Cell Survival In Response Research Articles

The Question of Survival or Death: What Is the Role of Autophagy in Acute Myeloid Leukemia (AML)?

Autophagy plays a critical role in balancing sources of energy in response to harsh conditions and nutrient deprivation. Autophagy allows cells to survive in harsh condition and also serve as a death mechanism. Any dysregulation in autophagy signaling may lead to several disorders. Autophagy has been proposed to explain chemotherapy resistance in acute myeloid leukemia (AML). This signaling pathway can either act as a tumor suppressive function or chemo-resistance mechanism. Conventional chemotherapy drugs enhance apoptosis and indicate clinical benefit, but in some cases, relapse and chemotherapy resistance are observed. In leukemia, autophagy may promote cell survival in response to chemotherapy drugs. Therefore, new strategies by inhibiting or activating autophagy may find a broad application for treating leukemia and may significantly enhance clinical outcomes. In this review, we discussed the dimensional role of autophagy in leukemia.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Effects of an enhanced acoustic environment on residual hearing following chronic cochlear implantation and electrical stimulation in the partially deafened cat

There is an increasing trend to provide cochlear implants for people with useful residual hearing, typically in the low frequency range (<2 kHz). These recipients typically use both electrical stimulation from their implant and acoustic stimulation that has been amplified with a hearing aid to access their residual hearing, so called electro-acoustic stimulation (EAS). However, a significant problem is the loss of residual hearing following implantation that can occur immediately following surgery or delayed over many months. One potential cause of the loss of residual hearing is the over stimulation of remaining hair cells due to the combination of an amplified acoustic input and direct electrical activation. This paper aims to test this hypothesis.Here, we have used a neonatal aminoglycoside-induced partial hearing cat model that resulted in a high frequency hearing loss (>4 kHz). Two separate cohorts of animals were implanted and received unilateral chronic electrical stimulation using clinical stimulators and speech processors over 5 months. To simulate potential over stimulation via a hearing aid, one cohort of animals were also exposed to an enhanced acoustic environment consisting of 80 dB SPL 4-talker babble presented 14 h per day. Hearing thresholds for both stimulated and unstimulated ears were measured throughout the implantation period. Cochleae were collected for histology to measure spiral ganglion neuron survival, hair cell survival and tissue response to chronic implantation and electrical stimulation. Consistent with clinical observations, cochlear implantation and stimulation resulted in an increase in threshold across the population. There was no significant effect of the enhanced acoustic environment on auditory thresholds or histological measures (hair cell survival, neuronal survival) of hearing, indicating that hair cell overstimulation was not a significant driver of loss of residual hearing.

Autophagy plays an essential role in ultraviolet radiation-driven skin photoaging.

Photoaging is characterized by a chronic inflammatory response to UV light. One of the most prominent features of cutaneous photoaging is wrinkling, which is due primarily to a loss of collagen fibers and deposits of abnormal degenerative elastotic material within the dermis (actinic elastosis). These changes are thought to be mediated by inflammation, with subsequent upregulation of extracellular matrix-degrading proteases and down-regulation of collagen synthesis. Autophagy is a vital homeostatic cellular process of either clearing surplus or damaged cell components notably lipids and proteins or recycling the content of the cells’ cytoplasm to promote cell survival and adaptive responses during starvation and other oxidative and/or genotoxic stress conditions. Autophagy may also become a means of supplying nutrients to maintain a high cellular proliferation rate when needed. It has been suggested that loss of autophagy leads to both photodamage and the initiation of photoaging in UV exposed skin. Moreover, UV radiation of sunlight is capable of regulating a number of autophagy-linked genes. This review will focus on the protective effect of autophagy in the skin cells damaged by UV radiation. We hope to draw attention to the significance of autophagy regulation in the prevention and treatment of skin photoaging.

TRIM26 Maintains Cell Survival in Response to Oxidative Stress through Regulating DNA Glycosylase Stability.

