Translational Induction Research Articles

Diet induced insulin resistance is due to induction of PTEN expression.

Type 2 Diabetes (T2D) is a condition that is often associated with obesity and defined by reduced sensitivity of PI3K signaling to insulin (insulin resistance), hyperinsulinemia and hyperglycemia. Molecular causes and early signaling events underlying insulin resistance are not well understood. Insulin activation of PI3K signaling causes mTOR dependent induction of PTEN translation, a negative regulator of PI3K signaling. We speculated that insulin resistance is due to insulin dependent induction of PTEN protein that prevent further increases in PI3K signaling. Here we show that in a diet induced model of obesity and insulin resistance, PTEN levels are increased in fat, muscle and liver tissues. Onset of hyperinsulinemia and PTEN induction in tissue is followed by hyperglycemia, hepatic steatosis and severe glucose intolerance. Treatment with a PTEN phosphatase inhibitor prevents and reverses these phenotypes, whereas an mTORC1 kinase inhibitor reverses all but the hepatic steatosis. These data suggest that induction of PTEN by increasing levels of insulin elevates feedback inhibition of the pathway to a point where downstream PI3K signaling is reduced and hyperglycemia ensues. PTEN induction is thus necessary for insulin resistance and the type 2 diabetes phenotype and a potential therapeutic target.

Abstract 524: Using acquired resistance to explore the mechanism of action of the integrated stress response/GCN2 activator NXP800 - A new developmental agent for platinum-resistant ARID1A mutant ovarian cancer

Abstract Background: NXP800 is a potent, oral activator of the Integrated Stress response (ISR), inhibitor of heat shock factor 1 (HSF1) activation and tumor cell proliferation, which is in early clinical studies in ARID1A-mutated, platinum resistant, clear cell ovarian cancer (NCT05226507). We discovered NXP800 using multiparameter medicinal chemistry optimization of a hit identified from a cell-based phenotypic screen. Owing to the unbiased nature of phenotypic screening, target identification is crucial to understand the biological and therapeutic activity of hit compounds. Methods and Results:: By RNAseq profiling human cancer cells treated with NXP800 we identified changes in expression of genes regulated by HSF1 or ATF4 - effects accompanied by eIF2alpha (eIF2a) phosphorylation and resulting activation of the ISR. To further understand this response, we explored whether NXP800 resistance models could inform on its mechanism of action (MoA). We used ARID1A mutant SK-OV-3 human ovarian carcinoma cells that are 1) highly sensitive to NXP800, 2) model the target patient population and 3) MSI-high so likely to have an elevated mutation rate contributing to acquisition of resistance. We generated two independent NXP800-resistant SK-OV-3 cell lines in which NXP800-mediated ATF4 induction and concomitant inhibition of global translation were abolished. By whole exome sequencing, we identified a heterozygous L99P mutation in the alpha subunit of eIF2B (eIF2Ba), the nucleotide exchange factor for eIF2a. Expression of eIF2BaL99P, but not wild-type, in parental SK-OV-3 cells reduced sensitivity to NXP800 to the same level as cells with NXP800-induced resistance. Using fluorescence recovery after photobleaching to monitor the dynamic association of the eIF2B:eIF2a complex, we elucidated that the eIF2BaL99P mutation reduces NXP800-mediated inhibition of eIF2a-GFP recycling through eIF2B bodies. Phosphorylation of eIF2a is regulated by four stress-controlled kinases GCN2, HRI, PKR and PERK. Using systematic siRNA knockdown or small-molecule inhibitors, we showed that GCN2 alone is required for ISR activation by NXP800 and that ISR induction inhibited HSF1 activation. Furthermore, inactivation of GCN2 reduced the antiproliferative activity of NXP800 to the same extent observed in NXP800-resistant SK-OV-3 cells. In contrast, exposure of resistant or parental SK-OV-3 cells expressing eIF2BaL99P to GCN2 inhibitors did not cause further reduction in NXP800 sensitivity - confirming the importance of the eIF2BaL99P mutation in the resistance mechanism. Conclusions: We have used acquired resistance to understand the MoA of NXP800 as a potent activator of GCN2 and the ISR pathway. Further studies are underway to determine the exact proximal molecular target of NXP800 and the mechanism of GCN2/ISR activation. Citation Format: Marissa V. Powers, Rachel Hodgson, Swee Y. Sharp, Toby Roe, K. Elizabeth Allen, Susan Campbell, Robert te Poele, Matthew Cheeseman, Keith Jones, Paul A. Clarke, Paul Workman. Using acquired resistance to explore the mechanism of action of the integrated stress response/GCN2 activator NXP800 - A new developmental agent for platinum-resistant ARID1A mutant ovarian cancer [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 524.

