Biology subjects

Sullivan, L. B.

Publications and source records attributed to Sullivan, L. B..

15 recordsLinked to original sources

bioRxiv2026

Cysteine-mediated bortezomib resistance is governed by α-ketoacid availability

Dissecting how metabolite availability impacts drug sensitivity is critical for understanding therapy resistance. While proteasome inhibitors like the boronic acid proteasome inhibitor bortezomib are cornerstones of therapy for multiple myeloma, the clinical utility of these drugs is often limited by development of resistance, the metabolic drivers of which remain poorly understood. In this study, we find that -ketoacids, including pyruvate, increase sensitivity to boronic acid proteasome inhibitors independent of their conventional roles in metabolism. Instead, -ketoacids directly react with intracellular cysteine to form thiazolidines, sequestering cysteine away from forming conjugates with boronic acid proteasome inhibitors and detoxifying these drugs. Preventing cysteine-mediated detoxification through -ketoacid supplementation increases the efficacy of boronic acid proteasome inhibitors, leading to greater proteasome inhibition and cytotoxicity that is reversed by cysteine supplementation. These findings suggest that modulating available cysteine through -ketoacid interactions can impact effective levels of some drugs in cells, and represent a potential strategy to overcome resistance and maximize the efficacy of boronic acid proteasome inhibitors.

cancer biology↗

bioRxiv2026

Adaptive plasticity of aspartate metabolism in succinate dehydrogenase-deficient cancer cells

Succinate dehydrogenase (SDH) supports cancer cell proliferation by enabling oxidative biosynthesis of the amino acid aspartate, yet SDH loss can also drive tumorigenesis. To cope with SDH loss, cancer cells can engage alternative aspartate synthesis pathways; however, the variables dictating pathway usage and adaptive mechanisms involved are incompletely understood. Here, we systematically profile the adaptation of SDH-knockout cancer cells and find that cells can adapt to SDH loss via at least two distinct mechanisms: suppression of respiratory complex I or upregulation of pyruvate carboxylase. Each route gives rise to distinct metabolic states with both shared and unique dependencies, but either route allows cells to overcome aspartate limitation, improve proliferative fitness, and mitigate pyrimidine-dependent replication stress. Overall, this work provides a comprehensive view of adaptive aspartate synthesis in SDH-deficient cancer cells, highlights a remarkable redox-constrained metabolic plasticity, and nominates potential metabolic vulnerabilities likely to be shared among SDH-deficient cancer cells.

cancer biology↗

bioRxiv2026

Ribosome-associated quality control of aberrant protein production during amino acid limitation

Amino acids can become limiting for protein synthesis through depletion of charged tRNAs, leading to ribosome stalling and disruption of translation elongation at specific codons. To assess whether this is a mechanism by which amino acid availability can directly influence gene expression, we designed a reporter library to measure translation disruption across all sense codons in the context of amino acid limitations. We found that arginine limitation consistently impairs translation at the arginine codon AGA, resulting in synthesis of proteins from endogenous transcripts. In contrast, GCN2 pathway activation suppresses translation disruption following depletion of most other amino acids. Genome-wide screens revealed that the ribosome quality control trigger (RQC-T) and RQC pathways, which resolve ribosome collisions on defective mRNAs, catalyze ribosome splitting and premature fall-off in response to arginine depletion. Additionally, the E3 ubiquitin ligase RNF14, recently shown to clear ribosome A-site obstructions, promotes translation disruption through both ribosome fall-off and frameshifting during arginine limitation. Together, these data show that the RQC machinery is engaged by tRNA-limited ribosomes and identify a new role for RNF14 as a regulator of translation upon arginine limitation.

genetics↗

bioRxiv2025

Recurrent Breast Cancer Cells Depend on De novo Pyrimidine Biosynthesis to Suppress Ferroptosis

