FOXO Target Research Articles

L-carnitine cause to increase cell proliferation of C-Kit+ hematopoietic progenitor cells via decreasing the PI3K and FOXO-1 protein expression

BackgroundStem cell-based therapy has emerged as an attractive approach for regenerative medicine. Poor survival and maintenance of the cells used in regenerative medicine are considered as serious barriers to enhance the efficacy of the cell therapy. Using some antioxidants has been reported to prevent the aging of stem cells, and finding effective factors to reduce the senescence of these cells has impressive potential in cell therapy. The PI3K pathway adversely regulates the transcription factors known as FOXO, which are thought to have an inhibitory influence on cell proliferation. By downregulating FOXO and other targets, PI3K signaling controls the growth of cells. For this reason, the aim of the present study is to investigate the effect of L-carnitine (LC) as antioxidant on the cell proliferation and the protein expression of PI3K and FOXO. MethodsFor understanding the in vitro effect of LC on the PI3K and FOXO-1 expression of C-kit+ hematopoietic progenitor cells, the bone marrow mononuclear cells were isolated, and C-kit+ cells was enriched by the magnetic-activated cell sorting (MACS). Next, the identification of enriched C-kit+ cells were done by flowcytometry and immunocytochemistry. Then, C-kit+ cells were treated with 0.2 mM LC, the cells were collected at the end of the treatment period (48 h), and the proteins were extracted. In the following, the protein expression of PI3K and FOXO-1 was measured by western blotting. In addition, flowcytometry was done to assess the Ki-67 expression as a key marker for cell proliferation investigation. Results0.2 mM LC cause to significantly decrease in the protein expression of PI3K and FOXO-1 (*P<0.05 and **P<0.01, respectively). Also, the expression of Ki-67 was significantly increased in the presence of 0.2 mM LC (***P<0.001). ConclusionBriefly, LC can be considered an effective factor in increasing the proliferation of C-kit+ cells via some signaling pathways.

FOXO-regulated OSER1 reduces oxidative stress and extends lifespan in multiple species

FOXO transcription factors modulate aging-related pathways and influence longevity in multiple species, but the transcriptional targets that mediate these effects remain largely unknown. Here, we identify an evolutionarily conserved FOXO target gene, Oxidative stress-responsive serine-rich protein 1 (OSER1), whose overexpression extends lifespan in silkworms, nematodes, and flies, while its depletion correspondingly shortens lifespan. In flies, overexpression of OSER1 increases resistance to oxidative stress, starvation, and heat shock, while OSER1-depleted flies are more vulnerable to these stressors. In silkworms, hydrogen peroxide both induces and is scavenged by OSER1 in vitro and in vivo. Knockdown of OSER1 in Caenorhabditis elegans leads to increased ROS production and shorter lifespan, mitochondrial fragmentation, decreased ATP production, and altered transcription of mitochondrial genes. Human proteomic analysis suggests that OSER1 plays roles in oxidative stress response, cellular senescence, and reproduction, which is consistent with the data and suggests that OSER1 could play a role in fertility in silkworms and nematodes. Human studies demonstrate that polymorphic variants in OSER1 are associated with human longevity. In summary, OSER1 is an evolutionarily conserved FOXO-regulated protein that improves resistance to oxidative stress, maintains mitochondrial functional integrity, and increases lifespan in multiple species. Additional studies will clarify the role of OSER1 as a critical effector of healthy aging.

Abstract 1093: Small molecule inhibitors of the PAX3::FOXO1 fusion protein in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. It is commonly divided into subtypes based on histopathological appearance. The oncogenic fusion proteins PAX3::FOXO1 or PAX7::FOXO1 are present in the alveolar subtype of RMS (ARMS). These chimeric proteins are formed by reciprocal chromosomal translocations and consist of an N-terminal portion of the PAX3 or PAX7 transcription factor fused to a C-terminal portion of the FOXO1 transcription factor. These fusion proteins have been shown to drive malignant transformation in ARMS, and patients with fusion-positive ARMS have a worse prognosis than patients with fusion-negative ARMS and embryonal RMS. We recently reported that piperacetazine, a phenothiazine derivative and first-generation antipsychotic, is capable of binding to and inhibiting the transcriptional activity of PAX3::FOXO1, reducing the expression of PAX3::FOXO1 target genes, and inhibiting the ability of fusion-positive RMS cells to grow in soft agar. We hypothesize that other phenothiazine derivatives may have stronger binding affinity for PAX3::FOXO1 and greater ability to reduce tumor growth in vivo. We employed a structure-activity relationship campaign to develop new phenothiazine derivatives that may be more potent inhibitors of PAX3::FOXO1 than piperacetazine. We synthesized and tested 10 new compounds, in addition to 9 existing FDA-approved phenothiazine derivatives, for their ability to bind to PAX3::FOXO1 and inhibit its transcriptional activity and target gene expression as well as inhibiting the growth of fusion-positive RMS cells in 3D culture. Seven derivatives inhibited PAX3::FOXO1 reporter activity better than piperacetazine, nine of them performed worse, and three of them showed comparable activity to piperacetazine. We demonstrated that piperacetazine does not alter PAX3::FOXO1 levels, subcellular localization, binding to a PAX3::FOXO1 target DNA sequence, or phosphorylation at Ser256 in the FOXO1 portion of the fusion protein. We therefore hypothesize that piperacetazine may be disrupting interactions between PAX3::FOXO1 and its partner proteins. We are employing a proximity-based biotinylation method to identify proteins that are competed away from PAX3::FOXO1 by piperacetazine. Our data suggest that it is possible to target PAX3::FOXO1 protein using small molecules that can directly bind to it and inhibit its activity in fusion-positive RMS cells. Citation Format: Taryn Shaw, Kay Nakazawa, Purushottam Tiwari, Eryn Nelson, Jeffrey Formen, Christian Wolf, Jeffrey Toretsky, Aykut Üren. Small molecule inhibitors of the PAX3::FOXO1 fusion protein in rhabdomyosarcoma [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 1093.

