RET Protein Research Articles

Variability in proliferative and migratory defects in Hirschsprung disease-associated RET pathogenic variants.

Despite the extensive genetic heterogeneity of Hirschsprung disease (HSCR; congenital colonic aganglionosis) 72% of patients harbor pathogenic variants in 10 genes that form a gene regulatory network (GRN) controlling the development of the enteric nervous system (ENS). Among these genes, the receptor tyrosine kinase gene RET is the most significant contributor, accounting for pathogenic variants in 12%-50% of patients depending on phenotype. RET plays a critical role in the proliferation and migration of ENS precursors, and defects in these processes lead to HSCR. However, despite the gene's importance in HSCR, the functional consequences of RET pathogenic variants and their mechanism of disease remain poorly understood. To address this, we investigated the proliferative and migratory phenotypes in a RET-dependent neural crest-derived cell line harboring one of five missense (L56M, E178Q, Y791F, S922Y, F998L) or three nonsense (Y204X, R770X, Y981X) pathogenic heterozygous variants. Using a combination of cDNA-based and CRISPR-based PRIME editing coupled with quantitative proliferation and migration assays, we detected significant losses in cell proliferation and migration in three missense (E178Q, S922Y, F998L) and all nonsense variants. Our data suggests that the Y791F variant, whose pathogenicity has been debated, is likely not pathogenic. Importantly, the severity of migration loss did not consistently correlate with proliferation defects, and the phenotypic severity of nonsense variants was independent of their position within the RET protein. This study highlights the necessity and feasibility of targeted functional assays to accurately assess the pathogenicity of HSCR-associated variants, rather than relying solely on machine learning predictions, which could themselves be refined by incorporating such functional data.

Abstract 6996: A role for the cell adhesion molecule NCAM1 in regulating activity of the receptor tyrosine kinase RET in cancer

Abstract RET (REarranged during Transfection) is a receptor tyrosine kinase with essential roles in early development; however, genetic alterations lead to aberrant RET activity that contributes to oncogenic processes in several cancers. While some of the mechanisms by which RET mediates these processes are known, the network of proteins facilitating its activity remains largely undiscovered. To address this, we performed a genome-wide synthetic dosage lethal screen which identified NCAM1 (Neural Cell Adhesion Molecule 1) as one potential candidate modulating RET activity. Using an NCAM1 knockdown (KD) in SH-SY5Y neuroblastoma cells, we show that loss of NCAM1 reduces total protein levels of RET with no effect on RET transcription. Despite reduced abundance of RET, we show that the mature form of RET has a prolonged half-life in the absence of NCAM1. Assessing the functional outcomes of reduced RET protein levels, we observe reduced activation of RET by its extracellular ligand GDNF (Glial cell line-derived neurotrophic factor) and a subsequent reduction in activation of proliferative signaling pathways downstream of RET. As a result, RET-mediated proliferation is diminished in the absence of NCAM1, with reduced viability of NCAM1 KD SH-SY5Y observed in response to GDNF. Together, our data suggest that the synthetic lethal relationship between RET and NCAM1 may arise from a role for NCAM1 in regulating total RET protein available for activation, thereby reducing RET-mediated cell viability and proliferation. Our work has uncovered a role for NCAM1 in modulating RET activity in cancer and validates the use of our synthetic dosage lethal approach in uncovering proteins facilitating oncogenic RET activity. Citation Format: Montdher H. Hussain, Brandy D. Hyndman, Frederick S. Vizeacoumar, Mathieu J. Crupi, Franco J. Vizeacoumar, Lois M. Mulligan. A role for the cell adhesion molecule NCAM1 in regulating activity of the receptor tyrosine kinase RET in cancer [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 6996.

