Common Beta Research Articles

Applications of Escherichia Coli Esterases for Bioremediation and Treatment of Wastewater Organic Chemical Pollutants

This study used computational techniques, including 3D enzyme structural modeling and molecular docking, to gain insight into the bioremediation of organic wastewater contaminants using E. coli esterase enzymes. Furthermore, a total of 24 wastewater organic chemicals belonging to different categories, such as pharmaceuticals, artificial sweeteners, pesticides/herbicides, endocrine disrupting chemicals, and persistent organic pollutants, were identified through the toxicology database. Comparative genetics and reported literature. Furthermore, 3D PDB and AlphaFold structures of 194 esterase enzymes from E. coli were retrieved by first identifying a common domain (Alpha Beta hydrolase domain) using the InterPro database. Molecular docking of esterase enzymes and pollutants was used, resulting in the best binding enzymes to their respective organic wastewater pollutants, including bezafibrate which showed the best binding with all enzymes ranging from -6.33 kcal/mol to -9.87 kcal/mol . Subsequently, the majority of the ligands (organic wastewater pollutants) reacted with enzymes such as the ORFC-like enzymes, which were computationally annotated in this study for the first time, yuaR (strain K12), menH (strain ETI89/UPEC), and menH (strain O157). :H7) has significant binding affinities and consists of a common Alpha Beta hydrolasefold-1 domain. This suggests that esterase enzymes containing the Alpha Beta hydrolasefold-1 domain may be involved in the efficient degradation of organic wastewater pollutants.

E3 ubiquitin ligase RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα

IL-3/STAT5 signaling pathway is crucial for the development and activation of immune cells, contributing to the cellular response to infections and inflammatory stimuli. Dysregulation of the IL-3/STAT5 signaling have been associated with inflammatory and autoimmune diseases characterized by inflammatory cell infiltration and organ damage. IL-3 receptor α (IL-3Rα) specifically binds to IL-3 and initiates intracellular signaling, resulting in the phosphorylation of STAT5. However, the regulatory mechanisms of IL-3Rα remain unclear. Here, we identified the E3 ubiquitin ligase RNF128 as a negative regulator of IL-3/STAT5 signaling by targeting IL-3Rα for lysosomal degradation. RNF128 was shown to selectively bind to IL-3Rα, without interacting with the common beta chain IL-3Rβ, which shares the subunit with GM-CSF. The deficiency of Rnf128 had no effect on GM-CSF-induced phosphorylation of Stat5, but it resulted in heightened Il-3-triggered activation of Stat5 and increased transcription of the Id1, Pim1, and Cd69 genes. Furthermore, we found that RNF128 promoted the K27-linked polyubiquitination of IL-3Rα in a ligase activity-dependent manner, ultimately facilitating its degradation through the lysosomal pathway. RNF128 inhibited the activation and chemotaxis of macrophages in response to LPS stimulation, thereby attenuating excessive inflammatory responses. Collectively, these results reveal that RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα. This study uncovers E3 ubiquitin ligase RNF128 as a novel regulator of the IL-3/STAT5 signaling pathway, providing potential molecular targets for the treatment of inflammatory diseases.

Structure of the interleukin-5 receptor complex exemplifies the organizing principle of common beta cytokine signaling

Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (βc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of βc dimers. These findings provide insights into IL-5 and βc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged βc signaling.

Dual inhibition of airway inflammation and fibrosis by common β cytokine receptor blockade

