Cell Compartment Research Articles

Treatment of high risk myelodysplastic syndrome.

The myelodysplastic syndrome (MDS) is considered to be a heterogeneous myeloid malignancy with a common origin in the hematopoietic stem cell compartment, generally divided into lower and higher risk. While treatment goal for lower risk MDS (LR-MDS) is to decrease transfusion burden and transformation into acute leukemia major aim for high risk MDS is to prolong survival and ultimately cure. While novel agents such as luspatercept or imetelstat have recently been approved as new treatment options for LR-MDS, hypomethylating agents (HMA) remain currently the only approved non-transplant option for HR-MDS and is the standard of care for non-transplant-eligible patients. Combinations with other drugs as first-line treatment has to date not proven more efficacious than monotherapy in HR-MDS, and outcome after HMA failure is poor. The only potential cure and standard of care for eligible patients is allogeneic stem cell transplantation (HSCT) and even if the number of transplanted - especially older - MDS patients increased over time due to a better management and donor availability the majority of MDS patients will not be eligible for this curative approach. Current challenges encompass to decrease the relapse risk, the main cause of HSCT failure. This review will summarize current knowledge of options of transplant- and non-transplant treatment approaches for these patients and demonstrate the unmet clinical need for more effective therapies.

Left Ventricular Hypertrophy in Aortic Stenosis: Early Cell and Matrix Regression 2 Months Post-Aortic Valve Replacement.

In aortic stenosis, the myocardium responds with left ventricular hypertrophy, which is characterized by increased left ventricular mass due to cellular hypertrophy and extracellular matrix expansion. Following aortic valve replacement (AVR), left ventricular hypertrophy regression occurs, but early cellular and extracellular dynamics are unknown. Patients with severe symptomatic aortic stenosis undergoing surgical or transcatheter AVR were prospectively recruited. Pre- and early post-AVR cardiac magnetic resonance imaging assessed left ventricular remodeling, global longitudinal strain, and T1 mapping to determine extracellular volume fraction and volume of cellular and extracellular compartments. In all, 39 patients (aged 71.4±9.8 years, male 79%, aortic valve peak velocity 4.4±0.5 m/s) underwent cardiac magnetic resonance before and at median 7.7 weeks post-AVR. Left ventricular mass index reduced significantly by 15.4% (P<0.001*), primarily driven by cellular compartment regression (18.7%, P<0.001*), with a smaller reduction in the extracellular compartment (7.2%, P<0.001*). This unbalanced regression led to an apparent increase in extracellular volume fraction (27.4±3.1% to 30.2±2.8%; P<0.001*). Although there was no significant change in global longitudinal strain post-AVR, an increase in extracellular volume fraction was associated with worsening of global longitudinal strain (Pearson r=0.41, P=0.01). Mode of intervention (transcatheter versus surgical) did not influence the above myocardial parameters post-AVR (all P>0.05). The asterisk in P values indicates a statistical significance of <0.05. Within 8 weeks of AVR for aortic stenosis, substantial left ventricular hypertrophy regression occurs involving both cellular and extracellular compartments, demonstrating the early myocardial adaptability to afterload relief. Cellular compartment regression is greater than extracellular regression, leading to an apparent increase in extracellular volume fraction. Mode of intervention did not affect degree of reverse remodeling, indicating that both are effective at resulting beneficial changes post-AVR. URL: https://www.isrctn.com; Unique identifier: NCT04627987.

Engineering a Silk Protein-Mediated Customizable Compartment for Modular Metabolic Synthesis.

