Neurotrophic Tyrosine Kinase Receptor Research Articles

Identification of potential inhibitors of tropomyosin receptor kinase B targeting CNS-related disorders and cancers

Tropomyosin receptor kinase B (TrkB), also known as neurotrophic tyrosine kinase receptor type 2 (NTRK2), is a protein that belongs to the family of receptor tyrosine kinases (RTKs). NTRK2 plays a crucial role in regulating the development and maturation of the central nervous system (CNS) and peripheral nervous system (PNS). Elevated TrkB expression levels observed in different pathological conditions make it a potential target for therapeutic interventions against neurological disorders, including depression, anxiety, Alzheimer’s disease, Parkinson’s disease, and certain types of cancer. Targeting TrkB using small molecule inhibitors is a promising strategy for the treatment of a variety of neurological disorders. In this research, a systematic virtual screening was carried out on phytoconstituents found in the IMPPAT library to identify compounds potentially inhibiting TrkB. The retrieved compounds from the IMPPAT library were first filtered using Lipinski’s rule of five. The compounds were then sorted based on their docking score and ligand efficiency. In addition, PAINS, ADMET, and PASS evaluations were carried out for selecting drug-like compounds. Finally, in interaction analysis, we found two phytoconstituents, Wedelolactone and 3,8-dihydroxy-1-methylanthraquinone-2-carboxylic acid (DMCA), which possessed considerable docking scores and specificity on the TrkB ATP-binding pocket. The selected compounds were further assessed employing molecular dynamics (MD) simulations and essential dynamics. The results revealed that the elucidated compounds bind well with the TrkB binding pocket and lead to fewer conformations fluctuations. This study highlighted using phytoconstituents, Wedelolactone and DMCA as starting leads in developing novel TrkB inhibitors. Communicated by Ramaswamy H. Sarma

NTRK gene aberrations in triple-negative breast cancer: detection challenges using IHC, FISH, RT-PCR, and NGS.

Triple-negative breast cancer (TNBC) is usually an aggressive disease with a poor prognosis and limited treatment options. The neurotrophic tyrosine receptor kinase (NTRK) gene fusions are cancer type-agnostic emerging biomarkers approved by the Food and Drug Administration (FDA), USA, for the selection of patients for targeted therapy. The main aim of our study was to investigate the frequency of NTRK aberrations, i.e. fusions, gene copy number gain, and amplification, in a series of TNBC using different methods. A total of 83 TNBCs were analyzed using pan-TRK immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (RT-PCR), and RNA-based next-generation sequencing (NGS). Of 83 cases, 16 showed pan-TRK positivity although no cases had NTRK-fusions. Indeed, FISH showed four cases carrying an atypical NTRK1 pattern consisting of one fusion signal and one/more single green signals, but all cases were negative for fusion by NGS and RT-PCR testing. In addition, FISH analysis showed six cases with NTRK1 amplification, one case with NTRK2 copy number gain, and five cases with NTRK3 copy number gain, all negative for pan-TRK IHC. Our data demonstrate that IHC has a high false-positive rate for the detection of fusions and molecular testing is mandatory; there is no need to perform additional molecular tests in cases negativity for NTRK by IHC. In conclusion, the NTRK genes are not involved in fusions in TNBC, but both copy number gain and amplification are frequent events, suggesting a possible predictive role for other NTRK aberrations.

Current perspectives on the management of patients with advanced RET-driven thyroid cancer in Europe.

The incidence of thyroid cancer is increasing worldwide with the disease burden in Europe second only to that in Asia. In the last several decades, molecular pathways central to the pathogenesis of thyroid cancer have revealed a spectrum of targetable kinases/kinase receptors and oncogenic drivers characteristic of each histologic subtype, such as differentiated thyroid cancer, including papillary, follicular, and medullary thyroid cancer. Oncogenic alterations identified include B-Raf proto-oncogene (BRAF) fusions and mutations, neurotrophic tyrosine receptor kinase (NTRK) gene fusions, and rearranged during transfection (RET) receptor tyrosine kinase fusion and mutations. Multikinase inhibitors (MKIs) targeting RET in addition to multiple other kinases, such as sorafenib, lenvatinib and cabozantinib, have shown favourable activity in advanced radioiodine-refractory differentiated thyroid cancer or RET-altered medullary thyroid cancer; however, the clinical utility of MKI RET inhibition is limited by off-target toxicity resulting in high rates of dose reduction and drug discontinuation. Newer and selective RET inhibitors, selpercatinib and pralsetinib, have demonstrated potent efficacy and favourable toxicity profiles in clinical trials in the treatment of RET-driven advanced thyroid cancer and are now a therapeutic option in some clinical settings. Importantly, the optimal benefits of available specific targeted treatments for advanced RET-driven thyroid cancer require genetic testing. Prior to the initiation of systemic therapy, and in treatment-naïve patients, RET inhibitors may be offered as first-line therapy if a RET alteration is found, supported by a multidisciplinary team approach.