Adaptive Darwinian off-target resistance mechanisms to selective RET inhibition in RET driven cancer

Patients treated with RET protein tyrosine kinase inhibitors (TKIs) selpercatinib or pralsetinib develop RET TKI resistance by secondary RET mutations or alterative oncogenes, of which alterative oncogenes pose a greater challenge for disease management because of multiple potential mechanisms and the unclear tolerability of drug combinations. A patient with metastatic medullary thyroid carcinoma (MTC) harboring a RET activation loop D898_E901del mutation was treated with selpercatinib. Molecular alterations were monitored with tissue biopsies and cfDNA during the treatment. The selpercatinib-responsive MTC progressed with an acquired ETV6::NTRK3 fusion, which was controlled by selpercatinib plus the NTRK inhibitor larotrectinib. Subsequently, tumor progressed with an acquired EML4::ALK fusion. Combination of selpercatinib with the dual NTRK/ALK inhibitor entrectinib reduced the tumor burden, which was followed by appearance of NTRK3 solvent-front G623R mutation. Preclinical experiments validated selpercatinib plus larotrectinib or entrectinib inhibited RET/NTRK3 dependent cells, whereas selpercatinib plus entrectinib was necessary to inhibit cells with RET/NTRK3/ALK triple alterations or a mixture of cell population carrying these genetic alterations. Thus, RET-altered MTC adapted to selpercatinib and larotrectinib with acquisition of ETV6::NTRK3 and EML4::ALK oncogenes can be managed by combination of selpercatinib and entrectinib providing proof-of-concept of urgency of incorporating molecular profiling in real-time and personalized N-of-1 care transcending one-size-fits-all approach.

RETooling the RET Inhibitor Pralsetinib for ESR1 Fusion-Positive Breast Cancer and Beyond.

Transcriptionally active fusions of ESR1 (ESR1-TAF) and somatic mutations in the estrogen receptor alpha (ERα) ligand-binding domain (LBD) cause endocrine therapy resistance in breast cancer. In searching for therapeutic target kinase(s) in these breast cancers, Gou and colleagues identified FLT4, RET, JAK1, and IGF1R as the top upregulated kinases induced by ESR1-TAFs and ERα LBD mutants in breast cancer cells. Among them, inhibition of RET by pralsetinib suppressed ESR1-TAF-driven and ERα LBD mutant-driven cell proliferation and patient-derived xenograft growth. Pralsetinib is an inhibitor of the RET protein tyrosine kinase that is approved for treating oncogenic RET mutation-positive and RET fusion-positive thyroid cancers and non-small cell lung cancer. The work by Gou and colleagues reinforces the knowledge of RET as an ESR1 target gene and highlights that RET interacts with ERα to promote breast cancer tumorigenesis and antiestrogen resistance. It also raises the prospect of repositioning pralsetinib to target wildtype RET in ER-positive breast cancer. See related article by Gou et al., p. 3237.

Screening Streptomyces secondary metabolites against non-small cell lung cancer using computational approaches

Secondary metabolites from bacterial sources are being seen as alternatives to identify new medicines for various disease conditions. Streptomyces is known to be a warehouse of secondary metabolites and is a promising source to be looked for the discovery and development of new anti-tumorigenic agents. Such identifications could alleviate the issue of lung cancer that takes over 2 million lives a year. In the current study, we have screened secondary metabolites from Streptomyces for their potential to act as drugs for non-small cell lung cancer. The metabolites were identified for their ability to inhibit RET protein. Gene fusion partners cause alteration to the RET protein that causes the cancer condition. The metabolites identified were based on docking, pharmacokinetics, toxicity and pass prediction. 16 metabolites from 13 different species of Streptomyces showed binding affinity to the target protein. Among the metabolites, melanin was the one that had strongest evident to act as a drug candidate for non-small cell lung cancer therapeutics.

