Cellular Pool Research Articles

Piezo2 interacts with E-cadherin in specialized gastrointestinal epithelial mechanoreceptors.

Piezo2 is a mechanically gated ion channel most commonly expressed by specialized mechanoreceptors, such as the enteroendocrine cells (EECs) of the gastrointestinal epithelium. A subpopulation of EECs expresses Piezo2 and functionally resembles the skin's touch sensors, called Merkel cells. Low-magnitude mechanical stimuli delivered to the mucosal layer are primarily sensed by mechanosensitive EECs in a process we term "gut touch." Piezo2 transduces cellular forces into ionic currents, a process that is sensitive to bilayer tension and cytoskeletal depolymerization. E-cadherin is a widely expressed protein that mediates cell-cell adhesion in epithelia and interacts with scaffold proteins that anchor it to actin fibers. E-cadherin was shown to interact with Piezo2 in immortalized cell models. We hypothesized that the Piezo2-E-cadherin interaction is important for EEC mechanosensitivity. To test this, we used super-resolution imaging, co-immunoprecipitation, and functional assays in primary tissues from mice and gut organoids. In tissue EECs and intestinal organoids, we observed multiple Piezo2 cellular pools, including one that overlaps with actin and E-cadherin at the cells' lateral walls. Further, E-cadherin co-immunoprecipitated with Piezo2 in the primary colonic epithelium. We found that E-cadherin knockdown decreases mechanosensitive calcium responses in mechanically stimulated primary EECs. In all, our results demonstrate that Piezo2 localizes to the lateral wall of EECs, where it physically interacts with E-cadherin and actin. They suggest that the Piezo2-E-cadherin-actin interaction is important for mechanosensitivity in the gut epithelium and possibly in tissues where E-cadherin and Piezo2 are co-expressed in epithelial mechanoreceptors, such as skin, lung, and bladder.

Unraveling the full impact of SPD_0739: a key effector in S. pneumoniae iron homeostasis.

S. pneumoniae obtains growth essential iron from hemoglobin and other host hemoproteins. Still, the bacterial mechanisms involved are only partially understood, and there are inconsistent reports regarding the function of several transporters implicated in iron uptake. In this study, we clarified the role of PnrA/Spbhp-37, a ligand-binding protein previously linked to nucleoside or heme by different studies. We present data supporting a role in nucleoside scavenging rather than heme import and reveal that PnrA/Spbhp-37 modulates iron and heme uptake, likely by influencing the nucleoside cellular pool. Hence, this work provides a new understanding of a process critical to the pathophysiology of a significant human pathogen. Moreover, PnrA/Spbhp-37 is an abundant and immunogenic surface protein that is highly conserved. Hence, this study also clarifies the function of a promising vaccine target.

Identification of Novel Target DCTPP1 for Colorectal Cancer Therapy with the Natural Small‐Molecule Inhibitors Regulating Metabolic Reprogramming

AbstractColorectal cancer (CRC) is one of the most common malignant tumors. Identification of new effective drug targets for CRC and exploration of bioactive small‐molecules are clinically urgent. The human dCTP pyrophosphatase 1 (DCTPP1) is a newly identified pyrophosphatase regulating the cellular nucleotide pool but remains unexplored as potential target for CRC treatment. Here, twelve unprecedented chemical architectures terpene‐nonadride heterodimers (1–12) and their monomers (13–20) were isolated from endophyte Bipolaris victoriae S27. Compounds 1–12 represented the first example of terpene‐nonadride heterodimers, in which nonadride monomers of 1 and 2 were also first example of 5/6 bicyclic nonadrides. A series of assays showed that 2 could repress proliferation and induce cell cycle arrest, apoptotic and autophagic CRC cell death in vitro and in vivo. Clinical cancer samples data revealed that DCTPP1 was a novel target associated with poor survival in CRC. DCTPP1 was also identified as a new target protein of 2. Mechanically, compound 2 bound to DCTPP1, inhibited its enzymatic activity, intervened with amino acid metabolic reprogramming, and exerted anti‐CRC activity. Our study demonstrates that DCTPP1 was a novel potential biomarker and therapeutic target for CRC, and 2 was the first natural anti‐CRC drug candidate targeting DCTPP1.