Multi-omics reveals largely distinct transcript- and protein-level responses to the environment in an intertidal mussel.

Organismal responses to stressful environments are influenced by numerous transcript- and protein-level mechanisms, and the relationships between expression changes at these levels are not always straightforward. Here, we used paired transcriptomic and proteomic datasets from two previous studies from gill of the California mussel, Mytilus californianus, to explore how simultaneous transcript and protein abundance patterns may diverge under different environmental scenarios. Field-acclimatized mussels were sampled from two disparate intertidal sites; individuals from one site were subjected to three further treatments (common garden, low-intertidal or high-intertidal outplant) that vary in temperature and feeding time. Assessing 1519 genes shared between the two datasets revealed that both transcript and protein expression patterns differentiated the treatments at a global level, despite numerous underlying discrepancies. There were far more instances of differential expression between treatments in transcript only (1451) or protein only (226) than of the two levels shifting expression concordantly (68 instances). Upregulated expression of cilium-associated transcripts (likely related to feeding) was associated with relatively benign field treatments. In the most stressful treatment, transcripts, but not proteins, for several molecular chaperones (including heat shock proteins and endoplasmic reticulum chaperones) were more abundant, consistent with a threshold model for induction of translation of constitutively available mRNAs. Overall, these results suggest that the relative importance of transcript- and protein-level regulation (translation and/or turnover) differs among cellular functions and across specific microhabitats or environmental contexts. Furthermore, the degree of concordance between transcript and protein expression can vary across benign versus acutely stressful environmental conditions.

HPV+ head and neck cancer-derived small extracellular vesicles communicate with TRPV1+ neurons to mediate cancer pain.

Severe pain is often experienced by patients with head and neck cancer and is associated with a poor prognosis. Despite its frequency and severity, current treatments fail to adequately control cancer-associated pain because of our lack of mechanistic understanding. Although recent works have shed some light of the biology underlying pain in HPV-negative oral cancers, the mechanisms mediating pain in HPV+ cancers remain unknown. Cancer-derived small extracellular vesicles (cancer-sEVs) are well positioned to function as mediators of communication between cancer cells and neurons. Inhibition of cancer-sEV release attenuated pain in tumor-bearing mice. Injection of purified cancer-sEVs is sufficient to induce pain hypersensitivity in naive mice that is prevented by QX-314 treatment and in Trpv1-/- mice. Cancer-sEVs triggered calcium influx in nociceptors, and inhibition or ablation of nociceptors protects against cancer pain. Interrogation of published sequencing data of human sensory neurons exposed to human cancer-sEVs suggested a stimulation of protein translation in neurons. Induction of translation by cancer-sEVs was validated in our mouse model, and its inhibition alleviated cancer pain in mice. In summary, our work reveals that HPV+ head and neck squamous cell carcinoma-derived sEVs alter TRPV1+ neurons by promoting nascent translation to mediate cancer pain and identified several promising therapeutic targets to interfere with this pathway.

Multiple paths of plant host toxicity are associated with the fungal toxin cercosporin.

