Signaling Markers Research Articles

Small-molecule AMPK activation preserves anabolic signaling in a C2C12 myotube cachexia model

AMP-activated protein kinase (AMPK) is a master metabolic regulator that responds to a decline in cellular energy status by promoting processes that generate ATP, while inactivating anabolic processes that consume ATP, thereby restoring the energy charge of the cell. In line with this general function, acute AMPK activation in healthy muscle cells potently inhibits anabolic signaling through the mechanistic target of rapamycin (mTORC1). However, treatment of atrophying muscle (from tumor-bearing mice and cells cultured in tumor cell-conditioned media) with the non-specific AMPK activator AICAR suggests that AMPK has a protective effect on muscle mass and anabolic signaling. We have identified MLX-0867 as a novel AMPK-activating small molecule (EC50 = 32.0 mM in C2C12 myotubes). We hypothesized that similar to AICAR, treatment with MLX-0867 would also preserve muscle mass and anabolic mTORC1 signaling in cultured muscle subjected to hyperinflammatory signaling. To test this, we treated C2C12 myotubes with control media (CM) or CM + 20 ng/mL TNF-ɑ and 100 U/mL interferon-γ (T/I) for 24 hours. Additionally, the cells were concurrently treated with 250 mM AICAR, 40 mM MLX-0867 (a novel AMPK activating small-molecule), or vehicle control during the treatment period. To visualize myotubes, myosin heavy chain (MHC) was detected by immunofluorescence, and average myotube diameter was measured. In a separate experiment, the protein was extracted from treated myotubes to determine the phosphorylation of p-70S6k and ribosomal protein S6 (rpS6) as markers of anabolic signaling through mTORC1. We found that T/I treatment significantly decreased myotube diameter. Neither AICAR nor MLX-0867 attenuated this atrophy. However, while T/I substantially reduced S6k and rpS6 phosphorylation, MLX-0867 (but not AICAR) partially preserved the phosphorylation of rpS6 and tended (p=0.11) to preserve p-70S6k phosphorylation under T/I treatment. In summary, MLX-0867 partially preserved anabolic signaling in T/I treated myotubes. However, this did not preserve muscle mass, at least under the conditions utilized in this experiment. A Brigham Young University Widtsoe Grant and a sponsored research agreement with Biolexis Therapeutics funded this research. This is the full abstract presented at the American Physiology Summit 2024 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.

Oxidative stress: A driving force in CKD induced heart failure

Background: Several clinical investigations have shown that individuals with renal impairment are more likely to experience heart failure. Cardiac remodeling happens relatively early in the course of kidney injury, yet research into identification of common markers of cardiorenal signaling remains insuffcient. This may be because studies conducted in small animal models do not accurately reflect the complexity of the disease. To bridge the translational gap, we developed a swine model for chronic kidney disease (CKD) Aim: We employed proteome studies to clarify the pathophysiological impact of chronic kidney disease (CKD) on the heart and correlate to cardiac function and perfusion in order to offer mechanistic insights. Methods: We used microspheres to embolise both kidneys of swine at 10-12 weeks of age while sham operated swine served as controls. Post 6 months, we performed pressure volume loops to assess cardiac function, while coronary flow reserve (CFR) was measured by intracoronary infusion of adenosine in control and CKD. (LC-MS-MS based proteomic analyses of left ventricular tissue was performed. Paraffn embedded myocardium and kidney tissues were histologically examined for interstitial fibrosis and oxidative stress. Results: Renal embolization resulted in mild chronic kidney injury as evidenced by increased fibrosis staining and urinary NGAL (24±4 vs 48±9 pg/ml). Hemodynamic parameters showed increased wall stress (20±3 vs 40±7 ml*mmHg/g) as well as increase of end diastolic volume (1.5±0.1 vs 1.9±0.1 ml/kg) indicating dilation of the left ventricle in CKD. Quantitative proteomic analysis detected significant differences in the left ventricle of the controls when compared to those with CKD and STRING pre-ranked functional analysis showed enrichments in pathways related to reactive oxygen species. Furthermore, we detected alterations of pathways associated with remodeling of the extracellular matrix (ECM), Oxidative stress and ECM remodeling were confirmed histologically. Interestingly, in CKD we found that mitochondrial proteins like cytochrome oxidase (MT-CO2) and ATP5 synthase subunit were downregulated suggesting mitochondrial dysfunction which was associated with higher basal coronary blood flow (0.48±0.05 vs 0.87±0.11 ml/min/g). Conclusions: Our proteomic data in swine model provides strong evidence that mild CKD can induce early alterations in mitochondrial function along with oxidative stress and ECM remodeling. Thus, we aim to further our understanding by applying our proteomic findings in conjunction with functional hemodynamic data in this swine model of cardiorenal disease. German Center for Cardiovascular Research (DZHK81Z0600207 to D.M. and P.S.). This is the full abstract presented at the American Physiology Summit 2024 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.