Oxidative DNA base lesions in DNA are repaired through the base excision repair (BER) pathway, which consequently plays a vital role in the maintenance of genome integrity and in suppressing mutagenesis. 8-oxoguanine DNA glycosylase (OGG1), endonuclease III-like protein 1 (NTH1), and the endonuclease VIII-like proteins 1–3 (NEIL1–3) are the key enzymes that initiate repair through the excision of the oxidized base. We have previously identified that the E3 ubiquitin ligase tripartite motif 26 (TRIM26) controls the cellular response to oxidative stress through regulating both NEIL1 and NTH1, although its potential, broader role in BER is unclear. We now show that TRIM26 is a central player in determining the response to different forms of oxidative stress. Using siRNA-mediated knockdowns, we demonstrate that the resistance of cells to X-ray radiation and hydrogen peroxide generated as a consequence of trim26 depletion can be reversed through suppression of selective DNA glycosylases. In particular, a knockdown of neil1 or ogg1 can enhance sensitivity and DNA repair rates in response to X-rays, whereas a knockdown of neil1 or neil3 can produce the same effect in response to hydrogen peroxide. Our study, therefore, highlights the importance of TRIM26 in balancing cellular DNA glycosylase levels required for an efficient BER response.

Pro-Inflammatory and Pro-Apoptotic Effects of the Non-Protein Amino Acid L-Azetidine-2-Carboxylic Acid in BV2 Microglial Cells.

L-Azetidine-2-carboxylic acid (AZE) is a toxic non-protein coding amino acid (npAA) that is highly abundant in sugar and table beets. Due to its structural similarity with the amino acid L-proline, AZE can evade the editing process during protein assembly in eukaryotic cells and be misincorporated into L-proline-rich proteins, potentially causing protein misfolding and other detrimental effects to cells. In this study, we sought to determine if AZE treatment triggered pro-inflammatory and pro-apoptotic responses in BV2 microglial cells. BV2 microglial cells exposed to AZE at increasing concentrations (0–2000 µM) at 0, 3, 6, 12 and 24 h were assayed for cell viability (MTT) and nitric oxide release (Griess assay). Annexin V-FITC/propidium iodide (PI) staining was used to assess apoptosis. Real-time qPCR, Western blot and immunocytochemistry were used to interrogate relevant pro- and anti-inflammatory and other molecular targets of cell survival response. AZE (at concentrations > 1000 µM) significantly reduced cell viability, increased BAX/Bcl2 ratio and caused cell death. Results were mirrored by a robust increase in nitric oxide release, percentage of activated/polarised cells and expression of pro-inflammatory markers (IL-1β, IL-6, NOS2, CD68 and MHC-2a). Additionally, we found that AZE induced the expression of the extracellular matrix degrading enzyme matrix metalloproteinase 9 (MMP-9) and brain derived neurotrophic factor (BDNF), two critical regulators of microglial motility and structural plasticity. Collectively, these data indicate that AZE-induced toxicity is associated with increased pro-inflammatory activity and reduced survival in BV2 microglia. This evidence may prompt for an increased monitoring of AZE consumption by humans.

Inhibition of mitochondrial respiration has fundamentally different effects on proliferation, cell survival and stress response in immature versus differentiated cardiomyocyte cell lines.

Myocardial tissue homeostasis is critically important for heart development, growth and function throughout the life course. The loss of cardiomyocytes under pathological conditions ultimately leads to cardiovascular disease due to the limited regenerative capacity of the postnatal mammalian heart. Inhibition of electron transport along the mitochondrial respiratory chain causes cellular stress characterized by ATP depletion as well as excessive generation of reactive oxygen species. Adult cardiomyocytes are highly susceptible to mitochondrial dysfunction whereas embryonic cardiomyocytes in the mouse heart have been shown to be resistant towards mitochondrial complex III inhibition. To functionally characterize the molecular mechanisms mediating this stress tolerance, we used H9c2 cells as an in vitro model for immature cardiomyoblasts and treated them with various inhibitors of mitochondrial respiration. The complex I inhibitor rotenone rapidly induced cell cycle arrest and apoptosis whereas the complex III inhibitor antimycin A (AMA) had no effect on proliferation and only mildly increased cell death. HL-1 cells, a differentiated and contractile cardiomyocyte cell line from mouse atrium, were highly susceptible to AMA treatment evident by cell cycle arrest and death. AMA induced various stress response mechanisms in H9c2 cells, such as the mitochondrial unfolded protein response (UPRmt), integrated stress response (ISR), heat shock response (HSR) and antioxidative defense. Inhibition of the UPR, ISR and HSR by siRNA mediated knock down of key components does not impair growth of H9c2 cells upon AMA treatment. In contrast, knock down of NRF2, an important transcriptional regulator of genes involved in detoxification of reactive oxygen species, reduces growth of H9c2 cells upon AMA treatment. Various approaches to activate cell protective mechanisms and alleviate oxidative stress in HL-1 cells failed to rescue them from AMA induced growth arrest and death. In summary, these data show that the site of electron transport interruption along the mitochondrial respiratory chain determines cell fate in immature cardiomyoblasts. The study furthermore points to fundamental differences in stress tolerance and cell survival between immature and differentiated cardiomyocytes which may underlie the growth plasticity of embryonic cardiomyocytes during heart development but also highlight the obstacles of cardioprotective therapies in the adult heart.

