Receptor Tyrosine Kinase Signaling Pathways Research Articles

Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases.

Ligands such as insulin, epidermal growth factor, platelet derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell- impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

Exploring Emerging Therapeutic Targets and Opportunities in Neuroendocrine Tumors: Updates on Receptor Tyrosine Kinases

Neuroendocrine tumors (NETs) represent a diverse group of neoplasms originating from neuroendocrine cells, presenting varied clinical behaviors and posing significant challenges in management. This review explores the emerging roles of receptor tyrosine kinases (RTKs) in the pathogenesis and progression of NETs, including vascular endothelial growth factor receptors (VEGFRs), insulin-like growth factor receptors (IGF-1R), RET, epidermal growth factor receptor (EGFR), and ALK. The dysregulation of RTK signaling pathways contributes to key cellular processes such as proliferation, survival, and invasion in NETs. We discuss the potential of targeting RTKs as therapeutic strategies in NETs, with a focus on recent developments in RET inhibitors and the therapeutic implications of RTK alterations.

Abstract 7048: Multiomics characterization of breast angiosarcoma from an Asian cohort reveals enrichment for angiogenesis signaling pathway and tumor-infiltrating macrophages

Abstract Recent epidemiological data suggests a rising incidence of breast angiosarcoma (AS-B) in the Western population, with over two-thirds related to irradiation or chronic lymphedema. However, unlike head and neck angiosarcoma (AS-HN), AS-B disease characteristics in Asia remains unclear. We examined clinical patterns of angiosarcoma patients (n = 176) seen in an Asian tertiary cancer centre from 1999 to 2021, and specifically investigated the molecular and immune features of AS-B in comparison to AS-HN. Data from whole genome sequencing (WGS) (AS-HN, n = 44; breast, n = 7), NanoString PanCancer IO360 gene expression profiling (AS-HN, n = 42; breast, n = 7) and 10X Visium spatial transcriptomics (AS-HN, n = 4; breast, n = 2) were analyzed. Majority of cases were AS-HN (n = 104; 59.1%), while AS-B (n = 16, all females) accounted for 9.1% of the cases. The median age at diagnosis was 43 years (range, 26 to 74). Four cases (25%) were related to prior irradiation, while 6 out of 8 cases evaluated were positive for human herpesvirus-7 on immunohistochemistry. Although all cases were non-metastatic at diagnosis and were treated with curative-intent surgery, 6 patients (37.5%) experienced rapid relapse within a median of 7.8 months. Based on WGS, 4 of the 7 AS-B had non-synonymous somatic variants in 48 genes (range, 2 to 28 per case). Among the notable variants found were in kinases (KDR, JAK2, FLT4, ATR, EPHB1 and PRKACA). Other variants were found in cancer-related genes (e.g. ATRX, FOXO1, SMAD3, PTCH1, NOTCH4, HIF1A and SETD1A). These genes were functionally annotated by performing over representation analysis (www.webgestalt.org) on publicly available databases (e.g. Gene Ontology and KEGG). They were enriched in cancer-related pathways such as regulation of cell differentiation, VEGFR and receptor tyrosine kinase signaling pathways. By NanoString profiling, compared to AS-HN, AS-B was enriched for angiogenesis, notch signaling and metastasis-associated matrix remodeling pathways. Conversely, AS-B was depleted of lymphoid compartment, immune cell adhesion and migration expression signatures. Based on the gene expression signatures, AS-B was enriched for macrophages, CD8+ T cells and was depleted for T-reg cells. Furthermore, spatial transcriptomics showed that compared to AS-HN, tumor-infiltrating leukocytes (TILs) in AS-B were enriched for macrophage and T-cells but were depleted for B-cells and NK cells. The location of TILs could provide information on the feasibility of immunotherapy in angiosarcoma. In conclusion, we observed a convergence of both mutational and expression signatures on angiogenesis signaling pathways in our AS-B cases. The spatial transciptomic data revealed that AS-B was enriched for tumor-infiltrating macrophages and T-cells which might have implications on the role of immunotherapy. Citation Format: Tun Kiat Ko, Zexi Guo, Bavani Kannan, Boon Yee Lim, Jing Yi Lee, Abner Herbert Lim, Zhimei Li, Elizabeth Chun Yong Lee, Cedric Chuan-Young Ng, Bin Tean Teh, Jason Yongsheng Chan. Multiomics characterization of breast angiosarcoma from an Asian cohort reveals enrichment for angiogenesis signaling pathway and tumor-infiltrating macrophages [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 7048.

