Activation Of pSTAT3 Research Articles

Development of Pro-resolving and Pro-efferocytic Nanoparticles for Atherosclerosis Therapy.

Atherosclerosis is a major contributor to cardiovascular diseases with a high global prevalence. It is characterized by the formation of lipid-laden plaques in the arteries, which eventually lead to plaque rupture and thrombosis. While the current lipid-lowering therapies are generally effective in lowering the risk of cardiovascular events, they do not address the underlying causes of disease. Defective resolution of inflammation and impaired efferocytosis are the main driving forces of atherosclerosis. Macrophages recognize cells for clearance by the expression of "eat me" and "do not eat me" signals, including the CD47-SIRPα axis. However, the "do not eat me" signal CD47 is overexpressed in atherosclerotic plaques, leading to compromised efferocytosis and secondary necrosis. In this context, prophagocytic antibodies have been explored to stimulate the clearance of apoptotic cells, but they are nonspecific and impact healthy tissues. In macrophages, downstream of signal regulatory protein α, lie protein tyrosine phosphatases, SHP 1/2, which can serve as effective targets for selectively phagocytosing apoptotic cells. While increasing the efferocytosis targets the end stages of lesion development, the underlying issue of inflammation still persists. Simultaneously increasing efferocytosis and reducing inflammation can be effective therapeutic strategies for managing atherosclerosis. For instance, IL-10 is a key anti-inflammatory mediator that enhances efferocytosis via phosphoSTAT3 (pSTAT3) activation. In this study, we developed a combination nanotherapy by encapsulating an SHP-1 inhibitor (NSC 87877) and IL-10 in a single nanoparticle platform [(S + IL)-NPs] to enhance efferocytosis and inflammation resolution. Our studies suggest that (S + IL)-NPs successfully encapsulated both agents, entered the macrophages, and delivered the agents into intracellular compartments. Additionally, (S + IL)-NPs decreased inflammation by suppressing pro-inflammatory markers and enhancing anti-inflammatory mediators. They also exhibited the potential for improved phagocytic activity via pSTAT3 activation. Our nanomedicine-mediated upregulation of the anti-inflammatory and efferocytic responses in macrophages shows promise for the treatment of atherosclerosis.

PSTAT3 activation of Foxl2 initiates the female pathway underlying temperature-dependent sex determination

Ovarian development was traditionally recognized as a "default" sexual outcome and therefore received much less scientific attention than testis development. In turtles with temperature-dependent sex determination (TSD), how the female pathway is initiated to induce ovary development remains unknown. In this study, we have found that phosphorylation of the signal transducer and activator of transcription 3 (pSTAT3) and Foxl2 exhibit temperature-dependent sexually dimorphic patterns and tempo-spatial coexpression in early embryos of the red-eared slider turtle (Trachemys scripta elegans). Inhibition of pSTAT3 at a female-producing temperature of 31 °C induces 64.7% female-to-male sex reversal, whereas activation of pSTAT3 at a male-producing temperature of 26 °C triggers 75.6% male-to-female sex reversal. In addition, pSTAT3 directly binds to the locus of the female sex-determining gene Foxl2 and promotes Foxl2 transcription. Overexpression or knockdown of Foxl2 can rescue the sex reversal induced by inhibition or activation of pSTAT3. This study has established a direct genetic link between warm temperature-induced STAT3 phosphorylation and female pathway initiation in a TSD system, highlighting the critical role of pSTAT3 in the cross talk between female and male pathways.

PLEK2 promotes migration and invasion in pancreatic ductal adenocarcinoma by MMP1 through IL-17 pathway.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis primarily due to metastasis. Accumulating evidence suggests that PLEK2 acts as an oncogene in various tumors. This study aimed to investigate the effects of PLEK2 on PDAC. Expression analysis of PLEK2 was conducted using qRT-PCR, Western blot, and immunohistochemistry in PDAC. Wound healing and transwell assays were performed to evaluate the impact of PLEK2 on cell migration and invasion. A xenograft tumor model was employed to assess the in vivo proliferation of PLEK2. Additionally, the downstream pathway of PLEK2 was analyzed through RNA-seq and confirmed by Western blot analysis. The results demonstrated the upregulation of PLEK2 expression in tumor specimens. High PLEK2 expression was significantly associated with poor overall survival and advanced TNM stages. Correlation analyses revealed positive correlations between PLEK2 and TGF-β, EGFR, and MMP1. Wound healing and transwell assays demonstrated that PLEK2 promoted PDAC cell migration and invasion, potentially through the activation of the epithelial-to-mesenchymal transition process. The in vivo experiment further confirmed that PLEK2 knockdown suppressed tumor growth. RNA-seq analysis revealed PLEK2's regulation of MMP1 and activation of p-ERK and p-STAT3, which were verified by Western blot analysis. Overall, the present study suggests that PLEK2 may play a tumor-promoting role in PDAC. These findings provide valuable insights into the molecular mechanisms of pancreatic cancer and highlight the potential of PLEK2 as a therapeutic target.

