Transmembrane Tyrosine Kinase Research Articles

MicroRNA expression signature in gastrointestinal stromal tumour & their molecular & histological features.

Background & objectives In gastrointestinal stromal tumour (GIST), not only genetic abnormalities are responsible for adverse clinical events, but epigenetic modifications also play a crucial role. MicroRNA (miRNA) dysregulation plays a significant role in carcinogenesis as miRNAs serve as natural silencer for their targets. Our study aimed to explore the miRNAs expression and its association with molecular and histopathological characteristics of GIST. Methods Fifty GIST samples, including 45 formalin fixed paraffin embedded (FFPE) and fresh tissues were included. Peripheral non-tumour tissues were used as controls. All the cases were confirmed using immunohistochemistry. RNA was extracted using miRNA-specific kit, and the expression was performed using RT-qPCR. The data were evaluated using AriaMx software version 1.5 (Agilent, US). MiRNAs expression was analyzed by using the relative quantification method (ΔΔCT). Results miR-221, miR-222, miR-494 and miR-34a showed significant down-regulation in tumours relative to non-tumour tissues. The expression levels of these miRNAs were significantly down-regulated in c-KIT (proto-oncogene encoding the tyrosine kinase transmembrane receptor)-positive tumours compared to c-KIT-negative. Further analysis revealed that reduced expression was associated with spindle subtypes and gastric localization. However, there was no significant correlation with other histological features. Additionally, miR-221/222, and miR-494 were down-regulated in most of the KIT exon 11 mutant subtypes, while miRNA-34a was associated with platelet derived growth factor receptor alpha (PDGFRA) mutations. Interpretation & conclusions The present study showed that the down-regulation of these miRNAs may help better molecular classification and characterization of GISTs. Our results offer new insight into the association between miRNAs and histological features, enabling a more thorough understanding of GISTs at the molecular level.

EGFR mutations and abnormal trafficking in cancers.

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor and a member of the ErbB receptor family. As a significant cancer driver, EGFR undergoes mutations such as gene amplification or overexpression in a wide range of malignant tumors and is closely associated with tumorigenesis. This review examines the aberrant expression of EGFR in several common cancers and summarizes the current therapeutic strategies developed for this receptor. Additionally, this review compares the differences in EGFR activation, internalization, endocytosis, and sorting in normal and cancer cells, and highlights some regulatory factors that influence its trafficking process.Kindly check and confirm the edit made in the title.Yes, correctAs per journal instructions structured abstract is mandatory kindly provideThe abstract format does not apply to Review articles.

Pyrazolo[1,5-a]pyrimidine as a Prominent Framework for Tropomyosin Receptor Kinase (Trk) Inhibitors-Synthetic Strategies and SAR Insights.

Tropomyosin receptor kinases (Trks) are transmembrane receptor tyrosine kinases named TrkA, TrkB, and TrkC and encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively. These kinases have attracted significant attention and represent a promising therapeutic target for solid tumor treatment due to their vital role in cellular signaling pathways. First-generation TRK inhibitors, i.e., Larotrectinib sulfate and Entrectinib, received clinical approval in 2018 and 2019, respectively. However, the use of these inhibitors was significantly limited because of the development of resistance due to mutations. Fortunately, the second-generation Trk inhibitor Repotrectinib (TPX-0005) was approved by the FDA in November 2023, while Selitrectinib (Loxo-195) has provided an effective solution to this issue. Another macrocycle-based analog, along with many other TRK inhibitors, is currently in clinical trials. Two of the three marketed drugs for NTRK fusion cancers feature a pyrazolo[1,5-a] pyrimidine nucleus, prompting medicinal chemists to develop numerous novel pyrazolopyrimidine-based molecules to enhance clinical applications. This article focuses on a comprehensive review of chronological synthetic developments and the structure-activity relationships (SAR) of pyrazolo[1,5-a]pyrimidine derivatives as Trk inhibitors. This article will also provide comprehensive knowledge and future directions to the researchers working in the field of medicinal chemistry by facilitating the structural modification of pyrazolo [1,5-a]pyrimidine derivatives to synthesize more effective novel chemotherapeutics as TRK inhibitors.