FoxO1 promotes ovarian cancer by increasing transcription and METTL14-mediated m6A modification of SMC4.

The transcription factor forkhead box protein O1 (FoxO1) is closely related to the occurrence and development of ovarian cancer (OC), however its role and molecular mechanisms remain unclear. Herein, we found that FoxO1 was highly expressed in clinical samples of OC patients and was significantly correlated with poor prognosis. FoxO1 knockdown inhibited the proliferation of OC cells invitro and invivo. ChIP-seq combined with GEPIA2 and Kaplan-Meier database analysis showed that structural maintenance of chromosome 4 (SMC4) is a downstream target of FoxO1, and FoxO1 promotes SMC4 transcription by binding to its -1400/-1390 bp promoter. The high expression of SMC4 significantly blocked the tumor inhibition effect of FoxO1 knockdown. Furtherly, FoxO1 increased SMC4 mRNA abundance by transcriptionally activating methyltransferase-like 14 (METTL14) and increasing SMC4 m6A methylation on its coding sequence region. The Cancer Genome Atlas dataset analysis confirmed a significant positive correlation between FoxO1, SMC4, and METTL14 expression in OC. In summary, this study revealed the molecular mechanisms of FoxO1 regulating SMC4 and established a clinical link between the expression of FoxO1/METTL14/SMC4 in the occurrence of OC, thus providing a potential diagnostic target and therapeutic strategy.

CRM1/XPO1-FOXO1 Pathway Is Associated with Clinical Outcome and Represents a Promising Target in B-Cell Acute Lymphocytic Leukemia

Background: Currently, a proportion of B-Cell Acute Lymphocytic Leukemia (B-ALL) patients still show no response to existing therapeutic drugs and existing therapeutic drugs cannot further improve the prognosis of refractory and relapsed B-ALL. Thus, the development of a new therapeutic target to battle this deadly disease is imperative. Exportin-1 (XPO1), also known as chromosomal region maintenance 1(CRM1), plays an important role in the progression of tumorigenesis. However, the prognostic and therapeutic values of XPO1 in B-ALL have not been reported. Methods: The difference in XPO1 and Foxo1 expression between normal B cells and leukemia cells was using the Human Protein Atlas (HPA) dataset and RT-qPCR results. Correlations between XPO1 expression level and clinical outcome were analyzed using the RNA-seq data of 110 newly diagnosed B−ALL patients in Nanfang Hospital. Cell proliferation assays, apoptosis assays, and cell cycle analysis were performed to analyze the anti-tumor effects of selinexor (KPT-330) in vitro. Bioinformatics analysis, western blot, and RT-qPCR were performed to explore the underlying mechanism. Results ： Higher XPO1 levels or Foxo1 levels were associated with poor prognosis in newly diagnosed B-ALL patients. Using bone marrow (BM)， peripheral blood (PB) cells from B-ALL and normal people as well as B-ALL cell lines, we found that XPO1 and Foxo1 expression was significantly increased in B-ALL patients and B-ALL cell lines. And notably, there was a positive correlation between the expression of Foxo1 and XPO1, which was verified by the data in the HPA database, and the results were consistent. Foxo1 is an important transcription factor in B-ALL, mainly localized in the nucleus, and is important for leukemia cell survival. KPT-330 (Selinexor), a selective Inhibitor of XPO1, mechanistically inhibited the expression of Foxo1, a leukemia transcription factor (reduced in both the nuclear and cytoplasmic compartments), and led to a strong repression of Foxo1 target genes associated with cell cycle, DNA replication, and transcriptional regulation. This induced growth inhibition, apoptosis, and cycle arrest in B-ALL cells. Moreover, in combination with Foxo1 Inhibitor (AS1842856), the proliferation inhibition and apoptosis-inducing effects of KPT-330 on B-ALL could be significantly enhanced. Furthermore, the KPT-330 strongly triggered apoptosis in leukemic but with minimal effects on normal cells implying that drugs of this class show promise for the targeted therapy of B-ALL leukemia cells. Conclusions: Our findings demonstrate that XPO1 is a promising prognostic indicator and a valid target for antitumor treatment with selective inhibitor KPT-330. This research was supported by the National Natural Science Foundation of China(NFSC82170163, 81970147), Clinical Study of Nanfang Hospital(LC2016ZD009/2019CR012).