Identification of Novel Target DCTPP1 for Colorectal Cancer Therapy with the Natural Small-Molecule Inhibitors Regulating Metabolic Reprogramming.

Colorectal cancer (CRC) is one of the most common malignant tumors. Identification of new effective drug targets for CRC and exploration of bioactive small-molecules are clinically urgent. The human dCTP pyrophosphatase 1 (DCTPP1) is a newly identified pyrophosphatase regulating the cellular nucleotide pool but remains unexplored as potential target for CRC treatment. Here, twelve unprecedented chemical architectures terpene-nonadride heterodimers (1-12) and their monomers (13-20) were isolated from endophyte Bipolaris victoriae S27. Compounds 1-12 represented the first example of terpene-nonadride heterodimers, in which nonadride monomers of 1 and 2 were also first example of 5/6 bicyclic nonadrides. A series of assays showed that 2 could repress proliferation and induce cell cycle arrest, apoptotic and autophagic CRC cell death in vitro and in vivo. Clinical cancer samples data revealed that DCTPP1 was a novel target associated with poor survival in CRC. DCTPP1 was also identified as a new target protein of 2. Mechanically, compound 2 bound to DCTPP1, inhibited its enzymatic activity, intervened with amino acid metabolic reprogramming, and exerted anti-CRC activity. Our study demonstrates that DCTPP1 was a novel potential biomarker and therapeutic target for CRC, and 2 was the first natural anti-CRC drug candidate targeting DCTPP1.

Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq.

Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-containing pseudoknot (t/PK) and the three-way junction (CR4/5). These two hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are thus essential for telomerase catalytic activity. Here, we probe the structure of hTR in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Unexpectedly, approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features thought to be required for hTERT binding. The proportion of hTR CR4/5 that is folded into the primary functional conformation does not require hTERT expression and the fraction of hTR that assumes a misfolded CR4/5 domain is not refolded by overexpression of its hTERT binding partner. This result suggests a functional role for an RNA folding cofactor other than hTERT during telomerase biogenesis. Mutagenesis demonstrates that stabilization of the alternative CR4/5 conformation is detrimental to telomerase assembly and activity. Moreover, the alternative CR4/5 conformation is not found in telomerase RNP complexes purified from cells via an epitope tag on hTERT, supporting the hypothesis that only the major CR4/5 conformer is active. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded. Thus, kinetic traps for RNA folding that have been so well-studied in vitro may also present barriers for assembly of ribonucleoprotein complexes in vivo.

Follicular Immune Landscaping Reveals a Distinct Profile of FOXP3hiCD4hi T Cells in Treated Compared to Untreated HIV.

Follicular helper CD4hi T cells (TFH) are a major cellular pool for the maintenance of the HIV reservoir. Therefore, the delineation of the follicular (F)/germinal center (GC) immune landscape will significantly advance our understanding of HIV pathogenesis. We have applied multiplex confocal imaging, in combination with the relevant computational tools, to investigate F/GC in situ immune dynamics in viremic (vir-HIV), antiretroviral-treated (cART HIV) People Living With HIV (PLWH) and compare them to reactive, non-infected controls. Lymph nodes (LNs) from viremic and cART PLWH could be further grouped based on their TFH cell densities in high-TFH and low-TFH subgroups. These subgroups were also characterized by different in situ distributions of PD1hi TFH cells. Furthermore, a significant accumulation of follicular FOXP3hiCD4hi T cells, which were characterized by a low scattering in situ distribution profile and strongly correlated with the cell density of CD8hi T cells, was found in the cART-HIV low-TFH group. An inverse correlation between plasma viral load and LN GrzBhiCD8hi T and CD16hiCD15lo cells was found. Our data reveal the complex GC immune landscaping in HIV infection and suggest that follicular FOXP3hiCD4hi T cells could be negative regulators of TFH cell prevalence in cART-HIV.

Pseudomonas aeruginosa Recombinant L-asparaginase: PEGylation with Low Molecular Weight Polyethylene Glycol, Molecular Dynamics Simulation, In vitro and In vivo Serum half-life and Biochemical Characterization.