Rational fusion design inspired by cell-penetrating peptide: SS31/S-14 G Humanin hybrid peptide with amplified multimodal efficacy and bio-permeability for the treatment of Alzheimer's disease

Alzheimer's disease is a neurodegenerative disease induced by multiple interconnected mechanisms. Peptide drug candidates with multi-modal efficacy generated from fusion strategy are suitable for addressing multi-facet pathology. However, clinical translation of peptide drugs is greatly hampered by their low permeability into brain. Herein, a hybrid peptide HNSS is generated by merging two therapeutic peptides (SS31 and S-14 G Humanin (HNG)), using a different approach from the classical shuttle-therapeutic peptide conjugate design. HNSS demonstrated increased bio-permeability, with a 2-fold improvement in brain distribution over HNG, thanks to its structure mimicking the design of signal peptide-derived cell-penetrating peptides. HNSS efficiently alleviated mitochondrial dysfunction through the combined effects of mitochondrial targeting, ROS scavenging and p-STAT3 activation. Meanwhile, HNSS with increased Aβ affinity greatly inhibited Aβ oligomerization/fibrillation, and interrupted Aβ interaction with neuron/microglia by reducing neuronal mitochondrial Aβ deposition and promoting microglial phagocytosis of Aβ. In 3× Tg-AD transgenic mice, HNSS treatment efficiently inhibited brain neuron loss and improved the cognitive performance. This work validates the rational fusion design-based strategy for bio-permeability improvement and efficacy amplification, providing a paradigm for developing therapeutic peptide candidates against neurodegenerative disease.

Protective effects of growth hormone – releasing hormone antagonists in the lungs of septic mice

Growth hormone-releasing hormone antagonists (GHRHAnt) have been associated with antitumor and antioxidative activities. The present study investigates for the first time the effects of those compounds towards pro-inflammatory cytokine expression in a murine model of cecal ligation and puncture (CLP) – induced sepsis. The results indicate that GHRHAnt JV-1-36 significantly suppressed IL-1α, IL-6, and pSTAT3 activation in septic lungs. Moreover, GHRHAnt treatment reduced bronchoalveolar lavage fluid (BALF) protein concentration, suggesting a protective effect of that compound in sepsis-induced lung edema. Based on those findings, it is suggested that GHRHAnt may represent an exciting new therapeutic possibility in sepsis-induced endotoxemia and lung injury.

Abstract PO1-25-08: Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive breast cancer

Abstract Background: The human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase overexpressed in approximately 20-25% of breast cancers with varying biological differences according to hormone receptor (HR) positive status. Combination of chemotherapy with dual HER2 blockade and endocrine therapy (adjuvant) or CDK4/6 inhibitor (CDK4/6i) therapy (metastatic) is standard of care for HER2-positive/HR-positive breast cancer. However, de-escalation of chemotherapy remains of significant interest given associated toxicities and, as a result, alternative therapeutic strategies are needed. One such strategy is targeting the signal transducer and activator of transcription 3 (STAT3) which is constitutively activated in the HER-2 positive setting. Moreover, there is preclinical evidence to suggest that phosphorylated STAT3 (pSTAT3) induces overexpression of cyclin D1 (CCND1), which along with CDK4 may mediate resistance to targeted therapy for HER2-positive breast cancer. However, it is unclear if pSTAT3 plays a significant role in tumorigenesis or the development of resistance to therapy in HER2-positive disease. We aim to evaluate the effect of CDK4/6i therapy with palbociclib on tumor growth in an inducible transgenic HER2-positive mouse model and the impact on pSTAT3 and cyclin D1 levels. Methods: Using an inducible transgenic mouse mammary tumor virus (MMTV) model of HER2-positive breast cancer, we evaluated STAT3 phosphorylation in tumors driven by HER2 expression in addition to the impact of CDK4/6 inhibition with palbociclib on tumorigenesis and pSTAT3 activation. To model tumor recurrence, doxycycline was withdrawn from tumor bearing mice and tumor regression was observed. Subsequently, the mice developed recurrent mammary tumors. Given recurrent tumors in this model are driven by a HER2-independent mechanism, we examined expression of cell cycle proteins together with STAT3 phosphorylation to determine association with recurrence. Result: In addition to expressing HER2, the primary tumors expressed estrogen receptor (ERα), Rb, cyclin D1, and CDK4/6 proteins. STAT3 phosphorylation was also observed. Treatment with palbociclib decreased Rb phosphorylation and decreased tumor growth compared to vehicle treated mice (%TGI 79.37% after 21 days). Furthermore, pSTAT3 levels were not altered with palbociclib therapy. When recurrent tumors were analyzed, they lacked HER2 expression but expressed cyclin D1 and CDK4/6 supporting that tumor growth is driven by cyclin D1/CDK4 pathway. Recurrent tumors also demonstrated pSTAT3 despite lack of HER2 expression suggesting that STAT3 phosphorylation in the recurrent tumors is mediated by an alternative mechanism, potentially the CDK4/6 pathway. Conclusion: We demonstrate STAT3 activation in a mouse model of HER2 driven breast cancer and show persistent activation in recurrent tumors lacking HER2 expression rendering them resistant to HER2-targeted therapy. We also demonstrate the anti-tumor effect of palbociclib in primary tumors with HER2-dependent proliferation. Taken together, our findings suggest that pSTAT3 is a viable therapeutic target and provides the rationale for combination therapy with CDK4/6 inhibition using palbociclib and STAT3 inhibition with a novel STAT3 inhibitor in HER2-positive breast cancer. This therapeutic approach may represent a potential chemotherapy de-escalation strategy. Citation Format: Taiwo Adesoye, Linjie Luo, Tuyen Bui, Serena Kim, Hannah Wingate, Debu Tripathy, Kelly Hunt, Khandan Keyomarsi. Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-08.

