Crosstalk Of Signaling Pathways Research Articles

NFκB pathway dysregulation due to reduced RelB expression leads to severe autoimmune disorders and declining immunity

BackgroundGenetic aberrations in the NFκB pathway lead to primary immunodeficiencies with various degrees of severity. We previously demonstrated that complete ablation of the RelB transcription factor, a key component of the alternative pathway, results in an early manifested combined immunodeficiency requiring stem cell transplantation. ObjectiveTo study the molecular basis of a progressive severe autoimmunity and immunodeficiency in three patients. MethodsWhole exome sequencing was performed to identify the genetic defect. Molecular and cellular techniques were utilized to assess the variant impact on NFκB signaling, canonical and alternative pathway crosstalk, as well as the resultant effects on immune function. ResultsPatients presented with multiple autoimmune progressive severe manifestations encompassing the liver, gut, lung, and skin, becoming debilitating in the second decade of life. This was accompanied by a deterioration of the immune system, demonstrating an age-related decline in naïve T cells and responses to mitogens, accompanied by a gradual loss of all circulating CD19+ cells. Whole exome sequencing identified a novel homozygous c. C1091T (P364L) transition in RELB. The P364L RelB protein was unstable, with extremely low expression, but retained some function and could be transiently and partially upregulated following Toll-like receptor stimulation. Stimulation of P364L patient fibroblasts resulted in a marked rise in a cluster of pro-inflammatory hyper-expressed transcripts consistent with the removal of RelB inhibitory effect on RelA function. This is likely the main driver of autoimmune manifestations in these patients. ConclusionIncomplete loss of RelB provided a unique opportunity to gain insights into NFκB's pathway interactions as well as the pathogenesis of autoimmunity. The P364L RelB mutation leads to gradual decline in immune function with progression of severe debilitating autoimmunity.

Multiple-Gene Regulation for Enhanced Antitumor Efficacy with Branch-PCR-Assembled TP53 and MYC Gene Nanovector.

Multiple proteins are involved in network regulation through the crosstalk of different signaling pathways in cancers. Here, we propose a novel strategy of genome therapy with branch-PCR-assembled gene nanovectors to perform network-based gene regulation at multiple levels for cancer therapy. To validate network-based multiplex-gene regulation for genome therapy, we chose to simultaneously target one tumor suppressor gene (TP53) and one oncogene (MYC) in two different signaling pathways. The results showed that, compared to gene nanovectors targeting single genes (NP-TP53 and NP-shMYC), branch-PCR-assembled gene nanovectors simultaneously expressing p53 proteins and MYC shRNA arrays (NP-TP53-shMYC) showed enhanced antitumor efficacy in both MDA-MB-231 cancer cells and an MDA-MB-231-tumor-bearing mouse model. These findings indicate the feasibility and effectiveness of genome therapy in cancer therapy.

Chemoresistance-Related Stem Cell Signaling in Osteosarcoma and Its Plausible Contribution to Poor Therapeutic Response: A Discussion That Still Matters.

Osteosarcoma is amongst the most prevalent bone sarcomas and majorly afflicts children and adolescents. Therapeutic regimens based on the triad of doxorubicin, cisplatin and methotrexate have been used as the state-of-the-art approach to clinical treatment and management, with no significant improvements in the general outcomes since their inception in the early 1970s. This fact raises the following problematic questions: Why do some patients still relapse despite an initial good response to therapy? Why do nearly 30% of patients not respond to neoadjuvant therapies? Does residual persistent disease contribute to relapses and possible metastatic dissemination? Accumulating evidence suggests that chemoresistant cancer stem cells may be the major culprits contributing to those challenging clinical outcomes. Herein, we revisit the maneuvers that cancer stem cells devise for eluding cell killing by the classic cytotoxic therapies used in osteosarcoma, highlighting studies that demonstrate the complex crosstalk of signaling pathways that cancer stem cells can recruit to become chemoresistant.

Structural insights into Ras regulation by SIN1

Over the years it has been established that SIN1, a key component of mTORC2, could interact with Ras family small GTPases through its Ras-binding domain (RBD). The physical association of Ras and SIN1/mTORC2 could potentially affect both mTORC2 and Ras-ERK pathways. To decipher the precise molecular mechanism of this interaction, we determined the high-resolution structures of HRas/KRas-SIN1 RBD complexes, showing the detailed interaction interface. Mutation of critical interface residues abolished Ras-SIN1 interaction and in SIN1 knockout cells we demonstrated that Ras-SIN1 association promotes SGK1 activity but inhibits insulin-induced ERK activation. With structural comparison and competition fluorescence resonance energy transfer (FRET) assays we showed that HRas-SIN1 RBD association is much weaker than HRas-Raf1 RBD but is slightly stronger than HRas-PI3K RBD interaction, providing a possible explanation for the different outcome of insulin or EGF stimulation. We also found that SIN1 isoform lacking the PH domain binds stronger to Ras than other longer isoforms and the PH domain appears to have an inhibitory effect on Ras-SIN1 binding. In addition, we uncovered a Ras dimerization interface that could be critical for Ras oligomerization. Our results advance our understanding of Ras-SIN1 association and crosstalk between growth factor-stimulated pathways.