Breast cancer recurrence remains a major clinical challenge, often associated with therapy resistance and altered metabolic states. To define metabolic vulnerabilities of recurrent disease, we performed a CRISPR knockout screen targeting 421 metabolic genes in paired primary and recurrent HER2-driven breast cancer cell lines. While both primary and recurrent tumors shared dependencies on core metabolic pathways, recurrent tumors exhibited selective essentiality for the de novo pyrimidine synthesis pathway, including Cad, Dhodh, and Ctps. Pharmacologic inhibition of the rate-limiting enzyme DHODH with BAY-2402234 selectively impaired the growth of recurrent tumor cells, while primary tumor cells were relatively resistant. BAY treatment robustly inhibited pyrimidine synthesis in all lines, but only recurrent cells underwent iron-dependent lipid peroxidation and ferroptotic cell death. Lipidomic profiling revealed enrichment of polyunsaturated ether phospholipids in recurrent cells, which may predispose them to ferroptosis. A sensitizer CRISPR screen in primary cells further identified nucleotide salvage and lipid metabolic pathways as modifiers of DHODH inhibitor sensitivity. Stable isotope tracing and nutrient depletion experiments showed that primary cells can compensate for DHODH inhibition through nucleotide salvage, whereas recurrent cells exhibit impaired salvage capacity, likely due to reduced expression of Slc28/Slc29 nucleoside transporters. Together, these findings reveal that breast cancer recurrence is associated with increased dependence on de novo pyrimidine synthesis to suppress ferroptosis, highlighting a therapeutically actionable metabolic vulnerability in recurrent disease.

cancer biology↗

bioRxiv2025

Succinate Dehydrogenase loss causes cascading metabolic effects that impair pyrimidine biosynthesis

Decreased availability of the amino acid aspartate can constrain cell function in diverse biological contexts, but the temporal interplay between aspartate, downstream metabolic changes, and functional effects remains poorly understood. Using an aspartate biosensor and live-cell imaging, we examine the interaction between aspartate abundance and cell proliferation in several models of aspartate limitation. While aspartate deficiencies intuitively interface with proliferation in some contexts, aspartate limitation from succinate dehydrogenase (SDH) inhibition causes strikingly nonintuitive dynamics resulting from an outsized impairment of pyrimidine synthesis. Mechanistically, we find that SDH loss impairs pyrimidine biosynthesis by decreasing aspartate and accumulating succinate, which competitively inhibits mammalian aspartate transcarbamylase (ATCase). This metabolic interaction persists in multiple models of SDH deficiency, causing pyrimidine insufficiency, replication stress, and sensitivity to ATR kinase inhibition. These findings define a novel role for succinate in modulating cellular nucleotide homeostasis, suggest a potential therapeutic vulnerability of SDH-deficient tumors, and demonstrate how cascading metabolic interactions can unfold to impact cell function.

cell biology↗

bioRxiv2025

Defined media reveals the essential role of lipid scavenging to support cancer cell proliferation

Fetal bovine serum (FBS) is a nearly ubiquitous, yet undefined additive in mammalian cell culture media whose functional contributions to promoting cell proliferation remain poorly understood. Efforts to replace serum supplementation in culture media have been hindered by an incomplete understanding of the environmental requirements fulfilled by FBS in culture. Here, we use a combination of live-cell imaging and liquid chromatography-mass spectrometry to elucidate the role of serum in supporting proliferation. We show that serum provides consumed factors that enable proliferation and demonstrate that the serum metal and lipid components are crucial to sustaining proliferation in culture. Importantly, despite access to a wide range of lipid classes, albumin-bound lipids are the primary species consumed during cancer cell proliferation. Furthermore, we find that combinations of the additive ITS, containing necessary metals, and albumin-associated lipid classes are sufficient to replace FBS in culture media. We show that serum-free media enables sensitive quantification of lipid consumption dynamics during cell proliferation, which indicate that fatty acids (FA) are consumed through a mass-action mechanism, with minimal competition from other lipid classes. Finally, we find that pharmacologic disruption of FA activation and incorporation into the cellular lipidome reduces uptake from the environment and impairs cell proliferation. This work therefore identifies metabolic contributions of serum in cell culture settings and provides a framework for building cell culture systems that sustain cell proliferation without the variable and undefined contributions of FBS.

cancer biology↗

bioRxiv2024

Converging cytokine and metabolite networks shape asymmetric T cell fate at the term human maternal-fetal interface

Placentation presents immune conflict between mother and fetus, yet in normal pregnancy maternal immunity against infection is maintained without expense to fetal tolerance. This is believed to result from adaptations at the maternal-fetal interface (MFI) which affect T cell programming, but the identities (i.e., memory subsets and antigenic specificities) of T cells and the signals that mediate T cell fates and functions at the MFI remain poorly understood. We found intact recruitment programs as well as pro-inflammatory cytokine networks that can act on maternal T cells in an antigen-independent manner. These inflammatory signals elicit T cell expression of co-stimulatory receptors necessary for tissue retention, which can be engaged by local macrophages. Although pro-inflammatory molecules elicit T cell effector functions, we show that additional cytokine (TGF-{beta}1) and metabolite (kynurenine) networks may converge to tune T cell function to those of sentinels. Together, we demonstrate an additional facet of fetal tolerance, wherein T cells are broadly recruited and restrained in an antigen-independent, cytokine/metabolite-dependent manner. These mechanisms provide insight into antigen-nonspecific T cell regulation, especially in tissue microenvironments where they are enriched.