A Case of Infant-type Hemispheric Glioma with NTRK1 fusion (P9-13.003)

<h3>Objective:</h3> Not applicable <h3>Background:</h3> The incidence of childhood central nervous system tumors in infants is about 6 per 100,000 children with glioma having the highest incidence at 1.38 per 100,000. A study by Wu et al. showed recurrent fusion of the <i>NTRK</i> gene in 10% of non-brainstem high grade glioma in very young children suggesting an oncogenic effect of the <i>NTRK</i> fusion genes. Here we present a rare case of a full-term neonate who was noted to have widely splayed sutures and a bulging fontanelle at birth who was found to have infant-type hemispheric glioma with <i>NTRK1</i> fusion. <h3>Design/Methods:</h3> Not applicable <h3>Results:</h3> A full-term infant with normal prenatal course, including a 20-week anatomy scan, was born with widely splayed sutures, bulging anterior fontanelle and head circumference &gt;99^th percentile (40 cm). Head ultrasound showed severe hydrocephalus and a large intracranial mixed echogenicity lesion in the left cerebral hemisphere with hemorrhage. He had sustained leftward gaze, and video EEG revealed seizures. MRI demonstrated a large 6×7cm lesion, hydrocephalus with subfalcine herniation and mass effect on the brainstem (figure, A–B). He underwent biopsy and ventriculoperitoneal shunt placement. MRI 2 weeks later showed tumor enlargement, obstructive hydrocephalus and severe brainstem compression (figure, C–D). Pathology was consistent with infant-type hemispheric glioma with <i>NTRK1</i> fusion. Due to the dismal prognosis, the family prioritized palliative care. <h3>Conclusions:</h3> Infant-type hemispheric glioma, previously termed glioblastoma (GBM), is a rare, rapidly-growing congenital tumor, and prenatal imaging is often normal. Mixed-age hemorrhages and diffusion restriction is suggestive of the diagnosis. The neurotrophic tyrosine receptor kinase (NTRK) genes <i>NTRK1, NTRK2</i>, and <i>NTRK3</i> are involved in infant-type hemispheric gliomas and are typically high grade in histology. A recent study by Torre et al. showed most <i>NTRK-</i>fused gliomas were hemispheric and had a higher prevalence in non-brainstem high grade gliomas in patients younger than 3 years old. <b>Disclosure:</b> Dr. Garcia has nothing to disclose. Dr. Bell has nothing to disclose. Dr. Nelson has nothing to disclose. Dr. Miller has nothing to disclose. Dr. Segal has nothing to disclose.

Abstract CT228: Concordance between tissue and circulating tumor DNA (ctDNA) testing for neurotrophic tyrosine receptor kinase (NTRK) gene fusions in larotrectinib (laro) clinical trials

Abstract Background: NTRK gene fusions are oncogenic drivers in various tumor types. Laro, a highly selective and central nervous system (CNS)-active tropomyosin receptor kinase (TRK) inhibitor, is approved for the treatment of patients (pts) with TRK fusion cancer. Laro demonstrated an objective response rate (ORR) of 69% in 244 pts with solid (non-primary CNS) tumors (Drilon et al. ASCO 2022). Here, we report on the concordance between baseline local tissue-based testing with central tissue-based and liquid biopsy-based ctDNA testing to identify NTRK gene fusions. Methods: Pts with non-primary CNS TRK fusion cancer who underwent independent review committee assessments by July 15, 2019, in three laro clinical trials (NCT02122913, NCT02576431, and NCT02637687) were included. Tumor NTRK gene fusion status was determined locally by a variety of methods, and centrally by next-generation sequencing in both tissue (using Illumina TruSight™ Oncology Comprehensive) and ctDNA (using Guardant360® or GuardantOMNI®) at baseline. Data cut-off: July 20, 2021. Results: Of the 164 pts with NTRK fusions identified by local testing, 117 had evaluable tissue samples for central testing, and 99 had evaluable liquid biopsy samples. In total, 81.2% (95/117) of tumors had the fusion confirmed by central tissue testing. ctDNA testing detected NTRK gene fusions in 33.3% (33/99) of patients. Positive predictive values for each NTRK gene fusion are shown in the Table. The ORR for pts with NTRK gene fusions determined by local and central testing will be presented. Conclusions: A high proportion of tumors with locally identified NTRK gene fusions were confirmed centrally. At present, analysis of ctDNA is significantly less sensitive at detecting NTRK gene fusions. A negative ctDNA result requires next-generation sequencing testing of a tissue biopsy. Further research is needed to improve the sensitivity of ctDNA gene fusion detection. Table NTRK gene fusion Tissue-based LT Tissue-based CT ctDNA-based CT with LB Fusion confirmed, n Fusion confirmed, n Fusion not detected, n Unavailable samples, n† PPV‡: CT vs tissue-based LT, % Fusion confirmed, n Fusion not detected, n Unavailable samples, n† PPV‡: LB vs tissue-based LT, % NTRK1 68 35 14 19 71.4 20 34 14 37.0 NTRK2 4 3 1 0 75.0 0 4 0 0 NTRK3 92§ 57 7 28 89.1 13 28 51 31.7 †Includes no sample available and sample QC failed. ‡Calculated as a percentage of the number of confirmed fusions over the number of available samples. §Includes 9 inferred NTRK3 fusions. CT, central testing; ctDNA, circulating tumor DNA; LB, liquid biopsy; LT, local testing; NTRK, neurotrophic tyrosine receptor kinase; PPV, positive predictive value; QC, quality control. Citation Format: Erin R. Rudzinski, Marc Ladanyi, Lauren L. Ritterhouse, Felix Sahm, Ricarda Norenberg, Hong Zheng, Chi Chen, Vadim Bernard-Gauthier, Nicoletta Brega, Cornelis M. van Tilburg, Jessica J. Lin, Marcia S. Brose, Ulrik N. Lassen, Ray McDermott, Theodore W. Laetsch, David S. Hong, Alexander Drilon, Sinchita Roy-Chowdhuri. Concordance between tissue and circulating tumor DNA (ctDNA) testing for neurotrophic tyrosine receptor kinase (NTRK) gene fusions in larotrectinib (laro) clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT228.

