Biology subjects

Linehan, W. M.

Publications and source records attributed to Linehan, W. M..

10 recordsLinked to original sources

bioRxiv2026

TFE3 fusions drive expression of CD44 and SPP1 in Translocation Renal Cell Carcinoma

Xp11.2 translocation RCC [Xp11.2 tRCC]) is an underdiagnosed and aggressive subtype of RCC with few specific or targeted therapies. The transmembrane glycoprotein CD44 is an emerging target in many advanced malignancies, and with its ligand OPN (SPP1), is a crucial driver of cancer progression, stemness, metastasis, and immune suppression. Here we show that common TFE3-fusions [ including SFPQ-TFE3, PRCC-TFE3, ASPSCR1-TFE3, and NONO-TFE3] are associated with upregulated expression of CD44 and SPP1, as observed in multiple human and murine bulk transcriptomic studies and a murine tRCC snRNA-Seq dataset. CD44 and/or SPP1 protein expression were also upregulated in murine models of transgenic tRCC kidneys and urine specimens, patient-derived cell lines and human tRCC cases, by immunoblotting and/or IHC. Transient deletion of CD44 was associated with profound and specific suppression of tRCC cell line growth, with decreased mTOR signaling. These data suggest that CD44 and/or SPP1 may potentially drive tumorigenesis in tRCC.

cancer biology↗

bioRxiv2025

Improved Tumor Blood Flow Enhances the Abscopal Effect: Preclinical Assessment in Mice Treated with Combined Radiation and PD-1 Blockade Therapy

The abscopal effect, where localized radiation therapy induces regression of distant metastatic lesions through immune activation, shows promise for treating metastatic cancer but occurs inconsistently. Here we demonstrate that tumor perfusion critically influences systemic immune responses to combination therapy with radiation and PD-1 blockade. Using multimodal imaging including DCE-MRI, EPR oximetry, and hyperpolarized 13C-MRI, we show that successful abscopal responses in MC38 tumors are characterized by enhanced perfusion, reduced hypoxia, decreased cellularity, and lower glycolytic activity in remote tumors. Notably, pre-treatment perfusion metrics (AUC1min) and extracellular volume (AUC10min) in primary tumors predict subsequent growth of remote tumors, while the same measurements in remote tumors lack predictive value. Based on these findings, we enhanced the abscopal effect by exposing mice to carbogen (95% O2 + 5% CO2) during radiation therapy. Carbogen exposure increased tumor perfusion by 71% (AUC1min) and significantly improved systemic responses in the checkpoint blockade responsive MC38 model but not in the poorly responsive B16.F10 tumors. The enhanced response correlated with increased activation of CD8+ T cells in tumor-draining lymph nodes and elevated serum HMGB-1 levels. RNA sequencing revealed significant extracellular matrix remodeling in carbogen-treated tumors. These results establish tumor perfusion as both a predictive biomarker and a modifiable determinant of systemic immune responses, suggesting that perfusion-based patient stratification and vascular modification strategies could improve outcomes in combination immunotherapy and radiation treatment.

cancer biology↗

bioRxiv2025

TEAD1 condensates are transcriptionally inactive storage sites on the pericentromeric heterochromatin

TEA domain transcription factor 1 (TEAD1), a Hippo pathway transcription factor important in cellular homeostasis and development, is increasingly implicated in cancer biology. Here, we reveal a novel role for TEAD1 in organizing nuclear condensates, independent of active transcription. Using high-resolution imaging, ChIP-seq, RNA-seq and proximity-based proteomics, we demonstrate that in patient-derived renal cell carcinoma cells, TEAD1 forms micron-sized condensates by binding to the heterochromatic pericentromeric regions using its DNA-binding domain. TEAD-specific MCAT motifs selectively enrich and cluster in the pericentromeric region, specifically seeding TEAD1 condensates. TEAD1 condensates do not activate transcription but instead serve as depots for excess TEAD1, and disrupting TEAD1 condensates leads to increases in YAP/TEAD target gene expression. This organization of TEAD1 contrasts with that observed in other genomic regions of both RCC and normal kidney cells, in which TEAD1 associates with markers of active transcription. Our findings provide a mechanistic framework for TEAD1s dual regulatory roles, offering new insights into its contribution to transcriptional dysregulation and tumor progression.