The Cercospora species of fungi are responsible for leaf spot disease affecting many key economic crops. Most of these fungi secrete a toxic photodynamic molecule, cercosporin, that reacts with light and oxygen to produce reactive singlet oxygen (1 O2 ) contributing to fungal virulence. We show similar cellular localization and aetiology of cercosporin in the non-host Arabidopsis and the host Nicotiana benthamiana. Cercosporin accumulates in cell membranes in an oxidized state and in plastids in a mixture of redox states in a manner that is dependent on ongoing photosynthetic processes. We observed that cercosporin rapidly compromised photosynthesis as measured by Fv /Fm , NPQ, and photosystem I (PSI) parameters. Stomatal guard cells in particular demonstrated rapid light-dependent membrane permeabilization that led to changes in leaf conductance. We showed that cercosporin-mediated 1 O2 generation oxidized RNA to form 8-oxoguanosine (8-oxoG), leading to translational attenuation and induction of 1 O2 signature gene transcripts. We also identified a subset of cercosporin-induced transcripts that were independent of the photodynamic effect. Our results point to the multimodal action of cercosporin that includes the inhibition of photosynthesis, the direct oxidation of nucleic acid residues and the elicitation of complex transcriptome responses.

Global translational induction during NLR-mediated immunity in plants is dynamically regulated by CDC123, an ATP-sensitive protein

The recognition of pathogen effectors by their cognate nucleotide-binding leucine-rich repeat (NLR) receptors activates effector-triggered immunity (ETI) in plants. ETI is associated with correlated transcriptional and translational reprogramming and subsequent death of infected cells. Whether ETI-associated translation is actively regulated or passively driven by transcriptional dynamics remains unknown. In a genetic screen using a translational reporter, we identified CDC123, an ATP-grasp protein, as a key activator of ETI-associated translation and defense. During ETI, an increase in ATP concentration facilitates CDC123-mediated assembly of the eukaryotic translation initiation factor 2 (eIF2) complex. Because ATP is required for the activation of NLRs as well as the CDC123 function, we uncovered a possible mechanism by which the defense translatome is coordinately induced during NLR-mediated immunity. The conservation of the CDC123-mediated eIF2 assembly suggests its possible role in NLR-mediated immunity beyond plants.

Efficient Delivery of GSDMD-N mRNA by Engineered Extracellular Vesicles Induces Pyroptosis for Enhanced Immunotherapy.

Pyroptosis is a newly discovered inflammatory form of programmed cell death, which promotes systemic immune response in cancer immunotherapy. GSDMD is one of the key molecules executing pyroptosis, while therapeutical delivery of GSDMD to tumor cells is of great challenge. In this study, an extracellular vesicles-based GSDMD-N mRNA delivery system (namely EVTx ) is developed for enhanced cancer immunotherapy, with GSDMD-N mRNA encapsulated inside, Ce6 (Chlorin e6 (Ce6), a hydrophilic sensitizer) incorporated into extracellular vesicular membrane, and HER2 antibody displayed onto the surface. Briefly, GSDMD-N mRNA is translationally repressed in donor cells by optimized puromycin, ensuring the cell viability and facilitating the mRNA encapsulation into extracellular vesicles. When targeted and delivered into HER2+ breast cancer cells by the engineered extracellular vesicles, the translational repression is unleashed in the recipient cells as the puromycin is diluted and additionally inactivated by sonodynamic treatment as the extracellular vesicles are armed with Ce6, allowing GSDMD-N translation and pyroptosis induction. In addition, sonodynamic treatment also induces cell death in the recipient cells. In the SKBR3- and HER2 transfected 4T1- inoculated breast tumor mouse models, the engineered EVTx efficiently induces a powerful tumor immune response and suppressed tumor growth, providing a nanoplatform for cancer immunotherapy.

Elongator and the role of its subcomplexes in human diseases.

The Elongator complex was initially identified in yeast, and a variety of distinct cellular functions have been assigned to the complex. In the last decade, several research groups focussed on dissecting its structure, tRNA modification activity and role in translation regulation. Recently, Elongator emerged as a crucial factor for various human diseases, and its involvement has triggered a strong interest in the complex from numerous clinical groups. The Elongator complex is highly conserved among eukaryotes, with all six subunits (Elp1-6) contributing to its stability and function. Yet, recent studies have shown that the two subcomplexes, namely the catalytic Elp123 and accessory Elp456, may have distinct roles in the development of different neuronal subtypes. This Commentary aims to provide a brief overview and new perspectives for more systematic efforts to explore the functions of the Elongator in health and disease.

