Utility of AlphaMissense predictions in Asparagine Synthetase deficiency variant classification.

AlphaMissense is a recently developed method that is designed to classify missense variants into pathogenic, benign, or ambiguous categories across the entire human proteome. Asparagine Synthetase Deficiency (ASNSD) is a developmental disorder associated with severe symptoms, including congenital microcephaly, seizures, and premature death. Diagnosing ASNSD relies on identifying mutations in the asparagine synthetase (ASNS) gene through DNA sequencing and determining whether these variants are pathogenic or benign. Pathogenic ASNS variants are predicted to disrupt the protein's structure and/or function, leading to asparagine depletion within cells and inhibition of cell growth. AlphaMissense offers a promising solution for the rapid classification of ASNS variants established by DNA sequencing and provides a community resource of pathogenicity scores and classifications for newly diagnosed ASNSD patients. Here, we assessed AlphaMissense's utility in ASNSD by benchmarking it against known critical residues in ASNS and evaluating its performance against a list of previously reported ASNSD-associated variants. We also present a pipeline to calculate AlphaMissense scores for any protein in the UniProt database. AlphaMissense accurately attributed a high average pathogenicity score to known critical residues within the two ASNS active sites and the connecting intramolecular tunnel. The program successfully categorized 78.9% of known ASNSD-associated missense variants as pathogenic. The remaining variants were primarily labeled as ambiguous, with a smaller proportion classified as benign. This study underscores the potential role of AlphaMissense in classifying ASNS variants in suspected cases of ASNSD, potentially providing clarity to patients and their families grappling with ongoing diagnostic uncertainty.

A method for measurement of human asparagine synthetase (ASNS) activity and application to ASNS protein variants associated with ASNS deficiency.

Human asparagine synthetase (ASNS) catalyzes the conversion of aspartate to asparagine in an ATP-dependent reaction that utilizes glutamine as a nitrogen source while generating glutamate, AMP, and pyrophosphate as additional products. Asparagine Synthetase Deficiency (ASNSD) is an inborn error of metabolism in which children present with homozygous or compound heterozygous mutations in the ASNS gene. These mutations result in ASNS variant protein expression. It is believed that these variant ASNS proteins have reduced enzymatic activity or stability resulting in a lack of sufficient asparagine production for cell function. Reduced asparagine production by ASNS appears to severely hinder fetal brain development. Although a variety of approaches for assaying ASNS activity have been reported, we present here a straightforward method for the in vitro enzymatic analysis by detection of AMP production. Our method overcomes limitations in technical feasibility, signal detection, and reproducibility experienced by prior methods like high-performance liquid chromatography, ninhydrin staining, and radioactive tracing. After purification of FLAG-tagged R49Q, G289A, and T337I ASNS variants from stably expressing HEK 293T cells, this method revealed a reduction in activity of 90, 36, and 96%, respectively. Thus, ASNS protein expression and purification, followed by enzymatic activity analysis, has provided a relatively simple protocol to evaluate structure-function relationships for ASNS variants reported for ASNSD patients.

Metabolomic Profiling of Asparagine Deprivation in Asparagine Synthetase Deficiency Patient-Derived Cells.

The natural amino acid asparagine (Asn) is required by cells to sustain function and proliferation. Healthy cells can synthesize Asn through asparagine synthetase (ASNS) activity, whereas specific cancer and genetically diseased cells are forced to obtain asparagine from the extracellular environment. ASNS catalyzes the ATP-dependent synthesis of Asn from aspartate by consuming glutamine as a nitrogen source. Asparagine Synthetase Deficiency (ASNSD) is a disease that results from biallelic mutations in the ASNS gene and presents with congenital microcephaly, intractable seizures, and progressive brain atrophy. ASNSD often leads to premature death. Although clinical and cellular studies have reported that Asn deprivation contributes to the disease symptoms, the global metabolic effects of Asn deprivation on ASNSD-derived cells have not been studied. We analyzed two previously characterized cell culture models, lymphoblastoids and fibroblasts, each carrying unique ASNS mutations from families with ASNSD. Metabolomics analysis demonstrated that Asn deprivation in ASNS-deficient cells led to disruptions across a wide range of metabolites. Moreover, we observed significant decrements in TCA cycle intermediates and anaplerotic substrates in ASNS-deficient cells challenged with Asn deprivation. We have identified pantothenate, phenylalanine, and aspartate as possible biomarkers of Asn deprivation in normal and ASNSD-derived cells. This work implies the possibility of a novel ASNSD diagnostic via targeted biomarker analysis of a blood draw.