Tyrosine kinase inhibitors in sarcoma treatment.

Sarcomas are a group of rare mesenchymal malignant tumors that arise from transformed cells of the mesenchymal connective tissue, which are challenging to treat. The majority of sarcomas are soft tissue sarcomas (STSs; 75%) and this heterogeneous group of tumors is further comprised of gastrointestinal stromal tumors (~15%) and bone sarcomas (10%). Although surgery remains the current primary therapeutic approach for localized disease, recurrent, metastatic and refractory sarcomas require cytotoxic chemotherapy, which usually yields poor results. Therefore the efficiency of sarcoma treatment imposes a difficult problem. Furthermore, even though progress has been made towards understanding the underlying molecular signaling pathways of sarcoma, there are limited treatment options. The aim of the present study was therefore to perform a systematic literature review of the available clinical evidence regarding the role of tyrosine kinase inhibitors (TKIs) in patients with recurrent or refractory STSs and bone sarcomas over the last two decades. Tyrosine kinases are principal elements of several intracellular molecular signaling pathways. Deregulation of these proteins has been implicated in driving oncogenesis via the crosstalk of pivotal cellular signaling pathways and cascades, including cell proliferation, migration, angiogenesis and apoptosis. Subsequently, small molecule TKIs that target these proteins provide a novel potential therapeutic approach for several types of tumor by offering significant clinical benefits. Among the eligible articles, there were 45 prospective clinical trials, primarily multicentric, single arm, phase II and non-randomized. Numerous studies have reported promising results regarding the use of TKIs, mainly resulting in disease control in patients with STSs. The lack of randomized clinical trials demonstrates the ambiguous efficiency of various studied treatment options, which therefore currently limits the approved drugs used in clinical practice. Research both in clinical and preclinical settings is needed to shed light on the underlying molecular drivers of sarcomagenesis and will identify novel therapeutic approaches for pretreated patients.

Semantic clustering analysis of E3-ubiquitin ligases in gastrointestinal tract defines genes ontology clusters with tissue expression patterns

BackgroundUbiquitin ligases (Ub-ligases) are essential intracellular enzymes responsible for the regulation of proteome homeostasis, signaling pathway crosstalk, cell differentiation and stress responses. Individual Ub-ligases exhibit their unique functions based on the nature of their substrates. They create a complex regulatory network with alternative and feedback pathways to maintain cell homeostasis, being thus important players in many physiological and pathological conditions. However, the functional classification of Ub-ligases needs to be revised and extended.MethodsIn the current study, we used a novel semantic biclustering technique for expression profiling of Ub-ligases and ubiquitination-related genes in the murine gastrointestinal tract (GIT). We accommodated a general framework of the algorithm for finding tissue-specific gene expression clusters in GIT. In order to test identified clusters in a biological system, we used a model of epithelial regeneration. For this purpose, a dextran sulfate sodium (DSS) mouse model, following with in situ hybridization, was used to expose genes with possible compensatory features. To determine cell-type specific distribution of Ub-ligases and ubiquitination-related genes, principal component analysis (PCA) and Uniform Manifold Approximation and Projection technique (UMAP) were used to analyze the Tabula Muris scRNA-seq data of murine colon followed by comparison with our clustering results.ResultsOur established clustering protocol, that incorporates the semantic biclustering algorithm, demonstrated the potential to reveal interesting expression patterns. In this manner, we statistically defined gene clusters consisting of the same genes involved in distinct regulatory pathways vs distinct genes playing roles in functionally similar signaling pathways. This allowed us to uncover the potentially redundant features of GIT-specific Ub-ligases and ubiquitination-related genes. Testing the statistically obtained results on the mouse model showed that genes clustered to the same ontology group simultaneously alter their expression pattern after induced epithelial damage, illustrating their complementary role during tissue regeneration.ConclusionsAn optimized semantic clustering protocol demonstrates the potential to reveal a readable and unique pattern in the expression profiling of GIT-specific Ub-ligases, exposing ontologically relevant gene clusters with potentially redundant features. This extends our knowledge of ontological relationships among Ub-ligases and ubiquitination-related genes, providing an alternative and more functional gene classification. In a similar way, semantic cluster analysis could be used for studding of other enzyme families, tissues and systems.