Characterization of gastric cancer-stimulated signaling pathways and function of CTGF in cancer-associated fibroblasts

BackgroundCancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME) that play an important role in cancer progression. Although the mechanism by which CAFs promote tumorigenesis has been well investigated, the underlying mechanism of CAFs activation by neighboring cancer cells remains elusive. In this study, we aim to investigate the signaling pathways involved in CAFs activation by gastric cancer cells (GC) and to provide insights into the therapeutic targeting of CAFs for overcoming GC.MethodsAlteration of receptor tyrosine kinase (RTK) activity in CAFs was analyzed using phospho-RTK array. The expression of CAFs effector genes was determined by RT-qPCR or ELISA. The migration and invasion of GC cells co-cultured with CAFs were examined by transwell migration/invasion assay.ResultsWe found that conditioned media (CM) from GC cells could activate multiple receptor tyrosine kinase signaling pathways, including ERK, AKT, and STAT3. Phospho-RTK array analysis showed that CM from GC cells activated PDGFR tyrosine phosphorylation, but only AKT activation was PDGFR-dependent. Furthermore, we found that connective tissue growth factor (CTGF), a member of the CCN family, was the most pronouncedly induced CAFs effector gene by GC cells. Knockdown of CTGF impaired the ability of CAFs to promote GC cell migration and invasion. Although the PDGFR-AKT pathway was pronouncedly activated in CAFs stimulated by GC cells, its pharmacological inhibition affected neither CTGF induction nor CAFs-induced GC cell migration. Unexpectedly, the knockdown of SRC and SRC-family kinase inhibitors, dasatinib and saracatinib, significantly impaired CTGF induction in activated CAFs and the migration of GC cells co-cultured with CAFs. SRC inhibitors restored the reduced expression of epithelial markers, E-cadherin and Zonula Occludens-1 (ZO-1), in GC cells co-cultured with CAFs, as well as CAFs-induced aggregate formation in a 3D tumor spheroid model.ConclusionsThis study provides a characterization of the signaling pathways and effector genes involved in CAFs activation, and strategies that could effectively inhibit it in the context of GC.DC5ErEG73tsyg3F8WfE1xiVideo

Integration of Multi-Omics Data for the Classification of Glioma Types and Identification of Novel Biomarkers

Glioma is currently one of the most prevalent types of primary brain cancer. Given its high level of heterogeneity along with the complex biological molecular markers, many efforts have been made to accurately classify the type of glioma in each patient, which, in turn, is critical to improve early diagnosis and increase survival. Nonetheless, as a result of the fast-growing technological advances in high-throughput sequencing and evolving molecular understanding of glioma biology, its classification has been recently subject to significant alterations. In this study, we integrate multiple glioma omics modalities (including mRNA, DNA methylation, and miRNA) from The Cancer Genome Atlas (TCGA), while using the revised glioma reclassified labels, with a supervised method based on sparse canonical correlation analysis (DIABLO) to discriminate between glioma types. We were able to find a set of highly correlated features distinguishing glioblastoma from lower-grade gliomas (LGGs) that were mainly associated with the disruption of receptor tyrosine kinases signaling pathways and extracellular matrix organization and remodeling. Concurrently, the discrimination of the LGG types was characterized primarily by features involved in ubiquitination and DNA transcription processes. Furthermore, we could identify several novel glioma biomarkers likely helpful in both diagnosis and prognosis of the patients, including the genes PPP1R8, GPBP1L1, KIAA1614, C14orf23, CCDC77, BVES, EXD3, CD300A, and HEPN1. Collectively, this comprehensive approach not only allowed a highly accurate discrimination of the different TCGA glioma patients but also presented a step forward in advancing our comprehension of the underlying molecular mechanisms driving glioma heterogeneity. Ultimately, our study also revealed novel candidate biomarkers that might constitute potential therapeutic targets, marking a significant stride toward personalized and more effective treatment strategies for patients with glioma.

Cell Membrane-Anchored DNA Nanoinhibitor for Inhibition of Receptor Tyrosine Kinase Signaling Pathways via Steric Hindrance and Lysosome-Induced Protein Degradation.