H2O2/DEM-Promoted Maft Promoter Demethylation Drives Nrf2/ARE Activation in Zebrafish.

The Nrf2/ARE signaling pathway is a cell survival response pathway in response to environmental stresses. The Nrf2/ARE signaling pathway can be activated by stimulating cysteine residues at different positions in the Keap1. However, the epigenetic mechanisms of the Nrf2/ARE pathway under different stimuli are still poorly understood. In this study, we found that both hydrogen peroxide (H2O2) and Diethyl Maleate (DEM) activated the Nrf2/ARE signaling pathway at 120 hpf in zebrafish. H2O2 regulated the demethylation of the maft promoter by inhibiting the expression of methyltransferase. This promotes the mRNA expression of the Nrf2 binding factor maft, thereby promoting the downstream antioxidant genes. The methylation of the Nrf2/ARE signaling pathway was not significantly regulated by DEM. However, under oxidative stress, the methyltransferase inhibitors (decitabine and azacitidine) demethylated the promoter region of maft. It activated the expression of the maft, further improving the Nrf2/ARE signal pathway. At last, antioxidant target genes were activated. It was shown that H2O2 and DEM cooperated with methyltransferase inhibitors, providing an important reference for the treatment of oxidative stress-related diseases and breaking new ground for the study of the mechanism of methyltransferase inhibitors in the process of tumor chemotherapy.

CXCR4 and CD74 together enhance cell survival in response to macrophage migration-inhibitory factor in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of small, mature CD5+ B lymphocytes in the blood, marrow, and lymphoid organs. Cell survival depends on interaction with the leukemic microenvironment. However, the mechanisms controlling CLL cell survival are still incompletely understood. Macrophage migration-inhibitory factor (MIF), a pro-inflammatory and immunoregulatory chemokine-like cytokine, interacts with CXCR4, a major chemokine receptor, as well as with CD74/invariant chain, a single-pass type II receptor. In this study, we analyzed the roles of CXCR4, CD74, and MIF in CLL. Mononuclear cells from patients with hematological malignancies were analyzed for coexpression of CXCR4 and CD74 by flow cytometry. Strong co- and overexpression of CXCR4 and CD74 were observed on B cells of CLL patients (n=10). Survival and chemotaxis assays indicated that CXCR4 and CD74 work together to enhance the survival and migration of malignant cells in CLL. Blockade of the receptors, either individually or in combination, promoted cell death and led to an abrogation of MIF-driven migration responses in murine and human CLL cells, suggesting that joint activation of both receptors is crucial for CLL cell survival and mobility. These findings indicate that the MIF/CXCR4/CD74 axis represents a novel therapeutic target in CLL.

Regulation of Claspin by the p38 stress-activated protein kinase protects cells from DNA damage.

Stress-activated protein kinases (SAPKs) enhance survival in response to environmental changes. In yeast, the Hog1 SAPK and Mrc1, a protein required for DNA replication, define a safeguard mechanism that allows eukaryotic cells to prevent genomic instability upon stress during S-phase. Here we show that, in mammals, the p38 SAPK and Claspin-the functional homolog of Mrc1-protect cells from DNA damage upon osmostress during S-phase. We demonstrate that p38 phosphorylates Claspin and either the mutation of the p38-phosphorylation sites in Claspin or p38 inhibition suppresses the protective role of Claspin on DNA damage. In addition, wild-type Claspin but not the p38-unphosphorylatable mutant has a protective effect on cell survival in response to cisplatin treatment. These findings reveal a role of Claspin in response to chemotherapeutic drugs. Thus, this pathway protects S-phase integrity from different insults and it is conserved from yeast to mammals.