Prognostic implications and functional enrichment analysis of LTB4R in patients with acute myeloid leukemia

To explore the expression patterns, prognostic implications, and biological role of leukotriene B4 receptor (LTB4R) in patients with acute myeloid leukemia (AML). We collected the data of mRNA expression levels and clinical information of patients with AML from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database for mRNA expression analyses, survival analyses, Cox regression analyses and correlation analyses using R studio to assess the expression patterns and prognostic value of LTB4R. The correlation of LTB4R expression levels with clinical characteristics of the patients were analyzed using UALCAN. The co-expressed genes LTB4R were screened from Linkedomics and subjected to functional enrichment analysis. A protein-protein interaction network was constructed using STRING. GSEA analyses of the differentially expressed genes (DEGs) were performed based on datasets from TCGA-LAML stratified by LTB4R expression level. We also collected peripheral blood mononuclear cells (PBMCs) from AML patients and healthy donors for examination of the mRNA expression levels of LTB4R and immune checkpoint genes using qRT-PCR. We also examined serum LTB4R protein levels in the patients using ELISA. The mRNA expression level of LTB4R was significantly increased in AML patients (4.898±1.220 vs 2.252±0.215, P < 0.001), and an elevated LTB4R expression level was correlated with a poor overall survival (OS) of the patients (P=0.004, HR=1.74). LTB4R was identified as an independent prognostic factor for OS (P=0.019, HR=1.66) and was associated with FAB subtypes, cytogenetic risk, karyotype abnormalities and NPM1 mutations. The co- expressed genes of LTB4R were enriched in the functional pathways closely associated with AML leukemogenesis, including neutrophil inflammation, lymphocyte activation, signal transduction, and metabolism. The DEGs were enriched in differentiation, activation of immune cells, and cytokine signaling. Examination of the clinical serum samples also demonstrated significantly increased expressions of LTB4R mRNA (P=0.044) and protein (P=0.008) in AML patients, and LTB4R mRNA expression was positively correlated with the expression of the immune checkpoint HAVCR2 (r= 0.466, P=0.040). LTB4R can serve as a novel biomarker and independent prognostic indicator of AML and its expression patterns provide insights into the crosstalk of leukemogenesis signaling pathways involving tumor immunity and metabolism.

The Cross-Talks Among Bone Morphogenetic Protein (BMP) Signaling and Other Prominent Pathways Involved in Neural Differentiation.

The bone morphogenetic proteins (BMPs) are a group of potent morphogens which are critical for the patterning, development, and function of the central nervous system. The appropriate function of the BMP pathway depends on its interaction with other signaling pathways involved in neural differentiation, leading to synergistic or antagonistic effects and ultimately favorable biological outcomes. These opposite or cooperative effects are observed when BMP interacts with fibroblast growth factor (FGF), cytokines, Notch, Sonic Hedgehog (Shh), and Wnt pathways to regulate the impact of BMP-induced signaling in neural differentiation. Herein, we review the cross-talk between BMP signaling and the prominent signaling pathways involved in neural differentiation, emphasizing the underlying basic molecular mechanisms regarding the process of neural differentiation. Knowing these cross-talks can help us to develop new approaches in regenerative medicine and stem cell based therapy. Recently, cell therapy has received significant attention as a promising treatment for traumatic or neurodegenerative diseases. Therefore, it is important to know the signaling pathways involved in stem cell differentiation toward neural cells. Our better insight into the cross-talk of signaling pathways during neural development would improve neural differentiation within in vitro tissue engineering approaches and pre-clinical practices and develop futuristic therapeutic strategies for patients with neurological disease.

A Bibliometric Analysis Based on Web of Science: Current Perspectives and Potential Trends of SMAD7 in Oncology.

Background: The number of publications on SMAD7 in the field of oncology is increasing rapidly with an upward tendency. In most cases, the mechanisms of carcinogenesis usually relate to disorders of signaling activity. Considering the crucial role of SMAD7 in the crosstalk of multiple signaling pathways, it is necessary to clarify and define the dominant research topics, core authors, and their cumulative research contributions, as well as the cooperative relationships among documents or researchers. Methods: Altogether, 3477 documents were retrieved from the Web of Science Core Collection with the following criteria: TS= (SMAD7 OR SMAD7-protein OR Small-Mothers-Against-Decapentaplegic-7) refined by WEB OF SCIENCE CATEGORY (ONCOLOGY) AND [excluding] PUBLICATION YEARS (2021) AND DOCUMENT TYPES (ARTICLE OR REVIEW) AND LANGUAGES (ENGLISH) AND WEB OF SCIENCE INDEX (Web of Science Core Collection, SCI), and the timespan of 2011–2020. Bibliometric visualization analysis was conducted with CiteSpace and VOSviewer. Results: The number of documents grew each year. A total of 2703 articles and 774 reviews were identified from 86 countries/regions, 3524 organizations, 928 journals, and 19,745 authors. China was the most prolific country, with 1881 documents. Contributions from China, the United States, and Germany were the most substantial. The most influential author was Lan Huiyao at The Chinese University of Hong Kong, with 24 publications and 2348 total citations. The bibliometric analysis showed that multilateral cooperation among diverse institutions or investigators was beneficial to high-quality outputs. The keyword “PPAR-gamma” exhibited the strongest burst in recent years, suggesting a potent research focus in the future. Conclusion: Research on SMAD7 in oncology is continuously developing. Bibliometrics is an interesting tool to present the characteristics of publication years, main authors, and productive organizations in a visualized way. It is worth mentioning that a prospective focus might be the specific mechanism of the interaction of PPAR-gamma with SMAD7 in oncology. In all, bibliometric analysis provides an overview and identifies potential research trends for further studies in this academic field.