Characterizing asparagine synthetase deficiency variants in lymphoblastoid cell lines.

Asparagine synthetase (ASNS) catalyzes the synthesis of asparagine (Asn) from aspartate and glutamine. Biallelic mutations in the ASNS gene result in ASNS Deficiency (ASNSD). Children with ASNSD exhibit congenital microcephaly, epileptic-like seizures, and continued brain atrophy, often leading to premature mortality. This report describes a 4-year-old male with global developmental delay and seizures with two novel mutations in the ASNS gene, c.614A > C (maternal) and c.1192dupT (paternal) encoding p.H205P and p.Y398Lfs*4 variants, respectively. We employed the novel use of immortalized lymphoblastoid cell lines (LCL) to show that the proliferation of the heterozygotic parental LCL was not severely affected by culture in Asn-free medium, but growth of the child's cells was suppressed by about 50%. Asn production by the LCL from both the father and the child was significantly decreased relative to the mother's cells. mRNA and protein analysis of the paternal LCL cells for the Y398Lfs*4 variant revealed reductions in both. Attempts to ectopically express the truncated Y398Lfs*4 variant in either HEK293T or ASNS-null cells resulted in little or no detectable protein. Expression and purification of the H205P variant from HEK293T cells revealed enzymatic activity similar to wild-type ASNS. Stable expression of WT ASNS rescued the growth of ASNS-null JRS cells in Asn-free medium and the H205P variant was only slightly less effective. However, the Y398Lfs*4 variant appeared to be unstable in JRS cells. These results indicate that co-expression of the H205P and Y398Lfs*4 variants leads to a significant reduction in Asn synthesis and cellular growth.

Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency.

Asparagine synthetase (ASNS) catalyzes synthesis of asparagine (Asn) and Glu from Asp and Gln in an ATP-dependent reaction. Asparagine synthetase deficiency (ASNSD) results from biallelic mutations in the ASNS gene. Affected children exhibit congenital microcephaly, continued brain atrophy, seizures, and often premature mortality. However, the underlying mechanisms are unclear. This report describes a compound heterozygotic ASNSD child with two novel mutations in the ASNS gene, c.1118G>T (paternal) and c.1556G>A (maternal), that lead to G373V or R519H ASNS variants. Structural mapping suggested that neither variant participates directly in catalysis. Growth of cultured fibroblasts from either parent was unaffected in Asn-free medium, whereas growth of the child's cells was suppressed by about 50%. Analysis of Asn levels unexpectedly revealed that extracellular rather than intracellular Asn correlated with the reduced proliferation during incubation of the child's cells in Asn-free medium. Our attempts to ectopically express the G373V variant in either HEK293T or JRS cells resulted in minimal protein production, suggesting instability. Protein expression and purification from HEK293T cells revealed reduced activity for the R519H variant relative to WT ASNS. Expression of WT ASNS in ASNS-null JRS cells resulted in nearly complete rescue of growth in Asn-free medium, whereas we observed no proliferation for the cells expressing either the G373V or R519H variant. These results support the conclusion that the coexpression of the G373V and R519H ASNS variants leads to significantly reduced Asn synthesis, which negatively impacts cellular growth. These observations are consistent with the ASNSD phenotype.