Receptor tyrosine kinase (RTK) plays a crucial role in cancer progression, and it has been identified as a key drug target for cancer targeted therapy. Although traditional RTK-targeting drugs are effective, there are some limitations that potentially hinder the further development of RTK-targeting drugs. Therefore, it is urgently needed to develop novel, simple, and general RTK-targeting inhibitors with a new mechanism of action for cancer targeted therapy. Here, a cell membrane-anchored RTK-targeting DNA nanoinhibitor is developed to inhibit RTK function. By using a DNA tetrahedron as a framework, RTK-specific aptamers as the recognition elements, and cholesterol as anchoring molecules, this DNA nanoinhibitor could rapidly anchor on the cell membrane and specifically bind to RTK. Compared with traditional RTK-targeting inhibitors, this DNA nanoinhibitor does not need to bind at a limited domain on RTK, which increases the possibilities of developing RTK inhibitors. With the cellular-mesenchymal to epithelial transition factor (c-Met) as a target RTK, the DNA nanoinhibitor can not only induce steric hindrance effects to inhibit c-Met activation but also reduce the c-Met level via lysosome-mediated protein degradation and thus inhibition of c-Met signaling pathways and related cell behaviors. Moreover, the DNA nanoinhibitor is feasible for other RTKs by just replacing aptamers. This work may provide a novel, simple, and general RTK-targeting nanoinhibitor and possess great value in RTK-targeted cancer therapy.

Prognostic model construction and target identification of Si-Wu-Tang against breast cancer

The targets and mechanisms of Si-Wu-Tang (SWT) against (Breast cancer) BRCA were identified and a survival model and nomogram was construted by network pharmacology, bioinformatic analysis and in vitro experiments. A total of 72 anti-breast cancer SWT targets were selected, among which eleven genes (MAOA、SQLE、CACNA2D1、GLI1、RORB、ITGB3、TACR1、NR3C2、CA3、RBP4 and PTK6) were used to construct a novel prognostic model of breast cancer. The anti-breast cancer activity of SWT was related to the modulation of the receptor tyrosine kinases signaling pathways. Moreover, two compounds, mairin and senkyunone were found to bind directly to ITGB3 and RORB proteins. Finally, mRNA and protein expression of ITGB3 and RORB was observed to be significantly down-regulated after incubation of MCF-7 cells with SWT. Overall, our results indicated that mairin and senkyunone were the key ingredients present in SWT, and ITGB3 as well as RORB proteins were the major targets affected by SWT. The prognostic model can be used to predict the outcome of BRCA patients.

Effect of Juanbi Qianggu Formula on biological behaviors of fibroblast-like synoviocytes in rheumatoid arthritis by regulating FGFR1 signaling pathway based on network pharmacology and cell function experiments

This study aimed to explore the molecular mechanism of Juanbi Qianggu Formula(JBQGF), an empirical formula formulated by the prestigious doctor in traditional Chinese medicine, in the treatment of rheumatoid arthritis based on network pharmacology and cell function experiments. The main active components and targets of JBQGF were obtained through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Encyclopedia of Traditional Chinese Medicine(ETCM), and the core targets underwent functional enrichment analysis and signaling pathway analysis. Cytoscape 3.6.0 was used to construct a visualized "active component-target-signaling pathway" network of JBQGF. After screening, nine potential pathways of JBQGF were obtained, mainly including G protein-coupled receptor signaling pathway and tyrosine kinase receptor signaling pathway. As previously indicated, the fibroblast growth factor receptor 1(FGFR1) signaling pathway was highly activated in active fibroblast-like synoviocytes(FLS) in rheumatoid arthritis, and cell and animal experiments demonstrated that inhibition of the FGFR1 signaling pathway could significantly reduce joint inflammation and joint destruction in collagen-induced arthritis(CIA) rats. In terms of the tyrosine kinase receptor signal transduction pathway, the analysis of its target genes revealed that FGFR1 might be a potential target of JBQGF for rheumatoid arthritis treatment. The biological effect of JBQGF by inhibiting FGFR1 phosphorylation was preliminarily verified by Western blot, Transwell invasion assay, and pannus erosion assay, thereby inhibiting matrix metalloproteinase 2(MMP2) and receptor activator of nuclear factor-κB ligand(RANKL) and suppressing the invasion of fibroblasts in rheumatoid arthritis and erosive effect of pannus bone. This study provides ideas for searching potential targets of rheumatoid arthritis treatment and TCM drugs through network pharmacology.