Abstract P4-02-08: Amcenestrant in combination with CDK4/6 inhibitor palbociclib demonstrates synergistic anti-tumor activity in ER+ endocrine-resistant breast cancer xenograft models

Abstract Nearly 70% of newly diagnosed cases of breast cancer (BC) are estrogen receptor positive (ER+) where endocrine therapy is a primary treatment. Despite initial efficacy seen with endocrine therapies, approximately 40% of patients develop acquired resistance which ultimately limits the use of these agents even as most of these tumors continue to require ERα. The strategies to combat these cancers with resistance are not yet defined and represent the next major clinical challenge in ER+ breast cancer. Amcenestrant is an optimized oral SERD (selective estrogen receptor degrader) with potent antagonist and degradation activity irrespective of ESR1 mutational status, and is currently being evaluated as monotherapy and in combination with CDK4/6 inhibitors and other targeted therapies for ER+ breast cancer patients in clinical trials. Here, we report that in preclinical models, amcenestrant achieved significant anti-tumor efficacy/regression as a monotherapy and strong synergistic activity when administered together with the CDK4/6 inhibitor, palbociclib, in subcutaneously xenografted endocrine resistant ER+ BC mouse models: ESR1-Y537S mutant-driven MCF7 tumor and HCI-013 PDX (patient-derived xenograft). Transcriptional profile analysis of xenograft tumor biopsies revealed the modulation of different pathways by SERD and palbociclib and highlighted crosstalk of ER and CDK4 signaling pathways. Interestingly, CDK4/6 inhibition by palbociclib induces partial activation of ER pathways as potential mechanism of tumor escape which is completely abolished by the combination treatment of amcenestrant with palbociclib. Early clinical data also showed that amcenestrant in combination with palbociclib, exhibited a favorable safety profile and encouraging antitumor activity in patients with endocrine resistant, ER+/HER2-, advanced breast cancer, which supports its potential to become a new endocrine backbone therapy for ER+ HER2- breast cancer. In conclusion, amcenestrant is an oral SERD, which exhibits significant anti-tumor activity as a single agent and shows synergistic activity with CDK4/6 inhibitor palbociclib in ER+ BC xenografts preclinically and in early clinical development. Citation Format: Maysoun Shomali, Zhuyan Guo, Jane Cheng, Amy Sullivan, Youssef El-Ahmad, Fangxian Sun, Sukhvinder Sidhu, Joon Sang Lee, Hui Cai, Jack Pollard, Laurent Debussche, Chris Soria, Monsif Bouaboula. Amcenestrant in combination with CDK4/6 inhibitor palbociclib demonstrates synergistic anti-tumor activity in ER+ endocrine-resistant breast cancer xenograft models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-08.

Abstract PD8-06: Acquired resistance to tucatinib is associated with EGFR amplification in HER2+ breast cancer (BC) models and can be overcome by a more complete blockade of HER receptor layer