BackgroundSevere asthmatics can present with eosinophilic type 2 (T2), neutrophilic or mixed inflammation that drives airway remodeling and exacerbations and represents a major treatment challenge. The common beta (βc) receptor signals for three cytokines GM-CSF, IL-5 and IL-3, that collectively mediate T2 and neutrophilic inflammation. ObjectivesTo determine the pathogenesis of βc receptor mediated inflammation and remodeling in severe asthma, and to investigate βc antagonism as a therapeutic strategy for mixed granulocytic airway disease. Methodsβc gene expression was analysed in bronchial biopsies from mild/moderate and severe asthmatics. House dust mite extract (HDM) and Aspergillus fumigatus extract (ASP) models were used to establish asthma-like pathology and airway remodeling in human βc transgenic mice. Lung tissue gene expression was analysed by RNA-sequencing. The monoclonal antibody CSL311 targeting the shared cytokine binding site of βc was used to block βc signaling. Resultsβc gene expression was increased in severe asthmatics. CSL311 potently reduced lung neutrophils, eosinophils and interstitial macrophages, and improved airway pathology and lung function in the acute steroid resistant HDM model. Chronic intranasal ASP exposure induced airway inflammation and fibrosis and impaired lung function that was inhibited by CSL311. CSL311 normalised the ASP-induced fibrosis-associated extracellular matrix gene expression network and strongly reduced signatures of cellular inflammation in the lung. Conclusionsβc cytokines drive steroid-resistant mixed myeloid cell airway inflammation and fibrosis. The anti-βc antibody CSL311 effectively inhibits mixed T2/neutrophilic inflammation and severe asthma-like pathology and reverses fibrosis gene signatures induced by exposure to commonly encountered environmental allergens.

Spectrum Of HBB Gene Variants And Major Endocrine Complications In Thalassemia Patients Of Pakistan.

To determine the molecular characterisation of beta-thalassemia major patients, pattern of major endocrine complications and its association with haemoglobin subunit beta gene variants. The cross-sectional study was conducted from November 2021 to November 2022 after approval from the ethics review committee of Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan, and comprised of 88 patients with beta thalassemia major aged >8 years and having serum ferritin level >1000 μg/L. The subjects were analysed for haemoglobin subunit beta gene variants and major endocrine complications, like growth retardation, hypogonadism, hypothyroidism, hypoparathyroidism and diabetic abnormalities using an automatic chemistry analyser, fully automatic chemiluminescence immunoassay analyser, enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Data was analysed using SPSS 25. Of the 88 subjects, 40(45.4%) were girls and 48(54.5%) were boys. The overall mean age was 12±2.81 years. Of the total, 55(62.5%) had growth retardation, 41(46.6%) were cases of hypogonadism, 16(18.1%) hypothyroidism, 5(5.7%) hypoparathyroidism, 3(3.4%) diabetes mellitus and 8 (9.1%) had impaired glucose tolerance. Also, 65 (73.9%) patients confronted at least one endocrine complication. Endocrine complications were strongly associated with serum ferritin levels (p=0.000). The most common haemoglobin subunit beta gene variant identified was IVSI-5 (G>C) in 36 (40.9%), and the least identified variant was cluster of differenctiation-CD26(G>A) 1(1.1%). The association between haemoglobin subunit beta gene variants with endocrine complications was statistically non-significant (p>0.05). IVSI-5 (G>C) was found to be the most frequent haemoglobin subunit beta gene variant among beta- thalassemia major patients.

(Invited) Effect of Gaseous Tritium Environments on Betavoltaic Device Longevity

Direct betavoltaic energy conversion is a specialized energy harvesting technology, converting beta radiation from a radiation source directly into electricity using a semiconductor. Of the most common beta emitting isotopes, tritium betavoltaics hold promise owing to the high specific activity of solid tritium compounds, low shielding requirements and relatively high availability. Betavoltaic devices offer great promise to produce continuous quantities of nanowatt to microwatt power over the course of several years, particularly for low-power sensor, medical, and space applications where sunlight is too sparse for solar cell use or where battery replacement is challenging.Canadian Nuclear Laboratories (CNL) is currently developing betavoltaic devices based on tritium. CNL has unique facilities to produce, fabricate and test betavoltaic devices. Computational techniques have been used to address challenges of longevity and electron-hole pair generation in semiconductor materials, in particular (In,Ga)P. Long-term studies on test wafers in different tritium-containing environments and the effects on power output and longevity will also be discussed.