Microbial L-asparaginase (L-ASNase, EC 3.5.1.1) is a pivotal biopharmaceutical drug-protein that catalyzes the hydrolysis of the non-essential amino acid L-asparagine (L-Asn) into L-aspartic acid (L-Asp) and ammonia , resulting in deplenishing the cellular L-Asn pool, which leads to the ultimate death of the L-asparagine synthetase (L-ASNS) deficient cancerous cells. This study aimed to investigate the impact of conjugating low molecular weight polyethylene glycol to recombinant P. aeruginosa L-ASNase by examining the pharmacokinetic properties, affinity towards the substrate, and enzyme stability prior to and following the reaction. The recombinant P. aeruginosa L-ASNase was affinity purified and then PEGylated by attaching polyethylene glycol (MW= 330 Da) site-specifically to the protein's N-terminus end. After which, the PEGylated L-ASNase was examined by SDS-PAGE (15%), FTIR, and UV/Vis spectrophotometry and subsequently biochemically characterized. The Km and Vmax values of free P. aeruginosa rL-ASNase were determined to be 0.318 ±1.76 mM and 2915 μmol min-1and following the PEGylation, they were found to be 0.396 ±1.736 mM and 3193 μmol min-1, respectively. Polyethylene glycol (330 Da) has markedly enhanced LASNase thermostability at 37, 45, 50, and 55 °C, as opposed to the free enzyme, which retained 19.5% after 1 h of incubation at 37 °C. The PEGylated L-ASNase was found to be stable upon incubation with human serum for 28 h, in contrast to the sharp decline in the residual bioactivity of the free rL-ASNase after 4 h incubation. Accordingly, an in vivo study was used for validation, and it demonstrated that PEGylated rL-ASNase exhibited longer bioactivity for 24 h, while the free form's activity vanished entirely from the rats' blood sera after 8 h. Molecular dynamics simulation indicated that PEG (330 Da) has affected the hydrodynamic volume of L-ASNase and increased its structural stability. Docking analysis has explored the position of PEG with respect to binding sites and predicted a similar binding affinity to that of the free enzyme. For the first time, recombinant L-ASNase was modified by covalently attaching PEG (330 Da). The resultant novel proposed PEGylated rL-ASNase with remarkably increased stability and prolonged in vivo half-life duration, which could be considered an alternative to mitigate the high molecular weight of PEGylation's drawbacks.

Glucose transporter 1 is essential for the resolution of methicillin-resistant S. aureus skin and soft tissue infections

Skin/soft tissue infections (SSTIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) pose a major healthcare burden. Distinct inflammatory and resolution phases comprise the host immune response to SSTIs. Resolution is a myeloid PPARγ-dependent anti-inflammatory phase that is essential for the clearance of MRSA. However, the signals activating PPARγ to induce resolution remain unknown. Here, we demonstrate that myeloid glucose transporter 1 (GLUT-1) is essential for the onset of resolution. MRSA-challenged macrophages are unsuccessful in generating an oxidative burst or immune radicals in the absence of GLUT-1 due to a reduction in the cellular NADPH pool. This translates invivo as a significant reduction in lipid peroxidation products required for the activation of PPARγ in MRSA-infected mice lacking myeloid GLUT-1. Chemical induction of PPARγ during infection circumvents this GLUT-1 requirement and improves resolution. Thus, GLUT-1-dependent oxidative burst is essential for the activation of PPARγ and subsequent resolution of SSTIs.

A model of time-dependent macromolecular and elemental composition of phytoplankton

Phytoplankton Chl:C:N:P ratios are important from both an ecological and a biogeochemical perspective. We show that these elemental ratios can be represented by a phytoplankton physiological model of low complexity that includes major cellular macromolecular pools. In particular, our model resolves time-dependent intracellular pools of chlorophyll, proteins, nucleic acids, carbohydrates/lipids, and N and P storage. Batch culture data for two diatom and two prasinophyte species are used to constrain parameters that represent specific allocation traits and strategies. A key novelty is the simultaneous estimation of physiological parameters for two phytoplankton groups of such different sizes. The number of free parameters is reduced by assuming (i) allometric scaling for maximum uptake rates, (ii) shared half-saturation constants for synthesis of functional macromolecules, (iii) shared exudation rates of functional macromolecules across the species. The rationale behind this assumption is that across the different species, the same or similar processes, enzymes, and metabolites play a role in key physiological processes. For the turnover numbers of macromolecular synthesis and storage exudation rates, differences between diatoms and prasinophytes need to be taken into account to obtain a good fit. Our model fits suggest that the parameters related to storage dynamics dominate the differences in the C:N:P ratios between the different phytoplankton groups. Since descriptions of storage dynamics are still incomplete and imprecise, predictions of C:N:P ratios by phytoplankton models likely have a large uncertainty.