Abstract 925: NTRK gene fusion in adults and pediatrics with solid tumors: a record linkage study of expression frequency and patient characteristics using the Auria Biobank in Finland

Abstract Introduction: Selective tropomyosin receptor kinase (TRK) inhibitors are targeted therapies for patients with solid tumors harboring a neurotrophic-receptor tyrosine kinase (NTRK) gene fusion. Estimates of the frequency of NTRK gene fusions and knowledge about characteristics of affected patients from population-based settings are limited. Methods: This was a retrospective cohort study using archival tissue samples from patients with histologically-confirmed solid tumors identified from the Auria Biobank in the Turku region of Finland (January 2005-December 2019). We evaluated all solid tumor types in pediatrics, and selected tumor types in adults (colorectal cancer [CRC], non-small cell-lung cancer [NSCLC], sarcoma, and salivary/parotid cancer). Expression of TRK protein was determined by pan-TRK immunohistochemistry (IHC) staining of tumor samples scored by a certified pathologist. NTRK gene fusion including fusion partners was then confirmed by next generation sequencing (NGS). Patients’ demographics, clinical characteristics, and survival status were determined through linkage to hospital electronic health records and the national vital statistics database; patients were followed from 1 year before cancer diagnosis until the end of follow-up/death. Results: NTRK gene fusion frequency and descriptions of patient and tumor characteristics are shown in the Table. The number of patients confirmed as NTRK positive after NGS as a proportion of those deemed NTRK positive after IHC staining was 80% (8/10) for CRC, 5% (1/21) for NSCLC, 6% (1/18) for salivary/parotid cancer, 0% (0/21) for sarcoma, and 25% (4/16) for pediatric solid tumors. Conclusions: This study shows that NTRK gene fusions are rare in adult CRC, NSCLC, parotid/salivary tumors, sarcoma, and pediatric solid tumors. Frequency of NTRK gene fusion in pediatric and select adult solid tumors, and characteristics of NTR NTRK gene fusion-positive NTRK gene fusion subtypes Fusion partners Median age (category) at diagnosis (years) Sex Tumor details Treatment/follow-up status Adults CRC (N=1151) 8 (0.7%) NTRK1 (n=6) NTRK2 (n=1) NTRK3 (n=1) NTRK1: TPM3 (n=3), LMNA (n=1), TPR (n=1), IRF2BP2 (n=1) NTRK2*: FXN (n=1), LPPR1 (n=1) NTRK3: GABRG1 (n=1) *In a single patient with two tumor samples NTRK2 was identified in both samples, but the fusion partner (and other genomic co-alterations) were different between the two samples. 60-69 3 male, 5 female All were grade 2-3 ECOG score 1 (n=4)† MSI: abnormal (n=4), normal (n=1), missing (n=1) †ECOG score was missing for the remaining patients. Chemotherapy: n=3 Radiotherapy: n=1 All alive at end of follow-up (range, 0.3-11.5 years) Sarcoma (N=379) 0 (0.0%) - - - - - - NSCLC (N=288) 1 (0.3%) NTRK3 (n=1) SGCZ (n=1) 60-69 Female Stage IVA, grade II, ECOG score 1 Chemotherapy but not radiotherapy Alive at 0.6 years‘ follow-up Salivary/parotid (N=114) 1 (0.9%) NTRK3 (n=1) ETV6 (n=1) 60-69 Male Salivary cancer, stage 1, T2N0M0 ECOG score was missing No chemotherapy/radiotherapy Alive after 13.9 years‘ follow-up Fibrosarcoma (n=1) Primitive neuroectodermal cancer (n=1) Ewing’s sarcoma (n=1) Papillary thyroid cancer (n=1) Pediatric solid tumors (N=127) 4 (3.1%) NTRK1 (n=1) NTRK2 (n=2) NTRK3 (n=1) NTRK1: TPM3 (n=1) NTRK2: DAB2IP (n=1), LPPR1 (n=1) NTRK3: RAD51B (n=1) NTRK1-TPM3; male, age category‡ 2-11 years, tumor stage unknown, no chemotherapy/radiotherapy. Alive at 15.1 years‘ follow-up. ‡According to US FDA age categories for pediatrics. NTRK2-DAB2IP; male, age category‡ 2-11 years, Grade III tumor, received both chemo- and radiotherapy. Died - survival time 0.9 years. ‡According to US FDA age categories for pediatrics. NTRK2-LPPR1; male, age category‡ 12-18 years, stage IV tumor, received both chemo- and radiotherapy. Died - survival time 1.1 years. ‡According to US FDA age categories for pediatrics. NTRK3-RAD51B; female, age category‡ 12-18 years, tumor stage unknown, no chemotherapy/radiotherapy. Alive at 7.4 years‘ follow-up. ‡According to US FDA age categories for pediatrics. Citation Format: Wei Zhang, Merja Perälä, Roosa E. Kallionpää, Niina Pitkänen, Korinna Jöhrens, Renate Schulze-Rath, Arndt A Schmitz, Helen Guo, Jihong Zong. NTRK gene fusion in adults and pediatrics with solid tumors: a record linkage study of expression frequency and patient characteristics using the Auria Biobank in Finland [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 925.

