Tyrosine Kinase Pathways Research Articles

BELLWAVE-008: A phase 3 study of the efficacy and safety of nemtabrutinib in previously untreated patients (pts) with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) without TP53 aberrations.

TPS7594 Background: Targeting the Bruton’s tyrosine kinase (BTK) pathway has been a revolutionary treatment option for first-line treatment of CLL/SLL. Nemtabrutinib is a noncovalent, reversible, potent BTK inhibitor that targets both wild-type and C481S-mutant BTK. Results of an ongoing phase 1/2 dose-escalation study (BELLWAVE-001) have demonstrated manageable safety and preliminary antitumor activity of nemtabrutinib in pts with CLL/SLL. BELLWAVE-008 is a randomized, open-label, phase 3 study (NCT05624554) designed to evaluate efficacy and safety of nemtabrutinib compared with investigator’s choice of chemoimmunotherapy (FCR or BR) in pts with previously untreated CLL/SLL without TP53 aberrations. Methods: Eligible pts are ≥18 years old with previously untreated CLL/SLL with active disease and without TP53 aberrations (17p deletion and/or TP53 mutation), with ECOG PS of 0-2 and adequate organ function. Pts with Richter transformation or active central nervous system involvement are excluded. Approximately 300 pts will be enrolled and randomly assigned 1:1 to receive nemtabrutinib (65 mg orally once daily until progressive disease, unacceptable toxicity, or discontinuation) or intravenous infusion of the investigator’s choice of chemoimmunotherapy for 6 cycles lasting 28 days each (FCR: fludarabine 25 mg/m2 and cyclophosphamide 250 mg/m2 on days 1, 2, and 3 of each cycle, plus rituximab 375 mg/m2 initially, followed by 500 mg/m2 on day 1 of each cycle; or BR: bendamustine 70-90 mg/m2 on days 1 and 2 of each cycle plus rituximab 375 mg/m2 initially, followed by 500 mg/m2 on day 1 of each cycle). Randomization will be stratified by risk (high risk: 11q deletion and/or IGHV unmutated vs low risk: absence of high-risk factors) and age ( < 65 years vs ≥65 years). Treatment response (imaging, physical examination, constitutional symptoms, hematologic evaluation, and bone marrow biopsy, as required) will be performed every 12 weeks up to and including cycle 25, and every 24 weeks thereafter. Adverse events (AEs) will be monitored up to 30 days after treatment cessation (90 days for serious AEs) and will be graded per National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0, and hematologic toxicities will be evaluated according to International Workshop on Chronic Lymphocytic Leukemia (iwCLL). Primary end point is progression-free survival per the iwCLL 2018 criteria as assessed by blinded independent central review. Secondary end points include overall survival, objective response rate (ORR), duration of response, and safety. Exploratory efficacy end points include resistance evaluations, ORR including partial response with lymphocytosis as a response category, pharmacokinetics/pharmacodynamics, and health-related quality of life. Enrollment is ongoing. Clinical trial information: NCT05624554 .

Identification of prognostic hub genes and therapeutic targets for selenium deficiency in chicks model through transcriptome profiling

Selenium deficiency is a prevalent micronutrient deficiency that poses a major health concern worldwide. This study aimed to shed light on the molecular mechanisms underlying selenium deficiency using a chick model. Chickens were divided into control and selenium deficient groups. Plasma samples were collected to measure selenium concentration and transcriptome analyse were performed on oviduct samples. The results showed that selenium deficiency led to a significant reduction in plasma selenium levels and altered the expression of 10,266 differentially expressed genes (DEGs). These DEGs primarily regulated signal transduction and cell motility. The molecular function includes GTPase regulatory activity, and KEGG pathway analysis showed that they were mainly involved in the signal transduction. By using Cytoscape and CancerGeneNet tool, we identified 8 modules and 10 hub genes (FRK, JUN, PTPRC, ACTA2, MST1R, SDC4, SDC1, CXCL12, MX1 and EZR) associated with receptor tyrosine kinase pathway, Wnt and mTOR signaling pathways that may be closely related to cancer. These hub genes could be served as precise diagnostic and prognostic candidate biomarkers of selenium deficiency and potential targets for treatment strategies in both animals and humans. This study sheds light on the molecular basis of selenium deficiency and its potential impact on public health.