Real-time detection of lysine decarboxylase activity and cellular energy pool under inducible CadA expression system in engineered Escherichia coli

BackgroundBiocatalysts hold significant promise in the production of 1,5-diaminopentane (cadAverine) due to its wide-ranging applications. However, the process of screening recombinant cells expressing lysine decarboxylase (LdcC or CadA) activity via high-performance liquid chromatography is labor-intensive and time-consuming. MethodsThis study has devised a straightforward colorimetric assay to quantify lysine decarboxylase activity. It utilizes the pH indicator bromocresol purple (BCP). As cadAverine is produced from l-lysine, it elevates the reaction pH, leading to a corresponding change in BCP color. Significant findingsReal-time monitoring of CadA activity was conducted using an inducible T7 promoter system, built upon the CadA/-CadB co-expression framework within Escherichia coli BL21(DE3). Analysis of Michaelis-Menten kinetics revealed that pyridoxal-5′-phosphate (PLP) was efficient in substrate conversion compared to pyridoxal (PL) and pyridoxine (PN) as cofactors. Supplementation of PN with ATP successfully restored Vmax to levels comparable with PLP. However, following freezer storage of biocatalysts, insufficient PLP generation from PN was observed due to variations in the cellular metabolic pool and ATP levels. The optimal conditions for CadA/B activity were achieved with 7 μM IPTG induction at 32 °C for 8 h, resulting in a maximum specific activity of 18.2 ± 1.1 mmol/min/g dried cell weight. The BCP-based colorimetric assay showcased its efficacy in rapidly detecting and quantifying CadA/B activity, thereby facilitating high-throughput screening of recombinant libraries.

Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and colocalization studies in S. cerevisiae.

Spatial and temporal tracking of fluorescent proteins (FPs) in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active FPs fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.

A Comprehensive Study of Light Quality Acclimation in Synechocystis Sp. PCC 6803.

Cyanobacteria play a key role in primary production in both oceans and fresh waters and hold great potential for sustainable production of a large number of commodities. During their life, cyanobacteria cells need to acclimate to a multitude of challenges, including shifts in intensity and quality of incident light. Despite our increasing understanding of metabolic regulation under various light regimes, detailed insight into fitness advantages and limitations under shifting light quality remains underexplored. Here, we study photo-physiological acclimation in the cyanobacterium Synechocystis sp. PCC 6803 throughout the photosynthetically active radiation (PAR) range. Using light emitting diodes (LEDs) with qualitatively different narrow spectra, we describe wavelength dependence of light capture, electron transport and energy transduction to main cellular pools. In addition, we describe processes that fine-tune light capture, such as state transitions, or the efficiency of energy transfer from phycobilisomes to photosystems (PS). We show that growth was the most limited under blue light due to inefficient light harvesting, and that many cellular processes are tightly linked to the redox state of the plastoquinone (PQ) pool, which was the most reduced under red light. The PSI-to-PSII ratio was low under blue photons, however, it was not the main growth-limiting factor, since it was even more reduced under violet and near far-red lights, where Synechocystis grew faster compared to blue light. Our results provide insight into the spectral dependence of phototrophic growth and can provide the foundation for future studies of molecular mechanisms underlying light acclimation in cyanobacteria, leading to light optimization in controlled cultivations.

HIV-1 Infection Reduces NAD Capping of Host Cell snRNA and snoRNA.

Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5' cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g., U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity.