Erythropoietin: function and therapeutic potential

Erythropoietin (EPO) exerts its effect on erythroid lineage cells through interaction with the EPO receptor (EPOR), the so-called canonical pathway, and through a complex consisting of EPOR and a common cytokine receptor beta subunit (CD131) – a non-canonical pathway for non-hematopoietic cells of the human and animal body. EPO realizes its effects through the launch of a signaling cascade, which begins with the phosphorylation of Janus kinase 2 (JAK2) and then with the involvement of phosphatidylinositol-3 kinase B (PI3K) or Ras-mitogen-activated protein kinase (MAPK) or signal transducers and transcription activators (STAT). EPO exhibits a direct cytoprotective effect through increased CD131 expression and subsequent development of anti-apoptotic and anti-inflammatory effects in target cells. In addition to its use in the treatment of anemia, EPO is increasingly being used in correction of inflammatory and degenerative processes, both in experimental and clinical studies. EPO promotes the engraftment of stem cells, differentiation of mesenchymal stem cells in the connective tissue direction, suppresses the inflammatory response and apoptosis of cells in the lesion. The article includes literature data concerning EPO and its clinical use in inflammatory and degenerative processes, based on data from eLibrary and the National Center for Biotechnological Information (NCBI) for the period 1998–2022.

Chromatin accessibility differences between alpha, beta, and delta cells identifies common and cell type-specific enhancers

BackgroundHigh throughput sequencing has enabled the interrogation of the transcriptomic landscape of glucagon-secreting alpha cells, insulin-secreting beta cells, and somatostatin-secreting delta cells. These approaches have furthered our understanding of expression patterns that define healthy or diseased islet cell types and helped explicate some of the intricacies between major islet cell crosstalk and glucose regulation. All three endocrine cell types derive from a common pancreatic progenitor, yet alpha and beta cells have partially opposing functions, and delta cells modulate and control insulin and glucagon release. While gene expression signatures that define and maintain cellular identity have been widely explored, the underlying epigenetic components are incompletely characterized and understood. However, chromatin accessibility and remodeling is a dynamic attribute that plays a critical role to determine and maintain cellular identity.ResultsHere, we compare and contrast the chromatin landscape between mouse alpha, beta, and delta cells using ATAC-Seq to evaluate the significant differences in chromatin accessibility. The similarities and differences in chromatin accessibility between these related islet endocrine cells help define their fate in support of their distinct functional roles. We identify patterns that suggest that both alpha and delta cells are poised, but repressed, from becoming beta-like. We also identify patterns in differentially enriched chromatin that have transcription factor motifs preferentially associated with different regions of the genome. Finally, we not only confirm and visualize previously discovered common endocrine- and cell specific- enhancer regions across differentially enriched chromatin, but identify novel regions as well. We compiled our chromatin accessibility data in a freely accessible database of common endocrine- and cell specific-enhancer regions that can be navigated with minimal bioinformatics expertise.ConclusionsBoth alpha and delta cells appear poised, but repressed, from becoming beta cells in murine pancreatic islets. These data broadly support earlier findings on the plasticity in identity of non-beta cells under certain circumstances. Furthermore, differential chromatin accessibility shows preferentially enriched distal-intergenic regions in beta cells, when compared to either alpha or delta cells.

Incidence of Βeta-Thalassemia Minor among Healthy Blood Donors

Background: Disorders with a markedly slowed rate of globin chain synthesis are referred to as thalassemia. Hemoglobinopathy is a word used to describe diseases that cause structurally aberrant hemoglobin. Iron deficiency is seen in beta-thalassemia minor, which may change the typically increased HbA2 levels. According to World Health Organization (WHO) statistics, 7% of the global population carries a hemoglobin problem. Thalassemia is the most prevalent of the hemoglobinopathies, which are significant genetic issues in Pakistan. The prevalence of -thalassemia in Pakistan is around 5%, whereas the prevalence of hemoglobin S or E is between 0.5% and 1%. The purpose of the study was to determine how common beta thalassemia minor was among blood donors. Methods: A total of 500 individuals were recruited in this cross-sectional study who were referred for hemoglobin electro-phoresis between September 2015 and March 2016 were the subject of this investigation. A thorough clinical history was taken, including information on the patient's age, sex, cast, family history, history of blood transfusions, and physical findings such as splenomegaly. Hb Electrophoresis tests were performed on all blood samples. Results: Out of total 500 patients, male were 83.1% (n=79) while female were 16.9% (n=16). Among total individuals, 18.3% (n=94) were found positive cases on Hb electrophoresis technique while remaining 406 patients were observed negative. All recruited individuals were categorised in four (04) different groups based on age; highest number of individuals were found in 18-30 years of age group with 69 patients, followed by 31-40 years of age group (n=19), 41-50 years of age group (n=5.3), and 51-60 years of age group (n=01). 94 (18.3%) had abnormal hemoglobin and beta thalassemia minor. Of them, 79 (84.4%) were men and 16 (17.2%) were women. Conclusion: Many groups in Pakistan continue to struggle with hemoglobin problems. Preventive interventions, such as pre-marriage carrier status identification and screening for beta thalassemia minor, are required to lower the incidence of beta thalassemia major by forcing couples with beta thalassemia minor to abort their pregnancies. Keywords: Hemoglobin Disorders, Thalassemia, β-Thalassemia, Healthy Blood Donors, Hemoglobinopathy