Role of Adiponectin in the Androgen-Mediated Cardiometabolic Complications in a Rat Model of Polycystic Ovary Syndrome

Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women and is associated with a high prevalence of obesity, adipose insulin resistance (adipose IR), and insulin resistance (IR). Adipose IR is an early metabolic defect in the development of whole-body IR. Specific evaluation of adipose IR and its consequences on the whole-body metabolic state in women with PCOS has not been thoroughly investigated. Adiponectin is a peptide secreted by adipocytes that increases insulin sensitivity. PEG-BHD1028 is a novel potent peptide adiponectin receptor agonist. Despite that PEG-BHD1028 modulates insulin sensitivity in different models of diabetes, the potential of PEG-BHD1028 to attenuate androgen-mediated adipose IR and obesity remains unknown. Hypothesis: We tested the hypothesis that PEG-BHD1028 attenuates adipose IR and obesity in a rat model of PCOS. Methods: To induce PCOS, four-week-old female Sprague Dawley rats were implanted with dihydrotestosterone (DHT) silastic tubes (7.5 mg) or placebo (PBO) for 90 days (n=4-8/group). During the final three weeks of DHT treatment, rats received PEG-BHD1028 (25 μg/kg/day) subcutaneously. Body weight (BW) by gravimetry, fat and lean mass were measured by Echo-MRI. Fasting plasma insulin was measured by ELISA. Fasting plasma glucose and non-esterified fatty acids (NEFA) were measured with a clinical chemistry analyzer. HOMA-IR, an index of systemic IR, and adipose IR (fasting insulin x fasting NEFA) were calculated. Protein levels of insulin signaling markers including IRS1, p-IRS1 (Ser1101), PI3 kinase p110, Akt, p-Akt (SER473), GLUT4, mTOR, p-mTOR (Ser2481) were quantified in the retroperitoneal fat by Western-blot. Results: DHT significantly (p<0.05) increased BW (352.67± 6.93 vs 250.88 ± 5.26 g), fat mass (20.82 ± 1.48 vs 9.44 ± 1.81 g), and lean mass (316.92 ± 4.15 vs 224.45 ± 4.79 g), compared to PBO group. DHT significantly (p<0.05) increased HOMA-IR and adipose IR by 75% and 80%, respectively, compared to PBO group. DHT downregulated retroperitoneal fat p-IRS-1, IRS-1, p-mTOR, mTOR, GLUT4, PI3 kinase and Akt protein expression by 50% to 70% while upregulating p-Akt by 1.8- fold compared to the PBO group. PEG-BHD1028 significantly decreased BW, fat mass and lean mass while attenuating IR by 34.7% reduction in HOMA-IR and significantly (p<0.05) decreased adipose IR by 51.6% in PCOS rats. Interestingly, PEG-BHD1028 significantly (p<0.05) upregulated p-IRS-1, IRS-1, p-mTOR, mTOR and p-AKT levels by 2.6-, 3-, 2.2-, 4-, 2.6- fold, respectively with no impact on the other insulin signaling markers compared to vehicle-treated group. Conclusion: PCOS rats exhibit obesity and lower adiponectin levels that are associated with both adipose and whole-body insulin resistance. Activating adiponectin receptors with PEG-BHD1028 attenuated PCOS-associated obesity and adipose IR. Significance: Targeting adiponectin signaling could be a novel and effective therapeutic approach to mitigate metabolic dysfunction in females with excess androgens. Supported by NIH-NIGMS grant P20GM121334 (L.L.Y.C. and D.G.R.), NIH-NIGMS grant P20GM121334, P20GM104357, NIH-NHLBI grant P01HL51971, NIH-NIMHD-P50MD017338 and University of Mississippi Medical Center Medical Student Research Program (R.C.) and the Robert M. Hearin Foundation (R.C.). This is the full abstract presented at the American Physiology Summit 2024 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.

Single-cell RNA sequencing reveals an immunomodulatory population of double negative T-cells in the diabetic NOD mouse

Abstract NOD mice are a widely used model of type 1 diabetes, an inflammatory autoimmune condition characterized by autoreactive T-cells that target insulin producing beta cells of the pancreatic islets. While T cell biology in the context of this disease is important it is unclear why autoreactive T-cells develop in this model or what they do within the islets themselves. We have designed a single cell sequencing experiment where enriched T-cells taken from diabetic and non-diabetic NOD mice are sequenced for mRNA expression and TCR sequence concurrently. The resulting sequencing data shows abnormal representation of Double Negative (DN) T-cells in all tissues and conditions (~30%). We have observed that like CD8+ and CD4+ T-cells, this population displays normal maturation phenotypes, but experience reduced exhaustion of effector subsets after infiltration of the pancreatic islets. Looking at clonal subpopulations of T-cells we have discovered a subset of CD8+ T-cells that have high expression of GzmA even after loss of the survival signal marker, IL7r. Through investigation of these aberrant T-cell populations, we have found that the diabetic NOD mouse pancreatic is highly immunosuppresive and expression of T-cell co-receptors may have a significant impact on diabetic onset.

Effect of Lampaya medicinalis Phil. (Verbenaceae) and Palmitic Acid on Insulin Signaling and Inflammatory Marker Expression in Human Adipocytes.

Aging and obesity are associated with insulin resistance (IR) and low-grade inflammation. Molecularly, IR is characterized by a reduction in glucose uptake and insulin signaling (IRS-1/Akt/AS160 pathway), while inflammation may result from upregulated NF-κB pathway after low Tyr-IκBα phosphorylation. Upregulated phosphatase activity of PTP1B is associated with impaired insulin signaling and increased inflammation. Plasma levels of palmitic acid (PA) are elevated in obesity, triggering inflammation and disruption of insulin signaling. Traditional medicine in Northern Chile uses oral infusions of Lampaya medicinalis Phil. (Verbenaceae) to treat inflammatory conditions. Significant amounts of flavonoids are found in the hydroethanolic extract of Lampaya (HEL), which may account for its biological activity. The aim of this work was to study the effect of HEL and PA on insulin signaling and glucose uptake as well as inflammatory marker expression in human adipocytes. We studied HEL effects on PA-induced impairment on insulin signaling, glucose uptake and inflammatory marker content in human SW872 adipocytes. HEL cytotoxicity was assessed in adipocytes at different concentrations (0.01 to 10 g/mL). Adipocytes were incubated or not with PA (0.4 mM, 24 h) with or without HEL (2 h pre-incubation), and then stimulated with insulin (10 min, 100 mM) or a vehicle. Phospho-IRS-1, phospho-Akt, phospho-AS160, phospho-NF-κB and phospho-IκBα, as well as protein levels of PTP1B, were assessed using Western blotting, and glucose uptake was evaluated using the 2-NBDG analogue. At the assessed HEL concentrations, no cytotoxic effects were observed. PA decreased insulin-stimulated phospho-Akt and glucose uptake, while co-treatment with HEL increased such markers. PA decreased phospho-IRS-1 and phospho-Tyr-IκBα. On the other hand, incubation with HEL+PA decreased phospho-AS160 and phospho-NF-κB compared with cells treated with PA alone. Our results suggest a beneficial effect of HEL by improving PA-induced impairment on molecular markers of insulin signaling, glucose uptake and inflammation in adipocytes. Further studies are necessary to elucidate whether lampaya may constitute a preventive strategy for people whose circulating PA levels contribute to IR and inflammation during aging and obesity.