Abstract Background: With recent approval of the irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (N) and the HER2-specific TKI tucatinib (T) in the advanced setting and their edging towards the early setting in HER2+ BC, resistance will likely emerge as a challenge, as illustrated by the HER2CLIMB study, where only one patient with brain metastasis remained progression free after 2 years on T. We set out to define the mechanisms of resistance to T and treatment strategies to overcome it. Materials and Methods: Our previously characterized HER2+ BT474 models with acquired resistance to lapatinib (L; LapR) or N (NrbR) (SABCS20-PD3-09), and our recently developed models of BT474 and SKBR3 with acquired resistance to T (TucaR) developed through long-term exposure to increasing doses of T (up to 200nM) and their naïve parental (P) were used. Genomic (DNA-seq), transcriptomic (RNA-seq), and proteomic (RPPA, western blot) characterization were performed. Drug efficacy studies involved cell growth assays by the imaging-based IncuCyte system. Results: We recently reported that while LapR is associated with acquisition of HER2 L755S mutation, which partially reactivates the HER pathway, NrbR is associated with the additional co-acquisition of a pathogenic PIK3CA mutation. Preliminary analysis of 2 BT474 TucaR models (ATCC and AZ) showed highly elevated levels of phosphorylated (p) and total (t) EGFR, suggesting EGFR signaling activation. Levels of pHER2, pHER3, and downstream pAKT and pS6 were also markedly higher in the TucaR models compared to P or short-term T. The TucaR but not LapR or NrbR models exhibited EGFR amplification, explaining the higher EGFR levels and signaling. Further, the elevated pEGFR, pHER2, pHER3, pAKT, and pS6 levels in TucaR models were substantially suppressed by the EGFR-specific TKI gefitinib (G) (50, 500nM) or even further when combined with T (500 nM G+200nM T). These results suggest that the higher pHER2 levels in TucaR models is probably due to heterodimerization of the amplified EGFR with HER2 and subsequent HER2 phosphorylation. In contrast to the P cells where the apoptotic marker cleaved (c)-PARP was not induced with G alone (50, 500nM), but with T (200nM) or 500nM G+T, in the TucaR model, 500nM G alone was enough to induce c-PARP, which was further enhanced when combined with T, implying the survival dependence of TucaR cells on EGFR signaling. The TucaR models were hypersensitive to G compared to P cells, and this growth inhibition was further enhanced with G+T. Whilst we previously reported that the LapR and NrbR cells were cross-resistant to T, the TucaR cells remained highly sensitive to the pan-HER TKIs N, poziotinib, and pyrotinib. Finally, in a second HER2+ model SKBR3, at 200nM TucaR, we observed elevated pEGFR, pHER2, pHER3, and pAKT levels, the underlying mechanism of which is under investigation by genomic and molecular analysis. In-depth characterization of our TucaR models to determine the differential gene expression and signatures is ongoing to gain additional mechanistic insights. Conclusions: Our findings suggest that whilst complete blockade of the HER layer using N is evaded by acquisition of HER and PIK3CA mutations, resistance to the HER2 TKI T is associated with EGFR amplification, a finding that underscores the HER signaling pathway redundancy and cross-talk between HER receptors to compensate for partial blockade of the pathway. Further, while resistance to L and N confers cross-resistance to T, resistance to T may be overcome using pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Together, our findings hold crucial implications in light of the current treatment landscape of HER2+ BC, with a particular emphasis on the considerations to strategize the treatment sequence of currently available TKIs. Citation Format: Jamunarani Veeraraghavan, Sreyashree Bose, Ragini Mistry, Pier Selenica, Sarmistha Nanda, Lanfang Qin, Andrea Gazzo, Yingjie Zhu, Michael A Mancini, Fabio Stossi, Britta Weigelt, Jorge S Reis-Filho, C. Kent Osborne, Mothaffar F Rimawi, Rachel Schiff. Acquired resistance to tucatinib is associated with EGFR amplification in HER2+ breast cancer (BC) models and can be overcome by a more complete blockade of HER receptor layer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD8-06.

Caffeine Therapy for Apnea of Prematurity: Role of the Circadian CLOCK Gene Polymorphism.

Standard-dose caffeine citrate has been routinely prescribed for apnea of prematurity (AOP) management; however, some preterm infants respond well to the therapy while others do not. The AOP phenotype has been attributed solely to the immature control of the respiratory system consequent to preterm birth, but there are also important genetic influences. Based on our previous report, we tested the hypothesis that the human circadian locomotor output cycles kaput (CLOCK) gene polymorphisms play a role in the response to caffeine citrate therapy in preterm infants. We also studied the interactions of the circadian clock with aryl hydrocarbon receptor (AHR) signaling pathways in preterm babies who received caffeine citrate. This single-center study collected data from 112 preterm infants (<35 weeks gestational age) between July 2017 and July 2018, including apnea-free (n = 48) and apneic (n = 64) groups. Eighty-eight candidate single nucleotide polymorphisms (SNPs) were tested using the MassARRAY system. Association analysis was performed using the PLINK Whole Genome Data Analysis Toolset and SNPStats software. Linkage disequilibrium (LD) and haplotype analyses were performed using Hapview software. No significant intergroup differences in allele distributions or genotype frequencies of CYP1A2, CYP3A4, CYP3A5, and CYP3A7 were detected in our study on preterm babies. Two more SNPs in AHR were found to be associated with determining the response to caffeine citrate therapy in our pediatric patients. Of the 46 candidate SNPs in the CLOCK gene, 26 were found to be associated with determining the response to caffeine treatment in these babies. Interestingly, a significant association was retained for 18 SNPs in the CLOCK gene after false discovery rate correction. Moreover, strong LD formed in those variants in AHR, ADORA2A, and CLOCK genes was confirmed to be significantly associated with a better response to standard-dose caffeine therapy. In summary, CLOCK gene polymorphisms play a role in determining the response to caffeine therapy in premature neonates with AOP. However, whether the AHR and CLOCK signaling pathways crosstalk with each other during caffeine treatment remains largely unclear. Future clinical studies including more immature babies and basic research are needed to explore the mechanism by which circadian rhythms affect the response to caffeine therapy.