Inhibition of human epidermal growth factor receptor-2 protein by some alkaloid inhibitors

BackgroundHuman Epidermal Growth Factor Receptor-2 (HER2), a transmembrane tyrosine kinase receptor, has been associated with several types of cancer, including breast, lung, ovarian, etc. Therefore, this receptor is targeted by a variety of therapeutic approaches for cancer treatments. The alkaloid and lead compounds are among the selective and potent HER2 inhibitors that have been reported so far. MethodsThe present work involves molecular docking, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations on alkaloid and lead compounds as HER2 inhibitors. Significant findingsThe docking results expressed that the alkaloids mainly interacted with Cys805, Val734, and Ala751 residues of HER2 protein. The MD simulation outcomes represented that all the complexes have adequate dynamic stability and flexibility based on the root mean square deviation, root mean square fluctuation, and radius of gyration. Furthermore, it is found that the strongest HER2 − ligand interaction belongs to the nocamycin I (NOI, ΔGbind = -12.84 kcal mol−1) molecule. The DFT calculations showed that electron density and the second − order perturbation stabilization energy values for HER2 − NOI interactions are higher than the other complexes. The molecular docking, MD simulation, and DFT calculation results are all in agreement and complementary. It is expected that the results obtained here can present very helpful information for the design of efficacious inhibitors for the treatment of HER2 − related cancer disease.

HER2-CD3-Fc Bispecific Antibody-Encoding mRNA Delivered by Lipid Nanoparticles Suppresses HER2-Positive Tumor Growth.

The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor and tumor-associated antigen abnormally expressed in various types of cancer, including breast, ovarian, and gastric cancer. HER2 overexpression is highly correlated with increased tumor aggressiveness, poorer prognosis, and shorter overall survival. Consequently, multiple HER2-targeted therapies have been developed and approved; however, only a subset of patients benefit from these treatments, and relapses are common. More potent and durable HER2-targeted therapies are desperately needed for patients with HER2-positive cancers. In this study, we developed a lipid nanoparticle (LNP)-based therapy formulated with mRNA encoding a novel HER2-CD3-Fc bispecific antibody (bsAb) for HER2-positive cancers. The LNPs efficiently transfected various types of cells, such as HEK293S, SKOV-3, and A1847, leading to robust and sustained secretion of the HER2-CD3-Fc bsAb with high binding affinity to both HER2 and CD3. The bsAb induced potent T-cell-directed cytotoxicity, along with secretion of IFN-λ, TNF-α, and granzyme B, against various types of HER2-positive tumor cells in vitro, including A549, NCI-H460, SKOV-3, A1847, SKBR3, and MDA-MB-231. The bsAb-mediated antitumor effect is highly specific and strictly dependent on its binding to HER2, as evidenced by the gained resistance of A549 and A1847 her2 knockout cells and the acquired sensitivity of mouse 4T1 cells overexpressing the human HER2 extracellular domain (ECD) or epitope-containing subdomain IV to the bsAb-induced T cell cytotoxicity. The bsAb also relies on its binding to CD3 for T-cell recruitment, as ablation of CD3 binding abolished the bsAb's ability to elicit antitumor activity. Importantly, intratumoral injection of the HER2-CD3-Fc mRNA-LNPs triggers a strong antitumor response and completely blocks HER2-positive tumor growth in a mouse xenograft model of human ovarian cancer. These results indicate that the novel HER2-CD3-Fc mRNA-LNP-based therapy has the potential to effectively treat HER2-positive cancer.

Effects of N361 Glycosylation on Epidermal Growth Factor Receptor Biological Function.

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase that is frequently modified by glycosylation post-translationally. In cancer, EGFR amplifications and hotspot mutations such as L858R that promote proliferation have been detected in a significant fraction of non-small cell lung carcinomas and breast adenocarcinomas. Molecular dynamic simulations suggested that glycosylation at asparagine residue 361 (N361) promotes dimerization and ligand binding. We stably expressed glycosylation-deficient mutant EGFR N361A, with or without the oncogenic mutation L858R. Immunofluorescence and flow cytometry demonstrated that the mutants were each well expressed at the cell membrane. N361A decreased proliferation relative to wild-type EGFR as well as decreased sensitivity to ligands. Proximity ligation assays measuring co-localization of EGFR with its binding partner HER2 in cells revealed that N361A mutations increased co-localization. N361A, located near the binding interface for the EGFR inhibitor necitumumab, desensitized cells expressing the oncogenic EGFR L858R to antibody-based inhibition. These findings underline the critical relevance of post-translational modifications on oncogene function.