Gab3 Plays an Essential Role in CD8 + T-Cell Expansion through Regulating IL-2-Mediated Activation of PI3K/AKT/mTOR and Erk/FoxO Pathways

Grb2-associated binding protein 3 (Gab3) is a member of the Gab family of scaffolding proteins. Its role in T cells is largely unknown. In the current study, we investigated its in vivo function in CD8 + T cells using the lymphocytic choriomeningitis virus (LCMV) Armstrong infection model. We created BM chimera mice with a specific deficiency of Gab3 in CD8 + T cells. This was achieved by transplanting a mixture of BM cells obtained from CD8-deficient mice and Gab3-deficient (Gab3 -/-) mice (1) into lethally irradiated CD45.1 mice. The control chimera mice were lethally irradiated CD45.1 mice transplanted with a mixture of BM cells from CD8-deficient mice and WT mice. Twelve weeks after the BM transplantation, the Gab3 -/- and control BM chimera mice were infected with Armstrong. On day eight following infection, Gab3 -/- CD8 + T cells exhibited reduced expansion compared to WT CD8 + T cells. Importantly, Gab3 -/- LCMV antigen specific CD8 + T cells, GP33 + and NP396 + CD8 + T cells, were significantly reduced compared to their WT counterparts. Furthermore, CD8 + T cells in Gab3 -/- BM chimera mice showed significant increase of apoptosis compared to that in WT BM chimera mice following LCMV infection. To explore the mechanism underlying the observed phenotype, we used a primary CD8 + T-cell culture system, in which we stimulated CD8 + T cells with anti-CD3 and anti-CD28 followed by expanding the cells in IL-2. We observed that Gab3 deficiency severely impaired IL-2-induced CD8 + T cell expansion. Gab3 -/- CD8 + T cells in this culture system displayed increased apoptosis and reduced proliferation compared to wild-type (WT) controls, which may account for the reduced CD8 + T cell expansion. We then performed Seahorse analysis of the cultured CD8 + T cells after IL-2 stimulation. We found that IL-2-mediated extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were largely impaired in Gab3 -/- CD8 + T cells compared to WT CD8 + T cells. This data indicates a crucial role of Gab3 in both glycolysis and mitochondrial respiration. We further investigated how Gab3 regulates the IL-2 signaling pathway by conducting transcriptomic and phosphoproteomic analysis. RNA-seq analysis revealed that genes associated with mTOR signaling, glucose metabolism, cell cycle, and Erk/MAPK targets were highly enriched in WT CD8 + T cells, whereas genes related to apoptosis, autophagy, and FoxO3 targets were highly enriched in Gab3 -/- CD8 + T cells. In contrast, the gene set enrichment analysis (GSEA) showed that IL-2-induced Stat5 target gene expression was comparable between WT and Gab3 -/- CD8 + T cells, indicating an important role of Gab3 specifically in the activation of the mTOR and Erk/MAPK pathways downstream of IL-2 signaling. Consistent with that observed in the transcriptomic analysis, phosphoproteomics analysis revealed a significant reduction in IL-2-induced phosphorylation of key components in ERK/FOXO signaling pathway, including RafA, MAPK1, FoxO3, AMPK, and mTOR downstream molecule Eif4B in Gab3 -/- CD8 + T cells compared to WT CD8 + cells. Furthermore, phosphorylation of Stat5 at different sites was comparable between Gab3 -/- and WT CD8 + T cells, further supporting an important role of Gab3 specifically in IL-2 mediated mTOR and Erk/MAPK activation. Finally, we validated the results obtained from the transcriptomic and phosphoproteomic analyses using western blot analysis. Our findings revealed that in Gab3 -/- CD8 + T cells, the IL-2-induced phosphorylation of signaling molecules in the mTOR pathway (S6K, S6, 4EBP1, AKT) and the Erk pathway (Erk) was impaired. Notably, the phosphorylation of Stat5 remained intact despite the absence of Gab3. In summary, our studies have demonstrated that Gab3 plays an essential role in CD8 + T-cell expansion by promoting cell proliferation and prohibiting cell apoptosis through regulating IL-2-mediated activation of PI3K/AKT/mTOR and Erk-FoxO3 pathways. (1) We gratefully acknowledge that Gab3-deficient mice were originally generated by Dr. Larry Rohrschneider at Fred Hutchinson Cancer Research Center ( Mol Cell Biol 2003 Apr;23(7):2415-24. doi: 10.1128/MCB.23.7.2415-2424.2003.). The mice were subsequently backcrossed to C57BL/6.

MiR-184 targeting FOXO1 regulates host-cell oxidative stress induced by Chlamydia psittaci via the Wnt/β-catenin signaling pathway.

Chlamydia psittaci is a human pathogen that causes atypical pneumonia after zoonotic transmission. We confirmed that C. psittaci infection induces oxidative stress in human bronchial epithelial (HBEs) cells and explored how this is regulated through miR-184 and the Wnt/β-catenin signaling pathway. miR-184 mimic, miR-184 inhibitor, FOXO1 siRNA, or negative control sequence was transfected into HBE cells cultured in serum-free medium using Lipofectamine 2000. Then, prior to the cells were infected with C. psittaci 6BC, and the cells were treated with or without 30 µM Wnt/β-catenin inhibitor ICG-001. Quantification of reactive oxygen species, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione was carried out according to the manufacturer's protocol using a corresponding assay kit. The outcome of both protein and gene was measured by western blotting or real-time fluorescence quantitative PCR. In C. psittaci-infected HBE cells, miR-184 was upregulated, while one of its target genes, FOXO1, was downregulated. ROS and MDA levels increased, while SOD and GSH contents decreased after C. psittaci infection. When miR-184 expression was downregulated, the level of oxidative stress caused by C. psittaci infection was reduced, and the Wnt/β-catenin signaling pathway was inhibited. The opposite results were seen when miR-184 mimic was used. Transfecting with FOXO1 siRNA reversed the effect of miR-184 inhibitor. Moreover, when the Wnt/β-catenin-specific inhibitor ICG-001 was used, the level of oxidative stress induced by C. psittaci infection was significantly suppressed. miR-184 can target FOXO1 to promote oxidative stress in HBE cells following C. psittaci infection by activation of the Wnt/β-catenin signaling pathway.