Genome-wide profiling of patient-derived glioblastoma stem-like cells reveals recurrent genetic and transcriptomic signatures associated with brain tumors

PurposePatient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs).Methods94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively.ResultsExome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S − adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways.ConclusionThe availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.

Targeting neddylation as a novel approach to lung cancer treatment (Review).

As a protein that resembles ubiquitin, neural precursor cell expressed developmentally downregulated 8 (NEDD8) takes part in neddylation, which modifies substrates in a manner similar to ubiquitination and alters the activity of target proteins. Neddylation may affect the activity of multiple signaling pathways, have a regulatory role in tumor formation, progression and metastasis, and influence the prognosis of cancer treatment. The present review summarizes the regulatory roles of NEDD8 in the MDM2‑p53, NF‑κB, PI3K/AKT/mTOR, hypoxia‑inducible factor, Hippo and receptor tyrosine kinase signaling pathways, as well as in the development and progression of lung cancer.

PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

Abstract 2659: Anti-HER3 antibody, HMBD-001, in combination with an EGFR inhibitor effectively inhibits tumor growth in biomarker-selected pre-clinical models of squamous cell carcinomas

Abstract Squamous cell carcinomas (SCC) originate from stratified squamous epithelium in diverse anatomical locations that share common histological features and genetic mutations. Non-melanoma skin cancer, head and neck cancer (HNSCC), esophageal cancer (ESCC), and non-small cell lung cancer (sqNSCLC) make up the majority of SCC cases and together represent the largest subtype of cancer. There is a significant unmet need for effective treatment options post-progression of standard-of-care therapies. Alterations in receptor tyrosine kinase signaling pathways are commonly found in SCCs, e.g. EGFR is expressed in 90% of HNSCC, 76% of ESCC and 82% of sqNSCLC. However, the combination of EGFR targeting antibody cetuximab with chemotherapy confers limited clinical benefit and even initially sensitive tumors often develop resistance. Studies in SCC have implicated HER3, a key dimerization partner of HER family members, as one likely cause of treatment failure, through HER3 heterodimer activation of PI3K/AKT and MAPK/ERK pathways. In this study, we investigated the potential of combining HER3 and EGFR inhibitors to improve efficacy and overcome resistance. Previous attempts to target HER3 have shown limited clinical efficacy due to suboptimal HER3 inhibition, especially the lack of efficacy in ligand-independent activation mechanisms. They also lacked biomarker stratification for patients where HER3 inhibition would confer benefit. HMBD-001 is a clinical-stage anti-HER3 antibody which is being evaluated in a first-in-human, open-label, multi-center, dose escalation and expansion Phase I/IIa trial in patients with HER3 expressing advanced solid tumors (NCT05057013). HMBD-001 was rationally developed to uniquely block the HER3 heterodimerization interface to potently inhibit all HER3 dimer formation, including ligand-dependent and independent HER3 dimerization. Here, we show that dual blockade of EGFR and HER3 using cetuximab and HMBD-001 robustly inhibits tumor growth and is superior to either monotherapy. In multiple cell and patient derived SCC xenograft models, including models of HNSCC, ESCC and sqNSCLC, selected based on a novel gene signature that is robustly predictive of response, HMBD-001 in combination with cetuximab demonstrated up to 100% tumor growth inhibition and the abrogation of PI3K signalling. This combination was well tolerated with no relapse observed, even after a prolonged period of over 100 days in selected models. This study lays the foundation for testing HMBD-001 in combination with EGFR inhibitors in clinical settings. Better combination strategies of SoC and targeted therapeutics alongside a potent anti-HER3 mAb, in biomarker-selected patient populations, have the potential to meaningfully improve patient outcomes and prolong clinical benefit. Citation Format: Weiyi Toy, Dipti Thakkar, Khang Luu, Bui Ngoc Linh Chi, Jason Lai, Debleena Ray, Namita Gandhi, Alejandro Mas, Konrad Paszkiewicz, Piers Ingram, Jerome Boyd-Kirkup. Anti-HER3 antibody, HMBD-001, in combination with an EGFR inhibitor effectively inhibits tumor growth in biomarker-selected pre-clinical models of squamous cell carcinomas [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 2659.