Current and Emerging Treatment Options for HER2‑Positive Gastroesophageal Cancer

Gastroesophageal cancer (GEC) is the fifth most common cancer and the second most common cause of cancer-related mortality, with 1.3 million annual deaths worldwide. The global incidence is increasing, particularly among younger patients. GEC can be classified into subtypes based on anatomic location, histology, molecular characteristics, or tumour biology and genomics. In approximately 20% of all GECs overexpression of HER2 is identified. The landscape of treatment options in this patient population is evolving rapidly. This review summarizes the progress of HER2-directed therapies for advanced disease and highlights future directions in targeting the disease. The epidermal growth factor receptor (EGFR) family of transmembrane tyrosine kinase receptors, EGFR/HER1, HER2/neu, HER3, and HER4, all have an extracellular ligand-binding domain, lipophilic transmembrane domain, and an intracellular domain with tyrosine kinase activity, binding to these receptors results in activation of downstream RAS/MAPK and PI3K/AKT pathways. In turn, this induces cell proliferation, differentiation, migration, and survival. The phase III Trastuzumab for Gastric Cancer (ToGA) trial reported the incidence of HER2-positive gastric cancer to be 22%. Therefore, targeting HER2 and its downstream signaling pathways holds important potential as a therapeutic strategy. Figure 1 illustrates potential targeting mechanisms that will be discussed in this review.

SCR-A002, a novel FGFR2b-targeting antibody-drug-conjugate for solid tumors.

e15019 Background: FGFR2b (fibroblast growth factor receptor 2 IIIb), a transmembrane tyrosine kinase, is overexpressed in various cancers, including gastric, esophageal, breast, hepatocellular, pancreatic, ovary, uterine, cervical, endometrial, lung, colorectal cancers. Bemarituzumab (FPA144), an afucosylated humanized IgG1 monoclonal antibody targeting FGFR2b with enhanced ADCC activity, showed promising clinical efficacy in phase 2 study of FGFR2b-selected gastric or gastroesophageal junction adenocarcinoma. We have developed a potential first-in-class FGFR2b-targeting ADC (SCR-A002) at IND-enabling status, displaying significantly superior anti-tumor potency compared to Bemarituzumab. Methods: SCR-A002 is comprised of a humanized anti-FGFR2b IgG1 monoclonal antibody conjugated with proprietary TOPO1 inhibitor payload (DAR 8) via a cleavable linker. Anti-FGFR2b antibody was generated by immunization of FGFR2b protein in mice followed by hybridoma technique. Binding activity and cytotoxicity were assessed in FGFR2b+ tumor cell line. And anti-tumor efficacy was evaluated in several CDX and PDX models. Results: SCR-A002 could specifically bind to FGFR2b on cancer cells and be internalized, and the TOPO1 inhibitor could be cleaved intracellularly to kill the cancer cells potently in vitro. After single-dose administration of SCR-A002, tumor regression was achieved in different FGFR2b expression level CDX and PDX models, even with big-size tumor or Bemarituzumab resistance. Conclusions: SCR-A002 showed potent anti-tumor efficacy in preclinical data, which suggest that SCR-A002 is expected to provide a new treatment option as single agent or combo with SoC for patients with FGFR2b-overexpressing solid tumor.

GASTRIC GASTROINTESTINAL STROMAL TUMOR WITH SUCCINATE DEHYDROGENASE B DEFICIENCY: CLINICAL CASE

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors found in the gastrointestinal tract, constituting a diverse group of c-Kit positive mesenchymal (stromal or connective tissue) tumors. Their development is primarily associated with mutations in the c-Kit proto-oncogene, leading to structural alterations in the type III transmembrane tyrosine kinase receptor (Kit). This receptor is crucial for regulating mitotic activity and cell differentiation. The activation of the Kit-receptor in tumor cells is identified through the expression of the immunohistochemical marker CD 117 (tyrosine kinase receptor protein), with approximately 80-90% of GISTs testing positive for CD 117. The objective of our study is to improve the diagnosis and treatment of gastrointestinal stromal tumors. The clinical case underscores the challenge of diagnosing gastrointestinal stromal tumors (GISTs) due to the absence of specific and definitive symptoms. In this instance, only radical surgical intervention facilitated an accurate diagnosis. Given the location of the tumor in the pyloric part of the gastric submucosal layer, a reliable biopsy during video esophagogastroduodenoscopy was unfeasible, as the gastric mucosa above the tumor appeared unaffected. Consequently, surgery with complete tumor excision and subsequent pathohistological and immunohistochemical analysis emerged as the primary diagnostic and therapeutic approach. These investigations confirmed the presence of a gastrointestinal stromal tumor of the stomach with succinate dehydrogenase B deficiency. In approximately 80% of GIST cases, succinate dehydrogenase deficiency plays a pivotal role. The absence of succinate dehydrogenase disrupts the Krebs cycle, leading to an accumulation of succinate, an oncometabolite that fuels carcinogenesis. Such tumors exhibit distinctive clinical characteristics, prognostic outcomes, and responsiveness to targeted therapies. Succinate dehydrogenase deficiency can arise from mutations or epigenomic alterations affecting gene expression. Notably, mutations or epigenomic disruptions in any succinate dehydrogenase subunit inevitably result in the loss of subunit B expression.