Abstract A004: The role of calcium in metastatic progression

Abstract Calcium is essential to the structural stability of integrin proteins implicated in platelet–cancer cell interactions, namely integrins GPIIb/IIIa and avb3, which share a common beta subunit (Pelletier et al., J Biol Chem, 1996), (Zhang and Chen, Cell Adh Migr, 2012). Additionally, calcium is essential for ligand binding to these integrins (e.g., fibrinogen) that bridge platelets and cancer cells (Raborn et al., Biochemistry, 2011) and for the downstream signaling following mechanical and chemical stimulation in the integrin microenvironment (Michelson, Platelets, 2013). Platelet-cancer cell interactions are key players in cancer progression as platelets support metastatic dissemination (Morris et al., Biochim. Biophys. Acta BBA - Rev. Cancer, 2022). Despite previous studies recognizing calcium’s role in integrin stability and function, calcium’s importance for platelet support in cancer progression is not well understood. Therefore, we aim to (1) characterize the receptor expression and ligand binding ability of GPIIb/IIIa and avb3 on respective cells via anti-CD41a (for platelets and breast cancer MDA-MB-231 cells) and anti-avb3 antibody (for lung cancer A549 cells), (2) characterize platelet function by assessing platelet aggregation using light transmission aggregometry and platelet adhesion on a thrombogenic surface, (3) characterize platelet-cancer cell interaction via flow cytometry, and (4) assess cancer cell invasion in the presence of platelets via transwell assay; all in the presence of an environment with calcium chelation and hypercalcemic conditions. Our results demonstrate that platelet GPIIb/IIIa, cancer cell GPIIb/IIIa (MDA-MB-231) and cancer cell integrin avb3 (A549) are modulated with calcium chelation. Additionally, platelet function, specifically aggregation is significantly decreased with calcium chelation and significantly increased with hypercalcemia. We also observed that the interaction between platelets and cancer cells is reduced with calcium chelation, platelet-MDA-MB-231 interaction specifically being significantly reduced in the presence of sodium citrate. Our overall hypothesis is that the observed effect of calcium levels of platelet and cancer cell integrins could have a significant influence on platelet-cancer cell interactions and cancer cell migration in hypercalcemia, often seen in patients with malignancy. A greater understanding of these interactions could have impact for the design of specific inhibitors with anti-metastatic effects and potentially lead to novel targeted therapies to combat metastatic dissemination. Citation Format: Kenise Morris, Anne-Laure Papa. The role of calcium in metastatic progression [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A004.

Bioaccumulation Behaviors of Copper Phthalocyanine Nano-pigments in Adult Zebrafish (Danio rerio)

Copper phthalocyanine (CuPc) has the largest application market of blue nano-pigments (NPs), which can release from relative products to aquatic environment during daily use. However, bioaccumulation of CuPc NPs in high trophic-level aquatic organisms is unclear. Therefore, bioaccumulation behaviors of CuPc in zebrafish as model aquatic organism were investigated. In this work, CuPc NPs were irregular in shape with an average size of 76.8 nm, whose crystal phase was a common beta phase according to the XRD spectrum. After 10 d of exposure to CuPc NPs, the weight of zebrafish at both low and high concentrations showed no obvious difference with con-trol, indicating little influence of CuPc on the survival of zebrafish. It was noted that amounts of Cu accumulated in the liver (982.84 ng/g) and skin (609.08 ng/g), and a small accumulation of Cu was observed in the gut, heart, and gill. CuPc NPs could not lead to Cu accumulation in the brain and muscle. This work helps to better understand the bioaccumulation behaviors and potential risks of CuPc NPs.