Microbial cell factories provide a nontoxic, economical way for the synthesis of various chemicals and drugs, garnering significant attention from researchers. However, excessive dispersion of enzymes and accumulation of intermediate metabolites in the production process will weaken the reaction efficiency of the pathway enzyme. In this study, a cellular compartment was constructed to isolate the enzyme reaction space and optimize the modular metabolic synthesis. First, a special spider silk protein was designed and constructed to form protein condensates in microbial cells, and its synthetic microcompartment effects were investigated. Second, the interaction of short peptide pairs or direct fusion based on the silk protein was used to recruit a variety of enzymes to improve the efficiency of enzyme catalysis. Third, the 2'-fucosyllactose (2'-FL) de novo synthesis pathway and its modular optimization were carried out to verify the mode. Finally, a synthetic compartment was introduced into the pathway to directly aggregate the 2'-FL synthesis pathway, thus obtaining synthetic-compartment-mediated multienzyme aggregates. The experimental results showed that the titer of 2'-FL was significantly improved compared with those of wild-type and modular-optimized free enzymes. The utilization of this cell microcompartment offers a novel avenue for the aggregation of diverse enzymes, thereby offering an innovative approach for enhancing the efficiency of the microbial modular metabolic pathway.

Structure of yeast RAVE bound to a partial V1 complex

Vacuolar-type ATPases (V-ATPases) are membrane-embedded proton pumps that acidify intracellular compartments in almost all eukaryotic cells. Homologous with ATP synthases, these multisubunit enzymes consist of a soluble catalytic V1 subcomplex and a membrane-embedded proton-translocating VO subcomplex. The V1 and VO subcomplexes can undergo reversible dissociation to regulate proton pumping, with reassociation of V1 and VO requiring the protein complex known as RAVE (regulator of the ATPase of vacuoles and endosomes). In the yeast Saccharomyces cerevisiae, RAVE consists of subunits Rav1p, Rav2p, and Skp1p. We used electron cryomicroscopy (cryo-EM) to determine a structure of yeast RAVE bound to V1. In the structure, RAVE is an L-shaped complex with Rav2p pointing toward the membrane and Skp1p distant from both the membrane and V1. Only Rav1p interacts with V1, binding to a region of subunit A not found in the corresponding ATP synthase subunit. When bound to RAVE, V1 is in a rotational state suitable for binding the free VO complex, but in the structure, it is partially disrupted, missing five of its 16 subunits. Other than these missing subunits and the conformation of the inhibitory subunit H, the V1 complex with RAVE appears poised for reassembly with VO.

The ABC transporter Opp imports reduced glutathione, while Gsi imports glutathione disulfide in Escherichia coli

Glutathione is the major thiol-based antioxidant in a wide variety of biological systems, ranging from bacteria to eukaryotes. As a redox couple, consisting of reduced glutathione (GSH) and its oxidized form, glutathione disulfide (GSSG), it is crucial for the maintenance of the cellular redox balance. Glutathione transport out of and into cellular compartments and the extracellular space is a determinant of the thiol-disulfide redox state of the organelles and bodily fluids in question, but is currently not well understood. Here we use the genetically-encoded, glutathione-measuring redox probe Grx1-roGFP2 to comprehensively elucidate the import of extracellular glutathione into the cytoplasm of the model organism Escherichia coli. The elimination of only two ATP-Binding Cassette (ABC) transporter systems, Gsi and Opp, completely abrogates glutathione import into E. coli’s cytoplasm, both in its reduced and oxidized form. The lack of only one of them, Gsi, completely prevents import of GSSG, while the lack of the other, Opp, substantially retards the uptake of reduced glutathione (GSH).

Monosaccharide coating modulate the intracellular trafficking of gold nanoparticles in dendritic cells