Transcriptomes of Wet Skin Biopsies Predict Outcomes after Ionizing Radiation Exposure with Potential Dosimetric Applications in a Mouse Model.

Countermeasures for radiation diagnosis, prognosis, and treatment are trailing behind the proliferation of nuclear energy and weaponry. Radiation injury mechanisms at the systems biology level are not fully understood. Here, mice skin biopsies at h2, d4, d7, d21, and d28 after exposure to 1, 3, 6, or 20 Gy whole-body ionizing radiation were evaluated for the potential application of transcriptional alterations in radiation diagnosis and prognosis. Exposure to 20 Gy was lethal by d7, while mice who received 1, 3, or 6 Gy survived the 28-day time course. A Sammon plot separated samples based on survival and time points (TPs) within lethal (20 Gy) and sublethal doses. The differences in the numbers, regulation mode, and fold change of significantly differentially transcribed genes (SDTGs, p < 0.05 and FC > 2) were identified between lethal and sublethal doses, and down and upregulation dominated transcriptomes during the first post-exposure week, respectively. The numbers of SDTGs and the percentages of upregulated ones revealed stationary downregulation post-lethal dose in contrast to responses to sublethal doses which were dynamic and largely upregulated. Longitudinal up/downregulated SDTGs ratios suggested delayed and extended responses with increasing IR doses in the sublethal range and lethal-like responses in late TPs. This was supported by the distributions of common and unique genes across TPs within each dose. Several genes with potential dosimetric marker applications were identified. Immune, fibrosis, detoxification, hematological, neurological, gastric, cell survival, migration, and proliferation radiation response pathways were identified, with the majority predicted to be activated after sublethal and inactivated after lethal exposures, particularly during the first post-exposure week.

Quantitation of the ataxia-telangiectasia-mutated and Rad3-related inhibitor elimusertib (BAY-1895344) in human plasma using LC-MS/MS.

Ataxia-telangiectasia-mutated and Rad3-related (ATR) is master regulator of the DNA-damage response that, through multiple mechanisms, can promote cancer cell survival in response to replication stress from sources, including chemotherapy and radiation. Elimusertib (BAY-1895344) is an orally available small-molecule ATR inhibitor currently in preclinical and clinical development for cancer treatment. To support these studies and define elimusertib pharmacokinetics, we developed a HPLC-MS method for its quantitation. A 50-μL volume of plasma was subjected to acetonitrile protein precipitation and then chromatographic separation using a Phenomenex Polar-RP column (2 × 50 mm, 4μm) and a gradient mobile phase consisting of 0.1% formic acid in acetonitrile and water during a 7-min run time. Mass spectrometric detection was achieved using a SCIEX 4000 triple-stage mass spectrometer with electrospray positive-mode ionization. With a stable isotopic internal standard, the assay was linear from 30 to 5000 ng/mL and proved to be both accurate (93.5-108.2%) and precise (<6.3% coefficient of variation) fulfilling criteria from the Food and Drug Administration guidance on bioanalytical method validation. This LC-MS/MS assay will support several ongoing clinical studies by defining elimusertib pharmacokinetics.

P38 MAPK-dependent phosphorylation of transcription factor SOX2 promotes an adaptive response to BRAF inhibitors in melanoma cells

Despite recent advances in the development of BRAF kinase inhibitors (BRAFi) for BRAF-mutant melanomas, development of resistance remains a major clinical problem. In addition to genetic alterations associated with intrinsic resistance, several adaptive response mechanisms are known to be rapidly activated to allow cell survival in response to treatment, limiting efficacy. A better understanding of the mechanisms driving resistance is urgently needed to improve the success of BRAF-targeted therapies and to make therapeutic intervention more durable. In this study, we identify the mitogen-activated protein kinase (MAPK) p38 as a novel mediator of the adaptive response of melanoma cells to BRAF-targeted therapy. Our findings demonstrate that BRAFi leads to an early increase in p38 activation, which promotes phosphorylation of the transcription factor SOX2 at Ser251, enhancing SOX2 stability, nuclear localization, and transcriptional activity. Furthermore, functional studies show that SOX2 depletion increases sensitivity of melanoma cells to BRAFi, whereas overexpression of a phosphomimetic SOX2-S251E mutant is sufficient to drive resistance and desensitize melanoma cells to BRAFi in vitro and in a zebrafish xenograft model. We also found that SOX2 phosphorylation at Ser251 confers resistance to BRAFi by binding to the promoter and increasing transcriptional activation of the ATP-binding cassette drug efflux transporter ABCG2. In summary, we unveil a p38/SOX2-mediated mechanism of adaptive response to BRAFi, which provides prosurvival signals to melanoma cells against the cytotoxic effects of BRAFi prior to acquiring resistance.