Beta inputs to motor neurons do not directly contribute to volitional force modulation.

Neural oscillatory activity in the beta band (13-30Hz) is prominent in the brain and it is transmitted partly linearly to the spinal cord and muscles. Multiple views on the functional relevance of beta activity in the motor system have been proposed. Previous simulation work suggested that pools of spinal motoneurons (MNs) receiving a common beta input could demodulate this activity, transforming it into low-frequency neural drive that could alter force production in muscles. This may suggest that common beta inputs to muscles have a direct role in force modulation. Here we report the experimental average levels and ranges of common beta activity in spinal MNs projecting to single muscles and use a computational model of a MN pool to test if the experimentally observed beta levels in MNs can influence force. When beta was modelled as a continuous activity, the amplitude needed to produce non-negligible changes in force corresponded to beta representation in the MN pool that was far above the experimental observations. On the other hand, when beta activity was modelled as short-lived events (i.e. bursts of beta activity separated by intervals without beta oscillations), this activity approximated levels that could cause small changes in force with estimated average common beta inputs to the MNs compatible with the experimental observations. Nonetheless, bursting beta is unlikely to be used for force control due to the temporal sparsity of this activity. It is therefore concluded that beta oscillations are unlikely to contribute to the voluntary modulation of force. KEY POINTS: It has been previously proposed that beta (13-30Hz) common inputs to a motor neuron pool may have a non-linear effect in voluntary force control. The needed strength of beta oscillations to modulate forces has not been analysed yet. Based on computer simulations, we show that sustained beta inputs to a spinal motoneuron pool at physiologically reported levels have minimal effect on force. Levels of sustained beta rhythmic activity that can cause a significant change in force are not compatible with experimental observations of intramuscular coherence in human skeletal muscles.

A Reference Source Circuit Design for Voltage Controlled Oscillator Chips

The basic principle and typical structure of the bandgap voltage reference source are introduced in detail in this paper. The performance index of the bandgap voltage reference is analyzed. The core circuit of a bandgap voltage reference source with good temperature characteristics and a high power supply rejection ratio (PSRR) is designed, in which a two-stage differential operational amplifier with a buffer stage is used, a start-up circuit without a capacitor is used to reduce the start-up time, and a common beta multiplication self-bias circuit is used to attenuate the effects of the power supply voltage. It is shown from the final HSPICE simulation results that the reference voltage temperature coefficient is approximately 7 ppm/°C within the range of -20 to 80°C in temperature, and the average PSRR can reach more than −56 dB within the range of 1 Hz to 10 kHz in frequency.

Abstract 3504: QL415, a tumor targeted IL-15 fusion protein stimulating both lymphoid and myeloid immune cells for optimal anti-tumor immune response