Binge alcohol induces NRF2-related antioxidant response in the skeletal muscle of female mice

BackgroundChronic alcohol enhances oxidative stress, but the temporal response of antioxidant genes in skeletal muscle following a binge drinking episode remains unknown. MethodsExperiment 1: C57BL/6Hsd female mice received an IP injection of saline (CON; n = 39) or ethanol (ETOH; n = 39) (5 g/kg). Gastrocnemius muscles were collected from baseline (untreated; n = 3), CON (n = 3), and ETOH (n = 3) mice every 4 h for 48 h. Experiment 2: Gastrocnemius muscles were collected from control-fed (CON-FED; n = 17), control-fasted (CON-FAST; n = 18), or alcohol-fed (ETOH-FED; n = 18) mice every 4hrs for 20hrs after saline or ethanol (5 g/kg). ResultsEtOH enhanced Superoxide dismutase 1 (Sod1) and NADPH Oxidase 4 (Nox4) from 24 to 48hr after the binge, while Sod2 and Nox2 were suppressed. Nuclear factor erythroid-derived 2-like 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) increased 12hrs after intoxication. Cytochrome P450 oxidoreductase (Por), Heme oxygenase 1 (Ho1), Peroxiredoxin 6 (Prdx6), Glutamate-cysteine ligase catalytic subunit (Gclc), Glutamate-cysteine ligase modifier subunit (Gclm), and Glutathione-disulfide reductase (Gsr) were increased by ETOH starting 12–16hrs post-binge. Fasting had similar effects on Nrf2 compared to alcohol, but downstream targets of NRF2, including Por, Ho1, Gclc, and Gclm, were differentially altered with fasting and EtOH. ConclusionThese data suggest that acute alcohol intoxication induced markers of oxidative stress and antioxidant signaling through the NRF2 pathway and that there were effects of alcohol independent of a possible decrease in food intake caused by binge intoxication.

Altered Glycolysis, Mitochondrial Biogenesis, Autophagy and Apoptosis in Peritoneal Endometriosis in Adolescents.

Energy metabolism plays a pivotal role in the pathogenesis of endometriosis. For the initial stages of the disease in adolescents, this aspect remains unexplored. The objective of this paper was to analyze the association of cellular and endosomal profiles of markers of glycolysis, mitochondrial biogenesis, apoptosis, autophagy and estrogen signaling in peritoneal endometriosis (PE) in adolescents. We included 60 girls aged 13-17 years in a case-control study: 45 with laparoscopically confirmed PE (main group) and 15 with paramesonephric cysts (comparison group). Samples of plasma and peritoneal fluid exosomes, endometrioid foci and non-affected peritoneum were tested for estrogen receptor (Erα/β), hexokinase (Hex2), pyruvate dehydrogenase kinase (PDK1), glucose transporter (Glut1), monocarboxylate transporters (MCT1 and MCT2), optic atrophy 1 (OPA1, mitochondrial fusion protein), dynamin-related protein 1 (DRP1, mitochondrial fission protein), Bax, Bcl2, Beclin1, Bnip3, P38 mitogen-activated protein kinase (MAPK), hypoxia-inducible factor 1 (Hif-1α), mitochondrial voltage-dependent anion channel (VDAC) and transforming growth factor (TGFβ) proteins as markers of estrogen signaling, glycolysis rates, mitochondrial biogenesis and damage, apoptosis and autophagy (Western-Blot and PCR). The analysis identified higher levels of molecules associated with proliferation (ERβ), glycolysis (MCT2, PDK1, Glut1, Hex2, TGFβ and Hif-1α), mitochondrial biogenesis (OPA1, DRP1) and autophagy (P38, Beclin1 and Bnip3) and decreased levels of apoptosis markers (Bcl2/Bax) in endometrioid foci compared to non-affected peritoneum and that in the comparison group (p < 0.05). Patients with PE had altered profiles of ERβ in plasma and peritoneal fluid exosomes and higher levels of Glut1, MCT2 and Bnip3 in plasma exosomes (p < 0.05). The results of the differential expression profiles indicate microenvironment modification, mitochondrial biogenesis, estrogen reception activation and glycolytic switch along with apoptosis suppression in peritoneal endometrioid foci already in adolescents.