Computational modeling and analysis of the morphogenetic domain signaling networks regulating C. elegans embryogenesis

Caenorhabditiselegans, often referred to as the ‘roundworm’, provides a powerful model for studying cell autonomous and cell–cell interactions through the direct observation of embryonic development in vivo. By leveraging the precisely mapped cell lineage at single cell resolution, we are able to study at a systems level how early embryonic cells communicate across morphogenetic domains for the coordinated processes of gene expressions and collective cellular behaviors that regulate tissue morphogenesis. In this study, we developed a computational framework for the exploration of the morphogenetic domain cell signaling networks that may regulate C. elegans gastrulation and embryonic organogenesis. We demonstrated its utility by producing the following results, i) established a virtual reference model of developing C. elegans embryos through the spatiotemporal alignment of individual embryo cell nuclear imaging samples; ii) integrated the single cell spatiotemporal gene expression profile with the established virtual embryo model by data pooling; iii) trained a Machine Learning model (Random Forest Regression), which predicts accurately the spatial positions of the cells given their gene expression profiles for a given developmental time (e.g. total cell number of the embryo); iv) enabled virtual 4-dimensional tomographic graphical modeling of single cell data; v) inferred the biology signaling pathways that act in each of morphogenetic domains by meta-data analysis. It is intriguing that the morphogenetic domain cell signaling network seems to involve some crosstalk of multiple biology signaling pathways during the formation of tissue boundary pattern. Lastly, we developed the Software tool ‘Embryo aligner version 1.0’ and provided it as an Open Source program to the research community for virtual embryo modeling, and phenotype perturbation analyses (https://github.com/csniuben/embryo_aligner/wiki and https://bioinfo89.github.io/C.elegansEmbryonicOrganogenesisweb/).

Crossed Pathways for Radiation-Induced and Immunotherapy-Related Lung Injury.

Radiation-induced lung injury (RILI) is a form of radiation damage to normal lung tissue caused by radiotherapy (RT) for thoracic cancers, which is most commonly comprised of radiation pneumonitis (RP) and radiation pulmonary fibrosis (RPF). Moreover, with the widespread utilization of immunotherapies such as immune checkpoint inhibitors as first- and second-line treatments for various cancers, the incidence of immunotherapy-related lung injury (IRLI), a severe immune-related adverse event (irAE), has rapidly increased. To date, we know relatively little about the underlying mechanisms and signaling pathways of these complications. A better understanding of the signaling pathways may facilitate the prevention of lung injury and exploration of potential therapeutic targets. Therefore, this review provides an overview of the signaling pathways of RILI and IRLI and focuses on their crosstalk in diverse signaling pathways as well as on possible mechanisms of adverse events resulting from combined radiotherapy and immunotherapy. Furthermore, this review proposes potential therapeutic targets and avenues of further research based on signaling pathways. Many new studies on pyroptosis have renewed appreciation for the value and importance of pyroptosis in lung injury. Therefore, the authors posit that pyroptosis may be the common downstream pathway of RILI and IRLI; discussion is also conducted regarding further perspectives on pyroptosis as a crucial signaling pathway in lung injury treatment.

Integrated analysis of mRNA and long non-coding RNA expression profiles reveals the potential roles of lncRNA-mRNA network in carp macrophage immune regulation

Long non-coding RNAs (lncRNAs) have emerged as a hot topic in research as mounting evidence has indicated their transcriptional or post-transcriptional regulatory potential in multiple biological processes. Previous studies have revealed the involvement of lncRNAs in the immunoregulation of mammalian macrophages by changing mRNA expression; however, studies on the lncRNAs in fish macrophages and their potential roles in the immune system remain unknown. Primary macrophages were isolated from the head kidney (HK) of red common carp (Cyprinus carpio) and high-throughput lncRNA-mRNA sequencing was performed using the Illumina HiSeq platform. The results revealed that the most highly expressed mRNAs in primary HK macrophages were mainly involved in immune-related signal pathways. Furthermore, the most enriched immune-related GO term and KEGG pathway of the mRNAs were "immune system development" and "chemokine signaling pathway," respectively. A total of 20,333 lncRNAs, composed of 10,512 known and 9821 novel lncRNAs, were identified, and functional enrichment analysis of the lncRNA-mRNA network indicated that the expressed lncRNAs in primary HK macrophages could be associated with the regulation of multiple immune-related signaling pathways. In addition, the expressions of several selected lncRNAs and their related mRNAs were determined in carp macrophages following a 6-h exposure to lipopolysaccharide (LPS) and Poly(I: C), the results of which confirmed the co-expression regulation of lncRNAs and target mRNAs in the immune response of carp macrophages. These results suggest the correlative of the lncRNA-mRNA network in fish macrophage immune response, which may further affect the cross-talk of various signaling pathways by interaction with other network genes. Here, we provided fundamental data about the transcriptome profiles of primary HK macrophages from red common carp by analysis of the lncRNA-mRNA network, and ultimately suggest the potential roles of lncRNA-mRNA networks in immune regulation in teleost fish.