A Recurrent ADPRHL1 Germline Mutation Activates PARP1 and Confers Prostate Cancer Risk in African American Families.

This study highlights a potential strategy for ADPRHL1 mutation detection in prostate cancer-risk assessment and a potential therapeutic application for individuals with prostate cancer in AA families.

MHY2251, a New SIRT1 Inhibitor, Induces Apoptosis via JNK/p53 Pathway in HCT116 Human Colorectal Cancer Cells.

Sirtuins (SIRTs) belong to the nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylase family. They are key regulators of cellular and physiological processes, such as cell survival, senescence, differentiation, DNA damage and stress response, cellular metabolism, and aging. SIRTs also influence carcinogenesis, making them potential targets for anticancer therapeutic strategies. In this study, we investigated the anticancer properties and underlying molecular mechanisms of a novel SIRT1 inhibitor, MHY2251, in human colorectal cancer (CRC) cells. MHY2251 reduced the viability of various human CRC cell lines, especially those with wild-type TP53. MHY2251 inhibited SIRT1 activity and SIRT1/2 protein expression, while promoting p53 acetylation, which is a target of SIRT1 in HCT116 cells. MHY2251 treatment triggered apoptosis in HCT116 cells. It increased the percentage of late apoptotic cells and the sub-G1 fraction (as detected by flow cytometric analysis) and induced DNA fragmentation. In addition, MHY2251 upregulated the expression of FasL and Fas, altered the ratio of Bax/Bcl-2, downregulated the levels of pro-caspase-8, -9, and -3 proteins, and induced subsequent poly(ADP-ribose) polymerase cleavage. The induction of apoptosis by MHY2251 was related to the activation of the caspase cascade, which was significantly attenuated by pre-treatment with Z-VAD-FMK, a pan-caspase inhibitor. Furthermore, MHY2251 stimulated the phosphorylation of c-Jun N-terminal kinase (JNK), and MHY2251-triggered apoptosis was blocked by pre-treatment with SP600125, a JNK inhibitor. This finding indicated the specific involvement of JNK in MHY2251-induced apoptosis. MHY2251 shows considerable potential as a therapeutic agent for targeting human CRC via the inhibition of SIRT1 and activation of JNK/p53 pathway.

Gallic Acid Improves Therapeutic Effects of Mesenchymal Stem Cells Derived from Adipose Tissue in Acute Renal Injury Following Rhabdomyolysis Induced by Glycerol.

Acute kidney injury (AKI) is identified by a progressive reduction in the glomerular filtration rate (GFR) and retention of nitrogenous waste products. Traumatic and nontraumatic rhabdomyolysis is recently considered the main cause of AKI. According to several studies, stem cell treatment is a promising therapeutic strategy for many types of disorders including AKI. The main limitation of mesenchymal stem cells (MSCs) therapy is reducing cell survival in response to oxidative stress products in injured organ areas. Gallic acid (GA) as a well-known antioxidant has been reported to confer potent-free radical scavenging and anti-inflammatory properties. Therefore, the aim of the current study was to assess the influence of MSCs and GA in acute renal injury following rhabdomyolysis induced by glycerol. A total of 70 healthy rats were divided into seven groups (10 in each group): control, AKI (glycerol, intramuscular), cell therapy (AKI + intravenous injection of mesenchymal stem cells derived from adipose tissue (AMCs), AKI + AMCs + GA (50, 100, and 200mg/kg, intraperitoneally, 3days a week for 3 consecutive weeks), and positive control group (the most effective dose of gallic acid). After the treatment, rats were sacrificed; blood, urine, and kidney tissues were collected; and qualitative and quantitative parameters (including blood urea nitrogen (BUN), creatine kinase (CK), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate transaminase (SGOT), oxidative stress markers kidney function parameters) and histopathological indexes were assayed. Our results revealed that co-treatment of AMCs plus GA into AKI rats decreased BUN and creatinine and ameliorated kidney injury parameters after 3weeks. Improved oxidative stress markers such as decreased MDA and increased SOD and CAT were significant in the GA + AMCs group compared to the AMCs alone in AKI rats. Also, the histopathological appearances of AKI rats including renal tubule cavity expansion and renal tubular epithelial cell edema, and interstitial inflammation, were alleviated using GA + AMCs treatment compared to the control. The obtained results of the current study documented that antioxidants could make mesenchymal stem cells more resistant to the condition in which they are supposed to be transplanted and probably improve the efficacy of stem cell therapy in AKI patients.