Interaction of Wild Type and V804l and V804m-Mutated Ret Protein Kinase with Emodin: In Silico Approach

Medullary thyroid carcinoma (MTC) is a malignant endocrine tumor originating from parafollicular calcitonin-producing C cells. The proto-oncogene RET (REarranged during Transfection) is known as the main actor in the development and outbreak of MTC. Gain of function mutations of RET constitutively activate the receptor which are found to be responsible for the high percentage of MTC cases. The two FDA-approved drugs used for MTC, vandetanib and cabozantinib, are resistant to two mutant variants of RET which are V804L and V804M. In this study, the interactions of emodin, a natural molecule found in plants, with wild-type as well as V804L and V804M-mutated RET kinase were investigated via molecular docking. Pymol was used to create point mutations on wild type RET. Vandetanib and cabozantinib were used as the reference drugs. The binding free energy of vandetanib with wild-type RET, V804L and V804M variants were found to be -9.3, -9.1 and -8.6 kcal/mol. Similarly, the binding free energy of cabozantinib with wild-type RET, V804L and V804M variants were found to be -10.6, -10.4, and -9.5 kcal/mol, respectively. Clearly, the binding affinity of vandetanib and cabozantinib to RET kinase was found to be reduced in mutated variants as compared to wild type. In the meantime, the binding energy between emodin and wild-type RET was shown to be -9.3 kcal/mol. Interestingly, the binding affinity of emodin to V804L and V804M variants was determined to be increased (-9.9 and -9.8 kcal/mol, respectively) compared to wild type. Furthermore, many H-bonds and hydrophobic interactions between emodin and mutated RET variants were shown. Therefore, strong binding affinity of emodin to wild-type and the mutated variants of RET was suggested in this study. In conclusion, emodin was found to be a potential molecule to inhibit RET kinase activity and could be used as a therapeutical agent against medullary thyroid carcinoma.

Molecular mechanisms of the tyrosine kinase inhibitor pralsetinib activity in in-vitro models of medullary thyroid carcinoma: Aberrant activation of the HH-Gli signaling pathway in acquired resistance

Medullary thyroid carcinoma (MTC) is a malignant tumor with challenging management. Multi-targeted kinase inhibitors (MKI) and tyrosine-kinase inhibitors (TKI) with high specificity for RET protein are approved for advanced MTC treatment. However, their efficacy is hindered by evasion mechanisms of tumor cells. Thus, the aim of this study was the identification of an escape mechanism in MTC cells exposed to a highly selective RET TKI. TT cells were treated with TKI, MKI, and/or the HH-Gli inhibitors, GANT61 and Arsenic Trioxide (ATO), in the presence or absence of hypoxia. RET modifications, oncogenic signaling activation, proliferation and apoptosis were assessed. Additionally, cell modifications and HH-Gli activation were also evaluated in pralsetinib-resistant TT cells. Pralsetinib inhibited RET autophosphorylation and RET downstream pathways activation in normoxic and hypoxic conditions. Additionally, pralsetinib impaired proliferation, induced the activation of apoptosis and, in hypoxic cells, downregulated HIF-1α. Focusing on escape molecular mechanisms associated with therapy, we observed increased Gli1 levels in a subset of cells. Indeed, pralsetinib stimulated the re-localization of Gli1 into the cell nuclei. Treatment of TT cells with both pralsetinib and ATO resulted in Gli1 down-regulation and impaired cell viability. Moreover, pralsetinib-resistant cells confirmed Gli1 activation and up-regulation of its transcriptionally regulated target genes. Altogether, we showed that pralsetinib impairs MTC cell growth and induces cell death, also in hypoxic conditions. The HH-Gli pathway is a new molecular mechanism of escape to pralsetinib therapy that can be overcome through combined therapy.

Targeting RET Solvent-Front Mutants with Alkynyl Nicotinamide-Based Inhibitors.