Dendritic cells (DCs) have emerged as a promising target for drug delivery and immune modulation due to their pivotal role in initiating the adaptive immune response. Gold nanoparticles (AuNPs) have garnered interest as a platform for targeted drug delivery due to their biocompatibility, low toxicity and precise control over size, morphology and surface functionalization. Our investigation aimed to elucidate the intracellular uptake and trafficking of AuNPs coated with different combinations of monosaccharides (mannose, galactose, and fucose) in DCs. We used 30 unique polymer-tethered monosaccharide combinations to coat 16 nm diameter spherical gold nanoparticles and investigated their effect on DCs phenotype, uptake, and intracellular trafficking.DCs internalised AuNPs coated with 100% fucose, 100% mannose, 90% mannose + 10% galactose, and 80% mannose + 20% galactose with highest efficiency. Flow cytometry analysis indicated that 100% fucose-coated AuNPs showed increased lysosomal and endosomal contents compared to other conditions and uncoated AuNPs. Imaging flow cytometry further demonstrated that 100% fucose-coated AuNPs had enhanced co-localisation with lysosomes, while 100% mannose-coated AuNPs exhibited higher co-localisation with endosomes.Furthermore, our data showed that the uptake of carbohydrate-coated AuNPs predominantly occurred through receptor-mediated endocytosis, as evidenced by a marked reduction of uptake upon treatment of DCs with methyl-β-cyclodextrins, known to disrupt receptor-mediated endocytosis. These findings highlight the utility of carbohydrate coatings to enable more targeted delivery of nanoparticles and their payload to distinct intracellular compartments in immune cells with potential applications in drug delivery and immunotherapy.

Determination of diffusion coefficients through gels with non-negligible finite-volume effects in the compartments of the diffusion cell

Diffusion cells are used to measure diffusion coefficients (DM) in gels. These measurements are of interest to understand and predict the availability of nutritive or toxic chemical species in waters, soils and sediments. When the diffusive flux from the donor to the acceptor compartment is constant (steady-state regime), DM is determined from the slope of the linear plot of the acceptor concentration vs time. However, at long enough times, there is a non-negligible concentration depletion in the donor compartment concomitant to a concentration increase in the acceptor compartment. Accordingly, the accumulation plot bends downwards preventing a linear fitting. This is the case of metals whose solubility (especially depending on pH values) limits the concentration in the donor compartment and the time required to reach concentrations above the limit of quantification in the acceptor compartment implies a non-negligible decrease of the concentration in the donor compartment.In this work, a simple linear regression is shown to provide the diffusion coefficient values from experiments exhibiting finite-volume effects. This expression is validated against rigorous numerical simulation as well as reported values in the literature. Diffusion coefficients of Zn, Ni and Pb in agarose cross-linked polyacrylamide (APA) gels (used in Diffusive Gradients in Thin-Film devices, DGT) are determined under finite-volume effects. The resulting values agree with those obtained under the standard linear regime.

Polymeric Vehicles for Nucleic Acid Delivery: Enhancing the Therapeutic Efficacy and Cellular Uptake.

Therapeutic gene delivery may be facilitated by the use of polymeric carriers. When combined with nucleic acids to form nanoparticles or polyplexes, a variety of polymers may shield the cargo from in vivo breakdown and clearance while also making it easier for it to enter intracellular compartments. Polymer synthesis design choices result in a wide variety of compounds and vehicle compositions. Depending on the application, these characteristics may be changed to provide enhanced endosomal escape, longer-lasting distribution, or stronger connection with nucleic acid cargo and cells. Here, we outline current methods for delivering genes in preclinical and clinical settings using polymers. Significant therapeutic outcomes have previously been attained using genetic material- delivering polymer vehicles in both in-vitro and animal models. When combined with nucleic acids to form nanoparticles or polyplexes, a variety of polymers may shield the cargo from in vivo breakdown and clearance while also making it easier for it to enter intracellular compartments. Many innovative diagnoses for nucleic acids have been investigated and put through clinical assessment in the past 20 years. Polymer-based carriers have additional delivery issues due to their changes in method and place of biological action, as well as variances in biophysical characteristics. We cover recent custom polymeric carrier architectures that were tuned for nucleic acid payloads such genomemodifying nucleic acids, siRNA, microRNA, and plasmid DNA. In conclusion, the development of polymeric carriers for gene delivery holds promise for therapeutic applications. Through careful design and optimization, these carriers can overcome various challenges associated with nucleic acid delivery, offering new avenues for treating a wide range of diseases.

Molecular principles of the assembly and construction of a carboxysome shell.