Whole exome and transcriptome analysis revealed the activation of ERK and Akt signaling pathway in canine histiocytic sarcoma

Histiocytic sarcoma (HS) is an incurable aggressive tumor, and no consensus has been made on the treatment due to its rare occurrence. Since dogs spontaneously develop the disease and several cell lines are available, they have been advocated as translational animal models. In the present study, therefore, we explored gene mutations and aberrant molecular pathways in canine HS by next generation sequencing to identify molecular targets for treatment. Whole exome sequencing and RNA-sequencing revealed gene mutations related to receptor tyrosine kinase pathways and activation of ERK1/2, PI3K-AKT, and STAT3 pathways. Analysis by quantitative PCR and immunohistochemistry revealed that fibroblast growth factor receptor 1 (FGFR1) is over-expressed. Moreover, activation of ERK and Akt signaling were confirmed in all HS cell lines, and FGFR1 inhibitors showed dose-dependent growth inhibitory effects in two of the twelve canine HS cell lines. The findings obtained in the present study indicated that ERK and Akt signaling were activated in canine HS and drugs targeting FGFR1 might be effective in part of the cases. The present study provides translational evidence that leads to establishment of novel therapeutic strategies targeting ERK and Akt signaling in HS patients.

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.

Advances in molecular evaluation of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders with uncontrolled proliferation of one or more hematopoietic cell types, including myeloid, erythroid and megakaryocytic lineages, and minimal defect in maturation. Most MPN are associated with well-defined molecular abnormalities involving genes that encode protein tyrosine kinases that lead to constitutive activation of the downstream signal transduction pathways and confer cells proliferative and survival advantage. Genome-wide sequencing analyses have discovered secondary cooperating mutations that are shared by most of the MPN subtypes as well as other myeloid neoplasms and play a major role in disease progression. Without appropriate management, the natural history of most MPN consists of an initial chronic phase and a terminal blast phase. Molecular aberrations involving protein tyrosine kinases have been used for the diagnosis, classification, detection of minimal/measurable residual disease, and target therapy. We review recent advances in molecular genetic aberrations in MPN with a focus on MPN associated with gene rearrangements or mutations involving tyrosine kinase pathways.

Multiple PIK3CA mutation clonality correlates with outcomes in taselisib + fulvestrant-treated ER+/HER2–, PIK3CA-mutated breast cancers

BackgroundMutations in the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), encoded by the PIK3CA gene, cause dysregulation of the PI3K pathway in 35–40% of patients with HR+/HER2– breast cancer. Preclinically, cancer cells harboring double or multiple PIK3CA mutations (mut) elicit hyperactivation of the PI3K pathway leading to enhanced sensitivity to p110α inhibitors.MethodsTo understand the role of multiple PIK3CAmut in predicting response to p110α inhibition, we estimated the clonality of multiple PIK3CAmut in circulating tumor DNA (ctDNA) from patients with HR+/HER2– metastatic breast cancer enrolled to a prospectively registered clinical trial of fulvestrant ± taselisib, and analyzed the subgroups against co-altered genes, pathways, and outcomes.ResultsctDNA samples with clonal multiple PIK3CAmut had fewer co-alterations in receptor tyrosine kinase (RTK) or non-PIK3CA PI3K pathway genes compared to samples with subclonal multiple PIK3CAmut indicating a strong reliance on the PI3K pathway. This was validated in an independent cohort of breast cancer tumor specimens that underwent comprehensive genomic profiling. Furthermore, patients whose ctDNA harbored clonal multiple PIK3CAmut exhibited a significantly higher response rate and longer progression-free survival vs subclonal multiple PIK3CAmut.ConclusionsOur study establishes clonal multiple PIK3CAmut as an important molecular determinant of response to p110α inhibition and provides rationale for further clinical investigation of p110α inhibitors alone or with rationally-selected therapies in breast cancer and potentially other solid tumor types.