Targeting of C-ROS-1 Activity Using a Controlled Release Carrier to Treat Craniosynostosis in a Preclinical Model of Saethre-Chotzen Syndrome

Saethre-Chotzen syndrome (SCS) is one of the most prevalent craniosynostosis, caused by a loss-of-function mutation in the TWIST-1 gene, with current treatment options relying on major invasive transcranial surgery. TWIST-1 haploinsufficient osteogenic progenitor cells exhibit increased osteogenic differentiation potential due to an upregulation of the transmembrane tyrosine kinase receptor, C-ROS-1, a TWIST-1 target gene known to promote bone formation. The present study assessed the efficacy of suppressing C-ROS-1 activity using a known chemical inhibitor to C-ROS-1, crizotinib, to halt premature coronal suture fusion in a preclinical mouse model of SCS. Crizotinib (1 μM, 2 μM, or 4 μM) was administered locally over the calvaria of Twist‐1del/+ heterozygous mice prior to coronal suture fusion using either a nonresorbable collagen sponge (quick drug release) or a resorbable sodium carboxymethylcellulose microdisk (slow sustained release). Coronal suture fusion rates and bone parameters were determined by μCT imaging and histomorphometric analysis of calvaria postcoronal suture fusion. Results demonstrated a dose-dependent increase in the efficacy of crizotinib to maintain coronal suture patency, with no adverse effects to brain, kidney, liver, and spleen tissue, or blood cell parameters. Moreover, crizotinib delivered on microdisks resulted in a greater efficacy at a lower concentration to reduce bone formation at the coronal suture sites compared to sponges. However, the bone inhibitory effects were found to be diminished by over time following cessation of treatment. Our findings lay the foundation for the development of a pharmacological nonsurgical, targeted approach to temporarily maintain open coronal sutures in SCS patients. This study could potentially be used to develop similar therapeutic strategies to treat different syndromic craniosynostosis conditions caused by known genetic mutations.

Abstract 2524: Molecular and clinicopathologic landscape of HER2 positive breast cancer: A single institution study

Abstract Background: Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase that is encoded by the ERBB2 gene, located on chromosome 17. Expression levels of HER2 are a key pathogenic and treatment-associated factor in breast carcinomas, with HER2 expression reported as per the ASCO/CAP scoring guidelines. Outside the intense focus on the HER2 overexpression few studies currently exist that look at the comprehensive molecular profile of these aggressive tumors. Design: Our institutional pathology database was searched for patients with breast carcinoma diagnosed between 2009 and 2013 and with HER2 scored as 3+ based on immunohistochemistry, or HER2 2+ with confirmatory positive FISH results. Comprehensive next-generation sequencing (NGS) was performed using the Illumina TruSight Oncology 523 gene panel, and clinicopathologic data were gathered from the institutional electronic health record system. A CoMutation plot was made using the CoMut python library. Results: The mean age of the patients at time of diagnosis was 56.1±12.7 years. The tumors were overwhelmingly high-grade, with 26/30 being grade 3 and the remaining 4/30 being grade 2 and a mean size of 2.6 cm. All were histologically invasive ductal carcinoma, and 26/30 were scored as HER2 3+ on IHC while 4/30 were HER2 2+ with positive FISH. ER was positive in 30% of cases (9/30), PR was positive in 23.3% (7/30), p53 was positive by IHC in 53.3% (16/30), and the mean Ki-67 proliferative index was 47.1%. 20 of the 30 (66%) cases meeting the inclusion criteria had pathogenic mutations detected by NGS testing. Several distinct molecular subgroups were seen on the CoMutation plot (figure 1). The mean tumor mutational burden (TMB) was 14.1 mutations per megabase. A significant subset of the tumors 70% (14/20) showed TP53 mutations, and 30% (6/20) show PIK3CA mutations. Of interest, only 1/14 tumors showed a co-occurring homologous recombination repair gene mutation and one tumor (1/20) showed mutations in 2 homologous recombination pathway genes (FANCA and BRCA1) as well as numerous additional mutations (tumor mutational burden of 65.3 mutations per megabase). Conclusion: Overall the mean TMB was 14.1 mutations/megabase in this HER2-positive breast cancer study set. Over 2/3 of our HER2-positive breast cancer cases also had TP53 mutations. In addition, 1/3 had PIK3CA mutations, suggesting that a subset of HER2-positive carcinomas also harbor other potentially actionable mutations which may increase the effectiveness of our existing interventions. Additional studies are needed to further subclassify genetically distinct subsets of HER2-positive breast cancers and to further understand clinical, pathologic and treatment-related differences in this aggressive breast cancer subset. Citation Format: Gokce Deniz Ardor, Zachary M. Coty-Fattal, Luis Z. Blanco, Kalliopi Siziopikou. Molecular and clinicopathologic landscape of HER2 positive breast cancer: A single institution study [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 2524.