Correction for: TGF-β and NF-κB signaling pathway crosstalk potentiates corneal epithelial senescence through an RNA stress response.

Correction for: TGF-β and NF-κB signaling pathway crosstalk potentiates corneal epithelial senescence through an RNA stress response.

GSK-3β, FYN, and DYRK1A: Master Regulators in Neurodegenerative Pathways.

Protein kinases (PKs) have been recognized as central nervous system (CNS)-disease-relevant targets due to their master regulatory role in different signal transduction cascades in the neuroscience space. Among them, GSK-3β, FYN, and DYRK1A play a crucial role in the neurodegeneration context, and the deregulation of all three PKs has been linked to different CNS disorders with unmet medical needs, including Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal lobar degeneration (FTLD), and several neuromuscular disorders. The multifactorial nature of these diseases, along with the failure of many advanced CNS clinical trials, and the lengthy approval process of a novel CNS drug have strongly limited the CNS drug discovery. However, in the near-decade from 2010 to 2020, several computer-assisted drug design strategies have been combined with synthetic efforts to develop potent and selective GSK-3β, FYN, and DYRK1A inhibitors as disease-modifying agents. In this review, we described both structural and functional aspects of GSK-3β, FYN, and DYRK1A and their involvement and crosstalk in different CNS pathological signaling pathways. Moreover, we outlined attractive medicinal chemistry approaches including multi-target drug design strategies applied to overcome some limitations of known PKs inhibitors and discover improved modulators with suitable blood–brain barrier (BBB) permeability and drug-like properties.

Abstract 3145: TRIPODD: a novel fluorescence imaging platform for in situ quantification of drug distribution and therapeutic response

Abstract Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells as well as signaling pathway cross talk. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this interaction network and identify therapeutic targets. Tyrosine kinase inhibitors (TKI) are a class of molecularly targeted therapeutics seeking to inhibit signaling pathways critical to sustaining proliferative signaling, resisting cell death, and other hallmarks of cancer. While tumors may initially respond to TKI therapy, disease progression is inevitable due to acquired resistance largely involving cellular signaling pathway reprogramming. With the ultimate goal of improved molecularly targeted therapeutic efficacy, we have developed a fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution), resulting in the only methodology capable of simultaneous quantification of single-cell drug target availability and protein expression with preserved tumor spatial context. TRIPODD combines intracellular paired agent imaging (iPAI) to quantify drug target interactions and oligonucleotide conjugated antibody cyclic immunofluorescence (cyCIF) to characterize proteomic response to therapy. Importantly, iPAI and cyCIF data is collected on the same tissue sections facilitating spatial registration of the two datasets. iPAI employs spectrally distinct, fluorescently labeled targeted and untargeted drug derivatives, which correct for untargeted uptake and facilitate quantitative in situ assessment of drug target engagement. cyCIF exploits in situ hybridization of complementary oligos for biomarker labeling, while oligo modifications facilitate signal removal for sequential rounds of fluorescent tagging. cyCIF is capable of generating multi-parametric images to quantify protein expression, distribution and phosphorylation. To date, we have quantified and evaluated our iPAI toolbox using a suite of functional assays, where in vitro screening confirmed biological functionality. Subsequent in vivo validation studies successfully performed ratiometric iPAI quantification after systemic iPAI probe administration, followed by sequential cyCIF imaging on the same xenograft tissue sections for single-cell quantification of drug target availability, EGFR pathway signaling and cell viability. TRIPODD will enable an improved mechanistic understanding of clinically-relevant treatment regimens through spatially resolved single-cell quantification of drug concentration and proteomic response to identify mechanisms of resistant subclonal population outgrowths driving resistance. Citation Format: Nathan P. McMahon, Allison Solanki, Jocelyn A. Jones, Lei G. Wang, Kenneth M. Tichauer, Kimberley S. Samkoe, Summer L. Gibbs. TRIPODD: a novel fluorescence imaging platform for in situ quantification of drug distribution and therapeutic response [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 3145.