Analysis of Enzyme Activity and Cellular Function for the N80S and S480F Asparagine Synthetase Variants Expressed in a Child with Asparagine Synthetase Deficiency.

Asparagine Synthetase Deficiency (ASNSD) is a disease caused by mutations in asparagine synthetase (ASNS). Newborns exhibit microcephaly, intractable epileptic-like seizures, progressive brain atrophy, and axial hypotonia. ASNSD results in global developmental delays and premature death. The present report describes a 9-year-old child who is a compound heterozygote with ASNS mutations c.1439C > T and c.239A > G leading to variants p.S480F and p.N80S, respectively. When grown in a complete culture medium, primary fibroblasts from the child contained ASNS mRNA and protein levels similar to an unrelated wild-type fibroblast cell line. When the child's fibroblasts were cultured for up to 72 h in a medium lacking asparagine, proliferation was reduced by about 50%. Purification of ASNS proteins harboring either the S480F or the N80S substitution had reduced enzymatic activity by 80% and 50%, respectively. Ectopic expression of either variant in ASNS-null Jensen rat sarcoma (JRS) cells did not support proliferation in the absence of medium-supplied asparagine, whereas expression of wild-type enzyme completely restored growth. These studies add to the list of pathogenic ASNS variants and use enzyme activity and protein expression in ASNS-null cells to expand our knowledge of the biological impact of mutations in the ASNS gene.

Sequencing of Argonaute-bound microRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a.

MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

Myeloma Cells Deplete Bone Marrow Glutamine and Inhibit Osteoblast Differentiation Limiting Asparagine Availability.

Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.

The role of asparagine synthetase on nutrient metabolism in pancreatic disease.

The pancreas avidly takes up and synthesizes the amino acid asparagine (Asn), in part, to maintain an active translational machinery that requires incorporation of the amino acid. The de novo synthesis of Asn in the pancreas occurs through the enzyme asparagine synthetase (ASNS). The pancreas has the highest expression of ASNS of any organ, and it can further upregulate ASNS expression in the setting of amino acid depletion. ASNS expression is driven by an intricate feedback network within the integrated stress response (ISR), which includes the amino acid response (AAR) and the unfolded protein response (UPR). Asparaginase is a cancer chemotherapeutic drug that depletes plasma Asn. However, asparaginase-associated pancreatitis (AAP) is a major medical problem and could be related to pancreatic Asn depletion. In this review, we will provide an overview of ASNS and then describe its role in pancreatic health and in the exocrine disorders of pancreatitis and pancreatic cancer. We will offer the overarching perspective that a high abundance of ASNS expression is hardwired in the exocrine pancreas to buffer the high demands of Asn for pancreatic digestive enzyme protein synthesis, that perturbations in the ability to express or upregulate ASNS could tip the balance towards pancreatitis, and that pancreatic cancers exploit ASNS to gain a metabolic survival advantage.

PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation.

The inability of cells to adapt to increased environmental tonicity can lead to inflammatory gene expression and pathogenesis. The Rel family of transcription factors TonEBP and NF-κB p65 play critical roles in the switch from osmoadaptive homeostasis to inflammation, respectively. Here we identified PACT-mediated PKR kinase activation as a marker of the termination of adaptation and initiation of inflammation in Mus musculus embryonic fibroblasts. We found that high stress-induced PACT-PKR activation inhibits the interaction between NF-κB c-Rel and TonEBP essential for the increased expression of TonEBP-dependent osmoprotective genes. This resulted in enhanced formation of TonEBP/NF-κB p65 complexes and enhanced proinflammatory gene expression. These data demonstrate a novel role of c-Rel in the adaptive response to hyperosmotic stress, which is inhibited via a PACT/PKR-dependent dimer redistribution of the Rel family transcription factors. Our results suggest that inhibiting PACT-PKR signaling may prove a novel target for alleviating stress-induced inflammatory diseases.