Activity-dependent translation dynamically alters the proteome of the perisynaptic astrocyte process.

Within eukaryotic cells, translation is regulated independent of transcription, enabling nuanced, localized, and rapid responses to stimuli. Neurons respond transcriptionally and translationally to synaptic activity. Although transcriptional responses are documented in astrocytes, here we test whether astrocytes have programmed translational responses. We show that seizure activity rapidly changes the transcripts on astrocyte ribosomes, some predicted to be downstream of BDNF signaling. In acute slices, we quantify the extent to which cues of neuronal activity activate translation in astrocytes and show that this translational response requires the presence of neurons, indicating that the response is non-cell autonomous. We also show that this induction of new translation extends into the periphery of astrocytes. Finally, synaptic proteomics show that new translation is required for changes that occur in perisynaptic astrocyte protein composition after fear conditioning. Regulation of translation in astrocytes by neuronal activity suggests an additional mechanism by which astrocytes may dynamically modulate nervous system functioning.

Induction of RAC1 protein translation and MKK7/JNK-dependent autophagy through dicer/miR-145/SOX2/miR-365a axis contributes to isorhapontigenin (ISO) inhibition of human bladder cancer invasion

Although our previous studies have identified that isorhapontigenin (ISO) is able to initiate autophagy in human bladder cancer (BC) cells by activating JNK/C-Jun/SESN2 axis and possesses an inhibitory effect on BC cell growth, association of autophagy directly with inhibition of BC invasion has never been explored. Also, upstream cascade responsible for ISO activating JNK remains unknown. Thus, we explored both important questions in the current study and discovered that ISO treatment initiated RAC1 protein translation, and its downstream kinase MKK7/JNK phosphorylation/activation, and in turn promoted autophagic responses in human BC cells. Inhibition of autophagy abolished ISO inhibition of BC invasion, revealing that autophagy inhibition was crucial for ISO inhibition of BC invasion. Consistently, knockout of RAC1 also attenuated induction of autophagy and inhibition of BC invasion by ISO treatment. Mechanistic studies showed that upregulation of RAC1 translation was due to ISO inhibition of miR-365a transcription, which reduced miR-365a binding to the 3’-UTR of RAC1 mRNA. Further study indicated that inhibition of miR-365a transcription was caused by downregulation of its transcription factor SOX2, while ISO-promoted Dicer protein translation increased miR-145 maturation, and consequently downregulating SOX2 expression. These findings not only provide a novel insight into the understanding association of autophagy induction with BC invasion inhibition by ISO, but also identify an upstream regulatory cascade, Dicer/miR145/SOX2/miR365a/RAC1, leading to MKK7/JNKs activation and autophagy induction.

Hypoxia-Mediated ATF4 Induction Promotes Survival in Detached Conditions in Metastatic Murine Mammary Cancer Cells.

Regions of hypoxia are common in solid tumors and drive changes in gene expression that increase risk of cancer metastasis. Tumor cells must respond to the stress of hypoxia by activating genes to modify cell metabolism and antioxidant response to improve survival. The goal of the current study was to determine the effect of hypoxia on cell metabolism and markers of oxidative stress in metastatic (metM-Wntlung) compared with nonmetastatic (M-Wnt) murine mammary cancer cell lines. We show that hypoxia induced a greater suppression of glutamine to glutamate conversion in metastatic cells (13% in metastatic cells compared to 7% in nonmetastatic cells). We also show that hypoxia increased expression of genes involved in antioxidant response in metastatic compared to nonmetastatic cells, including glutamate cysteine ligase catalytic and modifier subunits and malic enzyme 1. Interestingly, hypoxia increased the mRNA level of the transaminase glutamic pyruvic transaminase 2 (Gpt2, 7.7-fold) only in metM-Wntlung cells. The change in Gpt2 expression was accompanied by transcriptional (4.2-fold) and translational (6.5-fold) induction of the integrated stress response effector protein activating transcription factor 4 (ATF4). Genetic depletion ATF4 demonstrated importance of this molecule for survival of hypoxic metastatic cells in detached conditions. These findings indicate that more aggressive, metastatic cancer cells utilize hypoxia for metabolic reprogramming and induction of antioxidant defense, including activation of ATF4, for survival in detached conditions.

