentence Summary Global quantitative proteome and phosphoproteome analyses to examine altered signaling pathways in isogenic 3rd generation EGFR TKI sensitive and resistant cells.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2019

ERR agonism reverses mitochondrial dysfunction and inflammation in aging

BackgroundA gradual decline in renal function occurs even in healthy aging individuals. In addition to aging per se, concurrent metabolic syndrome and hypertension, which are common in the aging population, can induce mitochondrial dysfunction and inflammation, which collectively contribute to age-related kidney dysfunction and disease. Here we studied the role of the nuclear hormone receptors, the estrogen-related receptors (ERRs) in regulation of age-related mitochondrial dysfunction and inflammation. ERRs were decreased in aging human and mouse kidneys and were preserved in aging mice with lifelong caloric restriction (CR). MethodsA pan-ERR agonist was used to treat 21-month-old mice for 8-weeks. In addition, 21-month-old mice were treated with a STING inhibitor for 3 weeks. ResultsRemarkably, only an 8-week treatment with a pan-ERR agonist reversed the age-related increases in albuminuria, podocyte loss, mitochondrial dysfunction and inflammatory cytokines, including the cGAS-STING and STAT3 signaling pathways. A 3-week treatment of 21-month-old mice with a STING inhibitor reversed the increases in inflammatory cytokines and the senescence marker p21 but also unexpectedly reversed the age-related decreases in PGC-1, ERR, mitochondrial complexes and MCAD expression. ConclusionsOur studies identified ERRs as important modulators of age-related mitochondrial dysfunction and inflammation. These findings highlight novel druggable pathways that can be further evaluated to prevent progression of age-related kidney disease. Significance StatementThere is an increasing need for prevention and treatment strategies for age-related kidney disease. The hallmarks of aging kidneys are decreased mitochondrial function and increased inflammation. The expression of the nuclear hormone receptors estrogen-related receptors (ERRs) are decreased in aging human and mouse kidneys. This paper investigates the role of ERRs in the aging kidney. Treatment of aging mice with a pan-ERR agonist reversed the age-related increases in albuminuria and podocyte loss, mitochondrial dysfunction and inflammatory cytokines, including the cGAS-STING signaling pathways. Treatment of aging mice with a STING inhibitor decreased inflammation and increased mitochondrial gene expression. These findings identify ERRs as important modulators of age-related mitochondrial dysfunction and inflammation.

pathology↗

bioRxiv2018

Quantitative mass spectrometry to interrogate proteomic heterogeneity in metastatic lung adenocarcinoma and validate a novel somatic mutation CDK12-G879V

Lung cancer is the leading cause of cancer death both in men and women. Tumor heterogeneity is an impediment to targeted treatment of all cancers, including lung cancer. Here, we sought to characterize changes in tumor proteome and phosphoproteome by longitudinal, prospective collection of tumor tissue of an exceptional responder lung adenocarcinoma patient who survived with metastatic lung adenocarcinoma for more than seven years with HER2-directed therapy in combination with chemotherapy. We employed \"Super-SILAC\" and TMT labeling strategies to quantify the proteome and phosphoproteome of a lung metastatic site and ten different metastatic progressive lymph nodes collected across a span of seven years, including five lymph nodes procured at autopsy. We identified specific signaling networks enriched in lung compared to the lymph node metastatic sites. We correlated the changes in protein abundance with changes in copy number alteration (CNA) and transcript expression. To further interrogate the mass spectrometry data, patient-specific database was built incorporating all the somatic variants identified by whole genome sequencing (WGS) of genomic DNA from the lung, one lymph node metastatic site and blood. An extensive validation pipeline was built for confirmation of variant peptides. We validated 360 spectra corresponding to 55 germline and 6 somatic variant peptides. Targeted MRM assays demonstrated expression of two novel variant somatic peptides, CDK12-G879V and FASN-R1439Q, with expression in lung and lymph node metastatic sites, respectively. CDK12 G879V mutation likely results in a nonfunctional CDK12 kinase and chemotherapy susceptibility in lung metastatic sites. Knockdown of CDK12 in lung adenocarcinoma cells results in increased chemotherapy sensitivity, explaining the complete resolution of the lung metastatic sites in this patient.

cancer biology↗

bioRxiv2018

Integrated proteogenomic analysis of metastatic thoracic tumors identifies APOBEC mutagenesis and copy number alterations as drivers of proteogenomic tumor evolution and heterogeneity

Elucidation of the proteogenomic evolution of metastatic tumors may offer insight into the poor prognosis of patients harboring metastatic disease. We performed whole-exome and transcriptome sequencing, copy number alterations (CNA) and mass spectrometry-based quantitative proteomics of 37 lung adenocarcinoma (LUAD) and thymic carcinoma (TC) metastases obtained by rapid autopsy and found evidence of patient-specific, multi-dimensional heterogeneity. Extreme mutational heterogeneity was evident in a subset of patients whose tumors showed increased APOBEC-signature mutations and expression of APOBEC3 region transcripts compared to patients with lesser mutational heterogeneity. TP53 mutation status was associated with APOBEC hypermutators in our cohort and in three independent LUAD datasets. In a thymic carcinoma patient, extreme heterogeneity and increased APOBEC3AB expression was associated with a high-risk germline APOBEC3AB variant allele. Patients with CNA occurring late in tumor evolution had corresponding changes in gene expression and protein abundance indicating genomic instability as a mechanism of downstream transcriptomic and proteomic heterogeneity between metastases. Across all tumors, proteomic heterogeneity was greater than copy number and transcriptomic heterogeneity. Enrichment of interferon pathways was evident both in the transcriptome and proteome of the tumors enriched for APOBEC mutagenesis despite a heterogeneous immune microenvironment across metastases suggesting a role for the immune microenvironment in the expression of APOBEC transcripts and generation of mutational heterogeneity. The evolving, heterogeneous nature of LUAD and TC, through APOBEC-mutagenesis and CNA illustrate the challenges facing treatment outcomes.

cancer biology↗