Effect of moxibustion on immune function homeostasis in rats with diarrhea irritable bowel syndrome based on SCF/c-kit signaling pathway

To observe the effects of moxibustion on the stem cell factor (SCF)/tyrosine kinase receptor (c-kit) signaling pathway and immune function in rats with diarrhea irritable bowel syndrome (IBS-D), and to explore the mechanism of moxibustion for IBS-D. Among 52 young rats born from 6 healthy pregnant SPF rats, 12 rats were randomly selected into the normal group, and the remaining 40 rats were treated with the three-factor combination method of maternal separation, acetic acid enema and chronic restraint stress to establish the IBS-D rat model. Thirty-six rats with successful IBS-D model were randomly divided into a model group, a moxibustion group, and a medication group, 12 rats in each group. The rats in the moxibustion group were treated with suspension moxibustion at "Tianshu" (ST 25) and "Shangjuxu" (ST 37); the rats in the medication group were treated with intragastric administration of rifaximin suspension (150 mg/kg). All the treatments were given once a day for 7 consecutive days. The body mass, loose stool rate (LSR), the minimum volume threshold when abdominal withdrawal reflex (AWR) scored 3 were measured before acetic acid enema (35 days old), after modeling (45 days old), and after intervention (53 days old). After intervention (53 days old), HE staining was used to observe the morphology of colon tissue, and spleen and thymus coefficients were measured; ELISA method was used to detect serum inflammatory factors (tumor necrosis factor a [TNF-a], interleukin [IL]-10, IL-8), T-lymphocyte subsets (CD+4, CD+8, CD+45), value of CD+4/CD+8 and immune globulin (IgA, IgG, IgM); real-time PCR method and Western blot method was used to detect the expression of SCF, c-kit mRNA and protein in colon tissue; immunofluorescence staining method were used to detect positive expression of SCF and c-kit. After intervention, compared with the normal group, in the model group, the body mass and the minimum volume threshold when AWR scored 3 were decreased (P<0.01), LSR, spleen and thymus coefficients, serum levels of TNF-α, IL-8, CD+4, CD+45, CD+4/CD+8, IgA, IgG, IgM were increased (P<0.01), serum IL-10 level and protein and mRNA expression of SCF and c-kit in colon tissue were decreased (P<0.01), and the positive expression of SCF and c-kit was decreased (P<0.01). Compared with the model group, in the moxibustion group and the medication group, the body mass and the minimum volume threshold when AWR scored 3 were increased (P<0.01, P<0.05), LSR, spleen and thymus coefficients, serum levels of TNF-α, IL-8, CD+4, CD+8, CD+45, CD+4/CD+8, IgA, IgG, IgM were decreased (P<0.01, P<0.05), serum IL-10 level and protein and mRNA expression of SCF and c-kit in colon tissue were increased (P<0.01), and the positive expression of SCF and c-kit was increased (P<0.01). Compared with the medication group, in the moxibustion group, the level of serum CD+4 was decreased (P<0.05), the value of CD+4/CD+8 was increased (P<0.01), and there was no significant difference in other indexes (P>0.05). The expression of SCF and c-kit mRNA was positively correlated with the minimum volume threshold when AWR scored 3 and IL-10 (P<0.01), and negatively correlated with remaining indexes (P<0.01, P<0.05). Moxibustion could reduce visceral hypersensitivity, improve symptoms of abdominal pain and diarrhea in IBS-D rats, and its mechanism may be related to up-regulation of the expression of SCF/c-kit signaling pathway and improvement of IBS-D immune function.

Discovery of a Novel Series of Imidazopyrazine Derivatives as Potent SHP2 Allosteric Inhibitors.

Protein tyrosine phosphatase SHP2 is an oncogenic protein that can regulate different cytokine receptor and receptor tyrosine kinase signaling pathways. We report here the identification of a novel series of SHP2 allosteric inhibitors having an imidazopyrazine 6,5-fused heterocyclic system as the central scaffold that displays good potency in enzymatic and cellular assays. SAR studies led to the identification of compound 8, a highly potent SHP2 allosteric inhibitor. X-ray studies showed novel stabilizing interactions with respect to known SHP2 inhibitors. Subsequent optimization allowed us to identify analogue 10, which possesses excellent potency and a promising PK profile in rodents.

