Discovery of 9H-pyrimido[4,5-b]indole derivatives as dual RET/TRKA inhibitors.

Aberrant RET kinase signaling is activated in numerous cancers including lung, thyroid, breast, pancreatic, and prostate. Recent approvals of selective RET inhibitors, pralsetinib and selpercatinib, has shifted the focus of RET kinase drug discovery programs towards the development of selective inhibitors. However, selective inhibitors invariably lose efficacy as the selective nature of the inhibitor places Darwinian-like pressure on the tumor to bypass treatment through the selection of novel oncogenic drivers. Further, selective inhibitors are restricted for use in tumors with specific genetic backgrounds that do not encompass diverse patient classes. Here we report the identification of a pyrimido indole RET inhibitor found to also have activity against TRK. This selective dual RET/TRK inhibitor can be utilized in tumors with both RET and TRK genetic backgrounds and can also provide blockade of NTRK-fusions that are selected for from RET inhibitor treatments. Efforts towards developing dual RET/TRK inhibitors can be beneficial in terms of encompassing more diverse patient classes while also achieving blockade against emerging resistance mechanisms.

Gestational diabetes mellitus induces congenital anomalies of the kidney and urinary tract in mice by altering RET/MAPK/ERK pathway.

Gestational diabetes mellitus (GDM) presents a substantial population health concern. Previous studies have revealed that GDM can ultimately influence nephron endowment. In this study, we established a GDM mouse model to investigate the embryological alterations and molecular mechanisms underlying the development of congenital anomalies of the kidney and urinary tract (CAKUT) affected by GDM. Our study highlights that GDM could contribute to the manifestation of CAKUT, with prevalent phenotypes characterized by isolated hydronephrosis and duplex kidney complicated with hydronephrosis in mice. Ectopic ureteric buds (UBs) and extended length of common nephric ducts (CNDs) were noted in the metanephric development stage. The expression of Ret and downstream p-ERK activity were enhanced in UBs, which indicated the alteration of RET/MAPK/ERK pathway may be one of the mechanisms contributing to the increased occurrence of CAKUT associated with GDM.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Aberrant SOX10 and RET expressions in patients with Hirschsprung disease.

BACKGROUND: HSCR is a complex genetic disorder characterized by the absence of ganglion cells in the intestine, leading to a functional obstruction. It is due to a disruption of complex signaling pathways within the gene regulatory network (GRN) during the development of the enteric nervous system (ENS), including SRY-Box Transcription Factor 10 (SOX10) and REarranged during Transfection (RET). This study evaluated the expressions of SOX10 and RET in HSCR patients in Indonesia. METHODS: Total RNA of 19 HSCR ganglionic and aganglionic colons and 16 control colons were analyzed using quantitative real-time polymerase chain reaction for SOX10 and RET with GAPDH as the reference gene. Livak's method (2-ΔΔCT) was used to determine the expression levels of SOX10 and RET. RESULTS: Most patients were males (68.4%), in the short aganglionosis segment (78.9%), and had undergone transanal endorectal pull-through (36.6%). There were significant upregulated SOX10 expressions in both ganglionic (2.84-fold) and aganglionic (3.72-fold) colon of HSCR patients compared to controls' colon (ΔCT 5.21 ± 2.04 vs. 6.71 ± 1.90; p = 0.032; and ΔCT 4.82 ± 1.59 vs. 6.71 ± 1.90; p = 0.003; respectively). Interestingly, the RET expressions were significantly downregulated in both ganglionic (11.71-fold) and aganglionic (29.96-fold) colon of HSCR patients compared to controls' colon (ΔCT 12.54 ± 2.21 vs. 8.99 ± 3.13; p = 0.0004; and ΔCT 13.90 ± 2.64 vs. 8.99 ± 3.13; p = 0.0001; respectively). CONCLUSIONS: Our study shows aberrant SOX10 and RET expressions in HSCR patients, implying the critical role of SOX10 and RET in the pathogenesis of HSCR, particularly in the Indonesian population. Our study further confirms the involvement of SOX10-RET within the GNR during the ENS development.

Functional role of glial-derived neurotrophic factor in a mixed allergen murine model of asthma.