THU042 FOXO1 Stimulates Hsp90b1 Expression To Promote Efficient Glucocorticoid Signaling And Facilitate Maturation Of Somatotropes

Abstract Disclosure: P. Das: None. D.M. Abou-Jabal: None. B.S. Ellsworth: None. Glucocorticoids promote differentiation and maturation of many tissues. There is significant evidence that glucocorticoids are also important for maturation of pituitary somatotropes. We find that treating pregnant mice with the synthetic glucocorticoid, dexamethasone, leads to premature appearance of GH-positive cells, suggesting premature somatotrope maturation. Expression of Gh1 and Foxo1 are significantly increased in mice treated with dexamethasone compared to control mice. We have previously shown that deletion of the forkhead transcription factor, FOXO1, causes a delay in somatotrope maturation. Thus, both FOXO1 and glucocorticoid signaling affect the timing of somatotrope maturation. Interestingly, we find that the ability of glucocorticoids to promote somatotrope maturation is impaired in mice lacking FOXO1. Similar to what we find in vivo, deletion of Foxo1 from the somatotrope-derived MtT/S cells (MtT/SΔFoxo1) impairs glucocorticoid-induced expression of Gh1 suggesting FOXO1 may be required for glucocorticoid-induced somatotrope maturation. Glucocorticoid signaling depends on a number of chaperone proteins. Heat-shock protein 90 (HSP90) is essential for protecting the glucocorticoid receptor (NR3C1) from degradation and preparing it for ligand binding. Hsp90aa1, Hsp90ab1, and Hsp90b1 are all expressed in MtT/S cells. Chromatin immunoprecipitation sequencing (ChIPseq) for FOXO1 reveals FOXO1 binding at the promoter of Hsp90b1. Consistent with this, we find a reduction in Hsp90b1 expression in MtT/SΔFoxo1 cells. Thus, Hsp90b1 may be a direct transcriptional target of FOXO1. Expression of Hsp90ab1 is also reduced in the absence of FOXO1, however FOXO1 binding is not detected at its promoter suggesting Hsp90ab1 may be an indirect target of FOXO1. Together these data suggest that FOXO1 promotes somatotrope maturation by fostering efficient glucocorticoid signaling. Presentation: Thursday, June 15, 2023

SAT038 Dietary Trans-10,cis-12 Conjugated Linoleic Acid Regulates Glucose Levels In Mice With Severe Insulin Resistance: Potential Effects On The Liver Via HNF4α And FOX01

Abstract Disclosure: S.M. Lee: None. J.T. Muratalla: None. C.W. Liew: None. J. Cordoba-Chacon: None. Plasma trans-10 cis-12-conjugated linoleic acid CLA (t10c12-CLA) is inversely associated with increased risk of diabetes in humans. In mice, it is well-known that t10c12-CLA induces whole-body insulin resistance due to severe lipodystrophy (loss of whole-body adipose tissue). However, t10c12-CLA-fed mice show normal glucose levels and, t10c12-CLA improved glucose clearance in models with preexisting insulin resistance: Zucker fatty/diabetic rats, obese and insulin resistant Ldlr-/- mice, and diet-induced obese and insulin resistant male mice. In this study, we assessed whether supplementation of t10c12-CLA in a low-fat diet reduces glucose levels in adipocyte-specific insulin receptor knockout (IRFKO) mice, which is a mouse model of congenital lipodystrophy. In addition to lipodystrophy, IRFKO mice show liver steatosis and increased blood glucose levels that are consequences of whole-body insulin resistance and increased hepatic gluconeogenesis. Briefly, 5-6-week-old male and female control and IRFKO mice were fed with a low-fat diet supplemented with 0.8% t10c12-CLA for 5 weeks. In IRFKO mice, dietary t10c12-CLA did not reduce adipose tissue mass. However, dietary t10c12-CLA increased liver steatosis and plasma insulin levels. Surprisingly, dietary t10c12-CLA reduced glucose levels in both male and female IRFKO mice. Also, dietary t10c12-CLA reduced food intake and polydipsia, which were increased in IRFKO mice due to lipodystrophy and hyperglycemia, respectively. A transcriptomic analysis (RNA-seq) of livers of control and IRFKO male mice fed with a t10c12-CLA diet showed that t10c12-CLA reduced the expression of the key gluconeogenic and mitochondrial fatty oxidation genes. Previous studies have suggested that t10c12-CLA reduces the expression of Forkhead box 01 (Fox01) and Hepatocyte nuclear factor 4 alpha (HNF4α). Of note, the livers of t10c12-CLA-fed control and IRFKO male mice showed a downregulation of positive targets of FoxO1 and HNF4α. In addition, livers of t10c12-CLA-fed mice showed reduced total FOXO1 protein levels, and increased phosphorylation of FOXO1 in t10c12-CLA-fed IRFKO mice. These findings suggest that t10c12-CLA could be directly acting on the liver to alter HNF4α and FOXO1-dependent regulation of hepatic fatty acid oxidation and glucose production. These actions may explain why t10c12-CLA-fed mice do not show increased glucose levels and is potentially linked to the inverse association between t10c12-CLA levels and diabetes risk. Presentation: Saturday, June 17, 2023

Targeting cis-regulatory elements of FOXO family is a novel therapeutic strategy for induction of leukemia cell differentiation