Abstract Cytokines are potent stimulators of the immune system and have long been investigated and used as therapeutics to treat cancer. However, free cytokines have short half-life and the lack of tumor targeting often results in systemic toxicity. IL-15 is a stimulatory cytokine that shares the common beta and gamma receptors with IL-2. In contrast to IL-2, IL-15 is more selective for NK and effector memory T cells, with less proliferative effect on Tregs, making it a more preferred therapeutic candidate for cancer. QL415 is an anti-PD-L1 x IL-15 fusion protein designed to enhance tumor accumulation and prolong blood circulation, thereby widening the therapeutic window. IL-15 and IL-15 receptor alpha sushi domain are covalently linked to the C-terminus of our proprietary PD-L1 antibody. Fc-mediated effector functions were ablated using mutations in the CH2 domain, to prevent depletion of effector immune cells. In vitro studies showed QL415 to be highly potent in promoting proliferation and pSTAT5 signaling of IL-15 -responding NK92 and M07e cells. QL415 selectively induced proliferation of NK and CD8+ T cells from human PBMC in vitro and mouse CD8+ and NK1.1 cells in vivo. In a MC38 mouse colon cancer model expressing human PD-L1, QL401 was highly effective in suppressing tumor growth, in contrast to PD-L1 monoclonal antibody or a non-targeted IL-15 control. Follow up tumor rechallenge study showed protective memory against cognate MC38 but not B16F10. In a separate MC38 tumor model using a mouse surrogate molecule of QL415, immunophenotyping of antigen presenting cells offered preliminary evidence of enrichment of conventional dendritic cells 1 in the tumor draining lymph nodes. Therefore, QL415 not only directly stimulates effector immune cells, but also indirectly through antigen presenting cells, promotes robust anti-tumor response. In non-human primates, Q415 was tolerated up to 1.5 mg/kg, well above the effective therapeutic dose. In light of these favorable preclinical efficacy and safety results, a phase 1 dose escalation and expansion study has been initiated to evaluate the safety, tolerability, and early efficacy of QL415. Citation Format: Irene Tang, Lauren Schwimmer, Monica Jin, Shenda Gu, Wei Wei Prior, Arianne Capacio, Hieu V. Tran, Allan Chan, Anna McClain, Shihao Chen, Chuanzeng Cao, Xiaoran Wu, Yanling Xu, Dongmei Guan, Xi Chen, Xianli Su. QL415, a tumor targeted IL-15 fusion protein stimulating both lymphoid and myeloid immune cells for optimal anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3504.

Arab-Indian −530 β-distal promoter haplotype and sickle/Hb D heterozygosis in Badagas of Nilgiris: is it suggestive of Harappan origin?

Origin and ancestry of the Badagas of Nilgiris, an indigenous community of Nilgiris, Tamil Nadu in India, is a controversial topic. During our massive screening for haemoglobinopathies among Badagas, we identified a rare south Indian haemoglobin variant, the haemoglobin D (Hb D) Punjab (HBB: c.364G>C; rs33946267). The observed Hb D cases were double heterozygous with sickle cell trait (HbSD-Punjab) and manifested symptoms. The beta-globin distal promoter motif (-530 cap site BP1U) sequencing revealed a characteristic heterozygous combination of Arab-Indian sickle haplotype ((AT)9 (T)5) with universally common beta gene haplotype ((AT)7 (T)7). The report is a pioneer one informing the presence of Hb-D Punjab from an indigenous or aboriginal group of southern India. By the above information, we support the theory of unicentric tribal origin and distribution during Harappan civilization.

Blocking the human common beta subunit of the GM-CSF, IL-5 and IL-3 receptors markedly reduces hyperinflammation in ARDS models

Acute respiratory distress syndrome (ARDS) is triggered by various aetiological factors such as trauma, sepsis and respiratory viruses including SARS-CoV-2 and influenza A virus. Immune profiling of severe COVID-19 patients has identified a complex pattern of cytokines including granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-5, which are significant mediators of viral-induced hyperinflammation. This strong response has prompted the development of therapies that block GM-CSF and other cytokines individually to limit inflammation related pathology. The common cytokine binding site of the human common beta (βc) receptor signals for three inflammatory cytokines: GM-CSF, IL-5 and IL-3. In this study, βc was targeted with the monoclonal antibody (mAb) CSL311 in engineered mice devoid of mouse βc and βIL-3 and expressing human βc (hβcTg mice). Direct pulmonary administration of lipopolysaccharide (LPS) caused ARDS-like lung injury, and CSL311 markedly reduced lung inflammation and oedema, resulting in improved oxygen saturation levels in hβcTg mice. In a separate model, influenza (HKx31) lung infection caused viral pneumonia associated with a large influx of myeloid cells into the lungs of hβcTg mice. The therapeutic application of CSL311 potently decreased accumulation of monocytes/macrophages, neutrophils, and eosinophils without altering lung viral loads. Furthermore, CSL311 treatment did not limit the viral-induced expansion of NK and NKT cells, or the tissue expression of type I/II/III interferons needed for efficient viral clearance. Simultaneously blocking GM-CSF, IL-5 and IL-3 signalling with CSL311 may represent an improved and clinically applicable strategy to reducing hyperinflammation in the ARDS setting.