Abstract PO2-15-11: Kinetic Analysis of [5-11C] glutamine PET tracer data in breast cancer: Preclinical studies in mouse models

Abstract Background: Glutamine is an important metabolic substrate in many aggressive tumors, with comparable importance to glucose metabolism. Utilizing human breast cancer mouse xenograft models, we studied the kinetics of the PET imaging agent, [5-11C] glutamine, a biochemical authentic substrate for glutamine metabolism, to further characterize the metabolism of glutamine and downstream labeled metabolites. Studies were performed with and without inhibition of the enzyme, glutaminase (GLS), the first step in glutamine catabolism that generates glutamate, and key target for therapy directed to glutamine-metabolizing cancers. Methods: The study used xenograft mouse models for two breast cancer cell lines, HCC1806, a highly glutaminolytic triple-negative cell line, and MCF-7, a hormone receptor positive line with only low levels of glutaminolysis. Mice were injected with 5-[11C]L-glutamine, and the contributions of individual metabolites to the total [11C] signal in blood and tumor tissue were estimated at 10, 20, and 30 minutes after injection by assaying the fractional contributions of [5-11C] glutamine and labeled metabolites, specifically [5-11C] glutamate, [11C] CO2 and all other metabolites with [11C], in both blood and tumor tissue. This generated estimated time activity curves for [5-11C] glutamine and downstream metabolites in both HCC1806 and MCF-7 mouse models with and without CB-839 treatment, an inhibitor of GLS. We also investigated a compartment model that describes these data, including model simulations that fit the model to match the measured curves. Results: We found that, out to 30 minutes post-injection, the vast majority of tumor radioactivity was in the form of either 5-11C] glutamine or [5-11C] glutamate, with smaller amounts of radioactivity in the form of more downstream metabolites, including [11C]CO2. Systemic administration of the glutaminase inhibitor alters the arterial input curve by reducing the systemic rate of conversion of [5-11C] glutamine to [5-11C] glutamate. However, in GLS active tumors, [5-11C] glutamate retained in the large cellular glutamate pool represents a greater fraction of total radioactivity than in the blood and leads to a total tumor time-activity curve that is only marginally different than the total tumor time-activity curve after GLS inhibition. Data from blood and tissue analysis were used to test a hypothetical kinetic model for [5-11C] glutamine, which provided estimates of glutamate and glutamate tumor tissue concentrations and tumor GLS activity that agree with direct measurements in the animal models. Conclusion: The study of [ 5-11C] glutamine in breast cancer models leads to kinetic insight that are in line with biochemical studies and direct measurement taken for tumor tissue obtained from the animal models. Retention of metabolized [5-11C] glutamine as [5-11C] glutamate in a large tissue pool in tumors with high GLS activity makes non-invasive inference of GLS activity challenging without additional ex vivo analysis, supporting the use of non-metabolized glutamine analogs such as such as [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) to infer in vivo glutamine metabolism. Citation Format: Christopher Hensley, Prashanth Padakanti, Hoon Choi, Christina Dulal, Hsiaojiu Lee, Austin Pantel, Rong Zhou, David Mankoff. Kinetic Analysis of [5-11C] glutamine PET tracer data in breast cancer: Preclinical studies in mouse models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-15-11.

Disorders of fatty acid homeostasis.

Humans derive fatty acids (FA) from exogenous dietary sources and/or endogenous synthesis from acetyl-CoA, although some FA are solely derived from exogenous sources ("essential FA"). Once inside cells, FA may undergo a wide variety of different modifications, which include their activation to their corresponding CoA ester, the introduction of double bonds, the 2- and ω-hydroxylation and chain elongation, thereby generating a cellular FA pool which can be used for the synthesis of more complex lipids. The biological properties of complex lipids are very much determined by their molecular composition in terms of the FA incorporated into these lipid species. This immediately explains the existence of a range of genetic diseases in man, often with severe clinical consequences caused by variants in one of the many genes coding for enzymes responsible for these FA modifications. It is the purpose of this review to describe the current state of knowledge about FA homeostasis and the genetic diseases involved. This includes the disorders of FA activation, desaturation, 2- and ω-hydroxylation, and chain elongation, but also the disorders of FA breakdown, including disorders of peroxisomal and mitochondrial α- and β-oxidation.

Dominant role for pigment epithelial CRALBP in supplying visual chromophore to photoreceptors

Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.