Selpercatinib (LOXO292) and pralsetinib (BLU667) are RET protein tyrosine kinase inhibitors (TKIs) recently approved for treating RET-altered cancers. However, RET mutations that confer selpercatinib/pralsetinib resistance have been identified, necessitating development of next-generation RET TKIs. While acquired RET G810C/R/S/V mutations were reported in selpercatinib-treated patients, it was unclear whether all of these and other potential G810 mutants are resistant to selpercatinib and pralsetinib. Here, we profiled selpercatinib and pralsetinib on all six possible G810 mutants derived from single nucleotide substitution and developed novel alkynyl nicotinamide-based RET TKIs to inhibit selpercatinib/pralsetinib-resistant RET G810 mutants. Surprisingly, the G810V mutant found in a clinical study was not resistant to selpercatinib or pralsetinib. Besides G810C/R/S, G810D also conferred selpercatinib/pralsetinib resistance. Alkynyl nicotinamide compounds such as HSN608, HSL476, and HSL468 have better drug-like properties than alkynyl benzamides. Six of these compounds inhibited all six G810 solvent-front mutants and the V804M gatekeeper mutant with IC50 < 50 nmol/L in cell culture. Oral administration of HSN608 at a well-tolerated dose (30 mg/kg) gave plasma level > 30x the IC50s of inhibiting all G810 mutants in cell culture. In cell-derived xenograft tumors driven by KIF5B-RET (G810C) that contains the most frequently observed solvent-front mutant in selpercatinib-treated patients, HSN608, HSL476, and HSL468 significantly suppressed and caused regression of the selpercatinib-resistant tumors. This study clarifies the sensitivities of different RET solvent-front mutants to selpercatinib and pralsetinib and identifies novel alkylnyl nicotinamide-based RET TKIs for inhibiting selpercatinib/pralsetinib-resistant G810 mutants.

Adefovir Dipivoxil as a Therapeutic Candidate for Medullary Thyroid Carcinoma: Targeting RET and STAT3 Proto-Oncogenes.

Aberrant gene expression is often linked to the progression of various cancers, making the targeting of oncogene transcriptional activation a potential strategy to control tumor growth and development. The RET proto-oncogene's gain-of-function mutation is a major cause of medullary thyroid carcinoma (MTC), which is part of multiple endocrine neoplasia type 2 (MEN2) syndrome. In this study, we used a cell-based bioluminescence reporter system driven by the RET promoter to screen for small molecules that potentially suppress the RET gene transcription. We identified adefovir dipivoxil as a transcriptional inhibitor of the RET gene, which suppressed endogenous RET protein expression in MTC TT cells. Adefovir dipivoxil also interfered with STAT3 phosphorylation and showed high affinity to bind to STAT3. Additionally, it inhibited RET-dependent TT cell proliferation and increased apoptosis. These results demonstrate the potential of cell-based screening assays in identifying transcriptional inhibitors for other oncogenes.

The heterogeneous transition state of resistance to RET kinase inhibitors converges on ERK1/2-driven Aurora A/B kinases

AimWhile most patients with RET-altered cancer responded to the RET protein tyrosine kinase inhibitors (TKIs) pralsetinib (BLU667) and selpercatinib (LOXO292), few achieved a complete response. Heterogeneity in residual tumors makes it difficult to target their diverse genetic alterations individually. The aim of this study is to characterize the cancer cells that persist under continuous RET TKI treatment and identify the shared vulnerability of these cells. MethodsWe analyzed residual RET-altered cancer cells under prolonged RET TKI treatment by whole exome sequencing (WES), RNA-seq analysis, and drug-sensitivity screening. These were followed by tumor xenograft experiments of mono- and combinational drug treatments. ResultsBLU667- and LOXO292-tolerated persisters were cellularly heterogeneous, contained slowly proliferating cells, regained low levels of active ERK1/2, and displayed plasticity in growth rate, which we designated as in the transition state of resistance (TSR). TSR cells were genetically heterogeneous. Aurora A/B kinases were among the most significantly upregulated genes and that the MAPK pathway activity had significantly higher transcript footprints. MEK1/2 and Aurora kinase inhibitors were the most effective drugs when combined with a RET kinase inhibitor. In a TSR tumor model, combination of BLU667 with an Aurora kinase or a MEK1/2 kinase inhibitor caused TSR tumor regression. ConclusionOur experiments reveal that the heterogeneous TSR cancer cells under continuous RET TKI treatment converge on the targetable ERK1/2-driven Aurora A/B kinases. The discovery of the targetable convergent point in the genetically heterogeneous TSR points to an effective combination therapy approach to eliminate the residual tumors.