In vitro and in vivo exploration of the anti-atopic dermatitis mechanism of action of Tibetan medicine Qi-Sai-Er-Sang-Dang-Song decoction.

Tibetan medicine Qi-Sai-Er-Sang-Dang-Song Decoction(QSD, ཆུ་སེར་སེང་ལྡེང་སུམ་ཐང་།)is a traditional Tibetan medical formulation with demonstrated clinical benefits in atopic dermatitis (AD). However, its potential mechanism and molecular targets remain to be elucidated. This study aims to explore the activity and mechanism of QSD on AD in multiple dimensions by combining in vitro and in vivo experiments with network pharmacology. The AD effect of QSD was investigated by evaluating the levels of nitric oxide (NO) and interleukin-6 (IL-6) in the lipopolysaccharide (LPS) stimulated RAW264.7cells. AD-like skin lesions in female BALB/c mice were induced by 2,4-dinitrochlorobenzene (DNCB). QSD or dexamethasone (positive control) were gavagely administered daily for 15 consecutive days. The body weight and skin lesion severity were recorded throughout the study. Enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis were used to illuminate the molecular targets associated with the anti-AD effects of QSD. Meanwhile, the ingredients of QSD in the blood were revealed and analyzed by Ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) method. Network pharmacology was used to predict the targets and mechanism of active ingredient therapy for AD. In addition, the network pharmacology outcomes were further verified by molecular docking. After treatment with QSD, the levels of NO and IL-6 were decreased in the cell supernatant. Herein, QSD markedly decreased the eosinophil and mast cells infiltration in the dorsal skin of the 2,4-dinitrochlorobenzene. Moreover, QSD reconstructed the epidermal barrier by increasing the content of collagen fibers and changing the arrangement of DNCB-treated mice. QSD not only inhibited the levels of tumor necrosis factor-α (TNF-α) and interleukin-12 (IL-12) but also inhibited phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) proteins in the dorsal skin. Four active ingredients were identified through UPLC-Q-TOF/MS, including (-)-epicatechin, kaempferol-7-O-glucoside, cassiaside, and questin. After the network pharmacological analysis, six core targets of QSD closely related to AD were obtained, including TNF-α, IL-6, Caspase-3 (CASP3), Epidermal growth factor (EGFR), Peroxisome proliferator-activated receptor gamma (PPARG), and Neurotrophic Receptor Tyrosine Kinase 1 (NTRK1). Meanwhile, through Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, the Mitogen-activated protein kinase (MAPK) signaling pathway occupies an important position in the QSD treatment of AD. The molecular docking results showed that the six core targets are stable in binding to the four active ingredients as indicated by the molecular docking results. The anti-AD effect of QSD might be related to the reconstruction of the epidermal barrier and inhibition of inflammation, which regulated the MAPK pathway. Hence, it provided a promising idea for the study of Tibetan medicine prescriptions for the treatment of AD.

Unraveling the molecular mechanism of novel leukemia mutations on NTRK2 (A203T &amp; R458G) and NTRK3 (E176D &amp; L449F) genes using molecular dynamics simulations approach

Background: NTRK1, NTRK2, and NTRK3 are members of the neurotrophic receptor tyrosine kinases (NTRK) family, which encode TrkA, TrkB, and TrkC receptors, respectively. Hematologic cancers are also linked to point mutations in the NTRK gene's kinase domain. Trk fusions are the most common genetic change associated with oncogenic activity in Trk-driven liquid tumors. Thus, point mutations in NTRK genes may also play a role in tumorigenesis. The structural and functional effect of mutations in Trk-B &amp; Trk-C proteins remains unclear. Methods: In this research, Homology (threading-based approach) modeling and the all-atom molecular dynamics simulations approaches are applied to examine the structural and functional behavior of native and mutant Trk-B and Trk-C proteins at the molecular level. Results: The result of this study reveals how the mutations in Trk-B (A203T &amp; R458G) and Trk-C (E176D &amp; L449F) proteins lost their stability and native conformations. The Trk-B mutant A203T became more flexible than the native protein, whereas the R458G mutation became more rigid than the native conformation of the Trk-B protein. Also, the Trk-C mutations (E176D &amp; L449F) become more rigid compared to the native structure. Conclusions: This structural transition may interrupt the function of Trk-B and Trk-C proteins. Observing the impact of NTRK-2/3 gene alterations at the atomic level could aid in discovering a viable treatment for Trk-related leukemias.