Rocaglamide regulates iron homeostasis by suppressing hepcidin expression

The anemia of inflammation (AI) is characterized by the presence of inflammation and abnormal elevation of hepcidin. Accumulating evidence has proved that Rocaglamide (RocA) was involved in inflammation regulation. Nevertheless, the role of RocA in AI, especially in iron metabolism, has not been investigated, and its underlying mechanism remains elusive. Here, we demonstrated that RocA dramatically suppressed the elevation of hepcidin and ferritin in LPS-treated mice cell line RAW264.7 and peritoneal macrophages. In vivo study showed that RocA can restrain the depletion of serum iron (SI) and transferrin (Tf) saturation caused by LPS. Further investigation showed that RocA suppressed the upregulation of hepcidin mRNA and downregulation of Fpn1 protein expression in the spleen and liver of LPS-treated mice. Mechanistically, this effect was attributed to RocA's ability to inhibit the IL-6/STAT3 pathway, resulting in the suppression of hepcidin mRNA and subsequent increase in Fpn1 and TfR1 expression in LPS-treated macrophages. Moreover, RocA inhibited the elevation of the cellular labile iron pool (LIP) and reactive oxygen species (ROS) induced by LPS in RAW264.7 cells. These findings reveal a pivotal mechanism underlying the roles of RocA in modulating iron homeostasis and also provide a candidate natural product on alleviating AI.

Iron regulates the quiescence of naive CD4 T cells by controlling mitochondria and cellular metabolism

In response to an immune challenge, naive T cells undergo a transition from a quiescent to an activated state acquiring the effector function. Concurrently, these T cells reprogram cellular metabolism, which is regulated by iron. We and others have shown that iron homeostasis controls proliferation and mitochondrial function, but the underlying mechanisms are poorly understood. Given that iron derived from heme makes up a large portion of the cellular iron pool, we investigated iron homeostasis in T cells using mice with a T cell-specific deletion of the heme exporter, FLVCR1 [referred to as knockout (KO)]. Our finding revealed that maintaining heme and iron homeostasis is essential to keep naive T cells in a quiescent state. KO naive CD4 T cells exhibited an iron-overloaded phenotype, with increased spontaneous proliferation and hyperactive mitochondria. This was evidenced by reduced IL-7R and IL-15R levels but increased CD5 and Nur77 expression. Upon activation, however, KO CD4 T cells have defects in proliferation, IL-2 production, and mitochondrial functions. Iron-overloaded CD4 T cells failed to induce mitochondrial iron and exhibited more fragmented mitochondria after activation, making them susceptible to ferroptosis. Iron overload also led to inefficient glycolysis and glutaminolysis but heightened activity in the hexosamine biosynthetic pathway. Overall, these findings highlight the essential role of iron in controlling mitochondrial function and cellular metabolism in naive CD4 T cells, critical for maintaining their quiescent state.

Abstract 3267: Understanding the role of dNTP pool on double-strand break and resistant to therapy in quiescent hypoxic glioblastoma

Abstract Glioblastoma (GBM) is the deadliest type of primary brain tumor. After surgical resection of GBM, chemotherapy and radiotherapy (RT) are used as treatment modalities to induce double-strand breaks (DSBs), the most cytotoxic and lethal form of DNA damage. However, an inadequate oxygen supply and the high metabolic demand in rapidly growing solid tumors like GBM result in severe hypoxic regions, contributing to altered cellular metabolism and resistance to these cancer therapies. These subpopulations of resistant and refractory hypoxic cancer cells are clinically significant as they lead to poor prognosis and adverse clinical outcomes in patients. Ionizing radiation (IR) and chemotherapeutics such as temozolomide induce DNA damage, such as base deletions, single-strand breaks (SSBs), and DSBs. Rapidly replicating cells are more sensitive to these therapeutic agents partly due to replication-induced conversion of SSB and other manageable damages to highly lethal DSB. A balanced deoxyribonucleoside triphosphate (dNTP) is crucial for DNA replication, repair, and maintenance of genomic stability. Cellular dNTP is mainly regulated by ribonucleotide reductase (RNR) and the SAM- and HD-domain-containing protein (SAMHD1). RNR boosts the cellular dNTP pool through the de novo synthesis, while SAMHD1 depletes the pool by hydrolyzing dNTPs. Also, previous studies have shown that hypoxic cancer cells have low dNTP levels due to oxygen dependency of its rate-limiting RNR enzyme, which could result in reduced DNA replication and cell cycle arrest. Here, we show the direct relationships between dNTP levels, DNA replication, and DSB induction in hypoxic GBM cancer cells, which is currently unknown. To bridge this knowledge gap, we exposed GBM cells to severe hypoxia and utilized cellular dNTP analysis to demonstrate that their dNTP pool can be elevated. Our results show that elevating the cellular dNTP pool relieved hypoxia-induced cell cycle arrest as determined by flow cytometry and increased their DNA replication. A single-cell microscopic analysis also revealed that increasing the dNTP pool significantly enhanced the induction of DSBs following exposure to IR and bleomycin. Furthermore, we detected an increased efficacy of DSB-inducing agents in hypoxic GBM, as demonstrated by apoptosis pathway activation. Our result suggests that increasing the intracellular dNTP pool in hypoxic GBM will stimulate DNA replication, increase genotoxin-induced DSB, and improve DSB-inducing therapeutic efficacy. In conclusion, this study provides a preclinical justification for using cellular dNTP modulation as a novel therapeutic strategy and an adjunct to classical DSB-inducing agents in hypoxic GBM. Citation Format: Edidiong R. Usoro, Dominique Monroe, Kristen Carver, Emily Steinbeck, Arilyn Williams, Shelby Aycox, Waaqo Daddacha. Understanding the role of dNTP pool on double-strand break and resistant to therapy in quiescent hypoxic glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3267.

