Rearranged During Transfection Gene Research Articles

IN SILICO ANALYSIS OF IDENTIFIED MOST FREQUENT MUTATIONS IN EXON 10, CODON 618 OF THE HUMAN RET GENE

Background: The Rearranged during Transfection (RET) gene mutations are known to be responsible for Hirschsprung disease (HSCR), as well as the multiple endocrine neoplasia (MEN) types 2A, 2B and familial medullary thyroid carcinoma (FMTC). Aim: In this study we analyse mutations of codon 618 (Cys618Phe, Cys618Arg, Cys618Ser, Cys618Tyr) in exon 10, in terms of their structural similarities, stability, conservation and interaction with their close interactor GFRA3 protein. Material and Methods: In the present study, multiple bioinformatics software was used, including HGMD and UniProt for the data collection. The structural prediction of different mutations at codon 618 was performed by using PSIPRED for secondary structure prediction and I-TASSER for 3D structure modelling. The mutated proteins stability was determined, using I- Mutant and MuPro, while the evolutionary amino acids conservation was assessed with the ConSurf tool. The wildtype RET and mutant proteins were independently docked with their nearby interactor protein GFRA3.Findings: The analysis indicates that among the examined mutations, the Cys618Phe displayed the greatest structural and functional similarity to the wildtype RET protein. The interaction analysis suggested that the Cys618Phe demonstrated stronger binding with GFRA3 compared to other mutations. Conclusion: This study highlights the impact of codon 618 mutations in exon 10 of the RET gene on protein stability, structure, and interactions with GFRA3. We find mutation Cys618Phe comparatively similar to RET wildtype protein while dissimilar mutation Cys618Tyr. These findings could help a molecular geneticist in analyzing mutations at codon 618 for both research and diagnostic purpose.

A case of selpercatinib treatment for anaplastic thyroid carcinoma resulting in abscess formation.

Rearranged during transfection (RET) gene abnormality is a driver gene mutation that causes thyroid cancer, and selpercatinib has been shown to be useful for treating thyroid cancer with RET gene abnormalities. Anaplastic thyroid cancer is a disease with an extremely poor prognosis with no standard treatment established, and there are only one case reports of the efficacy of selpercatinib for RET fusion gene-positive anaplastic thyroid cancer. We herein report our experience treating an old Japanese woman with unresectable anaplastic thyroid cancer with selpercatinib. Surgical resection was initially attempted but was not possible due to adhesion to the common carotid artery. Postoperative genetic testing was positive for the RET fusion gene, and selpercatinib was administered. However, the administration had to be stopped due to the formation of an abscess on day 14 and a pharyngeal fistula on day 17, after which the tumor grew rapidly, and the patient died on day 65. Although selpercatinib has been reported to have a high safety profile with few adverse events, this case suggests that caution should be exercised when treating anaplastic thyroid cancer with invasion to vital organs.

30years of prophylactic thyroidectomy for hereditary medullary thyroid cancer : Amilestone in translational medicine

Medullary thyroid cancer (MTC) is the most frequent manifestation of multiple endocrine neoplasia type 2 (MEN2) that determines the oncological outcome. Germline mutations in the rearranged during transfection (RET) protooncogene, atumor suppressor gene on chromosome 10q11.2, were identified 30 years ago as the genetic basis of MEN2 and published in 1993 and 1994. These seminal findings gave rise to the concept of prophylactic thyroidectomy for asymptomatic gene mutation carriers based on apositive RET gene test, which has become the standard of care ever since. Clinical genetic investigations showed genotype-phenotype correlations with respect to the individual gene mutation regarding the penetrance and onset of MTC and to alesser extent also with respect to the other components of MEN2, pheochromocytoma and primary hyperparathyroidism. From this a clinically relevant risk stratification could be derived. Initially, the optimal timing of prophylactic thyroidectomy was primarily based on the RET genotype alone, which was not sufficient for a precise age recommendation and subsequently required additional consideration of calcitonin serum levels for fine tuning. Calcitonin levels first show the risk of lymph node metastasis when they exceed the upper normal limit of the assay independent of carrier age and RET mutation. Routine calcitonin screening of patients with nodular thyroid disease, screening of families on identification of MEN2 index patients, and pre-emptive thyroidectomy in carriers of gene mutations with normal calcitonin levels have led to the fact that nowadays, 30years after the first description of the gene mutations causing the disease, the life-threatening hereditary MTC has become curable: ashining example for the success of translational transnational medical research for the benefit of patients.