cell biology↗

bioRxiv2025

Low Field Magnetic Resonance Imaging to Detect Acute Kidney Injury

Renal oxygenation is essential for maintaining kidney function. Disruptions in oxygen delivery can lead to renal hypoxia, which can exacerbate kidney injury through multiple pathways, including inflammation, oxidative stress, and ischemia-reperfusion injury. Despite the recognized importance of oxygenation in renal pathology, non-invasive and reliable methods for assessing kidney oxygen levels are limited. Current techniques either lack sensitivity or involve invasive procedures, restricting their use in routine monitoring. Therefore, there is a pressing need for innovative approaches to assess renal oxygenation, particularly in kidney injury. This study evaluated Electron Paramagnetic Resonance (EPR)-based oxygen imaging using the paramagnetic tracer Ox071 to assess kidney oxygen levels in mice with cyclophosphamide-induced kidney injury. Urine pO2 was also assessed as a potential surrogate marker. EPR oximetry accurately measured kidney oxygen distribution, revealing a temporary increase in pO2 post-injury. Urine oximetry, however, did not reliably reflect changes in kidney oxygenation. Furthermore, EPR oximetry provided high-resolution spatial mapping of oxygen levels within the kidney, allowing for a detailed understanding of the impact of hypoxia on renal tissue. EPR oximetry is a promising, non-invasive tool for monitoring renal oxygenation, offering high-resolution mapping and longitudinal assessment. Its ability to provide detailed information about oxygen distribution within the kidney makes it a valuable tool for studying the pathophysiology of renal diseases and for developing novel therapeutic strategies. Translational Statement: Quantitative spatially resolved measurement of renal oxygenation has the potential to guide clinical decision making in renal disorders such as acute kidney injury. In this study we demonstrate the utility of electron paramagnetic resonance imaging to provide non-invasive and quantitative high-resolution mapping of kidney oxygen concentrations.

physiology↗

bioRxiv2024

Band-selective IR PRESS for brain tumor spectroscopy allows robust detection of lactate

Lactate plays a critical role in the tumor microenvironment, driving tumor progression, metastasis, and immune evasion. Despite its importance, in vivo quantification of lactate using magnetic resonance spectroscopy (MRS) has faced challenges, primarily due to the overlapping lipid signal at 1.3 ppm. Current clinical practice employs a long echo time to exploit differences in T2 relaxation between lactate and lipids; however, this approach significantly suppresses signals from other metabolites. Lipid has a notably different T1 relaxation time than lactate and other metabolites, which may be exploited by an inversion recovery sequence to better distinguish them. However, this method has not found wide use because of the loss of signal in other metabolites. Here we introduce a selective inversion pulse with a short echo time MRS method (SPIR-PRESS), which mitigates this issue. In phantom experiments, SPIR-PRESS successfully suppressed lipid signals that could be misinterpreted as lactate in short TE PRESS spectra, while maintaining sensitivity to the full metabolite profile. SPIR-PRESS demonstrated superior performance in quantifying lactate compared to long echo time PRESS, with [~] 60 % increase in sensitivity for lactate detection compared to conventional PRESS with a 288 ms TE. In a mouse glioma model, SPIR-PRESS clearly detected lactate and other key tumor metabolites (choline, creatine, NAA) in the tumor, which were not detectable in conventional long TE PRESS. These findings highlight SPIR-PRESS as a promising technique for improved lactate quantification and comprehensive metabolite profiling in tumor environments.

biochemistry↗

bioRxiv2023

Rewiring of RNA methylation by the oncometabolite fumarate in renal cell carcinoma

Metabolic reprogramming is a hallmark of cancer that facilitates changes in many adaptive biological processes. Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) lead to fumarate accumulation and cause hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC is a rare, inherited disease characterized by the development of non-cancerous smooth muscle tumors of the uterus and skin, and an increased risk of a highly metastatic and aggressive form of kidney cancer. Fumarate has been shown to inhibit 2-oxyglutarate-dependent dioxygenases (2OGDDs) involved in the hydroxylation of HIF1, as well as in DNA and histone demethylation. However, the link between fumarate accumulation and changes in RNA post-transcriptional modifications has not been defined. Here, we determine the consequences of fumarate accumulation on the activity of different members of the 2OGDD family targeting RNA modifications. By evaluating multiple RNA modifications in patient-derived HLRCC cell lines, we show that mutation of FH selectively alters the activity of demethylases acting upon N6-methyladenosine (m6A), while the demethylase acting upon N1-methyladenosine (m1A) and 5-formylcytosine (f5C) in mitochondrial RNA are unaffected. The observation that metabolites modulate specific subsets of RNA-modifying enzymes offers new insights into the intersection between metabolism and the epitranscriptome.