Abstract 3755: Impact of antigen retrieval and other parameters on detection of FGFR2b in various solid tumors on a BenchMark ULTRA automated staining platform: Development of a new IHC assay for FGFR2b

Abstract Background: Fibroblast growth factor receptor 2b (FGFR2b) is part of the FGFR transmembrane tyrosine kinase receptor family. This protein has gained more attention in recent years as a tumor progression biomarker in numerous solid tumors. The detection of FGFR2b by immunohistochemistry (IHC) has been used recently in immunotherapy clinical trials with multiple tumor types. During IHC assay development on formalin-fixed paraffin embedded tissue, a thorough study of antigen retrieval through cell conditioning or enzyme incubation, primary antibody incubation, and detection parameters is critical to evaluate staining performance. Design: A study of an investigational anti-FGFR2b (FPR2-D) assay developed on a BenchMark ULTRA automated staining platform explored the effect of antigen retrieval and detection parameters on staining performance. VENTANA System Software was used to modify steps in the IHC staining protocol resulting in 42 permutations. Ten different solid tumors were stained with these modifications to the assay and subsequently evaluated for FGFR2b expression. Result:s The results demonstrate that the staining clarity, intensity, and dynamic range within tissue cases of FGFR2b staining can vary widely between different solid tumor types. While most permutations of the initial assay parameters had minimal impact on the staining performance and readability by a pathologist, the order of the enzyme treatment and the cell conditioning steps (in addition to increased incubation time of primary antibody) had a significant impact when comparing across different solid tumors. Detection of FGFR2b seemed to have a greater susceptibility by tumor type to the antigen retrieval process than has traditionally been observed in other biomarkers. Conclusion: The overall results of this investigation show the importance of an extensive parameter screening approach when evaluating new antibody targets and indication combinations in IHC to ensure optimal staining performance. This is critical to downstream testing and the potential use of new assays in the context of in vitro diagnostics. Citation Format: Autumn Sky Watson, Catherine Le, Anne M. Wertheimer, Spencer Escobedo, Lisa So, Bhavani Bagevalu Siddegowda, John D. Palting, Susan Marion, Steven P. Stratton, Birte Aggeler. Impact of antigen retrieval and other parameters on detection of FGFR2b in various solid tumors on a BenchMark ULTRA automated staining platform: Development of a new IHC assay for FGFR2b [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 3755.

Abstract 2761: Activating RET mutations promotes osteoblastic bone metastasis by inhibiting osteoclastogenesis and stimulating osteoblast activity via regulation of osteoprotegerin