Real-world Experience With Neurotrophic Tyrosine Receptor Kinase Fusion-positive Tumors and Tropomyosin Receptor Kinase Inhibitors in Veterans.

Neurotrophic tyrosine receptor kinase 1-3 (NTRK1-3) gene fusions are found in a broad range of tumor types. Clinical trials demonstrated high response rates to tropomyosin receptor kinase (TRK) inhibitors in NTRK fusion-positive cancers, but few reports have described real-world experience with these targeted agents. We evaluated the prevalence of NTRK fusions and the outcomes with TRK inhibitor therapy in a real-world population of patients in the Veterans Health Administration. Patients with NTRK fusions or rearrangements were identified from the Veterans Affairs (VA) National Precision Oncology Program (NPOP), and patients who were prescribed TRK inhibitors were identified from the Corporate Data Warehouse. Baseline data and clinical outcomes were obtained by retrospective review of medical records. A total of 33 patients with NTRK fusions or rearrangements were identified, including 25 patients comprising 0.12% of all patients with solid tumors sequenced through VA NPOP. Twelve patients with NTRK fusions or rearrangements were treated with TRK inhibitors, none of whom had objective responses. Eight patients experienced toxicities leading to drug interruption, dose reduction, or discontinuation. In this retrospective study of VA patients, NTRK fusions and rearrangements were less common than in previous studies, and objective responses to TRK inhibitors were not observed. Real-world experience with TRK inhibitors differs markedly from clinical trial findings, possibly due to differences in patient demographics, tumor types, and sequencing methods. Our findings highlight the need to study TRK inhibitors in the real-world setting and in populations underrepresented in clinical trials.

Exploring the binding characteristics of bovine serum albumin with tyrosine kinase inhibitor entrectinib: Multi-spectral analysis and theoretical calculation

Entrectinib (ENB) is one of multi-target tyrosine kinase inhibitors, which is mainly used for treating neurotrophic tyrosine receptor kinase gene fusion positive solid tumors. The binding characteristics of ENB and bovine serum albumin (BSA) were studied by experiments and theoretical calculations. The steady-state fluorescence showed that ENB quenched the fluorescence of BSA through mixed quenching, and ENB was dominated by static quenching at low concentration. ENB and BSA had a moderate affinity, formed a complex with a stoichiometric ratio of 1:1 and the binding constant of about 105 M−1 at 298 K, and Förster non-radiative energy transfer occurs. According to the driving force competition experiment, thermodynamic parameter analysis and theoretical calculation, hydrogen bond, van der Waals force and hydrophobic force were the main factors affecting the stability of the ENB-BSA complex. Molecular docking and site markers competition showed that ENB spontaneously bound to the Site III of BSA so that ENB could make the skeleton of BSA loose, the spatial structure of BSA changed (α-helix decreased by 3.1%, random coil increased by 1.7%), and the microenvironment of Tyr and Trp residues changed. The existence of Co2+ metal ions can enhance the binding effect, thus prolonging the half-life of ENB in vivo, which may improve the efficacy of ENB, while Ca2+, Cu2+ and Mg2+ metal ions will reduce the efficacy of ENB.

Toll-like receptor 4 signaling in osteoblasts is required for load-induced bone formation in mice

In mature bone, NGF is produced by osteoblasts following mechanical loading and signals through resident sensory nerves expressing its high affinity receptor, neurotrophic tyrosine kinase receptor type 1 (TrkA), to support bone formation. Here, we investigated whether osteoblastic expression of Toll-like receptor 4 (TLR4), a key receptor in the NF-κB signaling pathway, is required to initiate NGF-TrkA signaling required for load-induced bone formation. Although Tlr4 conditional knockout mice have normal skeletal mass and strength in adulthood, the loss of TLR4 signaling significantly reduced lamellar bone formation following loading. Inhibition of TLR4 signaling reduced Ngf expression in primary osteoblasts and RNA sequencing of bones from Tlr4 conditional knockout mice and wild-type littermates revealed dysregulated inflammatory signaling three days after osteogenic mechanical loading. In total, our study reveals an important role for osteoblastic TLR4 in the skeletal adaptation of bone to mechanical forces.