High fructose Corn Syrup recast glucose transporter-5, Wnt, NF-κB signalling and mitochondrial apoptosis in an animal model of oral oncogenesis

BackgroundWnt signalling pathway, is mediated by members of T-cell factor (TCF) transcription factors family, is essential for the control of epithelial cell proliferation and death. Glucose transporter-5 (GLUT5), fructose-specific transporter, is also important in allowing transcellular fructose uptake. The goal of this work to determine how the High fructose Corn Syrup (HFCS) affected Wingless-related integration site (Wnt) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling in the 7,12 –dimethylbenzaanthracene (DMBA)-induced hamster buccal pouch carcinogenesis (HBPCs) model. MethodsFour groups of hamsters were created. For 12 weeks, 0.5 % DMBA was applied 3 times/week to the left side buccal pouches of the hamsters in groups (2 & 4). Additionally, the animals in groups (3 & 4) were given through drinking water of HFCS 25 %. The control animals were from group 1. By using western blot analysis, signalling network markers of the GLUT-5, Wnt, TCF-4, GSK-3β and NF-κB as well as mitochondrial apoptotic pathway marker expression B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cyclooxygenase – 2 (COX-2) was assessed. ResultsDrinking water uptake of 25% HFCS encouraged progress of HBP carcinomas by constitutive stimulating of the Wnt pathway via GSK-3β overexpression. HFCS suppressed Wnt signalling which contributed the NF-κB attenuation and changes the signalling markers in apoptotic network. ConclusionsOur hypothesis suggests a mechanically crosstalk between Wnt and NF-κB signalling pathways in HBP carcinomas that is developed by HFCS. HFCS that targets the Wnt pathway and its downstream signalling mediators could be additive reason for cancer development.

Abstract 6956: Role of soluble CD109 in the progression of head and neck squamous cell carcinoma

Abstract Introduction: About five percent of global cancer deaths are attributed to Head and Neck Squamous Cell Carcinoma (HNSCC), and its annual incidence is predicted to skyrocket by thirty percent by 2030 (according to GLOBOCAN estimates). HNSCC originates from squamous cells in the oral cavity, lips, tongue, larynx, pharynx, and salivary glands. Despite the comparatively easy tumor diagnosis, the number of patients diagnosed at an advanced stage with metastatic spread is substantial. Multiple cell-signaling pathways have been proven to be dysregulated in HNSCC, including the epidermal growth factor receptor (EGFR). Our group has identified CD109, a GPI-anchored membrane-associated protein, as a TGF-β co-receptor that inhibits TGF-β signaling. CD109 expression is upregulated in SCC, and its elevated expression in premalignant lesions is associated with an increased risk of metastatic progression. Recent studies by our team have revealed that CD109 promotes tumor growth in vivo, likely by enhancing EGFR signaling. Interestingly, we have also discovered that membrane CD109 (mCD109) is endogenously released from the cell surface; however, the mechanism of action of the released/soluble form of CD109 (sCD109) remains unknown. In the current study, we examined whether sCD109 opposes the mCD109/EGFR-mediated pro-tumorigenic signaling and HNSCC progression. Research Questions: 1.Does sCD109 regulate EGF-induced EGFR signaling and downstream stem cell marker expression?2.Does sCD109 regulate EGFR expression levels on the HNSCC cell surface?3.Does sCD109 affect the nuclear localization of EGFR?4.Does sCD109 promote EGFR internalization and degradation? Methodology: HNSCC cells and patient-derived primary cells were treated with/without sCD109 recombinant protein (1nM, 5nM, and 10nM) without and with the EGF induction (10ng/ml for 15 mins) and were analyzed by western blotting, immunofluorescence, and immunocytochemistry. The role of sCD109 in regulating membrane EGFR levels was assessed by flow cytometry and surface protein biotinylation assay. Results: Our findings demonstrated that sCD109 inhibited EGF-induced phosphorylation of EGFR (Y1068), STAT3, AKT, and ERK. The sCD109 suppressed the stem cell marker expression (Nanog, OCT4, SOX2). Additionally, sCD109 hindered the nuclear localization of activated EGFR. The sCD109 decreased the membrane EGFR expression levels and activated the phosphorylation of EGFR (Y1045), suggesting a role for sCD109 in promoting EGFR degradation. Conclusion: Our team has previously reported that mCD109 is pro-tumorigenic, and our current findings suggest that sCD109 opposes the pro-tumorigenic effects of mCD109. This study aimed to determine how sCD109 regulates cellular signaling pathways and will provide key insights into the molecular mechanisms underlying the progression of HNSCC, a highly prevalent malignancy. Citation Format: Varsha Reddy Durgempudi, Tenzin Kungyal, Anie Philip. Role of soluble CD109 in the progression of head and neck squamous cell carcinoma [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 6956.

Abstract 6056: Identification of first-in-class, orally bioavailable SOS1 bifunctional degraders for the treatment of KRAS- and RTK-driven cancers