Co-Expression and Functional Interaction between CXCR4 and CD74 in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by progressive accumulation of small, mature B-lymphocytes aberrantly expressing CD5. One of the crucial factors in B-CLL development is the decreased apoptosis of malignant B-Cells.The chemokine receptor CXCR4 and the MHC class II invariant chain (Ii) CD74, are expressed on CLL B-cells and play a role in cells' migration and survival. CXCL12, the cognate ligand of CXCR4, promotes homing of CLL cells into the bone marrow (BM) and survival within the niche. Furthermore, CXCR4 is activated by macrophage migration inhibitory factor (MIF), a chemokine-like inflammatory cytokine and non-cognate ligand of CXCR4. MIF and its homolog MIF2 are bona fide ligands of CD74.CD74 is highly expressed on CLL cells and expression is correlated with the adversely prognostic factor ZAP 70. Interestingly, MIF plays a role in the survival of CLL cells by binding to CD74. Furthermore, increased MIF levels are found in the plasma of CLL patients and genetic Mif deficiency leads to delayed development of CLL in the Eµ-TCL1 mouse model. Finally, the aim of our study was to investigate the expression and interaction between CXCR4 and CD74 as well as their potential contribution to B-Cell survival in B-CLL.An initial cohort of 350 patients with different backgrounds of hematological malignancies was analyzed for CXCR4 and CD74 expression on mononuclear cells. Interestingly, the highest coexpression of both receptors was observed in B-CLL (n=10) by flow cytometry (FCM), where cells were stained for CD3+, CD14+ and CD19+/CD5+ and compared to cells from healthy donors (n=20). For in vivo and in vitro studies cell lines and a mice model were chosen, which express both receptors CXCR4 and CD74 on the surface of B-Cells.The human CLL line JVM3 and primary CLL cells from aged leukemic Eμ-TCL1 mice were used to study the cross-talk of CXCR4 and CD74 in regulation of CLL survival after incubation with rhMIF, rhCXCL12, or rhMIF-2 in conjunction with Bcl2-inhibitor treatment (Venetoclax, 30µM) or pharmacological blockade of CXCR4 and/or CD74 (AMD3100 30µM, neutralizing CD74 antibody LN2 5µg/ml). Potential direct CXCR4/CD74 interactions were studied by confocal laser scanning microscopy (CLSM) and coimmunoprecipitation (CoIP). Chemotaxis towards CXCL12, MIF and MIF-2 were analyzed to understand homing behavior, which contributes to B-Cell survival.CXCR4 and CD74 were found significantly coexpressed on B-cells from CLL patients compared to controls (p< 0.0001), suggesting a functional interaction between these receptors. Colocalization was confirmed by CLSM and CoIP detected receptor complexes between CD74 and CXCR4 on B- Cells from the JVM3 and OSU CLL cell lineFunctionally, inhibition of either receptor substantially impaired survival rates that did not differ from a simultaneous co-blockade of CD74 and CXCR4, indicating a crosstalk and/or merging of downstream signaling pathways. Apoptosis assays indicated that inhibition of CXCR4 and/or CD74 triggered substantial cell death (78,17 +/- 1,84%). Of note, single vs. double blockade experiments confirmed that CD74 and CXCR4 functionally interact to control CLL survival. Inversely, pre-incubation of JVM3 cells with CXCL12, MIF and MIF-2 decreased apoptosis by 43, 36 % (MIF) and 51, 50 % (CXCL12) compared to the control.The ligands MIF, MIF2 and CXCL12 promoted chemotactic migration of JVM3 B-cells that contributes to the B-Cell survival mechanism.Together, our results suggest an important role of MIF proteins in the survival and homing of malignant CLL cells that is mediated through a functional interaction between the CXCR4 and CD74 survival receptors. In conclusion, high levels of MIF attract B-Cells with the coexpression of CXCR4 and CD74, which build a complex after binding to their ligands and activate survival pathways to inhibit cell death. The MIF/CXCR4/CD74 axis could represent a novel target for therapeutic intervention. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Koschmieder:Roche: Other: Clinical Trial participation; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding.

Crosstalk between Wnt/β-catenin signaling and NF-κB signaling contributes to apical periodontitis

In physiology conditions, the crosstalk of signaling pathways has been considered to extend the functions of individual pathways and results in a more complex regulatory network. The Wnt3a/β-catenin and NF-κB signaling pathways have been demonstrated involving in apical periodontitis (AP). As AP progresses, ultimately causes tooth loss. In the present study, we investigate the contribution of the crosstalk between the Wnt3a/β-catenin and NF-κB signaling pathways to the development of AP. Clinically, utilizing 60 human AP and healthy tissues (30 samples for each group), we found that the expression levels of Wnt3a/β-catenin and NF-κB were elevated in the Ap tissues compared to that in the healthy group. To further study the roles of Wnt3a/β-catenin and NF-κB signaling pathways in the development of AP, and the contribution of the crosstalk between these two signaling pathways to AP, we established the AP animal model and observed that, first, both pathways are activated in the AP group compared to the control group. Interestingly, by immunoprecipitation and western blot experiments, we revealed that there is greater interaction between NF-κB (phorspho-p65) and β-catenin in AP tissues compared to the control tissues. Importantly, when the NF-κB signaling pathway was blocked by its inhibitor, pyrrolidine dithiocarbamate (PDTC), the activity of the Wnt3a/β-catenin signaling pathway was abolished, and consequently led to the attenuation of the inflammation response in LPS-induced human periodontal ligament cells (hPDLCs). Thus, our data indicate that the crosstalk between Wnt3a/β-catenin and NF-κB signaling pathway contributes to the development of AP, and provide a therapeutic strategy for the treatment of AP as well.