Phosphatidylinositol kinases in GtoPdb v.2023.1

Phosphatidylinositol may be phosphorylated at either 3- or 4- positions on the inositol ring by PI 3-kinases or PI 4-kinases, respectively.Phosphatidylinositol 3-kinasesPhosphatidylinositol 3-kinases (PI3K, provisional nomenclature) catalyse the introduction of a phosphate into the 3-position of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol 4,5-bisphosphate (PIP2). There is evidence that PI3K can also phosphorylate serine/threonine residues on proteins. In addition to the classes described below, further serine/threonine protein kinases, including ATM (Q13315) and mTOR (P42345), have been described to phosphorylate phosphatidylinositol and have been termed PI3K-related kinases. Structurally, PI3Ks have common motifs of at least one C2, calcium-binding domain and helical domains, alongside structurally-conserved catalytic domains. wortmannin and LY 294002 are widely-used inhibitors of PI3K activities. wortmannin is irreversible and shows modest selectivity between Class I and Class II PI3K, while LY294002 is reversible and selective for Class I compared to Class II PI3K.Class I PI3Ks (EC 2.7.1.153) phosphorylate phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate and are heterodimeric, matching catalytic and regulatory subunits. Class IA PI3Ks include p110α, p110β and p110δ catalytic subunits, with predominantly p85 and p55 regulatory subunits. The single catalytic subunit that forms Class IB PI3K is p110γ. Class IA PI3Ks are more associated with receptor tyrosine kinase pathways, while the Class IB PI3K is linked more with GPCR signalling.Class II PI3Ks (EC 2.7.1.154) phosphorylate phosphatidylinositol to generate phosphatidylinositol 3-phosphate (and possibly phosphatidylinositol 4-phosphate to generate phosphatidylinositol 3,4-bisphosphate). Three monomeric members exist, PI3K-C2α, β and β, and include Ras-binding, Phox homology and two C2 domains.The only class III PI3K isoform (EC 2.7.1.137) is a heterodimer formed of a catalytic subunit (VPS34) and regulatory subunit (VPS15).Phosphatidylinositol 4-kinasesPhosphatidylinositol 4-kinases (EC 2.7.1.67) generate phosphatidylinositol 4-phosphate and may be divided into higher molecular weight type III and lower molecular weight type II forms.

Abstract 1003: The prognostic significance of prolyl 4-hydroxylase subunit alpha 1 and the therapeutic efficacy of diethyl-pythiDC, its small molecule inhibitor, in pancreatic ductal adenocarcinoma