Bi-functional IL2/CD25/IL10 fusion protein promotes Tregs, reduces inflammation, and is highly effective in limiting diabetes in NOD mice

Abstract Clinical trials indicate that low-dose IL-2 is a promising approach for patients with autoimmunity. To improve upon IL-2, the mouse (m) IL-2/CD25 fusion protein was developed, which has a long half-life, is selective to the high-affinity IL-2R and expands Tregs to limit diabetes in NOD mice. However, after an extended time off-therapy, many NOD mice exhibited T1D. To improve efficacy, we developed a bifunctional fusion protein, consisting of IL-2/CD25 covalently linked to IL-10 to increase Tregs and limit inflammation. With respect to IL-2 activity, IL2/CD25/IL10 supported IL-2-dependent proliferation of CTLL and STAT5 activation by Tregs. With respect to IL-10 activity, it supported IL-10-dependent STAT3 activation by RAW264.7 cells and limited IFNγ and IL-6 production by activated T and B cells, respectively. In vivo, IL2/CD25/IL10 supported expansion of Tregs as well as activation of pSTAT3 in macrophages and reduced LPS-dependent production of IL-6 and TNFα, indicative of bifunctional activity. When 12-week-old female NOD mice received IL2/CD25/IL10 for 5-weeks, IL2/CD25/IL10 (mean survival >50 week) was more effective than IL-2/CD25 (mean survival 26 weeks) in preventing progressive diabetes. Collectively, these data support the notion that a combination approach, where Tregs are increased and autoreactive T cells are limited, in this case through IL-10, will enhance the efficacy of a Treg-targeted immunotherapy of autoimmunity.

Dabrafenib mitigates the neuroinflammation caused by ferroptosis in experimental autoimmune encephalomyelitis by up regulating Axl receptor

Multiple sclerosis is an autoimmune disease that causes inflammatory damage to the central nervous system. At present, the pathogenesis of the disease is unknown. There is a lack of few effective therapy medications available. Therefore, it is necessary to further explore the pathogenesis of this illness and develop potential therapeutic drugs. Dabrafenib is potential therapeutic medicine for nervous system disease. In this study, we preliminarily studied the possible mechanism of dabrafenib in the treatment of multiple sclerosis from the perspective of ferroptosis. First, we observed that dabrafenib significantly improved symptoms of gait abnormalities, limb weakness or paralysis, and down-regulated levels of spinal cord inflammation in an experimental autoimmune encephalitis (EAE) model. Meanwhile, we also observed that dabrafenib could inhibit the proteins of ferroptosis in spinal cord tissue of EAE mice by Western blot. The results of immunohistochemical analysis showed that the effect of dabrafenib on ferroptosis mainly occurred in microglia. Second, dabrafenib was demonstrated to be able to inhibit the S phase of the cell cycle, reduce ROS levels, and reinstate mitochondrial activity in the LPS-induced BV2 inflammatory cell model. Futhermore, we found that dabrafenib inhibits P-JAK2 and P-STAT3 activation by acting Axl receptor, which in turn prevents neurogenic inflammation in microglia. The co-stimulated BV2 cell model with LPS and Erastin also verified these findings. Ultimately, the Axl knockout mice used to construct the EAE model allowed for the confirmation that dabrafenib prevented ferroptosis in microglia by up-regulating Axl receptor, which reduced the inflammatory demyelination associated with EAE. In summary, our research demonstrates the advantages of dabrafenib in multiple sclerosis treatment, which can prevent ferroptosis in microglia in multiple sclerosis through up-regulating Axl receptor, thus halting the progression of multiple sclerosis.

Incorporating IL7 receptor alpha signaling in the endodomain of B7H3-targeting chimeric antigen receptor T cells mediates antitumor activity in glioblastoma.

CAR-T-cell therapy has shown promise in treating hematological malignancies but faces challenges in treating solid tumors due to impaired T-cell function in the tumor microenvironment. To provide optimal T-cell activation, we developed a B7 homolog 3 protein (B7H3)-targeting CAR construct consisting of three activation signals: CD3ζ (signal 1), 41BB (signal 2), and the interleukin 7 receptor alpha (IL7Rα) cytoplasmic domain (signal 3). We generated B7H3 CAR-T cells with different lengths of the IL7Rα cytoplasmic domain, including the full length (IL7R-L), intermediate length (IL7R-M), and short length (IL7R-S) domains, and evaluated their functionality in vitro and in vivo. All the B7H3-IL7Rα CAR-T cells exhibited a less differentiated phenotype and effectively eliminated B7H3-positive glioblastoma in vitro. Superiority was found in B7H3 CAR-T cells contained the short length of the IL7Rα cytoplasmic domain. Integration of the IL7R-S cytoplasmic domain maintained pSTAT5 activation and increased T-cell proliferation while reducing activation-induced cell death. Moreover, RNA-sequencing analysis of B7H3-IL7R-S CAR-T cells after coculture with a glioblastoma cell line revealed downregulation of proapoptotic genes and upregulation of genes associated with T-cell proliferation compared with those in 2nd generation B7H3 CAR-T cells. In animal models, compared with conventional CAR-T cells, B7H3-IL7R-S CAR-T cells suppressed tumor growth and prolonged overall survival. Our study demonstrated the therapeutic potential of IL7Rα-incorporating CAR-T cells for glioblastoma treatment, suggesting a promising strategy for augmenting the effectiveness of CAR-T cell therapy.