Transcription factor Dmrt1 triggers the SPRY1-NF-κB pathway to maintain testicular immune homeostasis and male fertility.

Bacterial or viral infections, such as Brucella, mumps virus, herpes simplex virus, and Zika virus, destroy immune homeostasis of the testes, leading to spermatogenesis disorder and infertility. Of note, recent research shows that SARS-CoV-2 can infect male gonads and destroy Sertoli and Leydig cells, leading to male reproductive dysfunction. Due to the many side effects associated with antibiotic therapy, finding alternative treatments for inflammatory injury remains critical. Here, we found that Dmrt1 plays an important role in regulating testicular immune homeostasis. Knockdown of Dmrt1 in male mice inhibited spermatogenesis with a broad inflammatory response in seminiferous tubules and led to the loss of spermatogenic epithelial cells. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) revealed that Dmrt1 positively regulated the expression of Spry1, an inhibitory protein of the receptor tyrosine kinase (RTK) signaling pathway. Furthermore, immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP) analysis indicated that SPRY1 binds to nuclear factor kappa B1 (NF-κB1) to prevent nuclear translocation of p65, inhibit activation of NF-κB signaling, prevent excessive inflammatory reaction in the testis, and protect the integrity of the blood-testis barrier. In view of this newly identified Dmrt1- Spry1-NF-κB axis mechanism in the regulation of testicular immune homeostasis, our study opens new avenues for the prevention and treatment of male reproductive diseases in humans and livestock.

Pre-leukemic Cell Detection and Leukemic Transformation of a Normal Marrow Cell: A Mini review

The hematopoietic cells are multipotent primitive cells, which differentiate into either common myeloid and lymphoid progenitor. However, if there an abnormality in this process of differentiation, condition of leukemia arises, which is the 11th leading cause of cancer-related mortality worldwide in the year 2018. These abnormalities are brought about by array of mutations occurring at cellular level. According to the two-hit model hypothesis, key oncogenic events are classified into two classes: class I mutations and class II mutations. Class I mutations are those that causes activation of the receptor tyrosine kinase (RTK), FLT3, c-kit (KIT), and Ras signaling pathways thereby increasing proliferation rate of progenitor cells. Class II mutations include recurrent chromosomal abnormalities such as t(8; 21), inv(16), and t(15; 17), which result in fusion transcripts of RUNX1/ETO, CBF/MYH11, and PML/RAR, respectively that eventually impair hematopoietic differentiation. The factors associated with leukemia can be biological, chemical or socio-economical. The advancement in the researches on the topic have aided to the development of various technologies such as detection of DNMT3A and xenografts assays, in order to detect these mutations in pre-leukemic cells. This review aims to provide an introduction to the condition, its types and provide brief summary on genes and mutations responsible for the condition. The factors associated with leukemia and technologies involved in the detection of leukemia are also reviewed.

Outcomes of Hispanic Vs Non-Hispanic Philadelphia-like B Cell ALL Treated in the Central Valley of California