Selpercatinib-induced hepatotoxicity: A rare case report

Abstract Introduction/Objective Selpercatinib (Retevmo) is a highly selective rearranged during transfection (RET) kinase inhibitor indicated for treatment of solid tumors with RET gene mutation or fusion, such as medullary thyroid carcinoma or non-small cell lung carcinoma. Among selpercatinib’s side effects, severe hepatotoxicity was only seen in 3% of the patients. Here we report a case of selpercatinib-induced hepatotoxicity, which has not been well characterized before. Methods/Case Report The patient was a 56-year-old female who had medullary thyroid carcinoma with RET M918T mutation (pT3aN1b) status post total thyroidectomy. Recurrence occurred and selpercatinib treatment was initiated. Then the patient complained of mouth sores, tongue sensitivity, and lower extremity cramping. The dosage was reduced to half. The magnetic resonance imaging on day 67 of selpercatinib treatment showed heterogeneous enhancement in the right hepatic lobe, compatible with parenchymal liver disease/hepatitis, trace perihepatic ascites, and mild splenomegaly. On day 71, liver function abnormalities were detected (ALK 315 IU/L, ALT 745 IU/L, AST 899 IU/L, total bilirubin 7.7 mg/dL). Workups including autoimmune hepatitis serology, viral hepatitis, or other infections were all negative. She had no history of other drug use. On day 72, a liver biopsy was performed that demonstrated mild to moderate portal lymphocyte-predominant mixed inflammation with interface activity. The lobules showed marked inflammation with hepatocyte disorganization, acidophil bodies, hepatocyte dropout, and rare lipogranulomas. Bile duct injury and patchy canalicular cholestasis were also noted. Trichrome stain showed focal bridging fibrosis. Overall, the histologic findings were acute and chronic hepatitis pattern injury, favor selpercatinib-induced hepatotoxicity. Selpercatinib was discontinued. All liver enzymes gradually returned to normal levels on day 169. Results (if a Case Study enter NA) NA Conclusion Our study suggests that screening patients for abnormal liver function tests prior to and monitoring periodically during selpercatinib treatment is necessary. Based on the severity of hepatotoxicity, withholding, reducing the dose, or permanently discontinuing selpercatinib is recommended.

SAT535 ANKRD26-RET Fusion Positive Metastatic Papillary Thyroid Carcinoma - Advanced Presentation of a Rare Mutation

Abstract Disclosure: H. Hashmi: None. A. Ankireddypalli: None. L. Burmeister: None. A. Nelson: None. K. Amin: None. Background: While rearranged during transfection (RET) protooncogene mutations have been commonly associated with thyroid malignancy, ankyrin repeat domain containing protein 26 (ANKRD26) has been primarily linked to hematologic malignancy. Exon 29 of ANKRD26 (Chromosome [Chr] 10p12.1) has been shown to fuse with exon 12 of RET (Chr 10q11.21), resulting in a genetic fusion mutation ANKRD26:RET. This is a rare genetic mutation in papillary thyroid cancer with only one previously reported case. We report the first case in the US of this novel genetic mutation with a unique clinical presentation. Clinical Presentation: A 48-year-old, male patient presented with lower back pain radiating to the lower extremities of one year duration. MRI lumbar spine revealed an irregular mass lesion in the L1 vertebral body with extraosseous soft tissue extension, L1 compression fracture and extension into the T12 body. CT CAP revealed a 4.8 x 3.6 cm, heterogenous right thyroid lobe with tracheal compression and sub centimeter pulmonary nodules in the left lower and right middle lobes. Biopsy of the L1 paravertebral mass was diagnostic for metastatic thyroid carcinoma of a follicular pattern. Molecular testing with a next generation sequencing fusion profile assay was positive for ANKRD26:RET gene rearrangement. Thyroid ultrasound was notable for a solid, hypoechoic, 3.2 x 3.1 x 2.9 cm right lobe nodule with echogenic foci and level 4 lymph nodes with loss of reniform shape and fatty hilum. FNA of the nodule and right level 4 lymph node was suspicious for follicular neoplasm. The patient was admitted for acute cord compression necessitating extensive laminectomy, vertebral reconstruction and resection of the L1 paraspinal tumor. Pathology was positive for metastatic, follicular variant, papillary thyroid carcinoma. Total thyroidectomy, central and right modified radical neck dissection was performed. Pathology demonstrated a 4.2 cm papillary carcinoma, follicular type with tall cell variant; confined to the thyroid. Extensive angioinvasion (≥ 4 vessels) and lymphatic invasion was present. 10/32 positive lymph nodes. TNM grading was T3aN1bM1. Summary: The only other case of ANKRD26-RET fusion positive papillary thyroid cancer in the literature was reported by Staubitz et al in Germany in 2019. They reported a case of a 78-year-old male with follicular variant papillary thyroid carcinoma (pT3,pN0,M0). To our knowledge, our case is the second worldwide and the first to be reported in the United States of this rare fusion protein positive thyroid cancer. Such an aggressive presentation with distant osseous metastases has to our knowledge not been previously described. Conclusion: The clinical and therapeutic implications of this rare genetic mutation are not currently well elucidated. We present this case with the intent to expand the existing literature and increase recognition of this rare genetic mutation. Presentation Date: Saturday, June 17, 2023