A Single RET Mutation in Hirschsprung Disease Induces Intestinal Aganglionosis Via a Dominant-Negative Mechanism

Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of the enteric nervous system (ENS). HSCR potentially involves multiple gene aberrations and displays complex patterns of inheritance. Mutations of the RET gene, encoding the RET receptor tyrosine kinase, play a central role in the pathogenesis of HSCR. Although a wide variety of coding RET mutations have been identified, their pathogenetic significance invivo has remained largely unclear. We introduced a HSCR-associated RET missense mutation, RET(S811F), into the corresponding region (S812) of the mouse Ret gene. Pathogenetic impact of Ret(S812F) was assessed by histologic and functional analyses of the ENS and by biochemical analyses. Interactions of the Ret(S812F) allele with HSCR susceptibility genes, the RET9 allele and the Ednrb gene, were examined by genetic crossing in mice. RetS812F/+ mice displayed intestinal aganglionosis (incidence, 50%) or hypoganglionosis (50%), impaired differentiation of enteric neurons, defecation deficits, and increased lethality. Biochemical analyses revealed that Ret(S811F) protein was not only kinase-deficient but also abrogated function of wild-type RET in trans. Moreover, the Ret(S812F) allele interacted with other HSCR susceptibility genes and caused intestinal aganglionosis with full penetrance. This study demonstrates that a single RET missense mutation alone induces intestinal aganglionosis via a dominant-negative mechanism. The RetS812F/+ mice model HSCR displays dominant inheritance with incomplete penetrance and serves as a valuable platform for better understanding of the pathogenetic mechanism of HSCR caused by coding RET mutations.

CRISPR/Cas9 RET Gene Knockout in Medullary Thyroid Carcinoma Cell-lines: Optimization and Validation.

Medullary Thyroid Cancer (MTC) is a very aggressive type of thyroid carcinoma. Mutation in RET proto-oncogene is demonstrated in MTC development. We aimed to knock-out of RET-oncogene using CRISPR/Cas9 genome editing method in MTC cell-lines. This research was conducted in Shahid Beheshti University of Medical Sciences, Tehran, Iran during 2019-2020. Four different sgRNAs were designed to target exons one, two, and four of RET-oncogene in TT and MZ-CRC-1 cell-lines using bioinformatics tools, then the CRISPR/Cas9 constructs was made. About 72-hours after cell transfection, T7EI method and DNA sequencing were used to confirm the knock-out of RET-oncogene. Expression of RET, Calcitonin genes and RET protein were evaluated by Real-time PCR and ELISA, respectively. The results of T7E1, and DNA sequencing of transfected cells confirmed RET gene knock-out by CRISPR/Cas9. There was a significant decrease in RET gene expression and RET protein in transfected TT and MZ cells compared to controls. The rate of cell apoptosis in transfected cells was significantly increased. Calcitonin gene expression was also significantly reduced in transfected cells. p-RET, p-PI3K, p-AKT, p-MEK, p-ERK protein levels were significantly reduced in TT and MZ transfected cells. For the first time, knock-out of RET gene was performed and confirmed using CRISPR/Cas9. Inhibition of this gene leads to inhibition of the tyrosine kinase RET signal transduction pathway. Therefore, it can be one of the most effective and specific therapeutic goals in the field of Personalized Medicine in the treatment of diseases caused by over activity of RET molecular pathway.

A novel cell‐based Screening System Identifies Adefovir‐dipivoxil as Suppressor of RET expression in medullary thyroid carcinoma

The aberrant gene expression is often associated with the progression of several cancers and hence targeting the transcriptional activation of oncogenes using small‐molecules could be a potential strategy for controlling the tumor growth and development. The gain‐of‐function mutation in the RET proto‐oncogene has been identified as the major cause for the development of medullary thyroid carcinoma (MTC), which is a part of multiple endocrine neoplasia type 2 (MEN2) syndrome. In this study we utilized a cell‐based bioluminescence reporter system in which the luciferase gene expression is driven by the RET promoter to screen for drug‐like molecules that potentially suppress the transcription of this gene. Using this system, we identified adefovir‐ dipivoxil as a transcriptional inhibitor for the RET gene, which further suppressed the endogenous RET protein expression in MTC TT cells. Consistent with this effect, adefovir‐dipivoxil also inhibited RET dependent TT cell proliferation and increased apoptosis in these cells. These results highlight the potential of the cell‐based screening assay to identify transcriptional inhibitors for other oncogenes.