Effect of overdispersion of lethal lesions on cell survival curves

The linear-quadratic (LQ) model is the most commonly used mechanism to predict radiobiological outcomes. It has been used extensively to describe dose-response in vitro and in vivo. There are, however, some questions about its applicability in terms of its capacity to represent some profound mechanistic behaviour. Specifically, empirical evidence suggests that the LQ model underestimates the survival of cells at low doses while overestimating cell death at higher doses. It is believed to be driven from the usual LQ model assumption that radiogenic lesions are Poisson distributed. In this context, we use a negative binomial (NB) distribution to study the effect of overdispersion on the shapes and the possibility of reducing dose-response curvature at higher doses. We develop an overdispersion model for cell survival using the non-homologous end-joining (NHEJ) pathway double-strand break (DSB) repair mechanism to investigate the effects of the overdispersion on probabilities of repair of DSBs. The error distribution is customised to ensure that the refined overdispersion parameter depends on the mean of the distribution. The predicted cell survival responses for V79, AG and HSG cells exposed to protons, helium and carbon ions are compared with the experimental data in low and high dose regions at various linear energy transfer (LET) values. The results indicate straightening of dose-response and approaching a log-linear behaviour at higher doses. The model predictions with the measured data show that the NB modelled survival curves agree with the data following medium and high doses. Model predictions are not validated at very tiny and very high doses; the approach presented provides an analysis of mechanisms at the microscopic level. This may help improve the understanding of radiobiological responses of survival curves and resolve discrepancies between experimental and theoretical predictions of cell survival models.

Abstract 3040: The eIF2 kinase GCN2 controls expression of key amino acid transporters and is critical for prostate cancer growth and progression

Abstract The Integrated Stress Response (ISR) is central for adaptation and survival of tumor cells in response to exogenous and endogenous stresses. The ISR features a family of protein kinases that phosphorylate the eukaryotic translation initiation factor eIF2 in response to distinct stresses. While phosphoryation of eIF2 reduces global protein synthesis, there is paradoxical induced translation of select stress adapative gene transcripts, including ATF4, which is central for ISR-directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer adaptation to stress. Recently, eIF2 phosphorylation and ATF4 were suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinase(s), its mode of activation, and mechanisms by which the ISR facilitate PCa progression are not known.We discovered that the eIF2 kinase GCN2 (EIF2AK4) is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduced growth in androgen-sensitive and castration-resistant PCa cell lines in culture and cell line-derived and patient-derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2. A transcriptome analysis of PCa cells treated with a specific small molecular inhibitor of GCN2 indicates that this eIF2 kinase was critical for expression of multiple SLC genes, including amino acid transporters previously implicated in different cancers. We determined that GCN2 inhibition decreases intracellular amino acid levels, and reduced aminoacylation of select tRNAs, accounting for reduced growth in PCa cells. Using CRISPR-based phenotypic screens and genome-wide gene expression analyses of control and GCN2-depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is a key SLC gene induced by GCN2 and is essential for PCa proliferation. SLC3A2 has been reported to associate with many SLC7 family transporters, allowing for the localization of these transporters to the plasma membrane for amino acid uptake. Of importance, expression of SLC3A2 partially restored amino acid levels and growth due to loss of GCN2. Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including SLC3A2, which provides for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising new therapeutic targets for the treatment of PCa. Citation Format: Ricardo Cordova, Jagannath Misra, Nur P. Damayanti, Parth H. Amin, Kenneth R. Carlson, Angela J. Klunk, Emily T. Mirek, Marcus J. Miller, Tracy G. Anthony, Roberto Pili, Ronald C. Wek, Kirk A. Staschke. The eIF2 kinase GCN2 controls expression of key amino acid transporters and is critical for prostate cancer growth and progression [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 3040.