molecular biology↗

bioRxiv2022

Conditional covalent lethality driven by oncometabolite accumulation

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a cancer predisposition syndrome driven by mutation of the tumor suppressor fumarate hydratase (FH). Inactivation of FH causes accumulation of the electrophilic oncometabolite fumarate. In the absence of methods for reactivation, tumor suppressors can be targeted via identification of synthetic lethal interactions using genetic screens. Inspired by recent advances in chemoproteomic target identification, here we test the hypothesis that the electrophilicity of the HLRCC metabolome may produce unique susceptibilities to covalent small molecules, a phenomenon we term conditional covalent lethality. Screening a panel of chemically diverse electrophiles we identified a covalent ligand, MP-1, that exhibits FH-dependent cytotoxicity. Synthesis and structure-activity profiling identified key molecular determinants underlying the molecules effects. Chemoproteomic profiling of cysteine reactivity together with clickable probes validated the ability of MP-1 to engage an array of functional cysteines, including one lying in the Zn-finger domain of the tRNA methyltransferase enzyme TRMT1. TRMT1 overexpression rescues tRNA methylation from inhibition by MP-1 and partially attenuates the covalent ligands cytotoxicity. Our studies highlight the potential for covalent metabolites and small molecules to synergistically produce novel synthetic lethal interactions and raise the possibility of applying phenotypic screening with chemoproteomic target identification to identify new functional oncometabolite targets.

biochemistry↗

bioRxiv2020

Heterogeneous adaptation of cysteine reactivity to a covalent oncometabolite

ABSTRACTMetabolism and signaling intersect in the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the TCA cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand how metabolites function as covalent signals, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Building on previous studies, we applied a general reactivity probe to compile a dataset of cysteine residues sensitive to rescue of cellular FH activity. This revealed a broad upregulation of cysteine reactivity upon FH rescue, caused by an approximately equal proportion of transcriptional and posttranslational regulation in the rescue cell line. Gene ontology analysis highlights new targets and pathways potentially modulated by FH mutation. Comparison of the new dataset to literature studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation, and a foundation for future efforts to exploit this knowledge for cancer therapy.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2020

Glycolytic metabolism of pathogenic T cells enables early detection of GvHD by 13C-MRI

Graft-versus-host disease (GvHD) is a prominent barrier to allogeneic hematopoietic stem cell transplantation (HSCT). Definitive diagnosis of GvHD is invasive and biopsies of involved tissues pose a high risk of bleeding and infection. Our previous studies in a chronic GvHD mouse model demonstrated that alloreactive CD4+ T cells are distributed to target organs ahead of overt symptoms, meanwhile CD4+ T cell activation is tied to increased glycolysis. Thus, we hypothesized that metabolic imaging of glycolysis would allow non-invasive detection of insipient GvHD in target organs infiltrated by glycolytic effector memory CD4+ T cells. We metabolically characterized CD4+ T cell subsets on day 14 post-transplant before the onset of chronic GvHD in a pre-clinical mouse model and performed 13C hyperpolarized magnetic resonance imaging (MRI) to quantify glycolytic activity in the liver of mice over the course of the disease. Intracellular metabolic screening and ex vivo metabolic profiling of CD4+ T cell subsets at day 14 confirmed that activated CD4+ T cells were highly glycolytic. Concurrently, hyperpolarized 13C-pyruvate MRI of the liver showed high conversion of pyruvate to lactate, indicative of increased glycolytic activity, that distinguished allogeneic from syngeneic HSCT recipients prior to the development of overt chronic GvHD. Furthermore, single cell sequencing of T cells in patients undergoing allogeneic HSCT indicated that similar metabolic changes may play a role in acute GvHD, providing a rationale for testing this imaging approach in the clinical post-HSCT setting. Our imaging approach is amenable to clinical translation and may allow early, non-invasive diagnosis of GvHD.

immunology↗

bioRxiv2018

A chemoproteomic portrait of the oncometabolite fumarate

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth and survival. Understanding these mechanisms and developing biomarkers to identify their presence thus represents a vital goal. Towards this goal, here we report a chemoproteomic map of the covalent targets of fumarate, an oncometabolite whose accumulation marks the genetic cancer predisposition syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). First, we validate the ability of known and novel chemoproteomic probes to report on fumarate reactivity in vitro. Next, we apply these probes in concert with LC-MS/MS to identify cysteine residues sensitive to either fumarate treatment or fumarate hydratase (FH) mutation in untransformed and human HLRCC cell models, respectively. Mining this data to understand the structural determinants of fumarate reactivity reveals an unexpected anti-correlation with nucleophilicity, and the discovery of a novel influence of pH on fumarate-cysteine interactions. Finally, we show that many fumarate-sensitive and FH-regulated cysteines are found in functional protein domains, and perform mechanistic studies of a fumarate-sensitive cysteine in SMARCC1 that lies at a key protein-protein interface in the SWI-SNF tumor suppressor complex. Our studies provide a powerful resource for understanding the influence of fumarate on reactive cysteine residues, and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.

cancer biology↗