The RET gene encodes RET protein, which triggers intracellular signaling pathways for enteric neurogenesis, and RET mutation results in Hirschsprung's disease.

Enteric neurons and ganglia are derived from vagal and sacral neural crest cells, which undergo migration from the neural tube to the gut wall. In the gut wall, they first undergo rostrocaudal migration followed by migration from the superficial to deep layers. After migration, they proliferate and differentiate into the enteric plexus. Expression of the Rearranged During Transfection (RET) gene and its protein RET plays a crucial role in the formation of enteric neurons. This review describes the molecular mechanism by which the RET gene and the RET protein influence the development of enteric neurons. Vagal neural crest cells give rise to enteric neurons and glia of the foregut and midgut while sacral neural crest cells give rise to neurons of the hindgut. Interaction of RET protein with its ligands (glial cell derived neurotrophic factor (GDNF), neurturin (NRTN), and artemin (ARTN)) and its co-receptors (GDNF receptor alpha proteins (GFRα1-4)) activates the Phosphoinositide-3-kinase-protein kinase B (PI3K-PKB/AKT), RAS mitogen-activated protein kinase (RAS/MAPK) and phospholipase Cγ (PLCγ) signaling pathways, which control the survival, migration, proliferation, differentiation, and maturation of the vagal and sacral neural crest cells into enteric neurons. Abnormalities of the RET gene result in Hirschsprung's disease.

Glial cell line derived neurotrophic factor in brain repair after focal ischemic stroke.

Glial cell line derived neurotrophic factor in brain repair after focal ischemic stroke.

Deciphering the resistance mechanism of RET kinase mutant against vandetanib and nintedanib using molecular dynamics simulations

The RET protein is a transmembrane receptor tyrosine kinase (RTK) whose oncogenic mutations or fusions are closely related to human cancers such as thyroid and non-small cell lung cancer. Vandetanib as a clinical-approved protein-tyrosine kinase inhibitor (TKI) exhibits anti-cancer efficacy by blocking the RET ATP-binding site, but drug resistance was observed for the RETG810A mutant. Recent studies have identified another TKI nintedanib as an effective molecule to inhibit vandetanib-resistant RETG810A. However, there is no clear evidence of why nintedanib and vandetanib displayed different inhibitory activities towards RETG810A. Here, we exploited molecular dynamic (MD) simulations to compare the interactions of the RETG810A mutant with nintedanib and vandetanib. A higher structural flexibility of the activation loop was observed in the nintedanib-bound RETG810A, which may result in discrepant autophosphorylation activity in the nintedanib- and vandetanib-bound RET kinase, causing differentiated pharmacological effects of the two compounds. Molecular mechanics/Poisson-Bolzmann surface area method suggested that nintedanib had a higher affinity towards RETG810A over vandetanib, accounting for its better inhibitory effect as an ATP-competitive compound. These results depicted the underlying mechanism for the different inhibitory efficacy of nintedanib and vandetanib on RETG810A from both conformational and energetic aspects. Furthermore, we also found that both compounds maintained the ‘DFG-in, αC-helix-in, and activation loop-open’ conformation of RETG810A, which is the characteristic of the active state. Together, our results provide comprehensive mechanistic insights into nintedanib’s capability in inhibiting vandetanib-resistant RET mutant and enlighten future structural-based optimisation of RET TKIs to overcome drug resistance.

Live visualization of a functional RET-EGFP chimeric receptor in homozygous knock-in mice.