Differentiation therapy has been proposed as a promising therapeutic strategy for acute myeloid leukemia (AML); thus, the development of more versatile methodologies that are applicable to a wide range of AML subtypes is desired. Although the FOXOs transcription factor represents a promising drug target for differentiation therapy, the efficacy of FOXO inhibitors is limited in vivo. Here, we show that pharmacological inhibition of a common cis-regulatory element of forkhead box O (FOXO) family members successfully induced cell differentiation in various AML cell lines. Through gene expression profiling and differentiation marker-based CRISPR/Cas9 screening, we identified TRIB1, a complement of the COP1 ubiquitin ligase complex, as a functional FOXO downstream gene maintaining an undifferentiated status. TRIB1 is direct target of FOXO3 and the FOXO-binding cis-regulatory element in the TRIB1 promoter, referred to as the FOXO-responsive element in the TRIB1 promoter (FRE-T), played a critical role in differentiation blockade. Thus, we designed a DNA-binding pharmacological inhibitor of the FOXO-FRE-T interface using pyrrole-imidazole polyamides (PIPs) that specifically bind to FRE-T (FRE-PIPs). The FRE-PIPs conjugated to chlorambucil (FRE-chb) inhibited transcription of TRIB1, causing differentiation in various AML cell lines. FRE-chb suppressed the formation of colonies derived from AML cell lines but not from normal counterparts. Administration of FRE-chb inhibited tumor progression in vivo without remarkable adverse effects. In conclusion, targeting cis-regulatory elements of the FOXO family is a promising therapeutic strategy that induces AML cell differentiation.

Elevated CDKN1A (P21) mediates β-thalassemia erythroid apoptosis, but its loss does not improve β-thalassemic erythropoiesis

Abstractβ-thalassemias are common hemoglobinopathies due to mutations in the β-globin gene that lead to hemolytic anemias. Premature death of β-thalassemic erythroid precursors results in ineffective erythroid maturation, increased production of erythropoietin (EPO), expansion of erythroid progenitor compartment, extramedullary erythropoiesis, and splenomegaly. However, the molecular mechanism of erythroid apoptosis in β-thalassemia is not well understood. Using a mouse model of β-thalassemia (Hbbth3/+), we show that dysregulated expression of the FOXO3 transcription factor is implicated in β-thalassemia erythroid apoptosis. In Foxo3−/−/Hbbth3/+ mice, erythroid apoptosis is significantly reduced, whereas erythroid cell maturation, and red blood cell and hemoglobin production are substantially improved even with elevated reactive oxygen species in double-mutant erythroblasts. However, persistence of elevated reticulocytes and splenomegaly suggests that ineffective erythropoiesis is not resolved in Foxo3−/−/Hbbth3/+. We found the cell cycle inhibitor Cdkn1a (cyclin-dependent kinase inhibitor p21), a FOXO3 target gene, is markedly upregulated in both mouse and patient–derived β-thalassemic erythroid precursors. Double-mutant p21/Hbbth3/+ mice exhibited embryonic lethality with only a fraction of mice surviving to weaning. Notably, studies in adult mice displayed greatly reduced apoptosis and circulating Epo in erythroid compartments of surviving p21−/−/Hbbth3/+ mice relative to Hbbth3/+ mice, whereas ineffective erythroid cell maturation, extramedullary erythropoiesis, and splenomegaly were not modified. These combined results suggest that mechanisms that control β-thalassemic erythroid cell survival and differentiation are uncoupled from ineffective erythropoiesis and involve a molecular network including FOXO3 and P21. Overall, these studies provide a new framework for investigating ineffective erythropoiesis in β-thalassemia.

Maternal diabetes increases FOXO1 activation during embryonic cardiac development

Maternal diabetes is known to affect heart development, inducing the programming of cardiac alterations in the offspring's adult life. Previous studies in the heart of adult offspring have shown increased activation of FOXO1 (a transcription factor involved in a wide variety of cellular functions such as apoptosis, cellular proliferation, reactive oxygen species detoxification, and antioxidant and pro-inflammatory processes) and of target genes related to inflammatory and fibrotic processes. In this work, we aimed to evaluate the effects of maternal diabetes on FOXO1 activation as well as on the expression of target genes relevant to the formation of the cardiovascular system during organogenesis (day 12 of gestation). The embryonic heart from diabetic rats showed increased active FOXO1 levels, reduced protein levels of mTOR (a nutrient sensor regulating cell growth, proliferation and metabolism) and reduced mTORC2-SGK1 pathway, which phosphorylates FOXO1. These alterations were related to increases in the levels of 4-hydroxynonenal (an oxidative stress marker) and increased mRNA levels of inducible nitric oxide synthase, angiopoietin-2 and matrix metalloproteinase-2 (MMP2) (all FOXO1 target genes relevant for cardiac development). Results also showed increased extracellular and intracellular immunolocalization of MMP2 in the myocardium and its projection into the lumen of the cavity (trabeculations) together with decreased immunostaining of connexin 43, a protein relevant for cardiac function that is target of MMP2. In conclusion, increases in active FOXO1 induced by maternal diabetes initiate early during embryonic heart development and are related to increases in markers of oxidative stress and of proinflammatory cardiac development, as well to an altered expression of proteolytic enzymes that regulate connexin 43. These alterations may lead to an altered programming of cardiovascular development in the embryonic heart of diabetic rats.