Abstract Background: Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) has an oncogenic role in pancreatic ductal adenocarcinoma (PDAC). Since high expression of P4HA1 in PDAC is suggested to correlate with poor prognosis, targeting P4HA1 could have therapeutic benefits. Herein, we assessed the prognostic significance of P4HA1 expression at the RNA and protein levels in PDAC using publicly available data. We also evaluated the efficacy of diethyl-pythiDC, a small molecule inhibitor of P4HA1, in PDAC cells. Methods: UALCAN (ualcan.path.uab.edu), a comprehensive proteogenomic cancer data analysis portal, was used to obtain gene expression P4HA1 as transcripts per million reads (TPM) (PDAC n=178) from The Cancer Genome Atlas (TCGA); levels of P4HA1 protein expression data were obtained from (normal pancreas, n=74, PDAC n=137) the Clinical Proteomic Tumor Analysis consortium (CPTAC). Immunohistochemical analysis obtained from Human Protein Atlas (HPA) confirmed increased P4HA1 protein expression. Overall survival (OS) between patients with high and low/medium TPM (TPM values above and below upper quartile, respectively) and high and low protein expression score (based on staining intensity and fraction of stained cells) was calculated using the log-rank Wilcoxon test. To determine the efficacy of diethyl-pythiDC in PDAC cells (PANC-1 and BxPC-3), we performed clonogenic assay for colony formation, MTT assay for cell proliferation, flow cytometry for cell cycle analysis, and western blotting for markers of the epithelial-mesenchymal transition (EMT). Results: In the TCGA data, high P4HA1 expression (TPMs) was associated with a poor prognosis (high expression vs. low/medium expression, log-rank p=0.009). Analysis of HPA data showed that the 5-year OS of patients with high P4HA1 expression was 12% vs. 58% for patients with low P4HA1 (log-rank p<0.001). CPTAC data showed higher P4HA1 protein expression in patients with receptor tyrosine kinase (RTK) pathway (p<0.001), mammalian target of rapamycin (mTOR) pathway (p=0.045) and SWItch/Sucrose Non-Fermentable (SWI-SNF) complex (p=0.022) alterations. Diethyl-pythiDC treatment of PDAC cells reduced proliferation, decreased colony formation, and increased G2/M cell cycle arrest. In addition, diethyl-pythiDC increased protein expression of E-cadherin, an epithelial marker, and decreased the expression of N-cadherin and vimentin, the mesenchymal protein markers. Conclusions: Patients with high P4HA1 expression had poor OS. CPTAC data showed higher P4HA1 protein expression in patients with RTK, mTOR and SWI-SNF alterations. Targeting P4HA1 with diethyl-pythiDC reduced proliferation by arresting cells in G2/M phase of the cell cycle and modulated EMT markers. Thus, targeting of P4HA1 can be a viable strategy and may be considered in future PDAC clinical trials. Citation Format: Moh'd Khushman, Farrukh Afaq, Prachi Bajpai, Sameer Al Diffalha, Darryl Outlaw, Grant R. Williams, Rojymon Jacob, Kondal Rao Kyanam Kabir, Ali Ahmed, Peter Shajan, Sooryanarayana Varambally, Upender Manne. The prognostic significance of prolyl 4-hydroxylase subunit alpha 1 and the therapeutic efficacy of diethyl-pythiDC, its small molecule inhibitor, in pancreatic ductal adenocarcinoma [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 1003.

Abstract 4032: Discovery of a potent, selective, and orally available SHP2 inhibitor that can penetrate blood-brain barrier

Abstract Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2/PTPN11) is one of the key signaling proteins in many oncogenic receptor tyrosine kinase (RTK) pathways. It mediates RAS-driven downstream signaling and activates gene expression for tumorigenesis. Importantly, a critical role of SHP2 has been found during the process of temporal adaptation and ERK re-activation after the treatment of RAS/RAF/MEK/ERK inhibitors, which means that SHP2 could be a promising therapeutic target to shut down the oncogenic RAS pathway, especially in combination with RAS/RAF/MEK/ERK inhibitors. Here we present preclinical data of an allosteric small-molecule SHP2 inhibitor that is highly potent, selective, and brain-permeable. In vitro biochemical and cellular assays showed excellent potency against SHP2 protein. In addition, we confirmed exceptional in vivo activity in mouse xenograft models, and demonstrated combination effects with a KRAS G12C inhibitor. Most importantly, the BBB penetration and anti-tumor activity in brain was confirmed both in vitro and in vivo, supporting the great potential in treating brain-metastasized tumors. In summary, we have successfully discovered and developed a potent and blood-brain barrier permeable SHP2 inhibitor that provides a novel therapeutic option for diverse cancers associated in the RAS pathway. Citation Format: Miyeon Kim, Dongsu Kim, Kyeong Jin Yoon, Yeejin Jeon, Dohyun Park, Mijung Lee, Dahye Jeon, Soyeon Jang, Jinhwan Kim, Eunji Kim, Jihoon Park, Victor Hong, Sang Kyun Lim, Sungpil Choi. Discovery of a potent, selective, and orally available SHP2 inhibitor that can penetrate blood-brain barrier. [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 4032.