Generating in vitro models of NTRK-fusion mesenchymal neoplasia as tools for investigating kinase oncogenic activation and response to targeted therapy

The discovery of neurotrophic tyrosine receptor kinase (NTRK) gene fusions as pan-tumor oncogenic drivers has led to new personalized therapies in oncology. Recent studies investigating NTRK fusions among mesenchymal neoplasms have identified several emerging soft tissue tumor entities displaying various phenotypes and clinical behaviors. Among them, tumors resembling lipofibromatosis or malignant peripheral nerve sheath tumors often harbor intra-chromosomal NTRK1 rearrangements, while most infantile fibrosarcomas are characterized by canonical ETV6::NTRK3 fusions. However, appropriate cellular models to investigate mechanisms of how kinase oncogenic activation through gene fusions drives such a wide spectrum of morphology and malignancy are lacking. Progress in genome editing has facilitated the efficient generation of chromosomal translocations in isogenic cell lines. In this study we employ various strategies to model NTRK fusions, including LMNA::NTRK1 (interstitial deletion) and ETV6::NTRK3 (reciprocal translocation) in human embryonic stem (hES) cells and mesenchymal progenitors (hES-MP). Here, we undertake various methods to model non-reciprocal, intrachromosomal deletions/translocations by induction of DNA double strand breaks (DSBs) exploiting either the repair mechanisms of homology directed repair (HDR) or non-homologous end joining (NHEJ). Expression of LMNA::NTRK1 or ETV6::NTRK3 fusions in either hES cells or hES-MP did not affect cell proliferation. However, the level of mRNA expression of the fusion transcripts was significantly upregulated in hES-MP, and phosphorylation of the LMNA::NTRK1 fusion oncoprotein was noted only in hES-MP but not in hES cells. Similarly, an NTRK1-driven transcriptional profile related to neuronal and neuroectodermal lineage was upregulated mainly in hES-MP, supporting the importance of appropriate cellular context in modeling cancer relevant aberrations. As proof of concept of the validity of our in vitro models, phosphorylation was depleted by two TRK inhibitors, Entrectinib and Larotrectinib, currently used as targeted therapy for tumors with NTRK fusions.

The role of claudin 18.2 and HER-2 in pancreatic cancer outcomes.

To examine the prognostic value of claudin 18.2 (CLDN18.2) and human epidermal growth factor receptor-2 (HER-2) expression in patients with resected pancreatic ductal adenocarcinoma (PDAC). This study enrolled patients who underwent surgery and were diagnosed with PDAC at Suleyman Demirel University Hospital, Turkey between 2015 and 2019. Sixty-eight patients with resected PDAC treated at a medical oncology clinic were assessed. All patients were over the age of 18 years, underwent follow-up and treatment in our unit, and had pathology slides that we could access. Clinicopathological data were obtained from medical files, including the patients' age, sex, pathological parameters, and clinical stage according to the Eighth International Union against Cancer/American Joint Committee on Cancer. Patient survival and the period from the date of diagnosis to death were assessed in the follow-up data. There was no statistically significant difference between CLDN18.2 and HER-2 expression scores for samples and patient clinicopathological characteristics. No HER-2 expression scores of ≥2 were found in the samples. Only 25% (n = 17) of the samples had HER-2 expression scores of +1. CLDN18.2 expression was detected in 54.4% (n = 37) of the patient samples. CLDN18.2 expression scores were +1 in 30.8% (n = 21) of the patient samples, +2 in 16.2% (n = 11), and +3 in 7.4% (n = 5). When CLDN18.2 and HER-2 expression were compared, a statistically significant difference and moderate positive correlation were observed. No significant relationship between HER-2 expression and survival was observed in the survival analysis of PDAC patients; however, high CLDN18.2 expression was related to longer overall survival. Our study is the third to research CLDN18.2 expression in PDAC. HER-2 expression is low and CLDN18.2 expression is high in patients with PDAC. HER-2 expression is not related to overall survival but CLDN18.2 is related and may be used as a prognostic marker in patients with PDAC.

Entrectinib (Rozlytrek)

CADTH reimbursement reviews are comprehensive assessments of the clinical effectiveness and cost-effectiveness, as well as patient and clinician perspectives, of a drug or drug class. The assessments inform non-binding recommendations that help guide the reimbursement decisions of Canada's federal, provincial, and territorial governments, with the exception of Quebec. This review assesses Entrectinib (Rozlytrek) capsules, 100 mg and 200 mg, oral. Indication: For the treatment of adult patients who have unresectable, locally advanced or metastatic extracranial solid tumours (including brain metastases), that have a neurotrophic tyrosine receptor kinase (NTRK) gene fusion without a known acquired resistance mutation and no satisfactory treatment options.

Abstract TP227: Identification Of Somatostatin (sst) Interneuron-specific Micrornas And Transcriptome Associated With Cerebral Ischemia