immunology↗

bioRxiv2023

A robust method for measuring aminoacylation through tRNA-Seq

Current methods to quantify the fraction of aminoacylated tRNAs, also known as the tRNA charge, are limited by issues with either low throughput, precision, and/or accuracy. Here, we present an optimized charge tRNA-Seq method that combines previous developments with newly described approaches to establish a protocol for precise and accurate tRNA charge measurements. We verify that this protocol provides robust quantification of tRNA aminoacylation and we provide an end-to-end method that scales to hundreds of samples including software for data processing. Additionally, we show that this method supports measurements of relative tRNA expression levels and can be used to infer tRNA modifications through reverse transcription misincorporations, thereby supporting multipurpose applications in tRNA biology.

biochemistry↗

bioRxiv2023

A Compound that Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer

PurposeMetastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Non-specific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. Experimental designWe tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell cycle, metabolic and enzymatic assays were used to demonstrate their mechanism of action. A human PDX model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. ResultsWe demonstrate a new class of small molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. ConclusionThis work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.

cancer biology↗

bioRxiv2023

An engineered biosensor enables dynamic aspartate measurements in living cells

Intracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a GFP-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by mass spectrometry and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high throughput applications of variables that affect aspartate levels.

biochemistry↗

bioRxiv2023

Damage-induced pyroptosis drives endogenous thymic regeneration viainduction of Foxn1 by purinergic receptor activation

Endogenous thymic regeneration is a crucial process that allows for the renewal of immune competence following stress, infection or cytoreductive conditioning. Fully understanding the molecular mechanisms driving regeneration will uncover therapeutic targets to enhance regeneration. We previously demonstrated that high levels of homeostatic apoptosis suppress regeneration and that a reduction in the presence of damage-induced apoptotic thymocytes facilitates regeneration. Here we identified that cell-specific metabolic remodeling after ionizing radiation steers thymocytes towards mitochondrial-driven pyroptotic cell death. We further identified that a key damage-associated molecular pattern (DAMP), ATP, stimulates the cell surface purinergic receptor P2Y2 on cortical thymic epithelial cells (cTECs) acutely after damage, enhancing expression of Foxn1, the critical thymic transcription factor. Targeting the P2Y2 receptor with the agonist UTP{gamma}S promotes rapid regeneration of the thymus in vivo following acute damage. Together these data demonstrate that intrinsic metabolic regulation of pyruvate processing is a critical process driving thymus repair and identifies the P2Y2 receptor as a novel molecular therapeutic target to enhance thymus regeneration. SUMMARYO_LIThymocytes rapidly and transiently undergo pyroptosis after acute thymic damage and promote regeneration. C_LIO_LIDamage-induced redirection of pyruvate acutely enhances mitochondrial OXPHOS in thymocytes. C_LIO_LIElevated mitochondrial ROS promotes pyroptosis in thymocytes after acute insult by driving caspase 1 cleavage. C_LIO_LIExtracellular ATP release promotes Foxn1 expression in cTECs via activation of P2Y2 C_LIO_LITherapeutic targeting of the P2Y2 receptor promotes thymic regeneration. C_LI

immunology↗

bioRxiv2022

Fanconi anemia-isogenic head and neck cancer cell line pairs - a basic and translational science resource