Abstract LB089: Effective in vivo treatment of endometrial tumor models with coexistent mutant PI3K and PTEN inactivation with a selective bi-steric mTORC1 kinase inhibitor

Abstract Dysregulation of PI3K signaling is a common event in human cancer, but feedback inhibition of receptor kinase signaling and/or induction of PTEN translation by activated PI3K/AKT/mTOR signaling reduces pathway output and tumor dependency. In some cancers, including the majority of endometrial carcinomas, these lesions coexist and cause synergistic hyperactivation of pathway output, particularly mTORC1 kinase signaling. Although this suggests that PI3K signaling is a driver of these endometrial cancers, standard inhibitors have had only marginal activity in patients with advanced disease. We here show that, in experimental isogenic models and PDX models with PI3K and PTEN alterations, PI3K inhibitors and rapalogs are weak inhibitors of TORC1 targets and slow but do not suppress the in vivo growth of these tumors. Revolution Medicines has developed a series of bi-steric selective and potent mTORC1 inhibitors that inhibit the phosphorylation of 4EBP-1, S6K and other mTORC1 substrates at concentrations that do not affect TORC2 substrates and thus do not induce the hyperglycemia caused by downregulation of AKT signaling by pan-mTOR kinase inhibitors. We have explored the biologic and anti-tumor effects of these inhibitors in models of endometrial cancer with coexistent PI3K mutation and PTEN inactivation. The mTORC1 kinase selective bi-steric inhibitor RMC-6272 can potently suppress both mTORC1 activity and the growth of these tumor cells in vitro. Moreover, in contrast to PI3K inhibition, RMC-6272 effectively inhibited the phosphorylation of mTORC1 kinase substrates and completely suppressed the growth in vivo of multiple PDXs with this genotype without inducing hyperglycemia or causing weight loss in the mice. The results suggest that dysregulation of TORC1 activity is an important driver of endometrial carcinomas with coexistent PTEN inactivation and PI3K mutations and that selective TORC1 kinase inhibitors may prove to be useful therapeutics in this context. The bi-steric mTORC1 kinase inhibitor RMC-5552 is the first clinical candidate of this class and clinical testing is ongoing (NCT04774952). Citation Format: Hilla Solomon, Radha Mukherjee, Xiaoping Chen, HuiYong Zhao, Elisa de Stanchina, Britta Weigelt, Alison M. Schram, Carol Aghajanian, Mallika Singh, Jan Smith, Neal Rosen. Effective in vivo treatment of endometrial tumor models with coexistent mutant PI3K and PTEN inactivation with a selective bi-steric mTORC1 kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB089.

Integrative genome-wide analysis reveals EIF3A as a key downstream regulator of translational repressor protein Musashi 2 (MSI2).

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate decisions in normal and cancer stem cells. MSI2 appears to repress translation by binding to 3′ untranslated regions (3′UTRs) of mRNA, but the identity of functional targets remains unknown. Here, we used individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) to identify direct RNA binding partners of MSI2 and integrated these data with polysome profiling to obtain insights into MSI2 function. iCLIP revealed specific MSI2 binding to thousands of mRNAs largely in 3′UTRs, but translational differences were restricted to a small fraction of these transcripts, indicating that MSI2 regulation is not triggered by simple binding. Instead, the functional targets identified here were bound at higher density and contain more ‘UAG’ motifs compared to targets bound nonproductively. To further distinguish direct and indirect targets, MSI2 was acutely depleted. Surprisingly, only 50 transcripts were found to undergo translational induction on acute loss. Using complementary approaches, we determined eukaryotic translation initiation factor 3A (EIF3A) to be an immediate, direct target. We propose that MSI2 downregulation of EIF3A amplifies these effects on translation. Our results also underscore the challenges in defining functional targets of RBPs since mere binding does not imply a discernible functional interaction.