Abstract Bone metastases (BM) are a source of increased mortality and significant morbidity in patients with medullary thyroid cancer (MTC), with ~50% survival at five years post-diagnosis. MTC causes osteoblastic, osteolytic, or mixed lesions, yet the underlying mechanisms are unknown. The RET proto-oncogene encodes a transmembrane tyrosine kinase receptor and is the driver oncogene in ~60% of MTC cases where a germline or somatic activating mutation results in ligand-independent constitutive activation of the receptor. However, the role of RET mutations in the genesis of bone metastasis lesions is unknown. We show that activating RET mutations promotes an osteoblastic phenotype in mice implanted with patient-derived MTC cells by increasing bone formation and decreasing bone resorption. We found that the radiographic appearance of osteoblastic lesions differed between the types of RET mutations (C634W and M918T). MTC cells with RETC634W mutation showed a four-fold increase in bone mineral density, trabecular bone volume, number, and thickness with a decreased number of osteoblasts compared to the non-tumor-bearing femur. TRAP staining revealed that osteoclast numbers per long bone were reduced in the injected femur compared to the control. In contrast, the MTC-RETM918T mutation showed an increased cortical thickness and porosity with reduced medullary area and increased bone formation rate compared to the non-tumor bearing femur. MTC cells formed tumors with extensive mineralized tissue, composed of bone matrix surrounded by osteoblast-like cells and osteoid. The knockdown of RET in the MTC-RETM918T model significantly reduced cortical thickness and increased the number of osteoclasts per bone surface. We observed increased osteoprotegerin (OPG) expression in MTC-bearing femurs compared to controls, suggesting decreased bone resorption. The factors secreted by MTC cells, including OPG, inhibited osteoclast formation from primary bone marrow cells and Raw264 cells. In addition, RET knockdown by shRNA in MTC cells decreased OPG expression. Moreover, the MTC-conditioned media (CM) increased osteoblast differentiation and mineralization of the primary culture of pre-osteoblasts isolated from calvarial bone, long bones, and MC3T3 cells. The conditioned media of RET-depleted cells inhibits osteoblast differentiation and mineralization. The expression of alkaline phosphatase, osteocalcin, OPG, and osterix increased in pre-osteoblast incubated with MTC-CM. Compared to primary non-metastatic tumors, the OPG expression increases in tissues of sporadic MTC bone metastases harboring RETM918T mutation. These results suggest that MTC with RET-activating mutations induces osteoblastic bone metastasis due to decreased bone resorption and increased bone formation. Citation Format: Rozita Bagheri-Yarmand, Joseph L. Kidd, Gabriel M. Pagnotti, Trupti Trivedi, Leah Guerra, Xinhai Wan, Jian Song, Sue-Hwa Lin, Theresa A Guise. Activating RET mutations promotes osteoblastic bone metastasis by inhibiting osteoclastogenesis and stimulating osteoblast activity via regulation of osteoprotegerin [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 2761.

Abstract 3762: 11,000-plex aptamer based proteomics of bladder cancer urine for the discovery of novel disease biomarkers

Abstract In search of more accurate, non-invasive biomarkers of bladder cancer (BC), a comprehensive aptamer-based screen of urine proteins was conducted using urine samples from a BC cohort, followed by systems biology analyses. Following an initial 1317-plex screen, an extended 11,000 plex aptamer-based proteomic screen is in progress. Based on the initial screen, 30 urine proteins were next ELISA validated in an independent cohort of 68 subjects, and the results are shown in Table 1. Of 21 urine proteins that discriminated BC from urology clinic controls (UC), urine D-dimer displayed the highest ROC AUC value (0.96) and sensitivity (95%) (Table 1). Furthermore, 9 urine proteins significantly discriminated more advanced BC (T1-T4) from earlier stages (Ta-Tis), with IL-8 and MMP-12 being the best performers. Urine MMP12 exhibited the highest specificity of 80% at fixed sensitivity for identifying advanced BC. Systems biology analysis implicated molecular functions related to extracellular matrix, collagen, integrin, heparin and transmembrane tyrosine kinase signaling in BC susceptibility, with HNF4A and NFKB1 emerging as key molecular regulators. STEM analysis of the dysregulated pathways implicated a functional role for the immune system, complement and interleukins in BC disease progression. Urine C2, D-dimer, and Elastase were additionally ELISA validated in a second BC cohort of Chinese ethnicity. Thus, comprehensive proteomics and independent ELISA validation have uncovered novel urine biomarkers for initial BC diagnosis and subsequent disease progression, that warrant further head to head comparison with current diagnostic tests. Citation Format: Chandra Mohan, Kamala Vanarsa, Jessica Castillo, Long Wang, Yair Lotan. 11,000-plex aptamer based proteomics of bladder cancer urine for the discovery of novel disease biomarkers [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 3762.