Background: Philadelphia-like B cell ALL (Ph-like ALL) accounts for a majority of poor outcomes in patients with Ph-negative B cell ALL (Ph- ALL). Ph-like ALL is a heterogeneous subgroup characterized by mutations that involve the tyrosine kinase and cytokine receptor signaling pathways. Prior studies have suggested an increased incidence of B-ALL in the Hispanic community may be attributable to a higher incidence of Ph-like ALL. This is clinically relevant as Ph-like ALL is associated with worse outcomes. Advances in molecular testing have identified Ph-like sub-classifications of at least 4 unique groups. Ph-like ALL outcomes have not been well reported following the approval of novel treatment options such as bispecific antibodies and CAR-T cell therapy. Our prior study in 2020 reported mutational distributions of 8 Ph-like ALL patients out of 16 Ph- ALL cases sequenced. This is a follow up analysis of Ph- ALL patients treated in Fresno, CA (49.6% Hispanic population) both before and after the adoption of novel therapies. Methods: Patients diagnosed with Ph negative B-ALL from 01-01-2008 to 12-31-2021 at Community Medical Centers were included. Patients with available tissue and without molecular sequencing at diagnosis were sequenced by Neo-Genomics (14 patients), Foundation Medicine Hematology (23), other NGS (3), and no sequencing available (14). Demographic and disease relevant information was collected. Data was collected on incidence of cytogenetic and molecular abnormalities, disease spread (CNS involvement, organ involvement), CD20% expression, number of mutations (n<3 vs. n≥3), and number of treatment lines. Chi-square or Fisher's exact test and contingency table were used to compare characteristics of patients with Ph-like mutations vs those without. Kaplan-Meier curves were utilized to compare median progression free survival (PFS) after 1st line therapy and median overall survival (OS). Results: A total of 48 patients were included. 23 patients had Ph-like mutation present vs 25 without. 18 Hispanic patients had Ph-like mutation vs 15 without (p=0.183). The Ph-like group had higher BMI (36.4 vs 30.7, p=0.033) and more patients with ≥3 mutations compared to the non-Ph-like group (p=0.018). The distribution of mutations within the Ph like group were: CRLF (44%), JAK-STAT (39%), ABL (13%), and EPOR/JAK-2 (4%). There were no significant differences between the Ph-like and non-Ph-like groups in mean age at diagnosis (40.1 vs 35 years, p=0.27), CNS disease at diagnosis (17.4% vs 11.5%, p=0.350), CD20+ disease (17.4% vs 30.8%, p=0.098), complete response rates (60.9% vs 70.3%, p=0.223), or median PFS after 1st line therapy (284 vs 467 days, p=0.164). The non-Ph-like group had significantly higher median OS vs the Ph-like group (680.5 vs 377 days, p=0.018). Within the Ph-like group, Hispanic vs non-Hispanic patients did not differ significantly in terms of PFS after 1st line therapy (p=0.426) or OS (p=0.287). Survival analysis of patients before vs during/after the year 2017 showed significantly higher median PFS after 1st line therapy in patients treated during/after 2017 than before (1721 vs 631 days, p=0.037), however without significant median OS difference (1097 vs 819 days, p=0.712). There was no significant median PFS (p=0.166) or OS (p=0.731) difference among Ph-like patients treated before vs during/after 2017. Novel agent use in our study: 8/12 patients (Blinatumomab 7, Inotuzumab 1), 6/9 patients (Blinatumomab 2, Inotuzumab 2 CAR- T 1), 2/3 patients (Inotuzumab 2) in second, third and fourth line respectively. Conclusions: Ph-like ALL patients had worse OS outcomes compared to non-Ph-like patients. A significant proportion of both Ph-like and non-Ph-like patients consisted of Hispanics, confirming the high incidence of B-ALL in this ethnic subgroup reported in prior studies. Our Ph-like patients had significantly higher BMI and number of mutations which could be contributing factors to their worse outcomes compared to non-Ph-like patients. Those treated during/after 2017 had significantly improved PFS after induction; this could be due to the FDA approval of novel agents beginning that year. This however did not translate to a significant OS benefit regardless of Ph-like status. Overall, these results re-emphasize the need for further research involving ethnic subgroups and novel therapies for this high-risk and heterogeneous disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dehydromiltirone inhibits osteoclast differentiation in RAW264.7 and bone marrow macrophages by modulating MAPK and NF-κB activity.

Background: Osteoporosis is a type of systematic metabolic bone disease caused by the decrease in osteogenic activity or excessive resorption of bone with the relative enhancement of osteoclast function. As osteoporosis seriously affects the quality of patients’ life, effective drugs are needed to treat this disease. Based on the combination of network pharmacology and cellular studies, this study aimed to investigate the probable mechanism of Dehydromiltirone (DHT) in the treatment of osteoporosis. Method: The targets of DHT in osteoporosis were searched using the PharmGKB, OMIM, and Genecard platforms. The PPI core targets, and the GO and KEGG enrichment analysis results were obtained using Cytoscape software, and the David and Metascape databases, respectively. The network pharmacology results were also verified via in vitro cellular experiments. Results: Through network pharmacology and docking analysis, we found DHT was involved in peptide tyrosine phosphorylation, cell surface receptor tyrosine kinase signaling pathways, and MAPK signaling pathways. According to the molecular docking results, the binding of DHT to MAPK14 was more stable than other proteins, which suggests that DHT may affect osteoclast formation through the MAPK signaling pathway. Moreover, DHT was found to inhibit the expression of osteoclast-associated genes, including NFATc1, CTSK, c-Fos, Acp5, and MMP9; as well as the phosphorylation of P38, ERK, and JNK of the MAPK signaling pathway; and the degradation of IκB-α of NF-κB signaling pathway. Conclusion: DHT exhibited an anti-osteoclastogenesis effect by reducing the expression of related genes, ultimately inhibiting bone resorption in vitro.