Abstract 849: Deciphering the link between aging, lobular involution, and breast cancer risk: The role of LIFR/pSTAT3 signaling

Abstract Background: Age-related lobular involution (ARLI), characterized by regression of breast epithelial tissue during the peri-and post-menopausal period, plays a crucial role in breast cancer risk. Delayed or incomplete ARLI is linked with increased postmenopausal breast cancer susceptibility.This study focuses on unraveling the molecular mechanisms of ARLI, drawing parallels with the well-characterized postpartum involution (PPI). Our aim was to discover biomarkers differentiating women who progressed through ARLI from those with stagnant ARLI. Methods: We utilized the Mayo Clinic Benign Breast Disease (BBD) cohort, analyzing 108 perimenopausal women aged 45-55 who had a subsequent benign biopsy between 2-15 years later. We assessed Lobular Involution (Ll) status by comparing the number and size of lobules in initial and subsequent biopsies. RNA profiling of initial biopsies was performed using NanoString 10360 and BC360, identifying differentially expressed genes between ARLI progression and stagnation. Immunohistochemistry (IHC) mapped the expression of these biomarkers in epithelial lobules. Results: We identified 37 genes with significant differential expression, highlighting Leukemia Inhibitory Factor Receptor (LIFR) as upregulated in ARLI stagnant samples. LIFR, part of the IL-6 cytokine signaling pathway, is known for its role in the induction of apoptosis during PPI via pSTAT3 activation. Our IHC analyses of initial biopsies of women who differentially progress through ARLI confirmed differential expression of LIFR that is coordinately regulated with pSTAT3. Conclusion: Our findings reveal significant parallels between ARLI and PPI. Future research will assess how LIFR/pSTAT3 signaling is linked to induction of apoptotic markers in perimenopausal women and will investigate how anomalies in the LIFR/pSTAT3 pathway might contribute to heightened breast cancer risk in women with delayed or incomplete ARLI. This study not only sheds light on the complex biological processes of aging in breast tissue but also opens new avenues for breast cancer risk assessment and prevention strategies. Citation Format: Jaida C. Lue, Derek Radisky, Melody Stallings-Mann, Mark Sherman, Amy Degnim, Bryan McCauley, Tanya Hoskin. Deciphering the link between aging, lobular involution, and breast cancer risk: The role of LIFR/pSTAT3 signaling [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 849.

Epigallocatechin gallate suppresses mitotic clonal expansion and adipogenic differentiation of preadipocytes through impeding JAK2/STAT3-mediated transcriptional cascades

BackgroundMitotic clonal expansion (MCE) is a prerequisite for preadipocyte differentiation and adipogenesis. Epigallocatechin gallate (EGCG) has been shown to inhibit preadipocyte differentiation. However, the exact molecular mechanisms are still elusive. PurposeThis study investigated whether EGCG could inhibit adipogenesis and lipid accumulation by regulating the cell cycle in the MCE phase of adipogenesis and its underlying molecular mechanisms. Method3T3-L1 preadipocytes were induced to differentiate by a differentiation cocktail (DMI) and were treated with EGCG (25–100 μM) for 9, 18, and 24 h to examine the effect on MCE, or eight days to examine the effect on terminal differentiation. C57BL/6 mice were fed a high-fat diet (HFD) for three months to induce obesity and were given EGCG (50 or 100 mg/kg) daily by gavage. ResultsWe showed that EGCG significantly inhibited terminal adipogenesis and lipid accumulation in 3T3-L1 cells and decreased expressions of PPARγ, C/EBPα, and FASN. Notably, at the MCE phase, EGCG regulated the cell cycle in sequential order, induced G0/G1 arrest at 18 h, and inhibited the G2/M phase at 24 h upon DMI treatment. Meanwhile, EGCG regulated the expressions of cell cycle regulators (cyclin D1, cyclin E1, CDK4, CDK6, cyclin B1, cyclin B2, p16, and p27), and decreased C/EBPβ, PPARγ, and C/EBPα expressions at MCE. Mechanistic studies using STAT3 agonist Colivelin and antagonist C188–9 revealed that EGCG-induced cell cycle arrest in the MCE phase and terminal adipocyte differentiation was mediated by the inhibition of JAK2/STAT3 signaling cascades and STAT3 (Tyr705) nuclear translocation. Furthermore, EGCG significantly protected mice from HFD-induced obesity, reduced body weight and lipid accumulations in adipose tissues, reduced hyperlipidemia and leptin levels, and improved glucose intolerance and insulin sensitivity. Moreover, RNA sequencing (RNA-seq) analysis showed that the cell cycle changes in epididymal white adipose tissue (eWAT) were significantly enriched upon EGCG treatment. We further verified that EGCG treatment significantly reduced expressions of adipogenic factors, cell cycle regulators, and p-STAT3 in eWAT. ConclusionEGCG inhibits MCE, resulting in the inhibition of early and terminal adipocyte differentiation and lipid accumulation, which were mediated by inhibiting p-STAT3 nucleus translocation and activation.