Expert consensus on the diagnosis and treatment of RET gene fusion non-small cell lung cancer in China.

The rearranged during transfection (RET) gene is one of the receptor tyrosine kinases and cell-surface molecules responsible for transmitting signals that regulate cell growth and differentiation. In non-small cell lung cancer (NSCLC), RET fusion is a rare driver gene alteration associated with a poor prognosis. Fortunately, two selective RET inhibitors (sRETi), namely pralsetinib and selpercatinib, have been approved for treating RET fusion NSCLC due to their remarkable efficacy and safety profiles. These inhibitors have shown the ability to overcome resistance to multikinase inhibitors (MKIs). Furthermore, ongoing clinical trials are investigating several second-generation sRETis that are specifically designed to target solvent front mutations, which pose a challenge for first-generation sRETis. The effective screening of patients is the first crucial step in the clinical application of RET-targeted therapy. Currently, four methods are widely used for detecting gene rearrangements: next-generation sequencing (NGS), reverse transcription-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). Each of these methods has its advantages and limitations. To streamline the clinical workflow and improve diagnostic and treatment strategies for RET fusion NSCLC, our expert group has reached a consensus. Our objective is to maximize the clinical benefit for patients and promote standardized approaches to RET fusion screening and therapy.

Maternal Vitamin A Status as a Risk Factor of Hirschsprung Disease in the Child.

The gene-environment interaction of the REarranged during Transfection ( RET ) gene with vitamin A in the etiopathogenesis of Hirschsprung disease (HSCR) has been suggested in rodents. The aim of this study was to evaluate vitamin A status in mothers of children with HSCR and to assess its association with pathogenic variants of the RET gene in affected children. This was a case-control study of stable isotope-based vitamin A measurement stores of mothers of children diagnosed with HSCR (within 8 months from birth, n = 7) and age-matched mothers of normal children (n = 6). Next-generation sequencing of RET exons, along with their upstream promoter region, was performed in the 7 HSCR proband-parent triads to evaluate pathogenic variants. Maternal vitamin A stores in the HSCR group was almost 50% that of those in controls, tending toward significance (0.50 ± 0.17 vs 0.89 ± 0.51 μmol/g respectively, P = 0.079). Two novel pathogenic de novo mutations were identified in 2 cases, and a rare single-nucleotide deletion was detected in the 3.5-kb RET upstream region, in a heterozygous state, in all 7 proband-parent triads. Low-penetrance RET haplotypes associated with HSCR were detected in 5 cases. Mothers with children with HSCR had lower vitamin A liver stores than mothers with normal children, and the children who were affected had HSCR despite having no established pathogenic RET variants. Lower maternal vitamin A status may increase the penetrance of genetic mutations in RET , and vitamin-A mediated gene-environment interactions may underpin some of the etiology of HSCR.