An IGF-1R-mTORC1-SRPK2 signaling Axis contributes to FASN regulation in breast cancer

BackgroundFatty acid synthase (FASN) expression is associated with a more aggressive breast cancer phenotype and is regulated downstream of receptor tyrosine kinase (RTK) signaling pathways. Recently, post transcriptional regulation of lipogenic transcripts have been demonstrated as being mediated downstream of serine-arginine rich protein kinase 2 (SRPK2), which acts to phosphorylate serine-arginine rich splicing factors (SRSFs), resulting in RNA binding and various RNA regulatory processes. Though post-transcriptional regulation of FASN has been studied previously, the upstream mediators of these pathways have not been elucidated.MethodsWestern blotting and RT-qPCR were utilized to demonstrate alterations in FASN and mRNA expression upon modulation of the IGF-1-mTORC1-SRPK2 pathway by small molecule inhibitors or RNAi mediated silencing. RNA stability was accessed by using the transcriptional inhibitor actinomycin-D followed by RT-qPCR. Further, we employed RNA-immunoprecipitation to demonstrate the direct binding of SRSF-1 to FASN transcripts.ResultsIn the current study, we demonstrated an IGF-1 induced increase in FASN mRNA and protein expression that was attenuated by mTORC1 inhibition. This mTORC1 inhibition also resulted in decreases in total and nuclear p-SRPK2 in response to IGF-1 exposure. Upon SRPK2 knockdown and inhibition, we observed a decrease in FASN protein and mRNA stability, respectively, in response to IGF-1 exposure that was specific to triple negative and HER2+ breast cancer cell lines. As we explored further, IGF-1 exposure resulted in an altered localization of eGFP expressed SRSF-1, pEGFP-SRSF-1 that was rescued upon both SRPK2 knockdown and mTORC1 inhibition. Further, we observed an increase binding of SRSF-1 to FASN RNA upon IGF-1 exposure, which was abrogated by SRPK2 knockdown.ConclusionThese current findings establish a potential IGF-1-mTORC1-SRPK2-FASN axis in breast cancer, which could be a potential therapeutic target for cancers that overexpress FASN and components of the IGF-1R pathway.

Identification of immune subtypes to guide immunotherapy and targeted therapy in clear cell renal cell carcinoma.

Accumulating pieces of evidence suggested that immunotypes may indicate the overall immune landscape in the tumor microenvironment, which were closely related to therapeutic response. The purpose of this study was to classify and define the immune subtypes of clear cell renal cell carcinoma (ccRCC), so as to authenticate the potential immune subtypes that respond to immunotherapy. Transcriptome expression profile and mutation profile data of ccRCC, as well as clinical characteristics used in this study were obtained from TCGA database. There were significant differences in the infiltration of immune cells, immune checkpoints, and antigens between ccRCC and para-cancerous tissues. According to immune components, patients with ccRCC were divided into three immune subtypes, with different clinical and molecular characteristics. Compared with other subtypes, IS2 showed cold immune phenotype, and was associated with better survival. IS1 represented complex immune populations and was associated with poor overall survival (OS) and progression free survival (PFS). Further analysis indicated that expression of immune checkpoints also differed among the three subtypes, and was abnormally up-regulated in IS3. Pathway enrichment analysis indicated that the mTOR signaling pathway was abnormally enriched in IS3, while the TGF_BETA, ANGIOGENESIS and receptor tyrosine kinase signaling pathways were abnormally enriched in IS2. Furthermore, there was an abnormal enrichment of the epithelial-to-mesenchymal transition (EMT) signaling pathway in IS1, which may be associated with a higher rate of metastasis. Finally, SCG2 was screened as a specific antigen of ccRCC, which was not only related to poor prognosis, but also significantly associated with immune cells and immune checkpoints. In conclusion, the immune subtypes of ccRCC may provide new insights into the tumor biology and the precise clinical management of this disease.