Abstract Monotherapies which target nodes within the RTK-RAS-MAPK pathway often display early potency in the clinic, but their preliminary response is limited as tumors begin to regrow due to adaptive and acquired resistance mechanisms. Disrupting proteins that mitigate either type of resistance mechanism in combination with oncogene-targeted agents could help extend and enhance the clinical potential of these drugs. Son of Sevenless 1 (SOS1), a RAS guanine nucleotide exchange factor, is a highly attractable protein to target given its role in transducing inputs from upstream receptor tyrosine kinases (RTKs) to downstream RAS proteins to regulate signaling pathways involved in cell proliferation and survival. Additionally, SOS1 is subject to MAPK-mediated negative feedback inhibition and drugs targeting upstream and downstream nodes relieves SOS1 from this inhibition resulting in pathway reactivation. To address this need, we have developed orally bioavailable SOS1 bifunctional degraders for cancers driven by KRAS and RTKs. Our top two lead molecules demonstrate CRBN- and proteasomal-mediated SOS1 degradation with DC50s &amp;lt; 10nM and maximum degradation &amp;gt; 90%. Consistent with this notion, SOS1 degradation inhibited downstream signaling marker phopshoERK with IC50s &amp;lt; 5nM and were 26- or 53-fold more potent when compared to the clinical SOS1 inhibitor. Notably, the SOS1 degraders exhibited weaker SOS1 binding compared to other SOS1 inhibitors highlighting that the potency is heavily driven by the catalytic nature of degradation rather than occupancy-driven events. Both SOS1 degraders displayed CRBN- and SOS1-dependent antiproliferative activity in KRAS- and EGFR- mutant, solid tumor cell lines as well as in BCR-ABL driven, CML cells with IC50 values ranging from 0.5-10nM. Consistent with our in vitro data, oral administration of our SOS1 degraders in mice harboring H358 (KRAS G12C) and H441 (KRAS G12V) xenografts resulted in SOS1 degradation, phosphoERK reduction and significant tumor growth inhibition. While SOS1 degrader monotherapy demonstrated in vitro and in vivo potency, combination of mutant or allele specific EGFR and KRAS inhibitors and ABL inhibitors with our proof-of-concept degraders prevented pathway reactivation associated with these inhibitors and lead to a more durable response. These combinations also yielded synergistic effects in in vitro proliferation assays as well as enhanced tumor growth inhibition in KRAS-mutant xenograft models. Together, our SOS1 degraders alone and in combination with other targeted agents demonstrated anti-tumor activity in a variety of KRAS- and RTK-driven cancer cell lines and xenograft models. These results support the potential of orally bioavailable SOS1 degraders to help mitigate pathway reactivation for enhanced efficacy and prolonged response duration. Citation Format: Kyle Begovich, Angela Schoolmeesters, Navin Rajapakse, Elena Martinez, Qiao Liu, Arvind Shakya, Akinori Okano, Venkat Mali, Shenlin Huang, Aparajita Chourasia, Leah Fung. Identification of first-in-class, orally bioavailable SOS1 bifunctional degraders for the treatment of KRAS- and RTK-driven cancers [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 6056.

Abstract 3858: Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS)

Abstract Introduction: NeoICB is a promising approach for pts with DDLPS and UPS. However, biomarkers to identify which pts benefit from this treatment and understanding the mechanisms of response and resistance to ICB remains a challenge. Methods: We conducted a randomized phase 2 trial of neoICB (anti-PD1 +/- CTLA4) in pts with resectable DDLPS (n=17) and UPS (n=10). Pts with UPS received concurrent neoICB and radiation. The primary endpoint of the trial was pathological response; 3 DDLPS and 9 UPS pts were responders (R) and 14 DDLPS and 1 UPS pts were non-responders (NR). Peripheral blood mononuclear cells (PBMCs) were collected at baseline, prior to cycle 2 of neoICB (C2D1), and at the time of surgery (SURG). We performed mass cytometry (CyTOF) to identify immune cells associated with response and their dynamic changes with neoICB. We designed a custom 41-parameter panel using CD45 live cell barcoding with 31 markers for immune profiling, of which 23 were selected to detect key cell lineage and 8 to detect signaling markers. Batch correction and high dimensional data analyses were performed using CyCombine and CyTOF workflow R packages. Longitudinal comparisons between immune cell populations were conducted using Mann-Whitney U Test. Results: Of 79 available PBMC samples from 27 pts, 63 passed quality control after thawing (≥30% viability) and data clean-up (at least 10,000 events). Data was down-sampled to 5000 events for analysis. Of 19 immune cell types identified, monocytes were the most common followed by CD4+ and CD8+ T cells. There was an early increase in CD38+ effector/effector memory (EM) T cells at C2D1 in pts with DDLPS (CD8+ p=0.005, CD4+ p&amp;lt;0.001) and a similar increase observed in pts with UPS (CD8+ C2D1 p=NS, SURG p=0.034; CD4+ C2D1 p=0.024, SURG p=0.06) and these cells had higher Ki67 expression than CD38- T cells (p&amp;lt;0.005). At baseline, R pts with DDLPS had higher fractions of CD38+ EM CD8+ T cells than NR pts (p=0.044). Analysis in the UPS cohort was limited by the low number of NR pts. Higher expression of TIGIT was observed on CD38+ EM CD8+ T cells compared to CD38- counterparts at all time points. Conclusion: Peripheral CD38+ EM CD8+ T cells are associated with response to neoICB in pts with DDLPS. Longitudinal peripheral immune profiling reveals an increase in CD38+ EM CD8+ and CD4+ T cells with neoICB in pts with DDLPS and UPS. These cells are proliferative and express higher levels of TIGIT, which could be a future target for therapeutic exploration. Importantly, changes in the peripheral immune populations may precede changes in the tumor microenvironment (TME) as preliminary analyses of the TME did not identify early (C2D1) changes. TME imaging mass cytometry analyses are currently underway to associate peripheral and intratumor changes in immune populations. Citation Format: Elise F. Nassif, Hsinyi Lu, Michael Stanford, Karen Millerchip, Diana Romero, Jared Malke, Brenna Matejka, Jeffrey E. Gershenwald, Susmita Kumari, Duncan H. Mak, Angelique J. Lin, Jared K. Burks, Wei-Lien Wang, Alexander J. Lazar, Neeta Somaiah, Cara Haymaker, Christina L. Roland, Emily Z. Keung. Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS) [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 3858.