Abstract 262: Heat Map Analysis, a new integrated pathway scoring system for cancer genome profiling

Abstract Background: Cancer genome profiling using next-generation sequencing (NGS) has become widespread. Multiple genetic abnormalities including variant of unknown significance (VUS) have been detected in various cancer types. However, there is a lack of an integrated analysis system capable of identifying relationships among multiple genetic abnormalities and weighting them appropriately. Here, we report heat map analysis, a new integrated pathway scoring system for cancer genome profiling. Methods: In heat map analysis, approximately 180 cancer-related genes among 324 genes evaluated by FoundationOne CDxTM are scored based on gene annotation and variant allele fraction (VAF), and classified into 16 pathways. This system provides an overall picture of genetic abnormalities and easy recognition of abnormal pathways. To examine the usefulness of the heat map analysis, a retrospective observational study was conducted on 50 patients with solid tumors who underwent cancer genome profiling test at Saga University Hospital from June 2019 to February 2022. The primary end point was the presentation rate of pathways with significantly increased scores. Results: A total of 749 genetic abnormalities including VUS were detected in 50 patients with 15 cancer types: 571 single nucleotide variants (SNVs), 156 copy number variants (CNVs), and 22 fusions. The median number of abnormalities detected was 11 (2-20) SNVs, 2 (0-23) CNVs, and 0 (0-4) fusions, respectively. Heat map analysis presented active pathways with significant score elevation in 42 cases (84%). The TP53 pathway was the most common, followed by the Receptor Tyrosine Kinase pathway, PIK3-AKT, RAS, and DNA repair-related pathways. Conclusion: Heat map analysis could be a promising new method for integrated analysis using cancer genome profiling tests. Citation Format: Chiho Nakashima, Yukimasa Shiotsu, Yohei Harada, Hiroo Katsuya, Emi Ookuma, Masanori Nishi, Akemi Sato, Hideaki Nakamura, Naoko Sueoka-Aragane. Heat Map Analysis, a new integrated pathway scoring system for cancer genome profiling [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 262.

IL-37 Attenuates Platelet Activation and Thrombosis Through IL-1R8 Pathway.

IL-37 (interleukin-37), a natural suppressor of innate inflammatory and immune responses, is increased in patients with myocardial infarction. Platelets play an important role in the progress of myocardial infarction, but the direct effects of IL-37 on platelet activation and thrombosis, as well as the underlying mechanisms, still remain unclear. We evaluated the direct effects of IL-37 on agonists-induced platelet activation and thrombus formation, as well as revealed the underlying mechanisms using platelet-specific IL-1R8 (IL-1 receptor 8)-deficient mice. Using myocardial infarct model, we explored the effects of IL-37 on microvascular obstruction and myocardial injury. IL-37 directly inhibited agonists-induced platelet aggregation, dense granule ATP release, P-selectin exposure, integrin αIIbβ3 activation, platelet spreading, and clot retraction. IL-37 inhibited thrombus formation in vivo in a FeCl3-injured mesenteric arteriole thrombosis mouse model and ex vivo in a microfluidic whole-blood perfusion assay. Mechanistic studies using platelet-specific IL-1R8-deficient mice revealed that IL-37 bound to platelet IL-1R8 and IL-18Rα, and IL-1R8 deficiency impaired the inhibitory effects of IL-37 on platelet activation. Using PTEN (phosphatase and tensin homolog)-specific inhibitor and PTEN-deficient platelets, we found that IL-37 combined with IL-1R8 to enhance PTEN activity, inhibit Akt (protein kinase B), mitogen-activated protein kinases, and spleen tyrosine kinase pathways, as well as decrease the generation of reactive oxygen species to regulate platelet activation. Exogenous IL-37 injection suppressed microvascular thrombosis to protect against myocardial injury in wild-type mice but not in platelet-specific IL-1R8-deficient mice after permanent ligation of the left anterior descending coronary. Finally, a negative correlation between plasma IL-37 concentration and platelet aggregation was observed in patients with myocardial infarction. IL-37 directly attenuated platelet activation, thrombus formation, and myocardial injury via IL-1R8 receptor. Accumulated IL-37 in plasma inhibited platelet activation to ameliorate atherothrombosis and infarction expansion, and thus may have therapeutic advantages as potential antiplatelet drugs.

Response to acute hyperglycemia and high fructose in cultured tenocytes.