Background: Somatostatin (SST) lineage neurons, the second largest family of GABAergic interneurons in the neocortex, play a critical role in cortical activity and neuroplasticity after stroke. microRNAs (miRNAs) play an important role in mediating ischemic brain repair. Elucidating changes of miRNAs in SST neurons after stroke may provide molecular insight into SST neuron function. Methods: To systematically determine miRNA expression in SST neurons, we employed a miRNA tagging and affinity-purification (miRAP) method to select specific SST neurons via the Cre-loxp binary system in male SST-tAgo2 (tAgo2: a GFP-MYC-Ago2 fusion protein) mice subjected to 7 day middle cerebral artery occlusion (MCAO) (N=3/group). Results: Using miRNA sequencing, we found a total of 523 known and novel miRNAs with distinct profiles that were expressed in SST neurons isolated from ischemic hemispheres compared to miRNAs from sham operated mice. Among them, 70 and 7 miRNAs showed significantly increased and decreased expression, respectively, with a fold change above 2.0 and over 100 copy numbers. Top 3 upregulated miRNAs included miR-381-3p, miR-543-3p and miR-433-3p, while the top 3 downregulated ones were miR-9-5p, miR-423-5p and miR-486b-5p. Furthermore, using miRAP method in combination with RNA-sequencing, we uncovered over 1000 differential gene expression with at least 1.5-fold enrichment in ischemic SST neurons. Integrative analysis of miR-mRNA network by IPA algorithm revealed that 23 miRNAs were predicted to target deregulated mRNAs. Enriched pathways targeted by miRNAs were involved in NTRK3 (neurotrophic receptor tyrosine kinase 3), insulin, PI3K, EGFR1, mTOR signaling, etc, which are associated with neurodevelopment, neuronal growth and survival. These data highlight the importance of these gene regulatory mechanisms to SST neuron function after stroke. Summary/Conclusion: Taken together, our study provides comprehensive miRNA and transcriptional signatures for SST neurons after stroke. These data will facilitate a deeper understanding of the neuroplasticity after brain injury, potentially leading to new therapeutic targets against ischemic stroke. Funding: NIDDK Diacomp 21AU4165 and AHA/ASA 18IPA34170331 (X.S.L).

Design of novel anti-cancer drugs targeting TRKs inhibitors based 3D QSAR, molecular docking and molecular dynamics simulation

Tropomyosin receptor kinase (TRK) enzymes are responsible for different types of tumors caused by neurotrophic tyrosine receptor kinase gene fusion and have been identified as an effective target for anticancer therapy. The study of the mechanism between polo-like kinase (PLKs) and pyrazol inhibitors was performed using 3D-QSAR modeling, molecular docking, and MD simulations in order to design high-activity inhibitors. The HQSAR (Q 2 = 0.793, R 2 = 0.917, R 2 ext = 0.961), CoMFA (Q 2 = 0.582, R 2 = 0.722, R 2 ext = 0.951), CoMSIA/SE (Q 2 = 0.603, R 2 = 0.801, R 2 ext = 0.849), and Topomer CoMFA (Q 2 = 0.726, R 2 = 0.992, R 2 ext = 0.717) showed good reliability and predictability. All models have been successfully tested by external validation, so all five established models are reliable. The analysis of the different contour maps of different models gives structural information to improve the inhibitory function. Molecular docking results show that the amino acids Met 592, GLU 590, LEU 657, VAL 524, and PHE 589 are the active sites of the tropomyosin receptor TRKs. The results obtained by MD showed that compound 19i could form a more stable complex protein (PDB id: 5KVT). Based on these results, we developed new compounds and their expected inhibitory activities. The results of physicochemical and ADME-Tox properties showed that the four proposed molecules are orally bioavailable, and they are not toxic in the Ames test. Thus, these results would provide modeling information that could help experimental researchers find TRK type I inhibitors more efficiently. Communicated by Ramaswamy H. Sarma

Targets of total glucosides of paeony in the treatment of Sjogren syndrome: A network pharmacology study.

We aimed to explore the underlying mechanism of the total glucoside of peony (TGP) in treating Sjogren syndrome (SS) using the network pharmacology approach. The protein targets of TGP and SS were identified by database search. Then, the intersection of the two groups was studied. The drug-target network between TGP and the overlapping genes was constructed, visualized, and analyzed by Cytoscape software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment were performed to analyze these genes. Finally, the predictions of potential targets were evaluated by docking study. Forty-six overlapping genes were discovered. The results suggested that TGP used in the treatment of SS is associated with cellular tumor antigen p53, neurotrophic tyrosine kinase receptor type 1, and epidermal growth factor receptor, as well as their related 3372 protein networks, which regulate intrinsic apoptotic signaling pathway, cellular response to oxidative stress, rhythmic process, and other processes. Molecular docking analysis proved that hydrogen bonding is the main form of interaction. Our research provided the protein targets affected by TGP in SS treatment. The key targets (caspase 3, vascular endothelial growth factor A, glyceraldehyde-3-phosphate dehydrogenase, etc.), which involve 3372 proteins, are the multitarget mechanism of TGP in SS treatment.

Insights into the medical management of gastrointestinal stromal tumours: lessons learnt from a dedicated gastrointestinal stromal tumour clinic in North India.