Our previous study showed that glial-derived neurotrophic factor (GDNF) expression is upregulated in asthmatic human lungs, and GDNF regulates calcium responses through its receptor GDNF family receptor α1 (GFRα1) and RET receptor in human airway smooth muscle (ASM) cells. In this study, we tested the hypothesis that airway GDNF contributes to airway hyperreactivity (AHR) and remodeling using a mixed allergen mouse model. Adult C57BL/6J mice were intranasally exposed to mixed allergens (ovalbumin, Aspergillus, Alternaria, house dust mite) over 4 wk with concurrent exposure to recombinant GDNF, or extracellular GDNF chelator GFRα1-Fc. Airway resistance and compliance to methacholine were assessed using FlexiVent. Lung expression of GDNF, GFRα1, RET, collagen, and fibronectin was examined by RT-PCR and histology staining. Allergen exposure increased GDNF expression in bronchial airways including ASM and epithelium. Laser capture microdissection of the ASM layer showed increased mRNA for GDNF, GFRα1, and RET in allergen-treated mice. Allergen exposure increased protein expression of GDNF and RET, but not GFRα1, in ASM. Intranasal administration of GDNF enhanced baseline responses to methacholine but did not consistently potentiate allergen effects. GDNF also induced airway thickening, and collagen deposition in bronchial airways. Chelation of GDNF by GFRα1-Fc attenuated allergen-induced AHR and particularly remodeling. These data suggest that locally produced GDNF, potentially derived from epithelium and/or ASM, contributes to AHR and remodeling relevant to asthma.NEW & NOTEWORTHY Local production of growth factors within the airway with autocrine/paracrine effects can promote features of asthma. Here, we show that glial-derived neurotrophic factor (GDNF) is a procontractile and proremodeling factor that contributes to allergen-induced airway hyperreactivity and tissue remodeling in a mouse model of asthma. Blocking GDNF signaling attenuates allergen-induced airway hyperreactivity and remodeling, suggesting a novel approach to alleviating structural and functional changes in the asthmatic airway.

Impact of the overexpression of the tyrosine kinase receptor RET in the hematopoietic potential of induced pluripotent stem cells (iPSCs).

INTRODUCTION: Previous studies have suggested that the tyrosine kinase receptor RET plays a significant role in the hematopoietic potential in mice and could also be used to expand cord-blood derived hematopoietic stem cells (HSCs). The role of RET in human iPSC-derived hematopoiesis has not been tested so far. METHODS: To test the implication of RET on the hematopoietic potential of iPSCs, we activated its pathway with the lentiviral overexpression of RETWT or RETC634Y mutation in normal iPSCs. An iPSC derived from a patient harboring the RETC634Y mutation (iRETC634Y) and its CRISPR-corrected isogenic control iPSC (iRETCTRL) were also used. The hematopoietic potential was tested using 2D cultures and evaluated regarding the phenotype and the clonogenic potential of generated cells. RESULTS: Hematopoietic differentiation from iPSCs with RET overexpression (WT or C634Y) led to a significant reduction in the number and in the clonogenic potential of primitive hematopoietic cells (CD34+/CD38-/CD49f+) as compared to control iPSCs. Similarly, the hematopoietic potential of iRETC634Y was reduced as compared to iRETCTRL. Transcriptomic analyses revealed a specific activated expression profile for iRETC634Y compared to its control with evidence of overexpression of genes which are part of the MAPK network with negative hematopoietic regulator activities. CONCLUSION: RET activation in iPSCs is associated with an inhibitory activity in iPSC-derived hematopoiesis, potentially related to MAPK activation.

Architecture and regulation of a GDNF-GFRα1 synaptic adhesion assembly.

Glial-cell line derived neurotrophic factor (GDNF) bound to its co-receptor GFRα1 stimulates the RET receptor tyrosine kinase, promoting neuronal survival and neuroprotection. The GDNF-GFRα1 complex also supports synaptic cell adhesion independently of RET. Here, we describe the structure of a decameric GDNF-GFRα1 assembly determined by crystallography and electron microscopy, revealing two GFRα1 pentamers bridged by five GDNF dimers. We reconsitituted the assembly between adhering liposomes and used cryo-electron tomography to visualize how the complex fulfils its membrane adhesion function. The GFRα1:GFRα1 pentameric interface was further validated both in vitro by native PAGE and in cellulo by cell-clustering and dendritic spine assays. Finally, we provide biochemical and cell-based evidence that RET and heparan sulfate cooperate to prevent assembly of the adhesion complex by competing for the adhesion interface. Our results provide a mechanistic framework to understand GDNF-driven cell adhesion, its relationship to trophic signalling, and the central role played by GFRα1.