Cells are sensitive to changes in their environment. For example, maintaining normal salt levels in the blood, also called tonicity, is essential for the health of individual cells and the organism as a whole. Tonicity controls the movement of water in and out of the cell: high levels of salt inside the cell draw water in, while high levels of salt outside the cell draw water out. If salt levels in the environment surrounding the cells become too high, too much water will be drawn out, causing the cells to shrink. Changes in tonicity can cause the cell to become stressed. Initially, cells adapt to this stress by switching on sets of genes that help restore fluid balance and allow the cell to regain its normal shape and size. If the increase in tonicity exceeds tolerable stress levels and harms the cell, this initiates an inflammatory response which ultimately leads to cell death. However, it remained unclear how cells switch from adapting to responding with inflammation. Now, Farabaugh et al. have used an experimental system which mimics high salt to identify the mechanism that allows cells to switch between these two responses. The experiments showed that when salt levels are too high, cells switch on a stress sensing protein called PACT, which activates another protein called PKR. When PACT was deleted from mouse cells, this led to a decrease in the activity of inflammatory genes, and prevented the cells from self-destructing. Other proteins that are involved in the adaptive and inflammatory response are the NF-κB family of proteins and TonEBP. Farabaugh et al. found that under low intensity stress, when salt levels outside the cell are slightly too high, a family member of NF-κB works with TonEBP to switch on adaptive genes. But, if salt levels continue to rise, PACT activates and turns on PKR. This blocks the interaction between NF-κB and TonEBP, allowing another family member of NF-κB to interact with TonEBP instead. This switches the adaptive response off and the inflammatory response on. There are many diseases that involve changes in tonicity, including diabetes, cancer, inflammatory bowel disease, and dry eye syndrome. Understanding the proteins involved in the adaptive and inflammatory response could lead to the development of drugs that help to protect cells from stress-induced damage.

Asparagine Synthetase Is Highly Expressed at Baseline in the Pancreas Through Heightened PERK Signaling.

Asparaginase (ASNase) causes pancreatitis in approximately 10% of leukemia patients, and the mechanisms underlying this painful complication are not known. ASNase primarily depletes circulating asparagine, and the endogenously expressed enzyme, asparagine synthetase (ASNS), replenishes asparagine. ASNS was suggested previously to be highly expressed in the pancreas. In this study, we determined the expression pattern of ASNS in the pancreas and the mechanism for increased pancreatic ASNS abundance. Compared with other organs, ASNS was highly expressed in both the human and mouse pancreas, and, within the pancreas, ASNS was present primarily in the acinar cells. The high baseline pancreatic ASNS was associated with higher baseline activation of protein kinase R-like endoplasmic reticulum kinase (PERK) signaling in the pancreas, and inhibition of PERK in acinar cells lessened ASNS expression. ASNase exposure, but not the common pancreatitis triggers, uniquely up-regulated ASNS expression, indicating that the increase is mediated by nutrient stress. The up-regulation of acinar ASNS with ASNase exposure was owing to increased transcriptional rather than delayed degradation. Knockdown of ASNS in the 266-6 acinar cells provoked acinar cell injury and worsened ASNase-induced injury, whereas ASNS overexpression protected against ASNase-induced injury. In summary, ASNS is highly expressed in the pancreatic acinar cells through heightened basal activation of PERK, and ASNS appears to be crucial to maintaining acinar cell integrity. The implications are that ASNS is especially hardwired in the pancreas to protect against both baseline perturbations and nutrient deprivation stressors, such as during ASNase exposure.

Induction of early growth response gene 1 (EGR1) by endoplasmic reticulum stress is mediated by the extracellular regulated kinase (ERK) arm of the MAPK pathways.