Abstract 6060: Discovery of potent SHP2 protein degraders with strong anti-tumor activities in KRAS mutant cancers

Abstract Background: SHP2 plays a pivotal role in modulating the RTK/RAS/MAPK signaling pathway, which is frequently dysregulated in various cancers. Multiple allosteric SHP2 inhibitors have been under active development for the treatment of RTK/RAS/MAPK-dependent cancers, while certain limitations have come to light, such as their inability to target the active form of SHP2 and narrow therapeutic index necessitating intermittent dosing to mitigate toxicity. To overcome these challenges, we have discovered SHP2 protein degraders as next generation SHP2 targeting agents. Here, we present compelling profiles of our SHP2 degraders. Methods: We employed a variety of cancer cell lines harboring mutations in RTK, KRAS, or SHP2 for assessing in vitro activity of SHP2 degraders. For in vivo assessment of pharmacological profile, we utilized an NCI-H358 KRAS G12C mutant NSCLC xenograft. SHP2 protein levels in cell lysates and tumors were analyzed to assess SHP2 degradation efficiency. Simultaneously, ERK phosphorylation and DUSP6 mRNA levels were analyzed to evaluate downstream effects. Immunoprecipitation assays were used to assess the impact on SHP2's scaffolding roles. Results: A series of SHP2 degraders have been identified, demonstrating highly efficient SHP2 degradation activity with DC50 values in subnanomolar ranges. Robust inhibition of ERK phosphorylation, followed by remarkable anti-cancer effects in multiple KRAS mutant cancer cell lines were observed. Interestingly, in vitro cell growth inhibition by SHP2 degraders are 10 times more potent than SHP2 inhibitors currently under clinical development. Further characterization reveals several advantages of SHP2 degraders compared to SHP2 inhibitors. First, we observed a more efficient disruption of SHP2 scaffolding function, such as the formation of the SHP2-GRB2-GAB1 complex. Second, we detected effective degradation of activating mutants of SHP2, such as E76K and T507K, which are typically not targeted by SHP2 inhibitors. Experiments using a KRAS mutant CDX model demonstrate compelling in vivo profiles. A single administration of lead compounds resulted in Dmax values exceeding 95% in tumors, accompanied by the significant reduction of downstream signaling markers, such as p-ERK and DUSP6 mRNA. Notably, the reduced levels of SHP2 and downstream markers persist for several days. This suggests that utilizing intermittent dosing could be a viable approach with SHP2 degraders. Once-weekly intravenous administration achieved strong tumor growth inhibition without causing significant body weight loss. Conclusion: We have successfully discovered novel SHP2 degraders exhibiting remarkable activities. We demonstrate that SHP2 degraders have potential to overcome the limitations associated with allosteric SHP2 inhibitors and provide a promising therapeutic option for RTK/RAS/MAPK-dependent cancer patients. Citation Format: So Hyun Kim, Jihye Lee, Jiyoung Song, Geon-Tae Park, Jieun Jung, Jungtae Na, Dong Ho Lee, Soohyun Lee, Ji Youn Park, Jiyoung Kim, Onnuri Bae, Jeonghwa Han, Ju Young Kang, Mijin Moon, Yongje Shin, Je Ho Ryu, Song Hee Lee, Sook-Kyung Park. Discovery of potent SHP2 protein degraders with strong anti-tumor activities in KRAS mutant cancers [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 6060.

Abstract 6268: A study of orphan nuclear receptor SHP (NR0B2) in prostate cancer progression

Abstract Prostate cancer (PCa) is a significant male cancer in many countries accounting for around 14% of new cancer cases globally in 2020 and ranked as 3rd common cancer in males in Hong Kong. The major risk factors include old age, high-fat diet, and genetics. Compared to other cancers, PCa is unique as its initial and advanced growth is androgen dependent. As tumor progresses, it accumulates numerous genetic alterations. Main therapeutic options include surgery, radiotherapy and or active surveillance. Upon treatment failure or when metastasis develops, androgen-deprivation therapy (ADT) with the use of potent androgen receptor (AR) antagonists and androgen biosynthesis inhibitors (e.g. Enzalutamide and abiraterone acetate) aiming to suppress AR signaling axis are the standard treatment options. However, the effect of these treatments is short-lived, in which nearly all patients will inevitably relapse to a hormone-independent and metastatic stage known as metastatic castration-resistant prostate cancer (mCRPC) within few years and further drive progression to a more lethal subtype, described as treatment-induced neuroendocrine prostate cancer (NEPC). The treatment options are limited and therefore resulted in high mortality, representing an emerging clinical problem. Recent studies have illustrated that NEPC progression follows a lineage plasticity model with a phenotypic transition to AR loss and neuroendocrine transdifferentiation. Our preliminary study has identified a nuclear receptor NR0B2 (SHP, small heterodimer partner SHP), which is a unique ligand-independent orphan nuclear receptor without a DNA-binding domain, that displays a significant up-regulation in clinical NEPC and prostate cancer cells with NEPC phenotype. Mechanistic studies revealed that SHP could directly interact with AR utilizing its NR Box1 motif while inducing repression on the AR transactivation via its another NR Box2 motif. Based on this, we conducted in vitro growth studies and revealed that NR0B2 overexpression could promote growth differentially in AR-negative PCa cells. However, AR-positive PCa cells were insensitive to AR blockade, providing supporting evidence that NR0B2 may be involved in repressing AR activity in prostate cancer cells. Multiple key NEPC and oncogenic TGFβ signaling markers showed significant upregulation in gene expression. Based on this, we hypothesize that upregulation of NR0B2 could play an important role in driving the advanced progression of CRPC via its direct interference of AR signaling axis and through other AR-independent signaling pathways, including activation of TGFβ signaling. (Study supported by a CUHK Direct Grant for Research 2022-2023, Project No. 2022.081) Citation Format: Hau Tak Daniel Lam, Franky Leung Chan. A study of orphan nuclear receptor SHP (NR0B2) in prostate cancer progression [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 6268.