Abstract 5625: Targeting the nonsense mediated mRNA decay pathway to prevent the degradation of highly immunogenic frameshift mutated transcripts

Abstract Introduction: Frameshift insertions/deletions (fs-indels) can lead to the generation of immunogenic non-self-peptides which contribute to anti-tumor immune responses. However, fs-indels often generate premature termination codons (PTCs), leading to mRNA degradation by the nonsense mediated mRNA decay (NMD) pathway. Importantly, some PTC-containing transcripts naturally escape NMD degradation and produce highly immunogenic neoantigens. Therefore, we hypothesize that NMD inhibition could be used to increase the cellular pool of fs-indels derived neoantigens in cancer cells, and enhance anti-tumor immunogenicity. NMD also regulates other cellular processes by degrading a subset of non-PTC-containing self-transcripts. Therefore, by focusing NMD inhibition towards the degradation of PTC-transcripts only, toxicity may be limited in a clinical setting. We investigate how targeting different NMD proteins affects the degradation of fs-transcripts across several cancers types. To assess the clinical relevance of NMD inhibition, we study how it specifically affects p53 fs-transcripts, since p53 is the most mutated gene in cancer and fs-mutant p53 has been shown to be highly immunogenic. Methods: Core NMD proteins were depleted across a panel of cell lines from different cancer types and transcriptomics, flow cytometry, qPCR and western blot were used to evaluate changes in the expression of NMD-targeted transcripts and NMD members. Proteomics and immunopeptidomics were used to study protein expression and HLA-peptide presentation arising from fs-transcripts. A Translating RNA Imaging by Coat protein Knock-off (TRICK) assay was developed to study the translation of NMD-targeted p53 mutants upon NMD inhibition. This assay can precisely quantify the translation of p53 mutant RNA upon different treatments and cellular stresses at single-molecule resolution. Results: We found an increase in expression of PTC-transcripts upon silencing of multiple NMD members. Our data suggests that some NMD members are better targets for inhibition of NMD PTC-surveillance, whilst others are more appropriate for inhibition of NMD-mediated gene expression regulation. These data support the recently proposed hypothesis that NMD is a branched pathway, where each branch targets specific subsets of transcripts depending on the cell type. These results are clinically relevant as the method by which NMD is inhibited could help optimize the generation of immunogenic neoantigens whilst minimizing toxicity. The TRICK assay has shown that inhibiting NMD surveillance reduces the degradation of p53 fs-transcripts and increases their protein expression. Conclusion: We have identified NMD pathway members that can be targeted to efficiently inhibit the surveillance role of NMD and prevent the degradation of fs-transcripts that could generate highly immunogenic neoantigens. Citation Format: Shanila Fernandez Patel, Roberto Vendramin, Danwen Qian, Yue Zhao, Lorena Ligammari, Krupa Thakkar, Jun Murai, Eva Grönroos, Jeremy Carlton, Kevin Litchfield. Targeting the nonsense mediated mRNA decay pathway to prevent the degradation of highly immunogenic frameshift mutated transcripts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5625.