Endoplasmic reticulum (ER) stress activates three principal signaling pathways, collectively known as the unfolded protein response, leading to translational and transcriptional control mechanisms that dictate the cell's response as adaptive or apoptotic. The present study illustrates that for HepG2 human hepatocellular carcinoma cells the signaling pathways triggered by ER stress extend beyond the three principal pathways to include mitogen-activated protein kinase (MAPK) signaling, leading to activation of transcription from the early growth response 1 (EGR1) gene. Analysis provided evidence for a SRC-RAS-RAF-MEK-ERK cascade mechanism that leads to enhanced phosphorylation of the transcription factor ELK1. ELK1 and serum response factor (SRF) are constitutively bound to the EGR1 promoter and are phosphorylated by nuclear localized ERK. The promoter abundance of both phospho-SRF and phopsho-ELK1 was increased by ER stress, but the SRF phosphorylation was transient. Knockdown of ELK1 had little effect on the basal EGR1 mRNA content, but completely blocked the increase in response to ER stress. Conversely, knockdown of SRF suppressed basal EGR1 mRNA content, but had only a small effect on the induction by ER stress. This research highlights the importance of MAPK signaling in response to ER stress and identifies ELK1 as a transcriptional mediator and the EGR1 gene as a target.

Asparagine Synthetase in Cancer: Beyond Acute Lymphoblastic Leukemia.

Asparagine Synthetase (ASNS) catalyzes the synthesis of the non-essential amino acid asparagine (Asn) from aspartate (Asp) and glutamine (Gln). ASNS expression is highly regulated at the transcriptional level, being induced by both the Amino Acid Response (AAR) and the Unfolded Protein Response (UPR) pathways. Lack of ASNS protein expression is a hallmark of Acute Lymphoblastic Leukemia (ALL) blasts, which, therefore, are auxotrophic for Asn. This peculiarity is the rationale for the use of bacterial L-Asparaginase (ASNase) for ALL therapy, the first example of anti-cancer treatment targeting a tumor-specific metabolic feature. Other hematological and solid cancers express low levels of ASNS and, therefore, should also be Asn auxotrophs and ASNase sensitive. Conversely, in the last few years, several reports indicate that in some cancer types ASNS is overexpressed, promoting cell proliferation, chemoresistance, and a metastatic behavior. However, enhanced ASNS activity may constitute a metabolic vulnerability in selected cancer models, suggesting a variable and tumor-specific role of the enzyme in cancer. Recent evidence indicates that, beyond its canonical role in protein synthesis, Asn may have additional regulatory functions. These observations prompt a re-appreciation of ASNS activity in the biology of normal and cancer tissues, with particular attention to the fueling of Asn exchange between cancer cells and the tumor microenvironment.

Regulation of the ATF3 gene by a single promoter in response to amino acid availability and endoplasmic reticulum stress in human primary hepatocytes and hepatoma cells.

Activating transcription factor 3 (ATF3) is a highly regulated protein that is implicated in a wide range of pathological conditions including inflammation and transformation. Transcription from the ATF3 gene is induced by several stress-induced signaling pathways, including amino acid limitation (amino acid response, AAR) and ER stress (unfolded protein response, UPR). Induction of ATF3 transcription by these pathways is mediated by ATF4 and cJUN recruitment to enhancer elements within the ATF3 gene. Although a canonical promoter (promoter A) has been studied by numerous laboratories, a second promoter activity (promoter A1), 43 kb upstream of the first, has been reported to respond to stress-induced signaling and to be critical for ATF3 expression in certain transformed cells. The results of the present study show that in normal human hepatocytes and HepG2 human hepatoma cells both basal as well as AAR- and UPR-induced transcription occurs almost exclusively from promoter A. This selectivity between the two promoters correlated with increased binding of ATF4, recruitment of RNA polymerase II, and the expected histone modifications in the promoter A region of the gene. Time course studies of ATF3 transcription activity revealed that the temporal kinetics for ATF3 induction differ between the AAR and UPR, with the former being more transient than the latter. Collectively, the results document that ATF3 expression in normal and transformed human liver originates from the canonical promoter A that responds to multiple stress signals.