Intracellular compartmentalization enhances biological reactions, crucial for cellular function and survival. An example is the carboxysome, a bacterial microcompartment for CO2 fixation. The carboxysome uses a polyhedral protein shell made of hexamers, pentamers, and trimers to encapsulate Rubisco, increasing CO2 levels near Rubisco to enhance carboxylation. Despite their role in the global carbon cycle, the molecular mechanisms behind carboxysome shell assembly remain unclear. Here, we present a structural characterization of α-carboxysome shells generated from recombinant systems, which contain all shell proteins and the scaffolding protein CsoS2. Atomic-resolution cryo-electron microscopy of the shell assemblies, with a maximal size of 54 nm, unveil diverse assembly interfaces between shell proteins, detailed interactions of CsoS2 with shell proteins to drive shell assembly, and the formation of heterohexamers and heteropentamers by different shell protein paralogs, facilitating the assembly of larger empty shells. Our findings provide mechanistic insights into the construction principles of α-carboxysome shells and the role of CsoS2 in governing α-carboxysome assembly and functionality.

Identification of novel interacting partners of the TRIM29 E3-ubiquitin ligase in immortalized cells of normal prostate basal epithelium

BACKGROUND: Based on the most recent findings, the E3 ubiquitin ligase TRIM29 participates in numerous cellular processes and holds significance in both the formation of normal basal epithelium and prostate cancer. Exploring these molecular mechanisms could be pivotal in developing novel cancer therapies. AIM: Identification of protein partners of TRIM29 and its truncated forms, followed by functional analysis of these proteins. Identification of the key molecular processes involving TRIM29. METHODS: Cell cultures from normal basal epithelium of the prostate with overexpression of the chimeric protein TRIM29-FLAG or its truncated forms lacking the B-Box domain or the Coiled-Coil domain were conducted. Protein partners of TRIM29 and its truncated forms were identified through protein immunoprecipitation followed by proteomic (HPLC-MS/MS) analysis. The findings were validated through Western blot analysis and immunocytochemistry. RESULTS: TRIM29 interacts with 288 proteins in normal prostate basal epithelium. Deletion of the B-Box has minimal impact on TRIM29's protein interactions, while deletion of the Coiled-Coil domain significantly reduces its protein partners and disrupts its dimerization. Our research indicates that TRIM29 is present in both the nucleus and cytoplasm, and deletion of functional domains does not affect its localization but alters binding to compartment-specific proteins. TRIM29 binds to cytoskeletal proteins, cellular stress response proteins, and RNA-binding proteins. Furthermore, TRIM29 enhances cell resistance to genotoxic agents and influences RNA splicing. CONCLUSION: Proteomic analysis reveals that in normal prostate basal epithelium, the E3-ubiquitin ligase TRIM29 binds to a broad spectrum of proteins with distinct functions in various cell compartments. Our findings align with other studies demonstrating involvement of TRIM29 in cytoskeletal rearrangement, cellular response to viral infections, and DNA damage. Additionally, our research demonstrates the interaction of TRIM29 with RNA-binding proteins and its potential role in regulating RNA splicing, particularly through the Coiled-Coil domain.

Quantitative determination of the spatial distribution of components in single cells with CellDetail

The distribution of biomolecules within cells changes upon aging and diseases. To quantitatively determine the spatial distribution of components inside cells, we built the user-friendly open-source 3D-cell-image analysis platform Cell Detection and Analysis of Intensity Lounge (CellDetail). The algorithm within CellDetail is based on the concept of the dipole moment. CellDetail provides quantitative values for the distribution of the polarity proteins Cdc42 and Tubulin in young and aged hematopoietic stem cells (HSCs). Septin proteins form networks within cells that are critical for cell compartmentalization. We uncover a reduced level of organization of the Septin network within aged HSCs and within senescent human fibroblasts. Changes in the Septin network structure might therefore be a common feature of aging. The level of organization of the network of Septin proteins in aged HSCs can be restored to a youthful level by pharmacological attenuation of the activity of the small RhoGTPase Cdc42.