OP28 Defective STAT3 signaling in refractory Very Early Onset Inflammatory Bowel Disease is associated with a transcriptional signature which predicts response to anti-IL23-based therapies

Abstract Background Very early onset inflammatory bowel disease (VEOIBD) has a monogenic cause in >5% of cases. IL10R deficiency is among the more common causes of monogenic IBD and presents with severe disease refractory to conventional therapies. For most VEOIBD patients, the molecular basis of disease is unknown. We employed flow cytometry and bulk RNA-sequencing to localize pathway-level dysfunction in VEOIBD patients with unknown molecular basis. Methods VEOIBD patients prospectively enrolled in a biorepository were screened for IL10R deficiency using a flow cytometry assay. Patient peripheral blood mononuclear cells (PBMCs) were stimulated with IL10 and control cytokine IL21 and pSTAT3 activation was measured. RNA-sequencing was performed on 115 whole blood samples, comprising VEOIBD patients with either confirmed (n=35, IL10R deficient n=4), or unknown monogenic causes (n=70), and controls (n=10). Transcriptome analysis focused on patients with abnormal STAT3 activation as determined by flow cytometry. Results We identified 6 patients with refractory VEOIBD and defective phosphorylation of STAT3 upon stimulation with both IL10 and IL21 by flow cytometry (Fig 1A), a result confirmed by Western blot (Fig 1B). All six "STAT3-defective" (STAT3-def) patients lacked deleterious mutations in known VEOIBD genes as determined by whole exome sequencing. Principal component analysis of blood transcriptome data showed the STAT3-def and IL10R deficient patients generally clustered together (Fig 1C). Cell-type de-convolution analysis of the blood transcriptomes was used to infer cell-type abundance and cell-type specific differentially expressed genes (DEGs) for the STAT3-def and IL10R deficient groups relative to the rest. Genes downregulated in estimated M1 macrophages from the STAT3-def group were enriched in the Hallmark pathway "IL6-JAK-STAT3 signaling." STAT3-def M1 macrophage DEGs were then projected on an adult colonic Crohn’s disease Bayesian network and extended out a path length of 1. The resulting STAT3-def subnetwork of 440 genes included the known VEOIBD genes STAT3, STAT1, TYMP, and TRIM22 (Fig 1D) and the top enriched signaling pathways included IL23, IL6, and IL12. This is of significant interest given three STAT3-def patients with disease refractory to anti-TNF and vedolizumab achieved remission with anti-IL23-based therapies (Table 1). Conclusion We identified 6 patients with refractory VEOIBD and a shared biochemical phenotype and blood transcriptional signature. Network analysis revealed signaling pathways targeted by existing IBD medications, including anti-IL23-based therapies. Molecular analysis of IBD patient blood has exciting potential to localize pathway-level dysfunction and guide precision medicine approaches.

ALYREF-mediated RNA 5-Methylcytosine modification Promotes Hepatocellular Carcinoma Progression Via Stabilizing EGFR mRNA and pSTAT3 activation.

5-Methylcytosine (m5C) is one of the most ubiquitous modifications of mRNA and contributes to cancer pathogenesis. Aly/REF export factor (ALYREF), an m5C reader, is associated with the prognosis of liver hepatocellular carcinoma (LIHC). However, the effects of ALYREF on the progression of LIHC and the underlying molecular mechanisms remains elusive. Through an analysis of an online database and 3 independent LIHC cohorts, we found that ALYREF was markedly elevated in human liver cancer tissues and was significantly correlated with LIHC clinicopathological parameters, including Ki67+ cell rate, high-grade TNM stage, and poor prognosis. Several experiments were conducted to investigate the molecular basis and functional role of ALYREF-related progression in this study. ALYREF could enhance LIHC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro and tumor formation in vivo. Mechanistically, ALYREF promoted the progression of human LIHC through EGFR pathways. Furthermore, ALYREF could directly bind to the m5C modification site of EGFR 3' untranslated region (3' UTR) to stabilize EGFR mRNA. Collectively, ALYREF played a crucial oncogenic role in LIHC via the stabilization of EGFR mRNA and subsequent activation of the STAT3 signaling pathway. Our results may help to elucidate the potential mechanisms of ALYREF-induced m5C modification in the progression of human LIHC.

Distinct Cellular, Transcriptional, and Mechanistic Properties Determine HSC Fate during Emergency Granulopoiesis