Usefulness of an RNA extraction-free test for the multiplexed detection of ALK, ROS1, and RET Gene Fusions in Real Life FFPE Specimens of Non-Small Cell Lung Cancers.

BACKGROUND: ALK, ROS1 and RET rearrangements occur, respectively, in 5%, 2%, and 1% non-small cell lung cancers (NSCLC). ALK and ROS1 fusion proteins detection by immunohistochemistry (IHC) has been validated for rapid patient screening, but ROS1 fusions need to be confirmed by another technique and no RET IHC test is available for clinical use. RESEARCH DESIGN AND METHODS: We report herein the usefulness of the HTG EdgeSeq Assay, an RNA extraction-free test combining a quantitative nuclease protection assay with NGS, for the detection of ALK, ROS1 and RET fusions from 'real-life' small NSCLC samples. A total of 203 FFPE samples were collected from 11 centers. They included 143 rearranged NSCLC (87 ALK, 39 ROS1, 17 RET) and 60 ALK-ROS1-RET negative controls. RESULTS: The assay had a specificity of 98% and a sensitivity for ALK, ROS1 and RET fusions of 80%, 94% and 100% respectively. Among the 19 HTG-assay false negative samples, the preanalytical conditions were identified as the major factors impacting the assay efficiency. CONCLUSIONS: Overall, the HTG EdgeSeq assay offers comparable sensitivities and specificity than other RNA sequencing techniques, with the advantage that it can be used on very small and old samples collected multicentrically.

TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation.

The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma.

Targeting the RET tyrosine kinase in neuroblastoma: A review and application of a novel selective drug design strategy.

The RET (REarranged during Transfection) gene, which encodes for a transmembrane receptor tyrosine kinase, is an established oncogene associated with the etiology and progression of multiple types of cancer. Oncogenic RET mutations and rearrangements resulting in gene fusions have been identified in many adult cancers, including medullary and papillary thyroid cancers, lung adenocarcinomas, colon and breast cancers, and many others. While genetic RET aberrations are much less common in pediatric solid tumors, increased RET expression has been shown to be associated with poor prognosis in children with solid tumors such as neuroblastoma, prompting an interest in RET inhibition as a form of therapy for these children. A number of kinase inhibitors currently in use for patients with cancer have RET inhibitory activity, but these inhibitors also display activity against other kinases, resulting in unwanted side effects and limiting their safety and efficacy. Recent efforts have been focused on developing more specific RET inhibitors, but due to high levels of conservation between kinase binding pockets, specificity remains a drug design challenge. Here, we review the background of RET as a potential therapeutic target in neuroblastoma tumors and the results of recent preclinical studies and clinical trials evaluating the safety and efficacy of RET inhibition in adults and children. We also present a novel approach to drug discovery leveraging the chemical phenomenon of atropisomerism to develop specific RET inhibitors and present preliminary data demonstrating the efficacy of a novel RET inhibitor against neuroblastoma tumor cells.

Optogenetic dissection of RET signaling reveals robust activation of ERK and enhanced filopodia-like protrusions of regenerating axons.

RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light-inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.

Rapid and Cost-Efficient Detection of RET Rearrangements in a Large Consecutive Series of Lung Carcinomas.

RET-kinase-activating gene rearrangements occur in approximately 1-2% of non-small-cell lung carcinomas (NSCLCs). Their reliable detection requires next-generation sequencing (NGS), while conventional methods, such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) or variant-specific PCR, have significant limitations. We developed an assay that compares the level of RNA transcripts corresponding to 5'- and 3'-end portions of the RET gene; this test relies on the fact that RET translocations result in the upregulation of the kinase domain of the gene and, therefore, the 5'/3'-end expression imbalance. The present study included 16,106 consecutive NSCLC patients, 14,449 (89.7%) of whom passed cDNA quality control. The 5'/3'-end unbalanced RET expression was observed in 184 (1.3%) tumors, 169 of which had a sufficient amount of material for the identification of translocation variants. Variant-specific PCR revealed RET rearrangements in 155/169 (91.7%) tumors. RNA quality was sufficient for RNA-based NGS in 10 cases, 8 of which carried exceptionally rare or novel (HOOK1::RET and ZC3H7A::RET) RET translocations. We also applied variant-specific PCR for eight common RET rearrangements in 4680 tumors, which emerged negative upon the 5'/3'-end unbalanced expression test; 33 (0.7%) of these NSCLCs showed RET fusion. While the combination of the analysis of 5'/3'-end RET expression imbalance and variant-specific PCR allowed identification of RET translocations in approximately 2% of consecutive NSCLCs, this estimate approached 120/2361 (5.1%) in EGFR/KRAS/ALK/ROS1/BRAF/MET-negative carcinomas. RET-rearranged tumors obtained from females, but not males, had a decreased level of expression of thymidylate synthase (p < 0.00001), which is a known predictive marker of the efficacy of pemetrexed. The results of our study provide a viable alternative for RET testing in facilities that do not have access to NGS due to cost or technical limitations.