Abstract 3575: The estrogenic endocrine disrupting compounds bisphenol-a (BPA) and alpha-zeranol (aZAL) induce early mammary hyperplasia in female ACI rats

Abstract Endocrine disrupting compounds (EDCs) found in food-grade plastics, drinking water, and foodstuffs, pose a threat to human health. Estrogenic EDCs are capable of binding and activating the estrogen receptor (ER). Since breast tissue relies on estrogen signaling for growth and renewal, excessive estrogen stimulation is associated with breast cancer development. Breast cancer is a leading cause of cancer-related death in women. Only 5-10% of all breast cancer cases can be attributed to genetic causes, and the proportion of breast cancers of the luminal subtype are increasing in the United States for reasons unknown. Estrogenic EDCs may be able to initiate breast cancers through their estrogenic activities. The present study assessed two EDCs: the plasticizer bisphenol-A (BPA) and the mycotoxin alpha-zeranol (aZAL). To assess the estrogenic effects of these EDCs in an in vivo model, we utilized the ACI rat strain due to its sensitivity to estrogen-induced mammary carcinogenesis. The hypothesis of this work was that BPA and aZAL will induce mammary gland proliferation and estrogen signaling at early timepoints post-exposure. Estrogen, BPA, or aZAL were implanted into the backs of female ACI rats in silastic tubes at either a 9 gram doses. At a timepoint of 5 days post implantation, we saw no overt toxicity of our treatment. Both BPA and aZAL resulted in increased glandular proliferation and hyperplasia in a comparable manner to estrogen. Immunohistochemistry and qPCR revealed that the estrogen signaling marker progesterone receptor (PGR) was significantly upregulated in the estrogen, BPA, and aZAL treatment groups compared to control. The percent positivity of PGR staining in the mammary glands were 21.5% +/- 4.2%, 25.7% +/- 1.7%, 27.9% +/- 3.8%, and 8.18% +/- 2.5% respectively. Additionally, PCNA, a marker of cell proliferation, was significantly upregulated in the estrogen and aZAL treatment groups. The percent positivity of PCNA staining in the mammary glands were 7.67% +/- 2.56%, 49.6% +/- 5.2%, and 41.0% +/- 11.5% respectively. Additionally, the weight of the pituitary gland, an estrogen-sensitive endocrine organ, significantly increased in both the estrogen and aZAL treatment groups. Overall, this study demonstrated that the estrogenic EDCs BPA and aZAL induced mammary gland proliferation and hyperplasia in a comparable manner to estrogen. This is concerning due to the routine detection of these two compounds in human biosamples. More information is needed to assess the risk of exposure to these compounds as it pertains to breast cancer and other hormonal cancers. Citation Format: Cassandra Winz, Ba Liu, Caroline Xie, Shlok Rohatagi, Eric Li, Philip Furmanski, Nanjoo Suh. The estrogenic endocrine disrupting compounds bisphenol-a (BPA) and alpha-zeranol (aZAL) induce early mammary hyperplasia in female ACI rats [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 3575.

Carboxymethyl chitosan–modified UiO-66 for the rapid detection of fenpropathrin in grains

Fenpropathrin residues in grain are potentially harmful to humans. Therefore, a fluorimetric lateral flow immunoassay using a zirconium-based organic skeleton (UiO-66) as a signal marker was developed for detecting fenpropathrin. Herein, carboxymethyl chitosan (CMCS) was used to modify UiO-66 and improve its water solubility to facilitate stable binding with sodium fluorescein (NaFL). This resulted in formation of a new fluorescent probe that is more suitable for lateral flow immunoassay (LFIA). The materials were characterized via electron microscopy, Fourier-transform infrared spectroscopy, and powder X-ray diffraction. CMCS and NaFL were successfully bound to UiO-66. Under optimized conditions, the constructed NaFL/UiO-66@CMCS-LFIA exhibited a good linear relationship within the range of 0.98–62.5 μg/L, with a detection limit of 3.91 μg/L. This probe was fourfold more sensitive than traditional colloidal gold nanoparticle-based LFIA. Finally, NaFL/UiO-66@CMCS-LFIA was successfully applied to detect fenpropathrin in wheat and maize samples. The detection limit was 1.56 μg/kg and recoveries ranged from 96.58 % to 118.56 %. This study provides a sensitive, stable, and convenient method for the rapid detection of pesticide residues.

Steroidogenesis Upregulation through Mitochondria-Associated Endoplasmic Reticulum Membranes and Mitochondrial Dynamics in Rat Testes: The Role of D-Aspartate.

Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs) mediate the communication between the Endoplasmic Reticulum (ER) and the mitochondria, playing a fundamental role in steroidogenesis. This study aimed to understand how D-aspartate (D-Asp), a well-known stimulator of testosterone biosynthesis and spermatogenesis, affects the mechanism of steroidogenesis in rat testes. Our results suggested that D-Asp exerts this function through MAMs, affecting lipid trafficking, calcium signaling, ER stress, and mitochondrial dynamics. After 15 days of oral administration of D-Asp to rats, there was an increase in both antioxidant enzymes (SOD and Catalase) and in the protein expression levels of ATAD3A, FACL4, and SOAT1, which are markers of lipid transfer, as well as VDAC and GRP75, which are markers of calcium signaling. Additionally, there was a decrease in protein expression levels of GRP78, a marker of aging that counteracts ER stress. The effects of D-Asp on mitochondrial dynamics strongly suggested its active role as well. It induced the expression levels of proteins involved in fusion (MFN1, MFN2, and OPA1) and in biogenesis (NRF1 and TFAM), as well as in mitochondrial mass (TOMM20), and decreased the expression level of DRP1, a crucial mitochondrial fission marker. These findings suggested D-Asp involvement in the functional improvement of mitochondria during steroidogenesis. Immunofluorescent signals of ATAD3A, MFN1/2, TFAM, and TOMM20 confirmed their localization in Leydig cells showing an intensity upgrade in D-Asp-treated rat testes. Taken together, our results demonstrate the involvement of D-Asp in the steroidogenesis of rat testes, acting at multiple stages of both MAMs and mitochondrial dynamics, opening new opportunities for future investigation in other steroidogenic tissues.