The concept of natural genome reconstruction. Part 1. Basic provisions of the “natural genome reconstruction” concept. Changing the genome of hematopoietic stem cells using several natural cellular mechanisms that are inherent in the hematopoietic cell and determine its biological status as “the source of the body’s reparative potential”

We present a series of articles proving the existence of a previously unknown mechanism of interaction between hematopoietic stem cells and extracellular double-stranded DNA (and, in particular, double-stranded DNA of the peripheral bloodstream), which explains the possibility of emergence and fixation of genetic information contained in double-stranded DNA of extracellular origin in hematopoietic stem cells. The concept of the possibility of stochastic or targeted changes in the genome of hematopoietic stem cells is formulated based on the discovery of new, previously unknown biological properties of poorly differentiated hematopoietic precursors. The main provisions of the concept are as follows. The hematopoietic stem cell takes up and internalizes fragments of extracellular double-stranded DNA via a natural mechanism. Specific groups of glycocalyx factors, including glycoproteins/proteoglycans, glycosylphosphatidylinositol-anchored proteins and scavenger receptors, take part in the internalization event. The binding sites for DNA fragments are heparin-binding domains and clusters of positively charged amino acid residues that are parts of protein molecules of these factors. Extracellular fragments delivered to the internal compartments of hematopoietic stem cells initiate terminal differentiation, colony formation, and proliferation of hematopoietic precursors. The molecular manifestation of these processes is the emergence and repair of pangenomic single-strand breaks. The occurrence of pangenomic single-strand breaks and restoration of genome (genomic DNA) integrity are associated with activation of a “recombinogenic situation” in the cell; during its active phase, stochastic homologous recombination or other recombination events between extracellular fragments localized in the nucleus and chromosomal DNA are possible. As a result, genetic material of initially extracellular localization either integrates into the recipient genome with the replacement of homologous chromosomal segments, or is transitively present in the nucleus and can manifest itself as a new genetic trait. It is assumed that as a result of stochastic acts of homologous exchange, chromosome loci are corrected in hematopoietic stem cells that have acquired mutations during the existence of the organism, which are the cause of clonal hematopoiesis associated with old age. In this regard, there is a fundamental possibility of changing the hematopoietic status of hematopoietic stem cells in the direction of polyclonality and the original diversity of clones. Such events can form the basis for the rejuvenation of the blood-forming cell system. The results of the laboratory’s work indicate that other stem cells in the body capture extracellular DNA fragments too. This fact creates a paradigm for the overall rejuvenation of the body.

The Role of Metal Tolerance Proteins (MTPs) Associated with the Homeostasis of Divalent Mineral Elements in Ga-Treated Rice Plants.

Mineral elements typically act as transported substrates for metal tolerance proteins (MTPs). The chelation of MTPs with heavy metal ions is a suggestive detoxification pathway in plants; therefore, the trade-off between transporting mineral elements and chelating excess toxic metal ions is inevitable. Gallium (Ga) is an emerging pollutant associated with high-tech industries. This study investigated the impact of Ga stress on MTPs, subsequently altering the transport and distribution of mineral elements. Gallium exposure reduced rice seedling biomass, with roots accumulating more Ga than shoots. Ga stress also changed the rice plants' subcellular mineral element distribution. PCR assays showed that Ga stress negatively affected all genes belonging to the Mn group, except OsMTP9. While Mn accumulation in the rice cellular compartments did not respond positively to Ga stress, OsMTP8, OsMTP8.1, OsMTP11, and OsMTP11.1 were found to be intimately connected to Mn transport and repressed by increased Ga accumulation in roots. Mg and Cu accumulated in the cytosol and organelles of Ga-treated rice plants, while OsMTP9 expression increased, demonstrating its importance in transporting Mg and Cu. A positive link between Ga stress and Zn accumulation in the cytosol and organelles was found, and OsMTP7 and OsMTP12 expression was positive, suggesting that Ga stress did not impair their Zn transport. Notably, Ga exposure down-regulated Fe-transporting OsMTP1 and OsMTP6, wherein the subcellular concentrations of Fe showed negative responses to Ga accumulation. These findings provide valuable insights into elucidating the roles of OsMTPs in Ga tolerance and the transport of these mineral elements.