Response to Pralsetinib in Multi-Drug-Resistant Breast Cancer With CCDC6-RET Mutation.

Triple-negative breast cancers (TNBC) represent a pathological subtype of breast cancer, which are characterized by strong invasiveness, high metastasis rate, low survival rate, and poor prognosis, especially in patients who have developed resistance to multiline treatments. Here, we present a female patient with advanced TNBC who progressed despite multiple lines of treatments; next-generation sequencing (NGS) was used to find drug mutation targets, which revealed a coiled-coil domain-containing protein 6 (CCDC6)-rearranged during transfection (RET) gene fusion mutation. The patient was then given pralsetinib, and after one treatment cycle, a CT scan revealed partial remission and adequate tolerance to therapy. Pralsetinib (BLU-667) is a RET-selective protein tyrosine kinase inhibitor that can inhibit the phosphorylation of RET and downstream molecules as well as the proliferation of cells expressing RET gene mutations. This is the first case in the literature of metastatic TNBC with CCDC6-RET fusion treated with pralsetinib, an RET-specific antagonist. This case demonstrates the potential efficacy of pralsetinib in cases of TNBC with RET fusion mutations and suggests that NGS may reveal new opportunities and bring new therapeutic interventions to patients with refractory TNBC.

Assessment of In Silico and In Vitro Selpercatinib Metabolic Stability in Human Liver Microsomes Using a Validated LC-MS/MS Method.

Selpercatinib (SLP; brand name Retevmo®) is a selective and potent RE arranged during transfection (RET) inhibitor. On 21 September 2022, the FDA granted regular approval to SLP (Retevmo, Eli Lilly, and Company). It is considered the only and first RET inhibitor for adults with metastatic or locally advanced solid tumors with RET gene fusion. In the current experiment, a highly specific, sensitive, and fast liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying SLP in human liver microsomes (HLMs) was developed and applied to the metabolic stability evaluation of SLP. The LC-MS/MS method was validated following the bioanalytical methodology validation guidelines outlined by the FDA (linearity, selectivity, matrix effect, accuracy, precision, carryover, and extraction recovery). SLP was detected by a triple quadrupole detector (TQD) using a positive ESI source and multiple reaction monitoring (MRM) mode for mass spectrometric analysis and estimation of analytes ions. The IS-normalized matrix effect and extraction recovery were acceptable according to the FDA guidelines for the bioanalysis of SLP. The SLP calibration standards were linear from 1 to 3000 ng/mL HLMs matrix, with a regression equation (y = 1.7298x + 3.62941) and coefficient of variation (r2 = 0.9949). The intra-batch and inter-batch precision and accuracy of the developed LC-MS/MS method were -6.56-5.22% and 5.08-3.15%, respectively. SLP and filgotinib (FLG) (internal standard; IS) were chromatographically separated using a Luna 3 µm PFP (2) stationary phase (150 × 4.6 mm) with an isocratic mobile phase at 23 ± 1 °C. The limit of quantification (LOQ) was 0.78 ng/mL, revealing the LC-MS/MS method sensitivity. The intrinsic clearance and in vitro t1/2 (metabolic stability) of SLP in the HLMs matrix were 34 mL/min/kg and 23.82 min, respectively, which proposed an intermediate metabolic clearance rate of SLP, confirming the great value of this type of kinetic experiment for more accurate metabolic stability predictions. The literature review approved that the established LC-MS/MS method is the first developed and reported method for quantifying SLP metabolic stability.

A comprehensive overview of the relationship between RET gene and tumor occurrence.

RET gene plays significant roles in the nervous system and many other tissues. Rearranged during transfection (RET) mutation is related to cell proliferation, invasion, and migration. Many invasive tumors (e.g., non-small cell lung cancer, thyroid cancer, and breast cancer) were found to have changes in RET. Recently, great efforts have been made against RET. Selpercatinib and pralsetinib, with encouraging efficacy, intracranial activity, and tolerability, were approved by the Food and Drug Administration (FDA) in 2020. The development of acquired resistance is inevitable, and a deeper exploration should be conducted. This article systematically reviewed RET gene and its biology as well as the oncogenic role in multiple cancers. Moreover, we also summarized recent advances in the treatment of RET and the mechanism of drug resistance.