Abstract 4365: Mechanistic effects of N361 glycosylation in epidermal growth factor receptor protein

Abstract Glycosylation is a tightly regulated post-translational modification where structured sugar groups are added to protein amino acid backbones, most commonly on asparagine for N-glycosylation. The glycosylation-regulating metabolic enzyme uridine diphosphate-glucose pyrophosphorylase 2 (UGP2) is required in oncogene-transformed cancer cells, but not in normal cells. UGP2 heavily regulates glycosylation of epidermal growth factor receptor (EGFR) at asparagine 361 (N361), a key site near the ligand binding domain. EGFR is a transmembrane tyrosine kinase receptor that promotes cell proliferation via growth cascades. Mutations and amplifications in EGFR account for a significant fraction of non-small cell lung carcinomas and breast adenocarcinoma, with L858R being the most common oncogenic hotspot. Other glycosylation sites on EGFR have been implicated in ligand binding, dimerization, and phosphorylation of downstream targets. Molecular dynamic simulations suggested that N361 may be important for dimerization or ligand binding. However, the functional relevance of how glycosylation of EGFR at N361 impacts EGFR protein and cellular behaviors remains unclear. To determine the functional relevance of glycosylation at N361, we created glycosylation-defective EGFR N361A mutant, with or without an additional oncogenic EGFR L858R mutant. We expressed these constructs in MCF10A and 293T cells, which do not have pre-existing activation of this pathway. Immunofluorescence and flow cytometry showed that the mutants were each well expressed at the cell membrane. Proximity ligation assays measuring dimerization of EGFR in cells showed that EGFR N361A greatly increased dimerization relative to wildtype controls. Loss of glycosylation of EGFR N361 impacted cell viability when grown in dose courses of its high affinity ligand EGF or lower affinity ligand amphiregulin. Furthermore, EGFR N361A desensitized cells expressing the oncogenic EGFR L858R to inhibitors targeting the extracellular domain, possibly because the mutation alters the antibody binding interface. N361A sensitized EGFR L858R to inhibitors targeting the cytoplasmic domain of EGFR. These findings help us understand the intricate relationship between EGFR and N-glycosylation, reinforcing the critical functional relevance of post-translational modifications on oncogenes. Citation Format: Dennis Lam, Ariel N. Liberchuk, Brandon Arroyo, Andrew L. Wolfe. Mechanistic effects of N361 glycosylation in epidermal growth factor receptor protein [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 4365.

Targeting epidermal growth factor receptor and its downstream signaling pathways by natural products: A mechanistic insight.

The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase (RTK) that maintains normal tissues and cell signaling pathways. EGFR is overactivated and overexpressed in many malignancies, including breast, lung, pancreatic, and kidney. Further, the EGFR gene mutations and protein overexpression activate downstream signaling pathways in cancerous cells, stimulating the growth, survival, resistance to apoptosis, and progression of tumors. Anti-EGFR therapy is the potential approach for treating malignancies and has demonstrated clinical success in treating specific cancers. The recent report suggests most of the clinically used EGFR tyrosine kinase inhibitors developed resistance to the cancer cells. This perspective provides a brief overview of EGFR and its implications in cancer. We have summarized natural products-derived anticancer compounds with the mechanistic basis of tumor inhibition via the EGFR pathway. We propose that developing natural lead molecules into new anticancer agents has a bright future after clinical investigation.

Deplete and repeat: microglial CSF1R inhibition and traumatic brain injury.

Traumatic brain injury (TBI) is a public health burden affecting millions of people. Sustained neuroinflammation after TBI is often associated with poor outcome. As a result, increased attention has been placed on the role of immune cells in post-injury recovery. Microglia are highly dynamic after TBI and play a key role in the post-injury neuroinflammatory response. Therefore, microglia represent a malleable post-injury target that could substantially influence long-term outcome after TBI. This review highlights the cell specific role of microglia in TBI pathophysiology. Microglia have been manipulated via genetic deletion, drug inhibition, and pharmacological depletion in various pre-clinical TBI models. Notably, colony stimulating factor 1 (CSF1) and its receptor (CSF1R) have gained much traction in recent years as a pharmacological target on microglia. CSF1R is a transmembrane tyrosine kinase receptor that is essential for microglia proliferation, differentiation, and survival. Small molecule inhibitors targeting CSF1R result in a swift and effective depletion of microglia in rodents. Moreover, discontinuation of the inhibitors is sufficient for microglia repopulation. Attention is placed on summarizing studies that incorporate CSF1R inhibition of microglia. Indeed, microglia depletion affects multiple aspects of TBI pathophysiology, including neuroinflammation, oxidative stress, and functional recovery with measurable influence on astrocytes, peripheral immune cells, and neurons. Taken together, the data highlight an important role for microglia in sustaining neuroinflammation and increasing risk of oxidative stress, which lends to neuronal damage and behavioral deficits chronically after TBI. Ultimately, the insights gained from CSF1R depletion of microglia are critical for understanding the temporospatial role that microglia develop in mediating TBI pathophysiology and recovery.

Critical domains for NACC2-NTRK2 fusion protein activation.