Diminished lactate utilization in LDHB-deficient neurons leads to impaired long-term memory retention

Neurons' high energy demands for processing, transmitting, and storing information in the brain necessitate efficient energy metabolism to maintain normal neuronal function. The astrocyte-neuron lactate shuttle (ANLS) hypothesis suggests neurons preferentially use lactate from astrocytes over glucose for energy. This study investigated lactate dehydrogenase B (LDHB), which preferentially converts lactate to pyruvate, in neuronal energy metabolism and cognitive function. LDHB-deficient neurons showed reduced lactate-driven energy metabolism in culture, while LDHB-deficient brains accumulated lactate, both indicating decreased lactate utilization. This reduced lactate utilization was correlated with impaired long-term memory in LDHB-deficient mice, while short-term memory remained unaffected and overall neuropathology was only mildly disturbed. Unexpectedly, LDHB-deficient neurons maintain stable energy metabolism under physiological glucose conditions, indicating the presence of lactate dehydrogenase (LDH) activity in LDHB-deficient neurons. The observation of lactate dehydrogenase A (LDHA), which preferentially converts pyruvate to lactate but can also catalyze the reverse reaction less efficiently, in LDHB-deficient neurons may explain their stable energy metabolism and reduced lactate utilization. This study challenges the established concept of strict LDH isoform compartmentalization in brain cells, questioning the exclusive presence of LDHB in neurons and suggesting a more flexible neuronal metabolic profile than previously assumed by the ANSL hypothesis.

TET3 regulates terminal cell differentiation at the metabolic level

TET-family members play a critical role in cell fate commitment. Indeed, TET3 is essential to postnatal development due to yet unknown reasons. To define TET3 function in cell differentiation, we have profiled the intestinal epithelium at single-cell level from wild-type and Tet3 knockout mice. We have found that Tet3 is mostly expressed in differentiated enterocytes. In the absence of TET3, enterocytes exhibit an aberrant differentiation trajectory and do not acquire a physiological cell identity due to an impairment in oxidative phosphorylation, specifically due to an ATP synthase assembly deficiency. Moreover, spatial metabolomics analysis has revealed that Tet3 knockout enterocytes exhibit an unphysiological metabolic profile when compared with their wild-type counterparts. In contrast, no metabolic differences have been observed between both genotypes in the stem cell compartment where Tet3 is mainly not expressed. Collectively, our findings suggest a mechanism by which TET3 regulates mitochondrial function and, thus, terminal cell differentiation at the metabolic level.

Abstract C038: Harnessing NK cells to improve targeted therapy therapeutic efficacy in BRAF- mutant melanoma

Abstract Malignant melanoma is the most lethal form of skin cancer, with BRAFV600E as the most common driver mutation. Despite the success of BRAF/MEK inhibitors (BRAF/MEKi) as a standard-of-care, drug resistance occurs at high frequency and remains a concern to be overcome. In addition to suppressing cancer cell proliferation and survival, BRAF/MEKi treatment alters the tumor microenvironment to favor anti-tumor immunity, with extensive previous studies focusing on the T cell compartment. Investigating other immune components, such as natural killer (NK) cells, may offer new insights into improving the overall effectiveness of BRAF/MEKi. NK cells are innate immune cells that provide activating signals to cytotoxic T cells, in addition to their critical roles in tumor surveillance and metastasis. However, the role of NK cells as a component of the BRAF/MEKi response in melanoma remains largely unknown. To investigate the role of NK cells in BRAF/MEKi-mediated tumor control, we depleted NK cells in melanoma-bearing mice. We found that the tumors developed full resistance faster in the NK cell-depleted group than in controls. Moreover, quantification of tumor-infiltrating NK cells in different melanoma phases, including off-therapy growth, therapy-induced regression, drug- tolerant residual disease, and on-therapy regrowth and resistance, revealed a positive correlation between NK cell infiltration and the tumor response to BRAF/MEKi. We further increased NK cell infiltration during drug-tolerant residual disease by inhibiting Ptpn22, a potential negative regulator of activity in NK and other immune cell populations, resulting in a delayed onset of therapy resistance. These results demonstrate the indispensable role of NK cells in the BRAF/MEKi tumor response and their potential to improve the therapeutic efficacy of BRAF/MEKi. Citation Format: Chia-Hsin Hsu, Andrew C. White. Harnessing NK cells to improve targeted therapy therapeutic efficacy in BRAF- mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C038.