A Pseudorabies Virus Serine/Threonine Kinase, US3, Promotes Retrograde Transport in Axons via Akt/mToRC1.

ABSTRACTInfection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a lifelong infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete, and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified trichamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase US3 in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities in both the presence and absence of a functional US3 kinase; however, fewer nucleocapsids are moving when US3 is absent, and they move for shorter periods of time before stopping, suggesting that US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12 h later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1 h postinfection (hpi) through the stimulation of a phosphatidylinositol 3-kinase (PI3K)/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body.IMPORTANCE Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There is a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.

A Bioluminescence Reporter Assay for Retinoic Acid Control of Translation of the GluR1 Subunit of the AMPA Glutamate Receptor.

The present protocol describes a bioluminescence reporter assay developed to quantify the ability of synthetic agonists of retinoic acid receptors (RARs) to activate glutamate receptor subunit 1 (GluR1) translation. The reporter assay uses firefly luciferase under the control of the GluR1 5' untranslated region (5' UTR) which is bound by RARs to regulate its translation. This method is used to demonstrate the role of RARα in retinoic acid regulation of GluR1 translation. This method may also be used to screen drugs that influence RAR induction of GluR1 translation as an important mechanism controlling learning and memory in the brain.

Controlling selectivity of modular microbial biosynthesis of butyryl-CoA-derived designer esters

Short-chain esters have broad utility as flavors, fragrances, solvents, and biofuels. Controlling selectivity of ester microbial biosynthesis has been an outstanding metabolic engineering problem. In this study, we enabled the de novo fermentative microbial biosynthesis of butyryl-CoA-derived designer esters (e.g., butyl acetate, ethyl butyrate, butyl butyrate) in Escherichia coli with controllable selectivity. Using the modular design principles, we generated the butyryl-CoA-derived ester pathways as exchangeable production modules compatible with an engineered chassis cell for anaerobic production of designer esters. We designed these modules derived from an acyl-CoA submodule (e.g., acetyl-CoA, butyryl-CoA), an alcohol submodule (e.g., ethanol, butanol), a cofactor regeneration submodule (e.g., NADH), and an alcohol acetyltransferase (AAT) submodule (e.g., ATF1, SAAT) for rapid module construction and optimization by manipulating replication (e.g., plasmid copy number), transcription (e.g., promoters), translation (e.g., codon optimization), pathway enzymes, and pathway induction conditions. To further enhance production of designer esters with high selectivity, we systematically screened various strategies of protein solubilization using protein fusion tags and chaperones to improve the soluble expression of multiple pathway enzymes. Finally, our engineered ester-producing strains could achieve 19-fold increase in butyl acetate production (0.64 g/L, 96% selectivity), 6-fold increase in ethyl butyrate production (0.41 g/L, 86% selectivity), and 13-fold increase in butyl butyrate production (0.45 g/L, 54% selectivity) as compared to the initial strains. Overall, this study presented a generalizable framework to engineer modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable feedstocks.

Mitochondrial Electron Transport Chain Inhibition Promotes Resistance to Proteasome Inhibitors in Multiple Myeloma