The IL-3, IL-5, and GM-CSF common receptor beta chain mediates oncogenic activity of FLT3-ITD-positive AML

FLT3-ITD is the most predominant mutation in AML being expressed in about one-third of AML patients and is associated with a poor prognosis. Efforts to better understand FLT3-ITD downstream signaling to possibly improve therapy response are needed. We have previously described FLT3-ITD-dependent phosphorylation of CSF2RB, the common receptor beta chain of IL-3, IL-5, and GM-CSF, and therefore examined its significance for FLT3-ITD-dependent oncogenic signaling and transformation. We discovered that FLT3-ITD directly binds to CSF2RB in AML cell lines and blasts isolated from AML patients. A knockdown of CSF2RB in FLT3-ITD positive AML cell lines as well as in a xenograft model decreased STAT5 phosphorylation, attenuated cell proliferation, and sensitized to FLT3 inhibition. Bone marrow from CSF2RB-deficient mice transfected with FLT3-ITD displayed decreased colony formation capacity and delayed disease onset together with increased survival upon transplantation into lethally irradiated mice. FLT3-ITD-dependent CSF2RB phosphorylation required phosphorylation of the FLT3 juxtamembrane domain at tyrosines 589 or 591, whereas the ITD insertion site and sequence were of no relevance. Our results demonstrate that CSF2RB participates in FLT3-ITD-dependent oncogenic signaling and transformation in vitro and in vivo. Thus, CSF2RB constitutes a rational treatment target in FLT3-ITD-positive AML.

A Functional GM-CSF Receptor on Dendritic Cells Is Required for Efficient Protective Anti-Tumor Immunity

Dendritic cells (DC) play a major role during the priming phase of anti-tumor immunization, as they are required for an efficient tumor-associated antigens presentation. At least one dendritic cell-based therapy has already been successfully approved by regulators for clinical application in prostate cancer patients. Moreover, DC development is dependent on the granulocyte macrophage colony stimulating factor (GM-CSF), a cytokine that has been successfully used as a potent inducer of anti-tumoral immunity. To better understand the relation between DC and GM-CSF in anti-tumor immunity, we studied the DC function in mice lacking the cytokine receptor common subunit beta (βc-/-) for GM-CSF, IL-3 and IL-5 and immunized with irradiated tumor cells. Such immunization induces a protective, specific tumor immunization in wild-type mice, while βc-/- mice failed to mount an immune response. Upon in vitro stimulation, DC from βc-/- mice (DCβc-/-) are unable to undergo a full maturation level. In vivo experiments show that they lack the ability to prevent tumor growth, in contrast to DCWT. Moreover, matured DCWT rescued immunization in βc-/- mice. DC maturation is dependent on a functional pathway involving GM-CSF signaling through a biologically functional receptor. These findings may contribute to new strategies for efficient anti-tumor immunotherapies.

Acute Exercise Regulates Htert Alternative Splicing In Contractile Tissues Of Htert-bac Mice