High monosaccharide levels are intimately associated with diabetes and impact tendon cells through inflammation and impairment in metabolic homeostasis. Experiments were designed to understand the responses elicited by cultured tenocytes under monosaccharide stress induced by hyperglycemia and hyperfructosemia. We simulated hyperglycemia and hyperfructosemia in vitro by treating tenocytes with media containing sublethal concentrations of glucose and fructose, respectively. Exposure of tenocytes to high glucose and high fructose altered the levels of IL-1β, IL-2, IL-6, IL10 and IL-17A. AMPK expression was increased in high-glucose and decreased in high-fructose groups. High fructose increased the level of IRS-1 compared with the control. Increased mitochondrial superoxide levels and compromised mitochondrial membrane integrity were exhibited by both the groups. The findings from the network analysis revealed many altered genes that are related to pathways for enzyme-linked receptor protein signaling, positive regulation of metabolic processes, transmembrane receptor tyrosine kinase pathway, insulin receptor signaling and regulation of cytokine production. Overall, the data suggest that the tenocytes under high monosaccharide levels exhibit survival responses by altering the expression status of cytokines and metabolic mediators that are involved in the underlying pathogenesis of tendinopathy.

Proteomic characterisation of perhexiline treatment on THP-1 M1 macrophage differentiation.

Dysregulated inflammation is important in the pathogenesis of many diseases including cancer, allergy, and autoimmunity. Macrophage activation and polarisation are commonly involved in the initiation, maintenance and resolution of inflammation. Perhexiline (PHX), an antianginal drug, has been suggested to modulate macrophage function, but the molecular effects of PHX on macrophages are unknown. In this study we investigated the effect of PHX treatment on macrophage activation and polarization and reveal the underlying proteomic changes induced. We used an established protocol to differentiate human THP-1 monocytes into M1 or M2 macrophages involving three distinct, sequential stages (priming, rest, and differentiation). We examined the effect of PHX treatment at each stage on the polarization into either M1 or M2 macrophages using flow cytometry, quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Quantitative changes in the proteome were investigated using data independent acquisition mass spectrometry (DIA MS). PHX treatment promoted M1 macrophage polarization, including increased STAT1 and CCL2 expression and IL-1β secretion. This effect occurred when PHX was added at the differentiation stage of the M1 cultures. Proteomic profiling of PHX treated M1 cultures identified changes in metabolic (fatty acid metabolism, cholesterol homeostasis and oxidative phosphorylation) and immune signalling (Receptor Tyrosine Kinase, Rho GTPase and interferon) pathways. This is the first study to report on the action of PHX on THP-1 macrophage polarization and the associated changes in the proteome of these cells.

Bushen Huoxue Fang improves recurrent miscarriage in mice by down-regulating the JAK2/STAT3 pathway

To investigate the efficacy of Bushen Huoxue Fang (BSHXF, a traditional Chinese medicine formula) for improving recurrent spontaneous abortion (RSA) in mice and the role of tyrosine kinase (JAK2) and transcriptional activator (STAT3) signaling pathway in its therapeutic mechanism. Female CBA/J mice were caged with male DBA/2 mice to establish RSA mouse models, which were randomly divided into model group, dydrogesterone group and BSHXF group, with the female mice caged with male BALB/c mice as the control group (n=6). From the first day of pregnancy, the mice were subjected to daily intragastric administration of BSHXF, dydrogesterone, or distilled water (in control and model groups) for 12 days. After the treatments, serum levels of antithrombin III (AT-III), activated protein C (APC), tissue plasminogen activator (t-PA), progesterone, human chorionic gonadotropin (HCG), and estradiol (E2) were detected in each group using ELISA. HE staining was used to observe the morphological changes of the endometrium of the mice. Western blotting was performed to determine the expressions of p-JAK2, p-Stat3 and Bcl-2 in the placenta of the mice. Compared with the control mice, the mouse models of RSA showed a significantly increased embryo loss rate with decreased serum levels of AT-III, T-PA, progesterone, APC and HCG, increased placental expressions of p-JAK2, p-STAT3 and Bax, and decreased expression of Bcl-2 (P < 0.05). Treatments with BSHXF and dydrogesterone both increased serum levels of AT-III, t-PA and HCG in the mouse models; Serum APC level was significantly reduced in BSHXF group and serum progesterone level was significantly increased in dydrogesterone group (P < 0.05). BSHXF can improve the prethrombotic state and inhibit cell apoptosis by downregulating the JAK2/STAT3 pathway to increase the pregnancy rate in mouse models of RSA.