242-LB: A Novel FoxO Target, LRRC2, Preserves Mitochondrial Function in Muscle from Insulin-Deficient Mice

Uncontrolled diabetes impairs muscle strength and mitochondrial metabolism, leading to increased mortality. FoxOs are key mediators of insulin signaling and we demonstrated that FoxOs mediate muscle atrophy and mitochondrial dysfunction in mice with streptozotocin (STZ) diabetes. Unfortunately, FoxOs are also tumor suppressors and knockout of FoxOs in proliferative tissues causes cancer. We aimed to investigate FoxO targets restricted to skeletal muscle in hopes of improving strength in diabetic conditions. RNA-seq from STZ mice with/without FoxO1/3/4 triple knockout showed several diabetes-induced FoxO targets, with the most robust being LRRC2 (Leucine rich repeating containing 2). The function of LRRC2 is unknown, but gene expression databases show LRRC2 is restricted to muscle and heart, and LRRC2 is enriched in human heart with dilated cardiomyopathy. Adenoviral overexpression of LRRC2 in C2C12 cells localized to mitochondria and showed increase mRNA levels of biogenesis marker PGC1α, and antioxidant genes SOD2 and Gpx3. However, LRRC2 overexpression did not change basal respiration measured in Seahorse. To determine LRRC2's effect on exercise tolerance and mitochondrial bioenergetics in vivo, we created muscle specific LRRC2 knockout (LKO) mice and rendered them diabetic with STZ. Diabetic LKO mice did worse than their littermate controls on treadmill exercise to exhaustion. Soleus permeabilized fibers showed significantly decreased ATP production that was uncoupled from respiration with glutamate/malate. Interestingly, soleus muscles from LKO mice were significantly larger than their littermate controls, whereas extensor digitorum longus and quadriceps were smaller in the diabetic LKO mice. Lastly, mRNA expression of mitochondrial subunits was not changed and thus did not explain the mitochondrial defects. In conclusion, LRRC2 is a novel FoxO target that helps preserve mitochondrial function and exercise tolerance in a Type 1 diabetes mouse model. Disclosure C. M. Penniman: None. G. Bhardwaj: None. C. Nowers: None. B. T. O'neill: Stock/Shareholder; Bristol-Myers Squibb Company, Pfizer Inc., Johnson &amp; Johnson. Funding U.S. Department of Veterans Affairs (lO1BXOO4468)

PRMT5 inhibition drives therapeutic vulnerability to combination treatment with BCL-2 inhibition in mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) is an incurable B-cell malignancy that comprises up to 6% of non-Hodgkin lymphomas diagnosed annually and is associated with a poor prognosis. The average overall survival of patients with MCL is 5 years, and for most patients who progress on targeted agents, survival remains at a dismal 3 to 8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated to improve treatment outcomes and quality of life. The protein arginine methyltransferase 5 (PRMT5) enzyme is overexpressed in MCL and promotes growth and survival. Inhibition of PRMT5 drives antitumor activity in MCL cell lines and preclinical murine models. PRMT5 inhibition reduced the activity of prosurvival AKT signaling, which led to the nuclear translocation of FOXO1 and modulation of its transcriptional activity. Chromatin immunoprecipitation and sequencing identified multiple proapoptotic BCL-2 family members as FOXO1-bound genomic loci. We identified BAX as a direct transcriptional target of FOXO1 and demonstrated its critical role in the synergy observed between the selective PRMT5 inhibitor, PRT382, and the BCL-2 inhibitor, venetoclax. Single-agent and combination treatments were performed in 9 MCL lines. Loewe synergy scores showed significant levels of synergy in most MCL lines tested. Preclinical, in vivo evaluation of this strategy in multiple MCL models showed therapeutic synergy with combination venetoclax/PRT382 treatment with an increased survival advantage in 2 patient-derived xenograft models (P ≤ .0001, P ≤ .0001). Our results provide mechanistic rationale for the combination of PRMT5 inhibition and venetoclax to treat patients with MCL.

FOXO1 cooperates with C/EBPΔ and ATF4 to regulate skeletal muscle atrophy transcriptional program during fasting

Catabolic conditions, such as starvation, inactivity, and cancer cachexia, induce Forkhead box O (FOXO) transcription factor(s) expression and severe muscle atrophy via the induction of ubiquitin–proteasome system-mediated muscle proteolysis, resulting in frailty and poor quality of life. Although FOXOs are clearly essential for the induction of muscle atrophy, it is unclear whether there are other factors involved in the FOXO-mediated transcriptional regulation. As such, we identified FOXO–CCAAT/enhancer binding protein Δ (C/EBPΔ) signaling pathway as a novel proteolytic pathway. By comparing the gene expression profiles of FOXO1-transgenic (gain-of-function model) and FOXO1,3a,4–/–(loss-of-function model) mice, we identified several novel FOXO1-target genes in skeletal muscle including Redd1, Sestrin1, Castor2, Chac1, Depp1, Lat3, as well as C/EBPΔ. During starvation, C/EBPΔ abundance was increased in a FOXOs-dependent manner. Notably, knockdown of C/EBPΔ prevented the induction of the ubiquitin–proteasome system and decrease of myofibers in FOXO1-activated myotubes. Conversely, C/EBPΔ overexpression in primary myotubes induced myotube atrophy. Furthermore, we demonstrated that FOXO1 enhances the promoter activity of target genes in cooperation with C/EBPΔ and ATF4. This research comprehensively identifies novel FOXO1 target genes in skeletal muscle and clarifies the pathophysiological role of FOXO1, a master regulator of skeletal muscle atrophy. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