INTRODUCTIONProteasome inhibitors (PI) such as bortezomib (Velcade), carfilzomib (Kyprolis) and ixazomib (Ninlaro)) have been shown to be efficacious in multiple myeloma (MM) therapy. However, despite being an effective first line therapy, resistance to PI usually develops, leading to relapse and refractory disease. Previous studies have implicated a role of OXPHOS, glycolysis, antioxidants and serine metabolism in PI resistance. Mitochondrial metabolism plays a central role in malignant progression not only by generating ATP but also by providing precursors for synthesis of several biomolecules such as proteins, nucleotides, fatty acids and antioxidants that can influence the efficacy of MM therapies. We previously determined reduced mitochondrial electron transport chain (ETC) activity promotes sensitivity to the BCL-2 antagonist, venetoclax. However, the relationship between the metabolic state of the mitochondria and proteasome inhibitor (PI) sensitivity is not fully understood. Unexpectedly, we found ETC inhibition or reduced ETC activity to promote resistance to PIs. Here, we investigate the mechanistic basis for divergent effects of mitochondrial stress on sensitivity to PIs and venetoclax.METHODSWe have used RNA-Seq and carbon isotope tracing using labeled U 13C-glucose or U 13C-glutamine, flow cytometry and western blot analysis in MM cell lines treated with mitochondrial Complex I inhibitor, IACS-010759 (IACS) +/- Bortezomib; and immunostaining of MM patient samples and interrogation of Multiple Myeloma Research Foundation's CoMMpass Study (NCT01454297, Interim Analysis 15).RESULTSWe find that mitochondrial ETC (complex I-V) inhibition antagonizes bortezomib (BTZ) and carfilzomib (CFZ) induced cell death in MM in contrast to promoting sensitivity to venetoclax. Additionally, cell lines exhibiting intrinsically reduced ETC activity were more resistant to PI in comparison to cell lines with higher ETC activity. Evaluation of CoMMpass MM trial (NCT0145429, IA15) and serial samples from 50 patients before PI treatment and after relapse, show pathways related to OXPHOS and TCA cycle to be downregulated in poor survival patients, corroborating our in vitro observations on reduced ETC activity promoting resistance to PI. To elucidate the mechanistic basis of ETC-inhibition induced PI resistance we performed RNA-Seq and U 13C-glucose and glutamine tracing in L363 cells treated with the ETC Complex I inhibitor IACS +/- BTZ. RNA-Seq analysis and further confirmation by western blot analysis reveals integrated stress response (ISR) upregulation, ATF4 induction, and suppression of protein translation and global protein ubiquitination levels, likely responsible for resistance to proteasome inhibition in cells co-treated with IACS and BTZ compared to BTZ alone. Stable isotope tracing reveals an upregulation of reductive carboxylation; while RNA-Seq data and flow cytometry demonstrate increase in the cystine/glutamate transporter SLC7A11. The ensuing metabolic rewiring in mitochondrially suppressed MM induces several metabolic vulnerabilities including sensitivity to the SLC7A11 inhibitor, erastin. We also show that knockdown of ATF4 re-sensitizes ETC-inhibited cells to BTZ while ablating sensitivity to venetoclax (previously reported). Furthermore, examination of patient samples demonstrates inter-cellular heterogeneity in ATF4 expression. Our results thus, support the role ATF4 as key determinants of PI and BCL-2 antagonist efficacy.CONCLUSIONWe show mitochondrial ETC inhibition induces ISR mediated resistance to PI. These ETC-inhibited cells are however sensitive to BCL-2 antagonists and afford additional metabolic vulnerabilities that can be capitalized upon to target metabolic heterogeneity in MM. Our study underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic heterogeneity-mediated resistance. DisclosuresLonial: Janssen: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria. Boise: AstraZeneca: Consultancy, Research Funding; Abbvie: Consultancy. Jaye: Stemline Therapeutics: Honoraria. Nooka: Janssen Oncology: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Other: Travel expenses; Sanofi: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Oncopeptides: Consultancy; Adaptive technologies: Consultancy; Karyopharm Therapeutics: Consultancy.