Diagnostic and Therapeutic Pathway in Diffuse Malignant Peritoneal Mesothelioma.

Diffuse malignant peritoneal mesothelioma (DMPM) is a rare form of mesothelioma that carries a very poor prognosis. The 5-year overall survival is about 20% (±5.9). Survival is optimal for patients suitable for cytoreductive surgery (CRS) with Hyperthermic Intraperitoneal Chemotherapy (HIPEC), with a median OS ranging from 34 to 92 months. However, selecting patients for surgery remains a complex task and requires a careful preoperative workup, rational analysis of prognostic profiles, and risk prediction models. Systemic chemotherapy could be offered: (1) in the adjuvant setting for high-risk patients; (2) for patients not eligible for CRS; and (3) for those with recurrent disease. It mainly includes the combination of Platin compound with Pemetrexed or immunotherapy. The biology of DMPM is still largely unknown. However, progress has been made on some fronts, such as telomere maintenance mechanisms, deregulation of apoptosis, tyrosine kinase pathways, and mutation of BRCA1-associated protein 1 (BAP1). Future perspectives should include translational research to improve our understanding of the disease biology to identify druggable targets. We should also clear the role of immune checkpoint inhibitors and investigate new locoregional technologies, such as pressurized intraperitoneal aerosol chemotherapy (PIPAC) or normothermic intraperitoneal chemotherapy (NIPEC).

Whole-exome sequencing and bioinformatic analyses revealed differences in gene mutation profiles in papillary thyroid cancer patients with and without benign thyroid goitre background.

Papillary thyroid cancer (PTC) is the most common thyroid malignancy. Concurrent presence of cytomorphological benign thyroid goitre (BTG) and PTC lesion is often detected. Aberrant protein profiles were previously reported in patients with and without BTG cytomorphological background. This study aimed to evaluate gene mutation profiles to further understand the molecular mechanism underlying BTG, PTC without BTG background and PTC with BTG background. Patients were grouped according to the histopathological examination results: (i) BTG patients (n = 9), (ii) PTC patients without BTG background (PTCa, n = 8), and (iii) PTC patients with BTG background (PTCb, n = 5). Whole-exome sequencing (WES) was performed on genomic DNA extracted from thyroid tissue specimens. Nonsynonymous and splice-site variants with MAF of ≤ 1% in the 1000 Genomes Project were subjected to principal component analysis (PCA). PTC-specific SNVs were filtered against OncoKB and COSMIC while novel SNVs were screened through dbSNP and COSMIC databases. Functional impacts of the SNVs were predicted using PolyPhen-2 and SIFT. Protein-protein interaction (PPI) enrichment of the tumour-related genes was analysed using Metascape and MCODE algorithm. PCA plots showed distinctive SNV profiles among the three groups. OncoKB and COSMIC database screening identified 36 tumour-related genes including BRCA2 and FANCD2 in all groups. BRAF and 19 additional genes were found only in PTCa and PTCb. "Pathways in cancer", "DNA repair" and "Fanconi anaemia pathway" were among the top networks shared by all groups. However, signalling pathways related to tyrosine kinases were the most significantly enriched in PTCa while "Jak-STAT signalling pathway" and "Notch signalling pathway" were the only significantly enriched in PTCb. Ten SNVs were PTC-specific of which two were novel; DCTN1 c.2786C>G (p.Ala929Gly) and TRRAP c.8735G>C (p.Ser2912Thr). Four out of the ten SNVs were unique to PTCa. Distinctive gene mutation patterns detected in this study corroborated the previous protein profile findings. We hypothesised that the PTCa and PTCb subtypes differed in the underlying molecular mechanisms involving tyrosine kinase, Jak-STAT and Notch signalling pathways. The potential applications of the SNVs in differentiating the benign from the PTC subtypes requires further validation in a larger sample size.

Antimetastatic effect of fucoidan against non-small cell lung cancer by suppressing non-receptor tyrosine kinase and extracellular signal-related kinase pathway.