Neurotrophic receptor tyrosine kinases (NTRKs) belong to the receptor tyrosine kinase (RTK) family. NTRKs are responsible for the activation of multiple downstream signaling pathways that regulate cell growth, proliferation, differentiation, and apoptosis. NTRK-associated mutations often result in oncogenesis and lead to aberrant activation of downstream signaling pathways including MAPK, JAK/STAT, and PLCγ1. This study characterizes the NACC2-NTRK2 oncogenic fusion protein that leads to pilocytic astrocytoma and pediatric glioblastoma. This fusion joins the BTB domain (Broad-complex, Tramtrack, and Bric-a-brac) domain of NACC2 (Nucleus Accumbens-associated protein 2) with the transmembrane helix and tyrosine kinase domain of NTRK2. We focus on identifying critical domains for the biological activity of the fusion protein. Mutations were introduced in the charged pocket of the BTB domain or in the monomer core, based on a structural comparison of the NACC2 BTB domain with that of PLZF, another BTB-containing protein. Mutations were also introduced into the NTRK2-derived portion to allow comparison of two different breakpoints that have been clinically reported. We show that activation of the NTRK2 kinase domain relies on multimerization of the BTB domain in NACC2-NTRK2. Mutations which disrupt BTB-mediated multimerization significantly reduce kinase activity and downstream signaling. The ability of these mutations to abrogate biological activity suggests that BTB domain inhibition could be a potential treatment for NACC2-NTRK2-induced cancers. Removal of the transmembrane helix leads to enhanced stability of the fusion protein and increased activity of the NACC2-NTRK2 fusion, suggesting a mechanism for the oncogenicity of a distinct NACC2-NTRK2 isoform observed in pediatric glioblastoma.

SAM domain variants of EPHB4 associated with aberrant signaling are linked to lymphatic-related fetal hydrops and facial dysmorphology.

Variants in EPHB4 (Ephrin type B receptor 4), a transmembrane tyrosine kinase receptor, have been identified in individuals with various vascular anomalies including Capillary Malformation-Arteriovenous Malformation syndrome 2 and lymphatic-related (non-immune) fetal hydrops (LRHF). Here, we identify two novel variants in EPHB4 that disrupt the SAM domain in two unrelated individuals. Proband 1 presented within the LRHF phenotypic spectrum with hydrops, and proband 2 presented with large nuchal translucency prenatally that spontaneously resolved in addition to dysmorphic features on exam postnatally. These are the first disease associated variants identified that do not disrupt EPHB4 protein expression or tyrosine-kinase activity. We identify that EPHB4 SAM domain disruptions can lead to aberrant downstream signaling, with a loss of the SAM domain resulting in elevated MAPK signaling in proband 1, and a missense variant within the SAM domain resulting in increased cell proliferation in proband 2. This data highlights that a functional SAM domain is required for proper EPHB4 function and vascular development.

Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis

Helicobacter pylori (H. pylori) is the leading risk factor for gastric carcinogenesis. Fibroblast growth factor receptor 4 (FGFR4) is a member of transmembrane tyrosine kinase receptors that are activated in cancer. We investigated the role of FGFR4 in regulating the cellular response to H. pylori infection in gastric cancer. High levels of oxidative stress signature and FGFR4 expression were detected in gastric cancer samples. Gene set enrichment analysis (GSEA) demonstrated enrichment of NRF2 signature in samples with high FGFR4 levels. H. pylori infection induced reactive oxygen species (ROS) with a cellular response manifested by an increase in FGFR4 with accumulation and nuclear localization NRF2. Knocking down FGFR4 significantly reduced NRF2 protein and transcription activity levels, leading to higher levels of ROS and DNA damage following H. pylori infection. We confirmed the induction of FGFR4 and NRF2 levels using mouse models following infection with a mouse-adapted H. pyloristrain. Pharmacologic inhibition of FGFR4 using H3B-6527, or its knockdown, remarkably reduced the level of NRF2 with a reduction in the size and number of gastric cancer spheroids. Mechanistically, we detected binding between FGFR4 and P62 proteins, competing with NRF2-KEAP1 interaction, allowing NRF2 to escape KEAP1-dependent degradation with subsequent accumulation and translocation to the nucleus. These findings demonstrate a novel functional role of FGFR4 in cellular homeostasis via regulating the NRF2 levels in response to H. pylori infection in gastric carcinogenesis, calling for testing the therapeutic efficacy of FGFR4 inhibitors in gastric cancer models.