RHODIOLA ROSEA AND FERULIC ACID ACTIVATE EXPRESSION OF GENES RELATED TO AUTOPHAGY AND RESISTANCE TO HEAT SHOCK IN MICE OF DIFFERENT AGE

The aim of our study was testing whether R. rosea extract and ferulic acid activate expression of targets of FoxO, regulators of energy metabolism and autophagy in livers of young and old mice, and to what extent the effects of R. rosea extract and ferulic acid on the genes studied coincide. Methods. . C57BL/6J mice were reared at 22 ± 2 °С, 50-60% humidity, and 12/12 hour light/dark cycle. All groups were reared on a standard chow (4,8% fats, 21,8% protein, and 3,9% fibre). Experimental groups consumed water, supplemented with either sodium ferulate or R. rosea during 12 weeks prior sacrificing. The amounts of ferulate and R. rosea were adjusted to provide 4 mg of phenol-containing substances per 100 g weight, for a mouse, for 24 hours. We tested three-month-old (“young”) and twelve-month-old males (“old”). The levels of messenger ribonucleic acid (mRNA) were assessed using AriaMx real-time polymerase chain reaction (RT-PCR) instrument (Agilent). Ribonucleic acid was purified using the Monarch Miniprep kit (New England BioLabs (NEB), T2010), complementary deoxyribonucleic acid synthesis was performed using the ProtoScript II kit (NEB, E6560), and quantitative RT-PCR (qRT-PCR) was performed using the Luna Universal kit (NEB, E3003). The expression of genes ATG5 (an autophagy marker), HSPB8 (a small heat shock protein, an FoxO target), UCP2 (uncoupling protein 2, a senescence marker), CDKN2 (cell cycle regulator, a senescence marker), PDK2 and PDK4 (pyruvate dehydrogenase kinases 2 and 4, regulators of oxidative metabolism), and TFEB (transcription factor EB, a transcriptional regulator of autophagy) was evaluated. Results. Livers of young mice that consumed food supplemented with either sodium ferulate or R. rosea extract had 3.2-fold and 3.6-fold higher levels of mRNA of the small heat shock protein HspB8 than control mice, respectively. In old mice, the levels of mRNA for this protein were 3.3-fold higher in mice reared on the diet containing R. rosea extract as compared with the control. However, there was no significant difference between control mice and those that consumed ferulate-supplemented food. In young mice, ferulate and R. rosea extract induced synthesis of mRNA of PDK4 by 4.3 and 6.6 times from the control level, respectively. Ferulate and R. rosea extract also affected the levels of mRNA of ATG5 and PDK2 in the livers of old mice. The levels of PDK2 were 1.5-fold higher in the livers of mice that consumed ferulate-supplemented food than in control mice. Conclusions. Both, R. rosea extract and one of its active components – ferulic acid – promote increasing in the levels of mRNA for genes HSPB8 and PDK4, coding for small heat shock protein and pyruvate dehydrogenase kinase 4, respectively. In old mice, R. rosea promote expression of HSPB8, ATG5, PDK2, and PDK4. Thus, ferulic acid and R. rosea exert similar effects on gene expression by supposed activation of heat shock response and autophagy, and concomitant inhibition of mitochondrial metabolism via boosting expression of PDK2 and PDK4.

Juvenile hormone suppresses the FoxO-takeout axis to shorten longevity in male silkworm

Juvenile hormone (JH) plays a crucial endocrine regulatory role in insect metamorphosis, reproduction, and longevity in multiple organisms, such as flies, honeybees, and migratory monarch butterflies. However, the molecular mechanism of JH affecting longevity remains largely unknown. In this study, we showed that JH III and its analog methoprene shortened the survival days significantly in the adulthood of male silkworm. At the same time, the allatostatin, a neuropeptide that inhibits the secretion of JH by the corpora allata, could extend the survival days dramatically after adult eclosion in male silkmoth. Interestingly, a central pro-longevity FoxO transcription factor was reduced upon JH stimulation in silkworm individuals and BmN-SWU1 cells. Furthermore, the analysis of the upstream sequence of the FoxO gene identified a JH response element which suggested that FoxO might be regulated as a target of JH. Surprisingly, we identified a Bmtakeout (BmTO) gene that encodes a JH-binding protein and contains a FoxO response element. As expected, FoxO overexpression and knockdown up- and down-regulated the expression of BmTO respectively, indicating that BmTO functions as a FoxO target. BmTO overexpression could release the inhibitory effect of JH on the BmFoxO gene by reducing JH bioavailability to block its signal transduction. Collectively, these results may provide insights into the mechanism of the JH-FoxO-TO axis in aging research and pest control.

Combined low-pass whole genome and targeted sequencing in liquid biopsies for pediatric solid tumors

We designed a liquid biopsy (LB) platform employing low-pass whole genome sequencing (LP-WGS) and targeted sequencing of cell-free (cf) DNA from plasma to detect genome-wide copy number alterations (CNAs) and gene fusions in pediatric solid tumors. A total of 143 plasma samples were analyzed from 19 controls and 73 patients, including 44 bone or soft-tissue sarcomas and 12 renal, 10 germ cell, five hepatic, and two thyroid tumors. cfDNA was isolated from plasma collected at diagnosis, during and after therapy, and/or at relapse. Twenty-six of 37 (70%) patients enrolled at diagnosis without prior therapy (radiation, surgery, or chemotherapy) had circulating tumor DNA (ctDNA), based on the detection of CNAs from LP-WGS, including 18 of 27 (67%) patients with localized disease and eight of 10 (80%) patients with metastatic disease. None of the controls had detectable somatic CNAs. There was a high concordance of CNAs identified by LP-WGS to CNAs detected by chromosomal microarray analysis in the matching tumors. Mutations identified in tumor samples with our next-generation sequencing (NGS) panel, OncoKids®, were also detected by LP-WGS of ctDNA in 14 of 26 plasma samples. Finally, we developed a hybridization-based capture panel to target EWSR1 and FOXO1 fusions from patients with Ewing sarcoma or alveolar rhabdomyosarcoma (ARMS), respectively. Fusions were detected in the plasma from 10 of 12 patients with Ewing sarcoma and in two of two patients with ARMS. Combined, these data demonstrate the clinical applicability of our LB platform to evaluate pediatric patients with a variety of solid tumors.