Full length human telomerase reverse transcriptase (FL hTERT) is the rate-limiting, catalytic component necessary for telomerase enzyme activity and telomere length maintenance. Unlike rodents, humans evolved elongated introns with cis-elements that when bound by specific RNA binding proteins (RNAbp) splice hTERT to FL transcripts or produce inactive variants through alternative splicing (AS). Our working hypothesis is that hTERT AS regulates the amount of active telomerase. Evidence from human studies have shown that aerobic exercise maintains telomere length through increased hTERT expression and telomerase enzyme activity, however the impact of acute exercise on hTERT AS is unknown. PURPOSE: To examine hTERT AS regulation in response to acute treadmill running. METHODS: A bacterial artificial chromosome mouse model containing the full hTERT gene (5' and 3' regulatory elements; 16 exons and 15 introns) inserted into its genome (hTERT-BAC) was utilized. Gastrocnemius (Gas.) and heart (Hrt.) were excised from 10-week old hTERT-BAC mice prior to (PRE), upon cessation (POST), and during recovery (1, 24, 48, and 72H; n = 5/time point) from 30 min. of treadmill running (60% max. speed). Primers specific to hTERT were utilized to measure FL and the most common AS variant minus beta (-β, which skips exons 7 and 8) by two different RT-PCR methods - gel based and droplet digital PCR. Gene expression of the RNAbp PTBP1 was also measured. RESULTS: The expected human patterns of hTERT AS was recapitulated across all tissues examined. Compared to PRE, FL hTERT gene expression increased at POST before decreasing in the Gas. (48 and 72H; p ≤ 0.001) and Hrt. (24H; p = 0.004). Acute exercise also increased the percentage of FL hTERT in the Gas. at 1 and 72H (p ≤ 0.017), whereas a decrease was observed in the Hrt. at 1, 24, and 48H (p ≤ 0.041). Finally, Ptbp1 decreased at POST (Gas.) and at 1, 24, 48, and 72H (Gas. and Hrt.; p ≤ 0.05). CONCLUSIONS: Evolutionarily conserved mouse splicing machinery (RNAbp) recognize hTERT cis-elements to recapitulate human AS patterns and highlight the importance of the intronic elements in regulating hTERT splicing choice in humans. The hTERT-BAC mouse is a valuable resource to study the exercise-induced adaptive mechanisms that maintain telomere lengths in endurance trained individuals across the lifespan.

Abstract LB133: Targetting IL-6/gp130 signalling axis attenuates acquired drug resistance in medulloblastoma

Abstract Medulloblastoma (MB) is the most common pediatric brain tumour comprising of four distinct molecular subgroups exhibiting high level of intertumoural heterogeneity. The standard multimodal treatment yields a survival rate of 70% but the aggressive treatment affects the long-term sequelae of MB patients. The tumour microenvironment (TME) is a regulator of cancer progression and affects therapeutic efficacy in primary and metastatic brain malignancies. Mechanistic insights into the tumor-promoting role of the individual components of the brain TME will aid in identifying key survival pathways and design of potential therapeutics to combat drug resistance and pathogenesis of the disease. We hypothesize that tumor associated macrophages (TAMs) in the brain TME facilitate drug resistance via the IL-6/STAT3 signalling axis in MB. To understand the role of paracrine signaling resulting from TAMs, a co-culture system was used to evaluate the expression of pSTAT3 and correlated with drug sensitivity and/or resistance in Med8A cell line. When co-cultured with HMC3, a human microglia cell line, the chemosensitive Med8A-S cells exhibited enhanced pSTAT3 expression and acquired drug resistance. Interestingly, Med8A cells lacking expression of IL-6 receptor (IL6R-/-) also exhibited high pSTAT3 levels and drug resistance; suggesting that HMC3 may release soluble factors in addition to IL-6 sufficient to drive chemoresistance in Med8A variants. To assess this, we conditioned cells with stimulatory cytokines belonging to the IL-6 family. We found that oncostatin M (OSM), interleukin-11 (IL-11) and leukemia inhibitory factor (LIF) induced drug resistance and enhanced pSTAT3 levels in IL6R-/- cells. Receptors of the IL-6 family share a common signal transducing beta subunit, glycoprotein (gp130) that transduces the signal intracellularly and phosphorylates janus activated kinases (JAKs) and subsequently STAT3. Our results suggest that paracrine proinflammatory cytokines found in the TME can promote MB drug resistance by signalling through a common signal transducer gp130, hence targeting any one cytokine receptor of the IL-6 family of cytokines may not sufficiently abrogate acquired drug resistance. Currently, we are evaluating the efficacy of agents that target common downstream elements of the IL-6/STAT3 signalling axis, including gp130, JAKs and STAT3, as a potentially improved therapeutic strategy to circumvent acquired drug resistance in medulloblastoma. Citation Format: Lakshana Sreenivasan, Pascal Leclair, Chinten James Lim. Targetting IL-6/gp130 signalling axis attenuates acquired drug resistance in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB133.