Role of circular RNAs in DNA repair.

Circular RNAs (circRNAs) exhibit a wide range of activities that allow them to participate in numerous cellular processes and make them relevant in a variety of diseases. In this regard, a key process in which circRNAs are involved, and which is the focus of this article, is DNA damage repair (DDR). This study aims to illustrate how circRNAs influence different DNA repair pathways, with particular emphasis on the underlying mechanisms. In addition, the potential medical applications of this knowledge are discussed, particularly in the diagnosis, prognosis and treatment of diseases. In this sense, circRNAs were found to play a crucial role in DNA repair processes by regulating the expression and activity of proteins involved in various DNA repair pathways. They influence the expression of DNA repair proteins by interacting with their mRNAs, sponging miRNAs that target these mRNAs, regulating transcription factors that bind to their promoters, modulating upstream signalling pathways, and affecting mRNA translation. Furthermore, circRNAs regulate the activity of DNA repair proteins by interacting directly with them, sequestering them in specific cellular compartments and controlling activation signalling or upstream DDR signalling.

Abstract C047: AGX101: Preclinical evaluation of a TM4SF1-directed tubulin inhibitor conjugate with ongoing first-in-human trial

Abstract Introduction: TM4SF1 (Transmembrane-4 L-Six-Family-Member-1) is an endothelial marker with critical roles in angiogenesis, as well as a tumor cell antigen that contributes significantly to invasion and metastasis. TM4SF1 is upregulated 20-fold in angiogenic tumor vascular endothelium compared to normal vasculature endothelium and exhibits a unique nuclear internalization pathway. AGX101 is a novel tubulin inhibitor conjugate specifically directed against TM4SF1, delivering a potent maytansinoid payload directly to the nucleus of cells within the tumor microenvironment. Delivery to both the vascular and tumor cell compartments of the tumor results in three mechanisms of action (MoAs): (1) activation of tumor immune surveillance, (2) tumor blood supply deprivation, and (3) direct tumor cell killing. Methods: TM4SF1 expression was assessed across solid tumor samples using immunohistochemistry. Safety and pharmacokinetics of AGX101 were evaluated in non-human primates, with escalating doses to determine the highest non-severely toxic dose (HNSTD). Efficacy studies were also conducted in mouse models, enabling assessment of the minimum effective dose (MED) needed to engage each of the three MoAs. The combination of HNSTD and MED enables calculation of a therapeutic index (TI). Preclinical efficacy studies used either AGX101 (in human tumor xenograft models, to study the tumor cell killing MoA) or AGXB01, a rodent surrogate of AGX101, to study vascular and immune MoAs and all three MoAs in syngeneic mouse cancer models. The potential for synergy with immune checkpoint inhibitors (ICIs) was also investigated. A first-in-human study of AGX101 in patients with advanced solid tumors with the primary objective of safety and dose-limiting toxicities (DLTs) has initiated. Results: TMFS41 is broadly expressed across solid tumors with increasing scoring intensity in higher grade tumors. In non-human primates, AGX101 exhibited a favorable safety profile, being tolerated at relatively high doses and with dose dependent, monitorable, and reversible effects. In preclinical efficacy studies, AGXB01 and AGX101 as monotherapies demonstrated robust efficacy through each of the three MoAs in multiple syngeneic, xenograft, and orthotopic tumor models in mice and rats as well as non-tumor models of angiogenesis. Measured by exposure, TI was large. Additionally, AGXB01 was shown to potentiate the effects of ICIs in syngeneic mouse models (CT26, Renca, and B16-F10), suggesting a potential synergistic approach in cancer therapy. The first dose level of AGX101 monotherapy in humans has cleared without DLTs. Conclusion: AGX101, targeting the TM4SF1 antigen, represents a promising new approach in cancer therapy. The preclinical data suggest that AGX101 could provide a significant therapeutic benefit by novel and differentiated mechanisms of action, namely selectively targeting the tumor vasculature and potentiating immune-based therapies. Further clinical development of AGX101 is ongoing. Citation Format: Ildefonso Ismael Rodriguez Rivera, Meredith S. Pelster, Shou-Ching Jaminet, Paul Jaminet, Glen J. Weiss. AGX101: Preclinical evaluation of a TM4SF1-directed tubulin inhibitor conjugate with ongoing first-in-human trial [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C047.