The advent of molecular driver alterations has brought in a revolutionary transformation in the treatment landscape of gastrointestinal stromal tumour (GIST). However, there is a paucity of data regarding mutational testing prevalence and associated outcomes from India. It was a retrospective study. We reviewed the case records of all patients diagnosed with GIST in a tertiary care centre from 2015 to 2021. The clinicopathological, mutational analysis and treatment plans were recorded. The study cohort was characterised by descriptive statistics. Our study included 120 patients with a median age of 53 years (range: 28-77), with a male preponderance of 2:1. The most common site of the primary was the stomach (50%), followed by the small intestine (37%), with 55.8% of the patients having disseminated disease at presentation with a predominance of liver metastasis (67%). The prevalence of mutational analysis among patients prior to referral was 4%. 60.8% of the patients at our clinic had mutational analysis performed, and unavailability of analysis in the rest was due to financial constraints (12.5%), exhaustion of tissue (7.5%), reluctance to repeat biopsy (4.1%) and low-risk patients. We report c-kit in the majority (52%), platelet-derived growth factor receptor (PDGFR) in 19.2% and wild type in 16.4% along with the rarer subtypes: succinate dehydrogenase (SDH)-deficient GIST in 10.9% and Neurotrophic tyrosine receptor kinase (NTRK) fusion in 1.3%. Four of the eight SDH-deficient GIST patients had germline mutations (50%). The knowledge of driver mutations led to a change of treatment in 39.7% (29/73), i.e. stoppage of tyrosine kinase inhibitor (TKI) in 3, switch of TKI in 23, increase in TKI dose in 2 and upfront surgery in 1. The most common change was the use of sunitinib and regorafenib in patients with SDH-deficient GIST. Our study is one of the largest comprehensive series describing the clinical and mutational profile of GIST from India. The mutation testing rates at primary care centres continue to be low. Despite the hurdles, a large percentage of our patients underwent molecular testing, aiding in therapeutic decision-making.

EANO guideline on rational molecular testing of gliomas, glioneuronal, and neuronal tumors in adults for targeted therapy selection.

The mainstay of treatment for adult patients with gliomas, glioneuronal and neuronal tumors consists of combinations of surgery, radiotherapy, and chemotherapy. For many systemic cancers, targeted treatments are a part of the standard of care, however, the predictive significance of most of these targets in central nervous system (CNS) tumors remains less well-studied. Despite that, there is increasing use of advanced molecular diagnostics that identify potential targets, and tumor-agnostic regulatory approvals on targets also present in CNS tumors have been granted. This raises the question of when and for which targets it is meaningful to test in adult patients with CNS tumors. This evidence-based guideline reviews the evidence available for targeted treatment for alterations in the RAS/MAPK pathway (BRAF, NF1), in growth factor receptors (EGFR, ALK, fibroblast growth factor receptor (FGFR), neurotrophic tyrosine receptor kinase (NTRK), platelet-derived growth factor receptor alpha, and ROS1), in cell cycle signaling (CDK4/6, MDM2/4, and TSC1/2) and altered genomic stability (mismatch repair, POLE, high tumor mutational burden (TMB), homologous recombination deficiency) in adult patients with gliomas, glioneuronal and neuronal tumors. At present, targeted treatment for BRAF p.V600E alterations is to be considered part of the standard of care for patients with recurrent gliomas, pending regulatory approval. For approved tumor agnostic treatments for NTRK fusions and high TMB, the evidence for efficacy in adult patients with CNS tumors is very limited, and treatment should preferably be given within prospective clinical registries and trials. For targeted treatment of CNS tumors with FGFR fusions or mutations, clinical trials are ongoing to confirm modest activity so far observed in basket trials. For all other reviewed targets, evidence of benefit in CNS tumors is currently lacking, and testing/treatment should be in the context of available clinical trials.

Frequent CD30 Expression in an Emerging Group of Mesenchymal Tumors With NTRK, BRAF, RAF1, or RET Fusions

Neurotrophic tyrosine receptor kinase (NTRK) fusions define infantile fibrosarcomas in young children and NTRK-rearranged spindle-cell tumors in older children and adults, which share characteristic spindle-cell histology and CD34 or S100 protein expression. Similar phenotypes were identified in tumors with BRAF, RAF1, or RET fusions, suggesting a unifying concept of “spindle-cell tumors with kinase gene fusions.” In this study, we investigated CD30 expression in 38 mesenchymal tumors with kinase gene fusions using immunohistochemistry. CD30 was expressed in 15 of 22 NTRK-rearranged tumors and 12 of 16 tumors with BRAF, RAF1, or RET fusions. In total, CD30 was expressed in 27 of the 38 tumors (71%), with >50% CD30-positive cells in 21 tumors and predominantly moderate or strong staining in 24 tumors. CD34 and S100 protein were also expressed in 71% and 69% of the tumors, respectively. In contrast, CD30 was significantly less frequently expressed in other mesenchymal tumor types that histologically mimic kinase fusion–positive tumors (9 of 150 tumors, 6%), of which none showed >50% or predominantly strong staining. Among these mimicking tumors, malignant peripheral nerve sheath tumors occasionally (30%) expressed CD30, albeit in a weak focal manner in most positive cases. CD30 was also expressed in 3 of 15 separately analyzed ALK- or ROS1-positive inflammatory myofibroblastic tumors. Frequent expression of CD30 enhances the shared phenotype of spindle-cell tumors with NTRK and other kinase gene fusions, and its sensitivity seems similar to that of CD34 and S100 protein. Although moderate sensitivity hampers its use as a screening tool, CD30 expression could be valuable to rapidly identify high-yield candidates for molecular workup, particularly in communities that lack routine genetic analysis and/or for tumors with BRAF, RAF1, or RET fusions.