Characterization of a novel variant in siblings with Asparagine Synthetase Deficiency.

Asparagine Synthetase Deficiency (ASD) is a recently described inborn error of metabolism caused by bi-allelic pathogenic variants in the asparagine synthetase (ASNS) gene. ASD typically presents congenitally with microcephaly and severe, often medically refractory, epilepsy. Development is generally severely affected at birth. Tone is abnormal with axial hypotonia and progressive appendicular spasticity. Hyperekplexia has been reported. Neuroimaging typically demonstrates gyral simplification, abnormal myelination, and progressive cerebral atrophy. The present report describes two siblings from consanguineous parents with a homozygous Arg49Gln variant associated with a milder form of ASD that is characterized by later onset of symptoms. Both siblings had a period of normal development before onset of seizures, and development regression. Primary fibroblast studies of the siblings and their parents document that homozygosity for Arg49Gln blocks cell growth in the absence of extracellular asparagine. Functional studies with these cells suggest no impact of the Arg49Gln variant on basal ASNS mRNA or protein levels, nor on regulation of the gene itself. Molecular modelling of the ASNS protein structure indicates that the Arg49Gln variant lies near the substrate binding site for glutamine. Collectively, the results suggest that the Arg49Gln variant affects the enzymatic function of ASNS. The clinical, cellular, and molecular observations from these siblings expand the known phenotypic spectrum of ASD.

Asparagine synthetase: Function, structure, and role in disease.

Asparagine synthetase (ASNS) converts aspartate and glutamine to asparagine and glutamate in an ATP-dependent reaction. ASNS is present in most, if not all, mammalian organs, but varies widely in basal expression. Human ASNS activity is highly responsive to cellular stress, primarily by increased transcription from a single gene located on chromosome 7. Elevated ASNS protein expression is associated with resistance to asparaginase therapy in childhood acute lymphoblastic leukemia. There is evidence that ASNS expression levels may also be inversely correlated with asparaginase efficacy in certain solid tumors as well. Children with mutations in the ASNS gene exhibit developmental delays, intellectual disability, microcephaly, intractable seizures, and progressive brain atrophy. Thus far, 15 unique mutations in the ASNS gene have been clinically associated with asparagine synthetase deficiency (ASD). Molecular modeling using the Escherichia coli ASNS-B structure has revealed that most of the reported ASD substitutions are located near catalytic sites or within highly conserved regions of the protein. For some ASD patients, fibroblast cell culture studies have eliminated protein and mRNA synthesis or stability as the basis for decreased proliferation.

Surviving Stress: Modulation of ATF4-Mediated Stress Responses in Normal and Malignant Cells.

Activating transcription factor 4 (ATF4) is a stress-induced transcription factor that is frequently upregulated in cancer cells. ATF4 controls the expression of a wide range of adaptive genes that allow cells to endure periods of stress, such as hypoxia or amino acid limitation. However, under persistent stress conditions, ATF4 promotes the induction of apoptosis. Recent advances point to a role for post-translational modifications (PTMs) and epigenetic mechanisms in balancing these pro- and anti-survival effects of ATF4. We review here how PTMs and epigenetic modifiers associated with ATF4 may be exploited by cancer cells to cope with cellular stress conditions that are intrinsically associated with tumor growth. Identification of mechanisms that modulate ATF4-mediated transcription and its effects on cellular metabolism may uncover new targets for cancer treatment.

Disulfide bond disrupting agents activate the unfolded protein response in EGFR- and HER2-positive breast tumor cells.

Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus, new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors, and "Triple-Negative" Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor, Progesterone Receptor, and HER2. A significant fraction of TNBCs overexpress the HER2 family member Epidermal Growth Factor Receptor (EGFR). Thus agents that selectively kill EGFR+ and HER2+ tumors would provide new options for breast cancer therapy. We previously identified a class of compounds we termed Disulfide bond Disrupting Agents (DDAs) that selectively kill EGFR+ and HER2+ breast cancer cells in vitro and blocked the growth of HER2+ breast tumors in an animal model. DDA-dependent cytotoxicity was found to correlate with downregulation of HER1-3 and Akt dephosphorylation. Here we demonstrate that DDAs activate the Unfolded Protein Response (UPR) and that this plays a role in their ability to kill EGFR+ and HER2+ cancer cells. The use of breast cancer cell lines ectopically expressing EGFR or HER2 and pharmacological probes of UPR revealed all three DDA responses: HER1-3 downregulation, Akt dephosphorylation, and UPR activation, contribute to DDA-mediated cytotoxicity. Significantly, EGFR overexpression potentiates each of these responses. Combination studies with DDAs suggest that they may be complementary with EGFR/HER2-specific receptor tyrosine kinase inhibitors and mTORC1 inhibitors to overcome drug resistance.

The C/ebp-Atf response element (CARE) location reveals two distinct Atf4-dependent, elongation-mediated mechanisms for transcriptional induction of aminoacyl-tRNA synthetase genes in response to amino acid limitation.

The response to amino acid (AA) limitation of the entire aminoacyl-tRNA synthetase (ARS) gene family revealed that 16/20 of the genes encoding cytoplasmic-localized enzymes are transcriptionally induced by activating transcription factor 4 (Atf4) via C/ebp-Atf-Response-Element (CARE) enhancers. In contrast, only 4/19 of the genes encoding mitochondrial-localized ARSs were weakly induced. Most of the activated genes have a functional CARE near the transcription start site (TSS), but for others the CARE is downstream. Regardless of the location of CARE enhancer, for all ARS genes there was constitutive association of RNA polymerase II (Pol II) and the general transcription machinery near the TSS. However, for those genes with a downstream CARE, Atf4, C/ebp-homology protein (Chop), Pol II and TATA-binding protein exhibited enhanced recruitment to the CARE during AA limitation. Increased Atf4 binding regulated the association of elongation factors at both the promoter and the enhancer regions, and inhibition of cyclin-dependent kinase 9 (CDK9), that regulates these elongation factors, blocked induction of the AA-responsive ARS genes. Protein pull-down assays indicated that Atf4 directly interacts with CDK9 and its associated protein cyclin T1. The results demonstrate that AA availability modulates the ARS gene family through modulation of transcription elongation.

Influence of Amino Acid Metabolism on Embryonic Stem Cell Function and Differentiation.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promise in regenerative medicine because of their ability to differentiate into all 3 primary germ layers. This review describes recent advances in the understanding of the link between the metabolism of ESCs/iPSCs and their maintenance/differentiation in the cell culture setting, with particular emphasis on amino acid (AA) metabolism. ESCs are endowed with unique metabolic features with regard to energy consumption, metabolite flux through particular pathways, and macromolecular synthesis. Therefore, nutrient availability has a strong influence on stem cell growth, self-renewal, and lineage specification, both in vivo and in vitro. Evidence from several laboratories has documented that self-renewal and differentiation of mouse ESCs are critically dependent on proline metabolism, with downstream metabolites possibly serving as signal molecules. Likewise, catabolism of either threonine (mouse) or methionine (human) is required for growth and differentiation of ESCs because these AAs serve as precursors for donor molecules used in histone methylation and acetylation. Epigenetic mechanisms are recognized as critical steps in differentiation, and AA metabolism in ESCs appears to modulate these epigenetic processes. Recent reports also document that, in vitro, the nutrient composition of the culture medium in which ESCs are differentiated into embryoid bodies can influence lineage specification, leading to enrichment of a specific cell type. Although research designed to direct tissue specification of differentiating embryoid bodies in culture is still in its infancy, early results indicate that manipulation of the nutrient milieu can promote or suppress the formation of specific cell lineages.