Emergency granulopoiesis (EG) is the process responsible for the rapid and enhanced production of granulocytes during acute infections. Traditionally, EG was understood as a cellular mechanism mostly initiated and supported by myeloid progenitors, however, recent data suggest that hematopoietic stem cells (HSCs) might also participate in the process. To investigate whether and how HSCs contribute to the initial steps of EG, we performed scRNA-seq and ATAC-seq analysis of sorted murine HSCs 4 hours after in vivo lipopolysaccharide (LPS) administration, mimicking an acute bacterial infection. Strikingly, we observed radical transcriptional changes between HSCs isolated from PBS control and LPS treated mice, mostly marked by alterations in HSC lineage bias. We identified a steady state lymphoid-biased subpopulation of HSCs, marked by expression of Procr (CD201). Following LPS stimulation, the CD201 expression was lost and this population was transcriptionally rewired to a myeloid-biased HSC population. Accordingly, ATACseq data corroborated opening of myeloid-bias loci upon LPS administration rather than steady-state lymphoid-biased loci. Further, we confirmed the loss of CD201 expression in HSCs by flow cytometry in mice after LPS and G-CSF treatment, as well as Candida albicans infection, suggesting that the loss of CD201 expression is a general event during EG induced by different stimuli. Interestingly, the CD201 downregulation in HSCs was independent of the master regulator of EG C/EBPβ, while we observed the contribution of the TLR4-MyD88 signaling axis. Next, we functionally validated our scRNA-seq results in vitro and in vivo. When cultivated under myeloid differentiation conditions, the CD201- HSCs gave rise to mature granulocytes, whereas CD201+ HSCs remained rather immature. In transplantation settings, CD201+ HSCs showed increased engraftment and preferential bias to produce lymphoid cells, while CD201- HSC showed decreased engraftment ability and a bias towards myeloid production. Surprisingly, LPS challenge of BM chimeras transplanted with lymphoid-biased CD201+ HSCs led to EG response marked by rapid loss of lymphoid-biased multipotent progenitors (MPPs) and the expansion of myeloid-biased MPPs, thus confirming the lympho-myeloid switch observed in the scRNA-seq data. Interestingly, the treatment of WT mice with CD201 blocking antibody was able to partially impair the lympho-myeloid switch, suggesting that CD201 plays an active role in HSCs during EG. Mechanistically, we observed that both CD201+ and CD201- HSCs rely on different signaling pathways under EG. While CD201+ HSCs express higher levels of TLR4 and have higher activation of NF-κB signaling upon in vitro LPS stimulation, the CD201- HSCs express higher levels of G-CSF-R and have higher activation of pSTAT3 signaling upon G-CSF-stimulation. Moreover, while the LIP isoform of C/EBPβ, important for cell proliferation, is present in both CD201+ and CD201- HSCs, the LAP/LAP* isoform, important for myeloid differentiation, is present only in the CD201- HSCs fraction. Altogether, our data suggest that EG is supported by a population of HSCs which upon pathogen sensing undergo a radical transcriptional rewiring that promotes their myeloid output. Initially, the pathogen is directly sensed by TLR4 on the surface of a steady state lymphoid-biased CD201+ HSCs, causing a rapid activation of the downstream NF-κB signaling pathway. Subsequently, the lymphoid-myeloid transcriptional switch, marked by the loss of CD201 expression, occurs and EG is then supported by myeloid-biased CD201- HSCs. CD201- HSCs respond to the infection in an indirect manner through G-CSF-R on their surface and exhibit enhanced pSTAT3 activation and elevated LAP/LAP* C/EBPβ isoform, cellular mechanisms known to promote myeloid differentiation and granulocytic production. In conclusion, the switch from CD201+ to CD201- HSCs facilitates both fast and sustained EG leading to the supply of new granulocytes to fight the infection. This detailed understanding of the distinct cellular, transcriptional, and mechanistic properties that determine HSC fate during emergency granulopoiesis opens new venues to potentially modulate granulocytic production in certain clinical conditions such as sepsis. This work was partially supported by a GACR 22-18300S, GAUK 327722, and IMG institutional funding RVO 68378050.

Multi-Omics Analysis Reveals That TET2 Loss Epigenetically Primes Monocytes for Inflammatory Responses Via AP-1 Signaling

Loss of function mutations in TET2 are common in clonal hematopoiesis (CH) and myelodysplastic syndromes (MDS). Increased inflammation and stem cell capacity have been reported in Tet2 KO mouse models, but molecular mechanisms and cell type-specific contributions to explain this phenotype are lacking. To create models of CH-like TET2 deficiency, we transplanted Mx1-CRE Tet2 fl/fl (Tet2 KO) or Tet2 fl/fl (both CD45.1 +) in CD45.2 + recipientsfollowing radiation-free conditioning with ACK2 (anti-cKit) antibody injection. Six months post-transplant, we flow sorted CD45.1 +Tet2 KO and Tet2 fl/fl Lin -Sca-1 +cKit + (LSK) cells, granulocyte-monocyte progenitors (GMP), and monocytes for RNA and assay for transposase-accessible chromatin (ATAC) sequencing. Differential gene expression analysis identified 902 (468 up, 434 down) differentially expressed genes (DEGs) in LSK, 134 (95 up, 39 down) in GMP, and 474 (125 up, 349 down) in monocytes (p adj<0.05). Gene set enrichment analysis (GSEA) identified that the top Hallmark gene sets in Tet2 KOLSK cells were related to MYC targets, oxidative phosphorylation, and E2F targets. In contrast, GMP and monocytes were most enriched for IFN-alpha and gamma gene sets along with IL6/JAK/STAT3 signaling. ATACseq revealed increased accessibility of myeloid-related transcription factor (TF) binding motifs in Tet2 KOLSK cells (CEBPA, PU.1-IRF(ETS:IRF), and AP-1 TF FOSL2(bZIP)), while prediction of cis-regulating regions via GREAT identified downregulated peaks associated with stem cell maintenance and upregulated peaks associated with myeloid mediated immunity. Peaks upregulated in Tet2 KOGMP cells were enriched for the motifs recognized by CEBPE, ISRE(IRF)) and the AP-1 TF JUND, while upregulated peaks in monocytes were enriched for RUNX1 and CEBPA motifs, as well as AP-1 TF FOS::JUNB. GREAT analysis in GMPs identified cis-regulatory regions related to myeloid mediated immunity, while monocytes possessed those associated with biotic stimuli. To better dissect heterogeneity across primitive and mature cell types driven by TET2 loss, we performed scRNAseq on Tet2 KO and Tet2 WT wholebone marrow (WBM) and sorted LSK cells. We identified 18 transcriptionally distinct cell subsets in a combined analysis of WBM. Tet2 KOprogenitor and myeloid subsets were all enriched for gene sets related to IFN-alpha and gamma and immune signaling. Interestingly, primitive and cycling HSPCs were also enriched for MYC target and oxidative phosphorylation gene sets, while these were negatively enriched for GMP, monocyte progenitor, and monocyte Tet2 KO subsets. Further, the IL-6/JAK/STAT3 signaling gene set was enriched in later myeloid cell types, but not in early or cycling HSPCs. These observations led us to hypothesize that Tet2 KO BM cell populations have differentially enhanced phosphoprotein signaling responses to key cytokines. We used a 36-antibody mass cytometry panel to perform deep immunophenotyping and signaling response studies in Tet2 KO BM. FlowSOM analysis identified clusters corresponding to cKIT +CD34 + myeloid progenitors, a Ly6C +CD34 +cKIT + progenitor, and Ly6Chi monocyte clusters were enriched in Tet2 KO BM, while manual gating revealed expansion of LSK, MPP3, and monocytes. We found that activation of pSTAT3 and pSTAT1, in response to either IL-6 or IFNγ, respectively, were significantly higher in Tet2 KO Ly6Chi monocytes, while the more primitive Ly6C +CD34 +cKIT + progenitors demonstrated only an increased response to IFNγ at pSTAT1. Further, prolonged exposure to IL-6 in immortalized Tet2 KO Hoxb8-ER GMP-like cells led to significantly higher levels of pSTAT3 over 4 hours. Since AP-1 TFs are drivers of inflammation and were enriched in our ATACseq dataset, we tested the effects of an AP-1 inhibitor on the Tet2 KO Hoxb8-ER cells. We found that the AP-1 inhibitor T5224 dose dependently reduced the expansion of Tet2 KO Hoxb8 progenitor cells in base media and inhibited the IL-6 mediated expansion of Tet2 KO Hoxb8 cells. Our data show that TET2 loss rewires BM HSPCs towards an inflammatory, myeloid phenotype. Starting at the GMP stage, we see increases in phosphoprotein responses to IFNγ, and then the addition of hypersensitivity to IL-6 in monocytes. TET2 loss leads to an increase in accessible AP-1 motifs in Tet2 KO cells, while inhibition of AP-1 reduces fitness of cells, suggesting a possible therapeutic strategy for TET2-mutated myeloid malignancy.

AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages

Tumor-associated macrophages (TAMs) are integral to the development of complex tumor microenvironments (TMEs) and can execute disparate cellular programs in response to extracellular cues. However, upstream signaling processes underpinning this phenotypic plasticity remain to be elucidated. Here, we report that concordant AXL-STAT3 signaling in TAMs is triggered by lung cancer cells or cancer-associated fibroblasts in the cytokine milieu. This paracrine action drives TAM differentiation toward a tumor-promoting "M2-like" phenotype with upregulation of CD163 and putative mesenchymal markers, contributing to TAM heterogeneity and diverse cellular functions. One of the upregulated markers, CD44, mediated by AXL-IL-11-pSTAT3 signaling cascade, enhances macrophage ability to interact with endothelial cells and facilitate formation of primitive vascular networks. We also found that AXL-STAT3 inhibition can impede the recruitment of TAMs in a xenograft mouse model, thereby suppressing tumor growth. These findings suggest the potential application of AXL-STAT3-related markers to quantitatively assess metastatic potential and inform therapeutic strategies in lung cancer.

Exercise improves cardiac function and attenuates myocardial inflammation and apoptosis by regulating APJ/STAT3 in mice with stroke

Stroke can induce cardiac dysfunction without a primary cardiac disease. Exercise can promote the overall rehabilitation of stroke patients and be beneficial for all kinds of heart diseases. However, the mechanisms underlying the protective effects of exercise in stroke-induced cardiac dysfunction are poorly understood. Hence, we aimed to distinguish the different effects of acute and long-term exercise and further study the mechanism of protection against cardiomyopathy caused by stroke. Mice underwent a single acute session or long-term exercise for 30 days, followed by middle cerebral artery occlusion surgery. The expression of apoptosis-related proteins and proinflammatory factors in the heart was evaluated. Then, overexpression of apelin peptide jejunum (APJ) transfected adeno-associated virus type 9 (AAV9) and inhibition of signal transducer and activator of transcription 3 (STAT3) by Stattic were used in stroke mice or hypoxic cardiomyocytes. ML221 were used to inhibit APJ activity in exercise mouse. Thereafter, changes in apoptotic and proinflammatory factors were evaluated. The results demonstrated that chronic exercise prevented myocardial inflammation, apoptosis and cardiac dysfunction after stroke. However, acute exercise did not have similar effects. Exercise maintained the levels of APJ expression and decreased phosphorylated-STAT3 (p-STAT3) activation to protect cardiomyocytes. Moreover, APJ overexpression promoted cardiomyocyte survival and reduced p-STAT3 levels. STAT3 inhibition also reduced apoptosis and proinflammatory factors in mice hearts. Conversely, the protective effect of exercise was eliminated by APJ inhibition. This study showed that exercise can maintain APJ expression and inhibit p-STAT3, thus, conferring protection against myocardial inflammation and apoptosis induced by stroke.