Phosphorylation of cytochrome c at tyrosine 48 finely regulates its binding to the histone chaperone SET/TAF-Iβ in the nucleus.

Post-translational modifications (PTMs) of proteins are ubiquitous processes present in all life kingdoms, involved in the regulation of protein stability, subcellular location and activity. In this context, cytochrome c (Cc) is an excellent case study to analyze the structural and functional changes induced by PTMS as Cc is a small, moonlighting protein playing different roles in different cell compartments at different cell-cycle stages. Cc is actually a key component of the mitochondrial electron transport chain (ETC) under homeostatic conditions but is translocated to the cytoplasm and even the nucleus under apoptotic conditions and/or DNA damage. Phosphorylation does specifically alter the Cc redox activity in the mitochondria and the Cc non-redox interaction with apoptosis-related targets in the cytoplasm. However, little is known on how phosphorylation alters the interaction of Cc with histone chaperones in the nucleus. Here, we report the effect of Cc Tyr48 phosphorylation by examining the protein interaction with SET/TAF-Iβ in the nuclear compartment using a combination of molecular dynamics simulations, biophysical and structural approaches such as isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) and in cell proximity ligation assays. From these experiments, we infer that Tyr48 phosphorylation allows a fine-tuning of the Cc-mediated inhibition of SET/TAF-Iβ histone chaperone activity in vitro. Our findings likewise reveal that phosphorylation impacts the nuclear, stress-responsive functions of Cc, and provide an experimental framework to explore novel aspects of Cc post-translational regulation in the nucleus.

Integrated proteomics and metabolomics analyses reveal new insights into the antitumor effects of valproic acid plus simvastatin combination in a prostate cancer xenograft model associated with downmodulation of YAP/TAZ signaling

BackgroundDespite advancements in therapeutic approaches, including taxane-based chemotherapy and androgen receptor-targeting agents, metastatic castration-resistant prostate cancer (mCRPC) remains an incurable tumor, highlighting the need for novel strategies that can target the complexities of this disease and bypass the development of drug resistance mechanisms. We previously demonstrated the synergistic antitumor interaction of valproic acid (VPA), an antiepileptic agent with histone deacetylase inhibitory activity, with the lipid-lowering drug simvastatin (SIM). This combination sensitizes mCRPC cells to docetaxel treatment both in vitro and in vivo by targeting the cancer stem cell compartment via mevalonate pathway/YAP axis modulation.MethodsHere, using a combined proteomic and metabolomic/lipidomic approach, we characterized tumor samples derived from 22Rv1 mCRPC cell-xenografted mice treated with or without VPA/SIM and performed an in-depth bioinformatics analysis.ResultsWe confirmed the specific impact of VPA/SIM on the Hippo–YAP signaling pathway, which is functionally related to the modulation of cancer-related extracellular matrix biology and metabolic reprogramming, providing further insights into the molecular mechanism of the antitumor effects of VPA/SIM.ConclusionsIn this study, we present an in-depth exploration of the potential to repurpose two generic, safe drugs for mCRPC treatment, valproic acid (VPA) and simvastatin (SIM), which already show antitumor efficacy in combination, primarily affecting the cancer stem cell compartment via MVP/YAP axis modulation. Bioinformatics analysis of the LC‒MS/MS and 1H‒NMR metabolomics/lipidomics results confirmed the specific impact of VPA/SIM on Hippo–YAP.