Fanconi anemia (FA) is a heritable malformation, bone marrow failure and cancer predisposition syndrome that confers an exceptionally high risk of developing carcinomas arising in squamous mucosal epithelia lining the mouth, proximal esophagus, vulva and anus. The origin of these cancers is not understood, and no effective way has been identified to prevent or delay their appearance. FA-associated carcinomas are also therapeutically challenging, as they may be multi-focal and stage-advanced at diagnosis making surgical control challenging. Moreover, individuals with FA have systemic DNA damage hypersensitivity and thus an elevated risk of toxicity when treated with standard-of-care therapies such as DNA cross-linking drugs and ionizing radiation. We developed the Fanconi Anemia Cancer Cell Line Resource (FA-CCLR) in order to foster new research on the origins, treatment, and prevention of FA-associated cancers. The FA-CCLR consists of FANC-isogenic head and neck squamous cell carcinoma (HNSCC) cell line pairs from cancers arising in individuals with FA, or newly engineered from sporadic HNSCC cell lines. Molecular, cellular, and biochemical analyses were used to demonstrate the causal dependence of key FA-associated phenotypes on FANC genotype, expression and pathway activity. These FANC-isogenic cell line pairs are available to academic and non-profit investigators, with ordering information available at the Fanconi Anemia Research Materials Resource and Repository at Oregon Health & Sciences University, Portland OR. SignificanceWe have generated new isogenic cancer cell line models to investigate the origins, treatment and prevention of Fanconi anemia-associated squamous carcinomas that target the oral mucosa, proximal esophagus, and anogenital region.

cancer biology↗

bioRxiv2022

Mitochondrial Redox Adaptations Enable Aspartate Synthesis in SDH-deficient Cells

The oxidative tricarboxylic acid (TCA) cycle is a central mitochondrial pathway integrating catabolic conversions of NAD+ to NADH and anabolic production of aspartate, a key amino acid for cell proliferation. Several TCA cycle components are implicated in tumorigenesis, including loss of function mutations in subunits of succinate dehydrogenase (SDH), also known as complex II of the electron transport chain (ETC). Mechanistic understanding of how proliferating cells tolerate the metabolic defects of SDH loss is still lacking. Here, we identify that SDH supports cell proliferation through aspartate synthesis but, unlike other ETC impairments, is not restored by electron acceptor supplementation. Interestingly, we find aspartate production and cell proliferation are restored to SDH impaired cells by concomitant inhibition of ETC complex I (CI). We determine that the benefits of CI inhibition in this context are dependent on decreasing mitochondrial NAD+/NADH, which drives SDH-independent aspartate production. We also find that genetic loss or restoration of SDH selects for cells with concordant CI activity, establishing distinct modalities of mitochondrial metabolism for maintaining aspartate synthesis. Collectively, these data identify a metabolically beneficial mechanism for CI loss in proliferating cells and reveal that compartmentalized redox changes can impact cellular fitness.

biochemistry↗

bioRxiv2020

Cancer cells depend on environmental lipids for proliferation when electron acceptors are limited

It is not well understood how physiological environmental conditions and nutrient availability influence cancer cell proliferation. Production of oxidized biomass, which requires regeneration of the cofactor NAD+, can limit cancer cell proliferation1-5. However, it is currently unclear which specific metabolic processes are constrained by electron acceptor availability, and how they affect cell proliferation. Here, we use computational and experimental approaches to demonstrate that de novo lipid biosynthesis can impose an increased demand for NAD+ in proliferating cancer cells. While some cancer cells and tumors synthesize a substantial fraction of their lipids de novo6, we find that environmental lipids are crucial for proliferation in hypoxia or when the mitochondrial electron transport chain is inhibited. Surprisingly, we also find that even the reductive glutamine carboxylation pathway to produce fatty acids is impaired when cancer cells are limited for NAD+. Furthermore, gene expression analysis of 34 heterogeneous tumor types shows that lipid biosynthesis is strongly and consistently negatively correlated with hypoxia, whereas expression of genes involved in lipid uptake is positively correlated with hypoxia. These results demonstrate that electron acceptor availability and access to environmental lipids can play an important role in determining whether cancer cells engage in de novo lipogenesis to support proliferation.

cancer biology↗

bioRxiv2020

Increased demand for NAD+ relative to ATP drives aerobic glycolysis

Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is a hallmark of proliferative metabolism that is observed across many organisms and conditions. To better understand why aerobic glycolysis is associated with cell proliferation, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase mitochondrial pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the nicotinamide adenine dinucleotide cofactor ratio (NAD+/NADH). This change in NAD+/NADH ratio is caused by an increase in mitochondrial membrane potential that impairs mitochondrial electron transport and NAD+ regeneration. Uncoupling mitochondrial respiration from ATP synthesis or increasing ATP hydrolysis restores NAD+/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when the demand for NAD+ to support oxidation reactions exceeds the demand for ATP consumption in cells, NAD+ regeneration by mitochondrial respiration becomes constrained, promoting fermentation despite available oxygen. This argues that cells engage in aerobic glycolysis when the cellular demand for NAD+ is in excess of the cellular demand for ATP.

biochemistry↗