Selpercatinib monotherapy in a Chinese patient with RET fusion/ EGFR co-mutated nonsmall cell lung cancer from the Phase II LIBRETTO-321 study: a case report.

Rearranged during transfection ( RET ) fusions and epidermal growth factor receptor ( EGFR ) mutations are potent oncogenic drivers in patients with nonsmall cell lung cancer (NSCLC), but rarely co-exist. Concurrent RET/EGFR mutations have been reported in patients with NSCLC who develop resistance to EGFR tyrosine kinase inhibitors but are even less frequent in treatment-naïve patients. Consequently, there is no standard treatment for RET/EGFR -mutated NSCLC. We report a case of RET/EGFR mutant NSCLC successfully treated with the oral, potent, highly selective RET inhibitor selpercatinib (160 mg daily for 28-day cycles) in an ongoing phase II study in Chinese patients with NSCLC (LIBRETTO-321). The patient, a female nonsmoker, was diagnosed with de-novo left lung adenocarcinoma with neuroendocrine differentiation, and a RET fusion was detected by next-generation sequencing testing. The patient had two tumors in the pleura, a third in the subcarinal lymph node, and a nontarget tumor in the pleura. Pleural biopsy analysis confirmed a RET fusion KIF5B (K15;R12) and an EGFR exon 19 deletion. The patient achieved a partial response (PR) with selpercatinib (absence of target tumors in pleura and reduction in the size of lymph node tumor). The PR persisted for 14.7 months, with disease progression in the nontarget lesion in the pleura and a new lesion in the liver (the PR had persisted), resulting in the discontinuation of selpercatinib. The only notable adverse event was grade 3 elevated transaminase, that was effectively managed by dose reduction. These data may support the use of selpercatinib in patients with RET/EGFR co-mutated NSCLC.

Chromosomal alterations in sporadic medullary thyroid carcinoma and correlation with outcome.

Somatic copy number alterations (SCNA) involving either a whole chromosome or just one of the arms, or even smaller parts, have been described in about 88% of human tumors. This study investigated the SCNA profile in 40 well-characterized sporadic medullary thyroid carcinomas by comparative genomic hybridization array. We found that 26/40 (65%) cases had at least one SCNA. The prevalence of SCNA, and in particular of chromosome 3 and 10, was significantly higher in cases with a RET somatic mutation. Similarly, SCNA of chromosomes 3, 9, 10 and 16 were more frequent in cases with a worse outcome and an advanced disease. By the pathway enrichment analysis, we found a mutually exclusive distribution of biological pathways in metastatic, biochemically persistent and cured patients. In particular, we found gain of regions involved in the intracellular signaling and loss of regions involved in DNA repair and TP53 pathways in the group of metastatic patients. Gain of regions involved in the cell cycle and senescence were observed in patients with biochemical disease. Finally, gain of regions associated with the immune system and loss of regions involved in the apoptosis pathway were observed in cured patients suggesting a role of specific SCNA and corresponding altered pathways in the outcome of sporadic MTC.

Isthmin-1 (Ism1) modulates renal branching morphogenesis and mesenchyme condensation during early kidney development.