Selpercatinib: A Review in Advanced RET Fusion-Positive NSCLC.

Selpercatinib (Retevmo®/Retsevmo®) is an orally-administered, selective inhibitor of rearranged during transfection (RET) kinase approved for the treatment of advanced RET fusion-positive non-small cell lung cancer (NSCLC). In a pivotal phase 1/2 clinical trial in this population, selpercatinib treatment was associated with robust and durable responses, including intracranial responses, in patients previously treated with platinum-based chemotherapy, as well as in treatment-naïve patients. Selpercatinib had a manageable tolerability profile and an acceptable safety profile; adverse events could generally be managed with dose reductions and only a small proportion of patients discontinued selpercatinib due to treatment-related adverse events. The most common treatment-related adverse events that were grade 3-4 in severity were hypertension, elevated alanine aminotransferase and elevated aspartate aminotransferase. Thus, currently available evidence suggests that selpercatinib is a promising new RET-targeted therapy option for patients with advanced RET fusion-positive NSCLC.

RET Proto-Oncogene-Not Such an Obvious Starting Point in Cancer Therapy.

Mutations and fusions of RET (rearranged during transfection) gene are detected in a few common types of tumors including thyroid or non-small cells lung cancers. Multiple kinase inhibitors (MKIs) do not show spectacular effectiveness in patients with RET-altered tumors. Hence, recently, two novel RET-specific inhibitors were registered in the US and in Europe. Selpercatinib and pralsetinib showed high efficacy in clinical trials, with fewer adverse effects, in comparison to previously used MKIs. However, the effectiveness of these new drugs may be reduced by the emergence of resistance mutations in RET gene and activation of different activating signaling pathways. This review presents the function of the normal RET receptor, types of molecular disturbances of the RET gene in patients with various cancers, methods of detecting these abnormalities, and the effectiveness of modern anticancer therapies (ranging from immunotherapies, through MKIs, to RET-specific inhibitors).

AST-487 Inhibits RET Kinase Driven TERT Expression in Bladder Cancer.

Mutations in the promoter of the human Telomerase Reverse Transcriptase (hTERT) gene are common and associated with its elevated expression in bladder cancer, melanoma, and glioblastoma. Though these mutations and TERT overexpression are associated with aggressive disease and poor outcome, an incomplete understanding of mutant TERT regulation limits treatment options directed at this gene. Herein, we unravel a signaling pathway that leads to upregulated hTERT expression resulting from the −124 bp promoter mutation, the most frequent variant across human cancer. We employed engineered bladder cancer cells that harbor a GFP insertion at the TSS region on −124 hTERT promoter for high-content screening drug discovery using a focused library of ~800 kinase inhibitors. Studies using in vitro and in vivo models prioritized AST-487, an inhibitor of the wild-type, and mutant RET (rearranged during transfection) proto-oncogene as a novel drug inhibitor of both wild-type and mutant promoter-driven hTERT expression. We also identified the RET kinase pathway, targeted by AST-487, as a novel regulator of mutant hTERT promoter-driven transcription in bladder cancer cells. Collectively, our work provides new potential precision medicine approaches for cancer patients with upregulated hTERT expression, perhaps, especially those harboring mutations in both the RET gene and the hTERT promoter, such as in thyroid cancer.

Discrepancies of RET gene and risk of differentiated thyroid carcinoma.

Somatic variations in rearranged during transfection (RET) proto-oncogene acts to influence Thyroid cancer (TC) in a low penetrance manner, but their effects tend to vary between different populations. This case-control study was aimed to evaluate effect of RET G691S, S904S and L769L single nucleotide polymorphisms (SNPs) on the risk for differentiated thyroid carcinoma (DTC). A total of 180 patients and 220 controls were genotyped by Polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). Di-Deoxy Sanger sequencing was performed on 100 samples with variations and 20 wild samples for each amplified exon. In addition, In Silico tools were used to evaluate structural and functional impact of individual SNPs in disease progression. In RET G691S/L769L/S904S SNPs, frequency of variant genotypes in DTC cases was 61.1%, 54.4% and 76.6% as compared to 45.9%, 43.6% and 89.09% in controls respectively (P⩽ 0.05). In Silico analysis revealed that different protein formed due to G691S substitution decreases the stability of 3D structure of protein. The RET G691S and L769L SNP followed "Dominant" but RET S904S SNP confirmed an "Additive" mode of inheritance. RET G691S/L769L/S904S SNPs are significantly associated with DTC with G691S SNP declining the stability of final protein product.