Fucoidan, a polysaccharide content in brown algae, has been reported to inhibit the growth of cancer cells. The present study aimed to investigate the suppression effects of fucoidan on A549 non-small cell lung cancer cells migration. The anti-migratory activity of fucoidan in A549 cells was examined by wound healing assay and phalloidin-rhodamine staining in response to fucoidan (0-100 µg/mL) treatment for 48 h. Western blot analysis was performed to clarify the protein expressions relevant to migratory activity. Fucoidan (25-100 µg/mL) significantly suppressed A549 cells migration together with reduced the intensity of phalloidin-rhodamine which detect filopodia and lamellipodia protrusions at 48 h of treatment. The protein expression indicated that fucoidan significantly suppressed the phosphorylation of focal adhesion kinase (FAK), Src, and extracellular signal-related kinase (ERK). In addition, the phosphorylation of p38 in A549 cells was found to be increased. Our data conclude that fucoidan exhibits anti-migratory activities against lung cancer A549 cells mediated by inhibiting ERK1/2 and FAK-Src pathway.

Receptor Tyrosine Kinase Pathway and Infiltrating Urothelial Carcinoma

Receptor tyrosine kinase pathway is frequently searched for cancer causing mutations in tumors. Emerging targeted therapies are gleam of hope for them. Infiltrating urothelial carcinoma can have many morphological aspects according to their differentiation/variants. To evaluate KRAS, BRAF, and PIK3CA mutations and HER2, EGFR, and p16 expression, we divided urothelial carcinomas into two groups: differentiated/variants (n = 12) and conventional (n = 12). We compared results with clinical, demographic, histopathologic features and survival rates. No statistically significant results could be obtained in the comparison of histopathologic properties/survival rates with mutation analysis and EGFR, HER2, and p16 status. Differentiated/variants urothelial carcinoma showed higher EGFR expression (P < 0.001). Glandular differentiation was the most frequent type, followed by squamous and sarcomatoid differentiation. We observed the most common mutation at KRAS with a propensity for urothelial carcinoma with glandular differentiation. More than one mutation/high protein expression was seen in some tumors. Targeted therapies for KRAS mutation can be effective at urothelial carcinoma with glandular differentiation. Heterologous expression of relevant proteins and genes can be a cause for targeted treatment obstacle. The determination of the molecular characters of tumors is a guide in creating targeted treatment algorithms and in choosing the patient.

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.

Molecularly Defined Subsets of Ewing Sarcoma Tumors Differ in Their Responses to IGF1R and WEE1 Inhibition.

Targeted cancer therapeutics have not significantly benefited patients with Ewing sarcoma with metastatic or relapsed disease. Understanding the molecular underpinnings of drug resistance can lead to biomarker-driven treatment selection. Receptor tyrosine kinase (RTK) pathway activation was analyzed in tumor cells derived from a panel of Ewing sarcoma tumors, including primary and metastatic tumors from the same patient. Phospho-RTK arrays, Western blots, and IHC were used. Protein localization and the levels of key markers were determined using immunofluorescence. DNA damage tolerance was measured through PCNA ubiquitination levels and the DNA fiber assay. Effects of pharmacologic inhibition were assessed in vitro and key results validated in vivo using patient-derived xenografts. Ewing sarcoma tumors fell into two groups. In one, IGF1R was predominantly nuclear (nIGF1R), DNA damage tolerance pathway was upregulated, and cells had low replication stress and RRM2B levels and high levels of WEE1 and RAD21. These tumors were relatively insensitive to IGF1R inhibition. The second group had high replication stress and RRM2B, low levels of WEE1 and RAD21, membrane-associated IGF1R (mIGF1R) signaling, and sensitivity to IGF1R or WEE1-targeted inhibitors. Moreover, the matched primary and metastatic tumors differed in IGF1R localization, levels of replication stress, and inhibitor sensitivity. In all instances, combined IGF1R and WEE1 inhibition led to tumor regression. IGF1R signaling mechanisms and replication stress levels can vary among Ewing sarcoma tumors (including in the same patient), influencing the effects of IGF1R and WEE1 treatment. These findings make the case for using biopsy-derived predictive biomarkers at multiple stages of Ewing sarcoma disease management.