Abstract 3293: In vitro cellular and molecular effects of oncolytic vaccinia virus in clear and non-clear cell renal cell carcinoma

Abstract Oncolytic vaccinia virus has several advantages over other OV platforms, including an efficient life cycle, multiple mechanisms of viral spread, large genome, allowing acceptance of foreign therapeutic DNA inserts, and proven safety in humans, due to its use as smallpox vaccine. JX-594 (Pexavec, SillaJen) is an attenuated (TKdeactivated ) oncolytic vaccinia VV, engineered to express GM-CSF and to selectively target tumor over non-tumor tissues. It is currently being investigated as a novel therapeutic option in patients with advanced Renal Cell Carcinoma (RCC, NCT03294083). The present study was undertaken to investigate the effects of JX-954 in a panel of clear and non-clear cell RCC cell lines and characterize the potential mechanisms of JX-594 oncolysis in vitro. We evaluated the oncolytic potency of JX-594 in human clear cell RCCs (VHL mutant 786-0, A498, VHL wild type Caki-1), non-clear cell RCC (ACHN, papillary RCC, and UOK-262, an FH deficient papillary RCC cell) and murine renal cancer cells (RENCA). Potent and persistent cytotoxic effects were induced by JX-594 in all human RCC cells, regardless of the histological subtype, and by mJX-594 (a Western Reserve strain of vaccinia virus expressing murine GMCSF) in RENCA cells. Moreover, we observed efficient viral replication in human (786-0, ACHN) and murine (RENCA) cells, confirming RCC permissiveness to viral infection. Functional proteomics analysis of JX-594 treated 786-0 cells was performed by reverse phase protein array (RPPA) to gain knowledge on potential mechanisms of viral oncolysis. JX-594 treatment had significant effects on the expression levels of proteins related to inducing cell cycle, apoptosis, necrosis, Akt signaling, MAPK signaling, autophagy and stress induction. In particular, the proteins related to cell cycle (Wee1, Cdc25c, CHK1/2, and Cyclin B1) and Akt signaling (PI3K, mTOR, Rictor and FOXO), MAPK signaling (JAK2, PAK1, FAK and MEK1) were down regulated, while proteins involved in apoptosis and necrosis were upregulated. In summary, our studies show that human and murine renal cancer cells are permissive to infection and replication by JX-594 and mJX-594, which induce potent oncolytic effects in clear and non-clear cell RCC, effects associated with significant modulation of RCC pathways associated with cell cycle, proliferation, and survival and stress responses. Citation Format: Yuqi Jing, F Bustamante Guerrero, Jaime Merchan. In vitro cellular and molecular effects of oncolytic vaccinia virus in clear and non-clear cell renal cell carcinoma [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 3293.

Beneficial Effects of O-GlcNAc Stimulation in a Young Rat Model of Sepsis: Beyond Modulation of Gene Expression.

The young population, which is particularly at risk of sepsis, is, paradoxically, rarely studied. Acute stimulation of O-GlcNAcylation, a post-translational modification involved in metabolic regulation, cell survival and stress response, is beneficial in young rats with sepsis. Considering that sepsis impacts the gene expression profile and that O-GlcNAcylation is a regulator of transcription, the aims of this study are to (i) unveil beneficial mechanisms of O-GlcNAcylation and (ii) decipher the relationship between O-GlcNAcylation and transcription during sepsis. Endotoxemic challenge was induced in 28-day-old male rats using a lipopolysaccharide injection (E. coli O111:B4, 20 mg·kg−1) and compared to control rats (NaCl 0.9%). One hour after, rats were assigned to no therapy or fluidotherapy (NaCl 0.9%, 10 mL.kg−1) ± NButGT (10 mg·kg−1) to stimulate O-GlcNAc levels. Cardiac O-GlcNAcylation levels were evaluated via Western blot and gene transcription using 3′ SRP analysis. Lipopolysaccharide injection favorizes inflammatory state with the overexpression of genes involved in the NF-κB, JAK/STAT and MAPK pathways. NButGT treatment increased cardiac O-GlcNAcylation levels (p < 0.05). Yet, the mRNA expression was not impacted two hours after fluidotherapy or NButGT treatment. In conclusion, O-GlcNAc stimulation-induced beneficial effects are not dependent on the gene expression profile at the early phase of sepsis.