The outgrowth of epithelial bud followed by reiterated bifurcations during renal development is driven by the ligand-receptor interactions between the epithelium and the surrounding mesenchyme. Here, by exploring ligand-receptor interactions in E10.5 and E11.5 kidneys by single cell RNA-seq, we find that Isthmin1 (Ism1), a secreted protein, resembles Gdnf expression and modulates kidney branching morphogenesis. Mice deficient for Ism1 exhibit defective ureteric bud bifurcation and impaired metanephric mesenchyme condensation in E11.5 embryos, attributable to the compromised Gdnf/Ret signaling, ultimately leading to renal agenesis and hypoplasia/dysplasia. By HRP-induced proximity labelling, we further identify integrin α8ß1 as a receptor of Ism1 in E11.5 kidney and demonstrate that Ism1 promoted cell-cell adhesion through interacting with Integrin α8ß1, the receptor whose activation is responsible for Gdnf expression and mesenchyme condensation. Taken together, our work reveals Ism1 as a critical regulator of cell-cell interaction that modulates Gdnf/Ret signaling during early kidney development.

[RET Gene Alterations in Thyroid Cancer-Towards Treatment with Selective RET Inhibitors].

Rearranged during transfection(RET)is one of the driver genes in thyroid cancer, which encodes a receptor tyrosine kinase. There are 2 types of genomic alterations of RET seen in thyroid cancer. Fusions of the RET tyrosine kinase domain region with partner genes are observed in papillary thyroid cancer, whereas RET mutations are observed in hereditary and sporadic medullary thyroid cancers. These alterations constantly activate downstream signaling pathways, leading to oncogenesis. Recently, selective RET inhibitors have been developed and approved overseas and in Japan for the treatment of RET-altered thyroid and lung cancers, and it will be important to detect genomic alterations in the RET gene using methods including companion diagnostics in the future.

High expression of the RET receptor tyrosine kinase and its ligand GDNF identifies a high-risk subset of estrogen receptor positive breast cancer.

PURPOSE: Resistance to endocrine therapy is the primary cause of treatment failure and death in patients with ER-positive (ER +)/luminal breast cancer. Expression and activation of the RET receptor tyrosine kinase may be driving poor outcomes. We aim to identify high-risk patients and druggable pathways for biomarker-based clinical trials. METHODS: We obtained batch-normalized mRNA expression data from Breast Invasive Carcinoma-The Cancer Genome Atlas, PanCancer Atlas (BRCA-TCGA). To determine clinically significant cutoffs for RET expression, patients were grouped at different thresholds for Kaplan-Meier plotting. Differential gene expression (DGE) analysis and enrichment for gene sets was performed. transcriptomic dataset of antiestrogen-treated ER + tumors stratified by clinical response was then analyzed. RESULTS: High RET expression was associated with worse outcomes in patients with ER + tumors, and stratification was enhanced by incorporating GDNF expression. High RET/GDNF patients had significantly lower overall survival (HR = 2.04, p = 0.012), progression-free survival (HR = 2.87, p < 0.001), disease-free survival (HR = 2.67, p < 0.001), and disease-specific survival (HR = 3.53, p < 0.001) than all other ER + patients. High RET/GDNF tumors were enriched for estrogen-independent signaling and targetable pathways including NTRK, PI3K, and KRAS. Tumors with adaptive resistance to endocrine therapy were enriched for gene expression signatures of high RET/GDNF primary tumors. CONCLUSION: Expression and activation of the RET receptor tyrosine kinase may be driving poor outcomes in some patients with ER + breast cancer. ER + patients above the 75th percentile may benefit from clinical trials with tyrosine kinase inhibitors.

RET signaling in breast cancer therapeutic resistance and metastasis.

RET, a single-pass receptor tyrosine kinase encoded on human chromosome 10, is well known to the field of developmental biology for its role in the ontogenesis of the central and enteric nervous systems and the kidney. In adults, RET alterations have been characterized as drivers of non-small cell lung cancer and multiple neuroendocrine neoplasms. In breast cancer, RET signaling networks have been shown to influence diverse functions including tumor development, metastasis, and therapeutic resistance. While RET is known to drive the development and progression of multiple solid tumors, therapeutic agents selectively targeting RET are relatively new, though multiple multi-kinase inhibitors have shown promise as RET inhibitors in the past; further, RET has been historically neglected as a potential therapeutic co-target in endocrine-refractory breast cancers despite mounting evidence for a key pathologic role and repeated description of a bi-directional relationship with the estrogen receptor, the principal driver of most breast tumors. Additionally, the recent discovery of RET enrichment in breast cancer brain metastases suggests a role for RET inhibition specific to advanced disease. This review assesses the status of research on RET in breast cancer and evaluates the therapeutic potential of RET-selective kinase inhibitors across major breast cancer subtypes.