Activation of Angiopoietin-Tie2 Signaling Protects the Kidney from Ischemic Injury by Modulation of Endothelial-Specific Pathways.

Ischemia-reperfusion AKI (IR-AKI) is common and causes significant morbidity. Effective treatments are lacking. However, preclinical studies suggest that inhibition of angiopoietin-Tie2 vascular signaling promotes injury, whereas activation of Tie2 is protective. We show that kidney ischemia leads to increased levels of the endothelial-specific phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP; PTPRB), which inactivates Tie2. Activation of Tie2 through VE-PTP deletion, or delivery of a novel angiopoietin mimetic (Hepta-ANG1), abrogated IR-AKI in mice. Single-cell RNAseq analysis showed Tie2 activation promotes increased Entpd1 expression, downregulation of FOXO1 target genes in the kidney vasculature, and emergence of a new subpopulation of glomerular endothelial cells. Our data provide a molecular basis and identify a candidate therapeutic to improve endothelial integrity and kidney function after IR-AKI. Ischemia-reperfusion AKI (IR-AKI) is estimated to affect 2%-7% of all hospitalized patients. The significant morbidity and mortality associated with AKI indicates urgent need for effective treatments. Previous studies have shown activation of the vascular angiopoietin-Tie2 tyrosine kinase signaling pathway abrogates ischemia-reperfusion injury (IRI). We extended previous studies to (1) determine the molecular mechanism(s) underlying kidney injury and protection related to decreased or increased activation of Tie2, respectively, and (2) to test the hypothesis that deletion of the Tie2 inhibitory phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP) or injection of a new angiopoietin mimetic protects the kidney from IRI by common molecular mechanism(s). Bilateral IR-AKI was performed in VE-PTP wild-type or knockout mice and in C57BL/6J mice treated with Hepta-ANG1 or vehicle. Histologic, immunostaining, and single-cell RNA sequencing analyses were performed. The phosphatase VE-PTP, which negatively regulates the angiopoietin-Tie2 pathway, was upregulated in kidney endothelial cells after IRI, and genetic deletion of VE-PTP in mice protected the kidney from IR-AKI. Injection of Hepta-ANG1 potently activated Tie2 and protected the mouse kidney from IRI. Single-cell RNAseq analysis of kidneys from Hepta-ANG1-treated and vehicle-treated mice identified endothelial-specific gene signatures and emergence of a new glomerular endothelial subpopulation associated with improved kidney function. Overlap was found between endothelial-specific genes upregulated by Hepta-ANG1 treatment and those downregulated in HUVECs with constitutive FOXO1 activation, including Entpd1 / ENTPD1 that modulates purinergic receptor signaling. Our data support a key role of the endothelium in the development of IR-AKI, introduce Hepta-ANG1 as a putative new therapeutic biologic, and report a model to explain how IRI reduces Tie2 signaling and how Tie2 activation protects the kidney. This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_05_23_JSN_Ang_EP23_052323.mp3.

Investigating the Micrornas as Key Regulator in Diabetic Cardiomyopathy

Diabetic Cardiomyopathy (DCM) is a state in which cardiomyopathy occurs in patients due to diabetes mellitus without valvular and coronary artery disease. The DCM prevalence is about 50% in patients diagnosed with diabetes at either clinical or preclinical stages. DCM is characterized by structural and metabolic myocardial changes established in patients with diabetes mellitus. These changes are triggered by insulin resistance and hyperinsulinemia that lead to the dysregulation of different signaling pathways. Despite the improvement in pharmacological interventions in DCM, still, new reliable strategies are required. MicroRNAs are small non-coding molecules that play a significant role in the regulation of genes at transcriptional and translational levels. We have found the clinical tested genes involved in DCM from gene target registry (GTR), and different miRNAs targeting these genes were identified by using bioinformatics tools NCBI, miRanda, TargetScan, miRBase, and TarBase. From the clinical data of DCM patients, we found that the number of genes are dysregulated which can be the putative target of miRNAs as they directly target these genes by binding with 8mer unit i.e STAT3 (having target binding site for miR-124-3p.1, miR125-5p), MAPK14 (targeted by miR128-3p, miR-124-3p.2, miR-124.3p.1), PRKAA1 (target site for miR-137, miR-19-3p, miR-148-3p), FOXO1 (target binding site for miR-135-3p, miR-96-5p), STK11 (putative target of miR17-5p/20-5p), and SOCS3 can be a potential target of miR-30-5p, miR-19-3p, miR-218-5p. Conclusively, we put forward the different miRNAs which are targeting the genes involved in DCM which may serve as a platform for the development of a miRNA-based therapeutic or diagnostic strategy for the treatment of diabetic cardiomyopathy.