The GDNF Family Ligands (GFLs) regulate neural development and kidney organogenesis by activating the RET receptor tyrosine kinase. Many RET-dependent developmental processes involve long-distance cell-cell communications or cell polarity, which includes cell migration and axon guidance. This suggests that spatiotemporally regulated subcellular localization of RET protein and appropriate propagation of RET signaling in cells are essential for the physiological function of the GFLs. Little is known, however, about the dynamics of RET protein in cells. Addressing this issue requires development of a system that allows visualization of RET in living cells. In this study, we report generation of a novel knock-in mouse line in which the RET-EGFP chimeric receptor is expressed under the Ret promoter. Unlike Ret-deficient mice that die after birth due to the absence of the enteric nervous system (ENS) and kidneys, RetRET-EGFP/RET-EGFP mice were viable and grew to adulthood with no overt abnormality, which indicated that RET-EGFP exerts function comparable to RET. In neurons and ENS progenitors, RET-EGFP signals were detected both on the cell membrane and in the cytoplasm, the latter of which appeared as a punctate pattern. Time-lapse imaging of cultured neural cells and embryos revealed active transport of RET-EGFP puncta in neuronal axons and cell bodies. Immunohistochemical analyses detected RET-EGFP signals in early and recycling endosomes, indicating that RET-EGFP is trafficked via the endocytic pathway. RetRET-EGFP/RET-EGFP mice enable visualization of functional RET protein in vivo for the first time and provide a unique platform to examine the dynamics and physiology of RET trafficking.

RET Regulates Human Medullary Thyroid Cancer Cell Proliferation through CDK5 and STAT3 Activation.

Medullary thyroid cancer (MTC) is a neuroendocrine tumor that arises from the parafollicular C-cells, which produces the hormone calcitonin. RET is a transmembrane receptor protein-tyrosine kinase, which is highly expressed in MTC. Our previous studies reported that cyclin-dependent kinase 5 (CDK5) plays a crucial role in cancer progression, including MTC. However, the role of CDK5 in GDNF-induced RET signaling in medullary thyroid cancer proliferation remains unknown. Here, we investigated RET activation and its biochemically interaction with CDK5 in GDNF-induced medullary thyroid cancer proliferation. Our results demonstrated that GDNF stimulated RET phosphorylation and thus subsequently resulted in CDK5 activation by its phosphorylation. Activated CDK5 further caused STAT3 activation by its specific phosphorylation at Ser727. Moreover, we also found that GDNF treatment enhanced ERK1/2 and EGR1 activity, which is involved in p35 activation. Interestingly, we identified for the first time that CDK5 physically interacted with RET protein in MTC. Overall, our results provide a new mechanism for medullary thyroid cancer cell proliferation, suggesting that targeting CDK5 may be a promising therapeutic candidate for human medullary thyroid cancer in the near future.

Abstract PS16-05: Identification of pathogenic RET alterations in cell-free DNA (cfDNA) from patients with metastatic breast cancer