Advances in the Treatment of RET Fusion-positive Advanced Non-small Cell Lung Cancer

转染重排（rearranged during transfection, RET）融合阳性发生于0.7%-2%的非小细胞肺癌（non-small cell lung cancer, NSCLC）。RET基因与其他结构域之间的融合代表了NSCLC独特的生物学和临床病理学亚型。近些年以来，RET融合阳性晚期NSCLC治疗领域取得了重要进展。传统化疗能够带来一定的临床获益。在靶向药物临床应用之前或不适用的情况下，以铂类为基础的系统性化疗方案是患者初始治疗选择。免疫治疗在RET融合阳性人群中的报告数据较少且通常结果较差，不推荐免疫单药或免疫联合化疗方案作为这类患者的系统性治疗。多靶点激酶抑制剂在这类人群中的重新应用能够取得一定的治疗反应，目前仅卡博替尼和凡德他尼推荐在有限条件下作为初始或后续治疗。然而，依然存在不能满足的临床需求。普拉替尼（Pralsetinib）和塞尔帕替尼（Selpercatinib）作为RET选择性酪氨酸激酶抑制剂（tyrosine kinase inhibitor, TKI），显著改善了RET融合阳性NSCLC患者生存及预后。普拉替尼和塞尔帕替尼已确立成为这类患者一线或后续治疗的首选方案。与其他TKI治疗时观察到的类似，RET靶向抑制治疗也会导致耐药问题，而获得性耐药的出现将影响治疗的远期有效性，并限制后续的药物选择。因此，本文将围绕RET融合阳性晚期NSCLC治疗进展进行综述。

Precision medicine in aggressive thyroid cancer: Moving beyond multitargeted tyrosine kinase inhibitors

Precision medicine in aggressive thyroid cancer: Moving beyond multitargeted tyrosine kinase inhibitors

RET Proto-Oncogene Mutational Analysis in 45 Iranian Patients Affected with Medullary Thyroid Carcinoma: Report of a New Variant.

Background The aim of this study was to identify germline mutation of the RET (rearranged during transfection) gene in patients with medullary thyroid carcinoma (MTC) and their first-degree relatives to find presymptomatic carriers for possible prophylactic thyroidectomy. Methods/Patients. We examined all six hot spot exons (exons 10, 11, 13, and 14–16) of the RET gene by PCR and bidirectional Sanger sequencing in 45 Iranian patients with MTC (either sporadic or familial form) from 7 unrelated kindred and 38 apparently sporadic cases. First-degree relatives of RET positive cases were also genotyped for index mutation. Moreover, presymptomatic carriers were referred to the endocrinologist for further clinical management and prophylactic thyroidectomy if needed. Results Overall, the genetic status of all of the participants was determined by RET mutation screening, including 61 affected individuals, 22 presymptomatic carriers, and 29 genetically healthy subjects. In 37.5% (17 of 45) of the MTC referral index patients, 8 distinct RET germline mutations were found, including p.C634R (35.3%), p.M918T (17.6%), p.C634Y (11.8%), p.C634F (5.9%), p.C611Y (5.9%), p.C618R (5.9%), p.C630R (5.9%), p.L790F (5.9%), and one uncertain variant p.V648I (5.9%). Also, we found a novel variant p.H648R in one of our apparently sporadic patients. Conclusion RET mutation detection is a promising/golden screening test and provides an accurate presymptomatic diagnostic test for at-risk carriers (the siblings and offspring of the patients) to consider prophylactic thyroidectomy. Thus, according to the ATA recommendations, the screening of the RET proto-oncogene is indicated for patients with MTC.

Higher RET Gene Expression Levels Do Not Represent anAlternative RET Activation Mechanism in Medullary Thyroid Carcinoma.