5,7-Dimethoxyflavone enhances barrier function by increasing occludin and reducing claudin-2 in human intestinal Caco-2 cells

• Dimethoxyflavone increases the integrity of intestinal tight junction barrier. • The barrier effect is due to the increased occludin and decreased claudin-2. • Translation of occludin protein is promoted by dimethoxyflavone. • Claudin-2 mRNA is destabilized and reduced via a microRNA by dimethoxyflavone. Defects in intestinal tight junction (TJ) barrier cause intestinal inflammation. We investigated the effects of 5,7-dimethoxyflavone (DMF), abundantly found in black ginger, on the TJ barrier in human intestinal Caco-2 cells. DMF reinforced TJ barrier integrity, indicated by increased transepithelial electrical resistance and reduced dextran permeability in Caco-2 cells. Immunoblot analysis revealed that the increases in the barrier-forming TJ molecules occludin and claudin-1 and the decrease in pore-forming claudin-2 in the cytoskeletal fraction of the cells were responsible for the TJ regulation. Increased occludin expression was sensitive to cycloheximide (an inhibitor of protein translation) and rapamycin (mechanistic target of rapamycin [mTOR] inhibitor). DMF reduced Cldn2 mRNA levels without suppressing its transcriptional activity; the reduction was associated with the upregulation of miR-16-5p. Thus, DMF-mediated reinforcement of intestinal TJ barrier was partly involved in the induction of occludin protein translation via mTOR and silencing Cldn2 mRNA via miR-16-5p.

Combined single-cell transcriptional, translational, and genomic profiling reveals HIV-1 reservoir diversity.

Although understanding the diversity of HIV-1 reservoirs is key to achieving a cure, their study at the single-cell level in primary samples remains challenging. We combine flow cytometric multiplexed fluorescent in situ RNA hybridization for different viral genes with HIV-1 p24 protein detection, cell phenotyping, and downstream near-full-length single-cell vDNA sequencing. Stimulation-induced viral RNA-positive (vRNA+) cells from viremic and antiretroviral-therapy (ART)-suppressed individuals differ in their ability to produce p24. In participants on ART, latency-reversing agents (LRAs) induce a wide variety of viral gene transcription and translation patterns with LRA class-specific differences in reactivation potency. Reactivated proviruses, including in p24+ cells, are mostly defective. Although LRAs efficiently induce transcription in all memory cell subsets, we observe induction of translation mostly in effector memory cells, rather than in the long-lived central memory pool. We identify HIV-1 clones with diverse transcriptional and translational patterns between individual cells, and this finding suggests that cell-intrinsic factors influence reservoir persistence and heterogeneity.

Abstract 2325: Hypoxia-mediated ATF4 induction promotes survival in detached conditions in metastatic murine mammary cancer cells

Abstract Regions of hypoxia are common in solid tumors and drive changes in gene expression that increase risk of cancer metastasis. Tumor cells must respond to the stress of hypoxia by activating genes to modify cell metabolism and antioxidant response to improve survival. The goal of the current studies was to determine the effect of hypoxia on cell metabolism and markers of oxidative stress in non-metastatic M-Wnt compared to metastatic metM-Wntlung murine mammary cancer cell lines. We show that hypoxia increased expression of genes involved in antioxidant response in metastatic compared to non-metastatic cells, including glutamate cysteine ligase catalytic and modifier subunits and malic enzyme 1. Further, hypoxia induces a greater suppression of glutamine to glutamate conversion in metastatic cells (15% in metastatic cells compared to 9% in non-metastatic cells). Interestingly, hypoxia increased the mRNA level of the transaminase glutamic pyruvic transaminase 2 (Gpt2, 7.7-fold) only in metM-Wntlung cells. The change in Gpt2 expression was accompanied by transcriptional (4.2-fold) and translational (6.5-fold) induction of the integrated stress response effector protein activating transcription factor 4 (ATF4), which mediated survival of hypoxic metastatic cells in detached conditions, as shown by genetic ATF4 depletion. These data indicate that more aggressive, metastatic cancer cells rely on metabolic reprogramming and induction of antioxidant defense, including activation of ATF4, in hypoxia for survival in detached conditions. Citation Format: Violet A. Kiesel, Stephen D. Hursting, Dorothy Teegarden. Hypoxia-mediated ATF4 induction promotes survival in detached conditions in metastatic murine mammary cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2325.