Abstract Background: RET is an important proto-oncogene involved in the development of various cancers for which we have FDA approved therapies. RET alterations are a known mechanism of resistance against breast cancer systemic therapies and higher RET expression has been demonstrated in breast cancer brain metastases compared to their primary tumors. While the genomic characterization of RET alterations has occurred from bulk breast tumors, the frequency and type of RET alterations in metastatic breast cancer (MBC) have not been fully characterized from cfDNA. The purpose of this study was to identify the incidence of RET genomic alterations occurring in cfDNA from patients with MBC and elucidate which RET alterations may increase RET kinase activity or may function as mechanisms of resistance against the FDA approved RET inhibitor, selpercatinib. Methods: We queried 16,053 reports from Guardant Health between June 2015 - October 2019 to identify the incidence of RET alterations detected in cfDNA from MBC. We classified each alteration type into the following categories: fusions, single nucleotide variants (SNVs), or indels. Focus was placed on characterizing RET SNVs. Amino acid changes occurring at conserved regions across multiple species were identified. We compared known activating mutations in EGFR and ERBB2 with homologous regions in RET. In silico modeling with PyRx was used to dock selpercatinib onto the RET kinase (PDB 6NJA). Three-dimensional in silico analyses with ChimeraX was utilized to further determine which alterations may increase RET kinase activity or may induce resistance against selpercatinib. Results: Nonsynonymous RET alterations from the Guardant Health breast cancer database were found in 162 samples from a cohort of 16,053 patients indicating an overall incidence of 1.0%. Alterations included: 3 (1.9%) RET-CCDC6 fusions, 2 (1.2%) RET-KIF5B fusions, 6 (3.7%) indels, and 151 (93.2%) SNVs. Of the 151 samples with SNVs, we identified 37 (23%) and 63 (38.9%) point mutations occurring in the transmembrane/juxtamembrane and kinase domains, respectively, and 77 occurred at highly conserved regions across species. The most frequent hotspot mutations occurring in 4 or more unique breast cancers included H594P, 6 (4.0%); S696L, 4 (2.6%); C634Y/G, 5 (3.3%); R813W/Q, 4 (2.6%); and M918T, 8 (5.3%). We aligned RET with homologous kinases and found 8 (5.3%) RET mutations (R721Q, V804L, M868I, R873Q, G894S) and (A641T, S891L, D925H) that corresponded to known activating mutations in EGFR (E709A/K, T790M, R831H, R836C, G857V) and ERBB2 (V659E, T862A/S, R896C), respectively. Three-dimensional analyses indicate that RET alterations occurring within the hinge (E805Q), HRD (H872R, R873Q), and DFG (D892Y, G894S) regions, and regulatory spine (D933N) of the RET kinase are postulated to induce resistance against selpercatinib. Conclusions: We found that a modest incidence of RET genomic alterations occur in cfDNA from patients with MBC. Novel somatic alterations in RET were identified from Guardant Health that were not detected in the public domain. A portion of RET SNVs occurred at highly conserved regions across species or at known homologous kinase activating mutations suggesting these specific RET mutations may increase RET kinase activity and might be actionable therapeutic targets in breast cancers harboring these mutations. Three dimensional analyses of the RET protein tyrosine kinase further illustrate which specific alterations may increase RET kinase activity or induce resistance against selpercatinib. Citation Format: Mary Love Taylor, Benjamin Mayro, Leylah Drusbosky, Carey Anders, Jeremy Force. Identification of pathogenic RET alterations in cell-free DNA (cfDNA) from patients with metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-05.

Aberrantly expressed GFRα-1/RET in patients with lacrimal adenoid cystic carcinoma is associated with high recurrence risk: a retrospective study of 51 LACC cases.

Objective:Because of the poor prognosis of lacrimal adenoid cystic carcinoma (LACC), we aimed to investigate the effects of perineural invasion (PNI) and consequent aberrations in GDNF/GFRα-1/RET protein expression on LACC recurrence.Methods:Clinicopathological data for 51 histologically confirmed patients with LACC enrolled between 2001 and 2017 were retrospectively analyzed. Hematoxylin and eosin staining was applied to assess PNI. Tissue-based immunohistochemistry (IHC) detection of GDNF, GFRα-1, and RET proteins was performed on LACC formalin-fixed, paraffin-embedded specimens. We generated semi-quantitative data of the IHC results and compared them with the clinicopathological data for the 51 patients.Results:Of the 51 patients, 19 (37.3%) were PNI positive. Recurrence was more common for LACC with than without PNI (73.7% vs. 37.5%, P = 0.01). GDNF, GFRα-1, and RET proteins were expressed in 62.7%, 62.7%, and 54.9% of the 51 patients with LACC, respectively. The expression of all 3 proteins was more common in patients with than without PNI. In agreement with previous findings, PNI-associated GFRα-1 and RET positivity, as detected by IHC, remained significantly associated with recurrence, whereas GDNF expression, as detected by IHC, was not correlated with LACC recurrence. Specifically, patients with concurrent GFRα-1 and RET expression may have a high risk of PNI (89.5% positivity rate) and recurrence (84.2% positivity rate).Conclusions:PNI may contribute to LACC recurrence. The concurrent expression of GFRα-1 and RET proteins, as detected by IHC, may potentially be associated with LACC PNI and recurrence.