This study was designed to investigate whether RET (rearranged during transfection) mRNA over-expression could be considered an alternative driver event for the development of medullary thyroid carcinoma (MTC), and if different RET isoforms could play a role in MTC tumorigenesis. Eighty-three MTC patients, whose mutational profile was previously identified by next-generation sequencing (NGS) IONS5, were included in this study. Expression analysis was performed by the quantitative reverse transcription-polymerase chain reaction technique. RET expression levels were found to be significantly higher in cases with RET somatic mutations than in cases that were negative for RET somatic mutations (p = 0.003) as well as in cases with a somatic mutation, either in RET or RAS than in cases negative for both these mutations (p = 0.01). All cases were positive for the RET51 isoform expression while only 72/83 (86.7%) were positive for RET9 isoform expression. A statistically significant higher expression of the RET51 isoform was found in cases positive for RET somatic mutation than in cases either positive for RAS mutation (p = 0.0006) or negative for both mutations (p = 0.001). According to our data, RET gene over-expression does not play a role in MTC tumorigenesis, neither as an entire gene or as an isoform. At variance, the RET gene, and in particular the RET51 isoform, is expressed higher in RET mutated cases. On the basis of these results we can hypothesize that the overexpression of RET, and in particular of RET51, could potentiate the transforming activity of mutated RET, making these cases more aggressive.

Targeting Rearranged during Transfection in Cancer: A Perspective on Small-Molecule Inhibitors and Their Clinical Development.

Rearranged during transfection (RET) is a receptor tyrosine kinase essential for the normal development and maturation of a diverse range of tissues. Aberrant RET signaling in cancers, due to RET mutations, gene fusions, and overexpression, results in the activation of downstream pathways promoting survival, growth, and metastasis. Pharmacological manipulation of RET is effective in treating RET-driven cancers, and efforts toward developing RET-specific therapies have increased over the last 5 years. In 2020, RET-selective inhibitors pralsetinib and selpercatinib achieved clinical approval, which marked the first approvals for kinase inhibitors specifically developed to target the RET oncoprotein. This Perspective discusses current development and clinical applications for RET precision medicine by providing an overview of the incremental improvement of kinase inhibitors for use in RET-driven malignancies.

Abstract 2450: A systemic approach to decipher the interactome of RET receptor isoforms

Abstract The REarranged during Transfection (RET) receptor tyrosine kinase is pivotal for normal tissue development, but is also an oncogene driver involved in several human cancers. Alternative splicing at the 3' end of the RET gene leads to expression of two conserved protein isoforms, RET9 and RET51, that differ in their subcellular localization and protein trafficking, as well as their functional roles in tumorigenesis and metastatic processes. Importantly, RET9 and RET51 have unique C-terminal phospho-tyrosine binding sites, suggesting that they may also differ in their interactomes. We used a combination of cell-based screening and in silico approaches to identify novel potential interaction partners of RET isoforms. We performed a Mammalian Membrane Two-Hybrid (MaMTH) screen using a library of SH2 domain-containing adaptor and signaling proteins, to identify interactions with each RET isoform. We complemented these studies by using sequence homology detection models (HMM, PSSM), based on SH2 domain sequences known to interact with RET, to rank the SH2 library members and predict novel interactions. Independently, we compared published consensus binding sequences for each SH2 domain library member with predicted RET phosphotyrosine motifs to identify potential interactors. Predicted interactions were validated in co-immunoprecipitation assays. We confirmed previously known interactions of RET with SH2 domain proteins including SHC1, GRB2 and GRB10, and identified additional novel RET-binding proteins, a subset of which showed differential interactions that were mediated through RET isoform-specific docking sites. Our results suggest that combinations of distinct interaction partners may contribute to RET isoform-specific functions. Together, our research has developed a systematic approach to map and characterize RET isoform interactions. Our data suggest that no single method identified all confirmed RET interactions, and that a combination of multiple approaches improves characterization of growth factor receptor interactomes. Citation Format: Samira Kheitan, Annika E. Pedersen, Brandy D. Hyndman, Luka Drecun, Punit Saraon, Igor Stagliar, Lois M. Mulligan. A systemic approach to decipher the interactome of RET receptor isoforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2450.