A structural blueprint for interleukin-21 signal modulation

Interleukin-21 (IL-21) plays a critical role in generating immunological memory by promoting the germinal center reaction, yet clinical use of IL-21 remains challenging because of its pleiotropy and association with autoimmune disease. To better understand the structural basis of IL-21 signaling, we determine the structure of the IL-21-IL-21R-γc ternary signaling complex by X-ray crystallography and a structure of a dimer of trimeric complexes using cryo-electron microscopy. Guided by the structure, we design analogs of IL-21 by introducing substitutions to the IL-21-γc interface. These IL-21 analogs act as partial agonists that modulate downstream activation of pS6, pSTAT3, and pSTAT1. These analogs exhibit differential activity on T and B cell subsets and modulate antibody production in human tonsil organoids. These results clarify the structural basis of IL-21 signaling and offer a potential strategy for tunable manipulation of humoral immunity.

SGLT2 inhibitor dapagliflozin alleviates intramyocardial hemorrhage and adverse ventricular remodeling via suppressing hepcidin in myocardial ischemia-reperfusion injury

Intramyocardial hemorrhage (IMH), a reperfusion therapy-associated complication, is the extravasation of red blood cells caused by severe microvascular injury. IMH is an independent predictor of adverse ventricular remodeling (AVR) after acute myocardial infarction (AMI). Hepcidin, a major regulator of iron uptake and systemic distribution, is a key factor affecting AVR. However, the role of cardiac hepcidin in the development of IMH has not been completely elucidated. This study aimed to explore if sodium-dependent glucose co-transporter 2 inhibitor (SGLT2i) exerts therapeutic effects on IMH and AVR by suppressing hepcidin and to elucidate the underlying mechanisms. SGLT2i alleviated IMH and AVR in the ischemia-reperfusion injury (IRI) mouse model. Additionally, SGLT2i downregulated the cardiac levels of hepcidin in IRI mice, suppressed M1-type macrophage polarization, and promoted M2-type macrophage polarization. The effects of hepcidin knockdown on macrophage polarization were similar to those of SGLT2i in RAW264.7 cells. SGLT2i treatment or hepcidin knockdown inhibited the expression of MMP9, an inducer of IMH and AVR, in RAW264.7 cells. Regulation of macrophage polarization and reduction of MMP9 expression by SGLT2i and hepcidin knockdown is achieved through activation of pSTAT3. In conclusion, this study demonstrated that SGLT2i alleviated IMH and AVR by regulating macrophage polarization. The potential mechanism through which SGLT2i exerted its therapeutic effect seems to involve the downregulation of MMP9 via the hepcidin-STAT3 pathway.

Abstract 2902: Off-the-Shelf CAR-NK cells engineered to express calibrated release IL-15 exhibit enhanced persistence and anti-tumor activities

Abstract Chimeric antigen receptor (CAR) natural killer (NK) cell therapy is an attractive immunotherapy strategy due to potential for increased anti-tumor activity and favorable safety. Allogeneic donor derived NK cells have shown promising clinical outcomes in both hematological and solid tumor malignancies but have a short lifespan in the absence of cytokine support. Interleukin (IL)-15 is a pleiotropic cytokine that promotes the survival, proliferation, and cytotoxicity of NK cells. In this study, we engineered human peripheral blood-derived NK cells to co-express a CAR and calibrated release (cr) IL-15 and studied how crIL-15 affected CAR-NK cell functions. Senti Bio has designed the calibrated release technology to simultaneously produce membrane bound and secreted cytokines, such as IL-15 and IL-12, to provide autocrine and paracrine activity. We further validated crIL-15 activity in vitro and demonstrated enhanced NK cell persistence, tumor killing and autocrine activity in CAR NK cells as well as paracrine stimulation of neighboring T cells and NK cells via pSTAT5 activation. Additionally, we examined in vivo persistence and biodistribution of NK cells engineered with crIL-15 and a nanoluc reporter. crIL-15 NK cells were intravenously (i.v.) injected into NSG mice. These NK cells were detected in the lung, femur, spleen, liver and stomach 1 day post injection by bioluminescence imaging and polymerase chain reaction (PCR) and lasted up to 20 days in the lung. In addition, crIL-15 increased CAR-NK cells persistence in a dose-dependent manner compared to unengineered NK cells. Injection of higher CAR-NK cell numbers (15× 106 vs 7.5× 106 CAR+ cells per mouse) or multiple NK dosages (1 vs 3 doses) both contributed to prolonged persistence. Finally, we studied how crIL-15 affected CAR-NK cell anti-tumor function in acute myeloid leukemia (AML) xenograft models. MV4-11-Fluc AML tumor cells were co-injected with engineered NK cells expressing a FLT3 and/or CD33 bivalent activating CAR, a EMCN inhibitory CAR and crIL-15 into NSG-Tg (Hu-IL15) mice via i.v. injection. Our data showed that these CAR-NK cells significantly reduced MV4-11 tumor burden and prolonged mouse survival compared to unengineered NK cells. The improved anti-tumor functions correlated with IL-15 levels produced by the CAR-NK cells. In conclusion, our results demonstrated that crIL-15 can improve persistence and anti-tumor activity of CAR-NK cells. Citation Format: Chen-Ting Lee, Michelle Hung, Andrew Banicki, Wenqi Song, Niran Almudhfar, Deepika Kaveri, Priscilla Wong, Lawrence Naitmazi, Marcela Guzman, Alice Lam, Gozde Yucel, Timothy Lu, Alba G. Junca, Brian Garrison, Philip Lee. Off-the-Shelf CAR-NK cells engineered to express calibrated release IL-15 exhibit enhanced persistence and anti-tumor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2902.