Interleukin-16 is increased in obesity and alters adipogenesis and inflammation in vitro.

Obesity is a chronic condition associated with low-grade inflammation mainly due to immune cell infiltration of white adipose tissue (WAT). WAT is distributed into two main depots: subcutaneous WAT (sWAT) and visceral WAT (vWAT), each with different biochemical features and metabolic roles. Proinflammatory cytokines including interleukin (IL)-16 are secreted by both adipocytes and infiltrated immune cells to upregulate inflammation. IL-16 has been widely studied in the peripheral proinflammatory immune response; however, little is known about its role in adipocytes in the context of obesity. We aimed to study the levels of IL-16 in WAT derived from sWAT and vWAT depots of humans with obesity and the role of this cytokine in palmitate-exposed 3T3-L1 adipocytes. The results demonstrated that IL-16 expression was higher in vWAT compared with sWAT in individuals with obesity. In addition, IL-16 serum levels were higher in patients with obesity compared with normal-weight individuals, increased at 6 months after bariatric surgery, and at 12 months after surgery decreased to levels similar to before the intervention. Our in vitro models showed that IL-16 could modulate markers of adipogenesis (Pref1), lipid metabolism (Plin1, Cd36, and Glut4), fibrosis (Hif1a, Col4a, Col6a, and Vegf), and inflammatory signaling (IL6) during adipogenesis and in mature adipocytes. In addition, lipid accumulation and glycerol release assays suggested lipolysis alteration. Our results suggest a potential role of IL-16 in adipogenesis, lipid and glucose homeostasis, fibrosis, and inflammation in an obesity context.

Machine learning predictions of T cell antigen specificity from intracellular calcium dynamics.

Adoptive T cell therapies rely on the production of T cells with an antigen receptor that directs their specificity toward tumor-specific antigens. Methods for identifying relevant T cell receptor (TCR) sequences, predominantly achieved through the enrichment of antigen-specific T cells, represent a major bottleneck in the production of TCR-engineered cell therapies. Fluctuation of intracellular calcium is a proximal readout of TCR signaling and candidate marker for antigen-specific T cell identification that does not require T cell expansion; however, calcium fluctuations downstream of TCR engagement are highly variable. We propose that machine learning algorithms may allow for T cell classification from complex datasets such as polyclonal T cell signaling events. Using deep learning tools, we demonstrate accurate prediction of TCR-transgenic CD8+ T cell activation based on calcium fluctuations and test the algorithm against T cells bearing a distinct TCR as well as polyclonal T cells. This provides the foundation for an antigen-specific TCR sequence identification pipeline for adoptive T cell therapies.

‘Three Birds With One Stone’ functionalized metal–organic frameworks for facile and efficient chemiluminescent immunoassays

The luminol–H2O2 system is crucial in the field of chemiluminescence (CL) analysis. However, luminol exhibits improved solubility and luminescence only in an alkaline environment, and its preparation process is cumbersome. This inconvenience highlights the need for developing a simple and universally applicable method in this regard. This study integrates luminol and a catalyst into metal–organic frameworks (MOFs) for fabrication of functionalised MOF nanoparticles. Further, PEGylated hematin (PEG = polyethylene glycol) was prepared. This modification with PEG enhanced the catalytic properties of hematin and facilitated the formation of zeolitic imidazole framework-8 nanoparticles. Furthermore, the built-in catalyst enhanced the CL in terms of strength and persistence. These MOF nanoparticles were used as signal markers to fabricate a sandwich-type CL immunosensor for detection of carcinoembryonic antigen, which is a disease marker, following which highly satisfactory analytical test results are obtained. This approach of integrating luminophores and catalysts offers a new idea for generating novel materials in the CL analysis field.

Co-Delivery of Aceclofenac and Methotrexate Nanoparticles Presents an Effective Treatment for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a common acute inflammatory autoimmune connective tissue arthropathy. The genetic studies, tissue analyses, experimental animal models, and clinical investigations have confirmed that stromal tissue damage and pathology driven by RA mounts the chronic inflammation and dysregulated immune events. We developed methotrexate (MTX)-loaded lipid-polymer hybrid nanoparticles (MTX-LPHNPs) and aceclofenac (ACE)-loaded nanostructured lipid carriers (ACE-NLCs) for the efficient co-delivery of MTX and ACE via intravenous and transdermal routes, respectively. Bio-assays were performed using ex-vivo skin permeation and transport, macrophage model of inflammation (MMI) (LPS-stimulated THP-1 macrophages), Wistar rats with experimental RA (induction of arthritis with Complete Freund's adjuvant; CFA and BCG), and programmed death of RA affected cells. In addition, gene transcription profiling and serum estimation of inflammatory, signaling, and cell death markers were performed on the blood samples collected from patients with RA. Higher permeation of ACE-NLCs/CE across skin layers confirming the greater "therapeutic index" of ACE. The systemic delivery of MTX-loaded LPHNPs via the parenteral (intravenous) route is shown to modulate the RA-induced inflammation and other immune events. The regulated immunological and signaling pathway(s) influence the immunological axis to program the death of inflamed cells in the MMI and the animals with the experimental RA. Our data suggested the CD40-mediated and Akt1 controlled cell death along with the inhibited autophagy in vitro. Moreover, the ex vivo gene transcription profiling in drug-treated PBMCs and serum analysis of immune/signalling markers confirmed the therapeutic role co-delivery of drug nanoparticles to treat RA. The animals with experimental RA receiving drug treatment were shown to regain the structure of paw bones and joints similar to the control and were comparable with the market formulations. Our findings confirmed the use of co-delivery of drug nanoformulations as the "combination drug regimen" to treat RA.