Dual targeting of RET and SRC synergizes in RET fusion-positive cancer cells.

Mechanisms of resistance to selective RET tyrosine kinase inhibitors in RET fusion-positive non-small-cell lung cancer

The rearranged during transfection (RET) gene encodes a receptor tyrosine kinase that is involved in the activation of several signal transduction pathways. Various RET fusions and point mutations were shown to play a role as oncogene drivers in different cancers. In 2020, two selective RET tyrosine kinase inhibitors (TKIs), pralsetinib and selpercatinib, were approved by the FDA for treatment of RET fusion-positive non-small cell lung cancer, RET-mutant medullary thyroid cancer and RET fusion-positive thyroid cancer. These drugs demonstrate a significant advance in efficacy and safety over previous, multi-targeted TKIs for the treatment of RET-positive patients. However, development of acquired resistance to these agents has been observed through the appearance of on-target secondary solvent front mutations in the RET G810 residue. Moreover, 5 different types of amino acid changes - G810A/C/R/S/V have been identified in this position, of which G810C, G810R and G810S are better characterized but their effect on protein activity is lacking. We used a high-throughput functional assay, in which the mutated protein is expressed together with a fluorescently labeled reporter in a cell-based assay. Using this system, we systematically assessed the activity of different combinations of RET fusions or oncogenic point mutations with one of three different common solvent front mutations and compared them to the activity of the individual mutations. Our results show that when a solvent front mutation is expressed on a RET oncogenic point mutation background, the solvent front mutation consistently decreases the oncogenic protein activity. This is supported by our observation that the individual solvent front mutations expressed on a RET WT background show an activity that is lower than that of the RET WT protein. On a RET fusion background the effect of solvent front mutations is more variable, with some RET fusion-solvent front mutation combinations showing a decreased activity and some not affecting the activity of the fusion protein. Across all mutations and fusions tested, the G810R and G810C mutations had a larger effect on RET activity than that of the G810S mutation. We also compared the effect of drug treatment (pralsetinib and selpercatinib) on RET oncogenic activity in the presence of the different solvent front mutations and observed that on all mutational backgrounds tested, the solvent front mutations have an effect on the response dynamics to the drug, and that the extent of the effect depends on the type of solvent front mutation, with G810R and G810C decreasing sensitivity to drug treatment to a larger extent compared to G810S. Together, our findings provide a better understanding of the differential effects of solvent front mutations on RET activity under different oncogenic contexts. Citation Format: Shay Rotkopf, DIkla Haham, Lea Birnbaum, Zohar Barbash, Gabi Tarcic. Analysis of RET solvent front mutations shows an effect on protein activity in tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 403.

Further Advances in Genetically Informed Lung Cancer Medicine

A Single-Tube Multiplexed Assay for Detecting ALK, ROS1, and RET Fusions in Lung Cancer

The receptor tyrosine kinase rearranged during transfection (RET) is an oncogenic driver activated in multiple cancers, including non-small cell lung cancer (NSCLC), medullary thyroid cancer (MTC), and papillary thyroid cancer. No approved therapies have been designed to target RET; treatment has been limited to multikinase inhibitors (MKI), which can have significant off-target toxicities and limited efficacy. BLU-667 is a highly potent and selective RET inhibitor designed to overcome these limitations. In vitro, BLU-667 demonstrated ≥10-fold increased potency over approved MKIs against oncogenic RET variants and resistance mutants. In vivo, BLU-667 potently inhibited growth of NSCLC and thyroid cancer xenografts driven by various RET mutations and fusions without inhibiting VEGFR2. In first-in-human testing, BLU-667 significantly inhibited RET signaling and induced durable clinical responses in patients with RET-altered NSCLC and MTC without notable off-target toxicity, providing clinical validation for selective RET targeting.Significance: Patients with RET-driven cancers derive limited benefit from available MKIs. BLU-667 is a potent and selective RET inhibitor that induces tumor regression in cancer models with RET mutations and fusions. BLU-667 attenuated RET signaling and produced durable clinical responses in patients with RET-altered tumors, clinically validating selective RET targeting. Cancer Discov; 8(7); 836-49. ©2018 AACR.See related commentary by Iams and Lovly, p. 797This article is highlighted in the In This Issue feature, p. 781.

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。

The rearranged during transfection (RET) gene is a well-known proto-oncogene and encodes a single-pass transmembrane receptor tyrosine kinase. RET fusions and point mutations are oncogenic drivers in NSCLC, medullary thyroid cancer and other solid tumors, and therefore potential targets for cancer therapy. Multikinase inhibitors targeting the RET mutations have been tested in clinical trials with moderate efficacy in terms of tumor shrinkage and PFS.Moreover, multikinase inhibitors are characterized by poor tolerability due to off-target kinase inhibitory activities. To widen the therapeutic index, a selective RET kinase inhibitor is highly desirable. TAS0286/HM05 is a novel highly selective and potent RET kinase inhibitor.RET kinase assay was performed by homogeneous time-resolved fluorescence (HTRF) method. In-vitro proliferation studies and pharmacodynamics analyses were conducted in cancer cell lines with the RET fusions and RET activating mutations. The antitumor efficacy of TAS0286/HM05 was evaluated using mice xenograft models implanted with cancer cells with various RET gene abnormality. TAS0286/HM05 was orally administered for 14 or 28 consecutive days after grouping. In-vitro and in-vivo RET phosphorylation was detected using western blot analysis. The IC50 value for RET kinase of TAS0286/HM05 was below 1 nM. TAS0286/HM05 showed highly selective RET inhibitory activity among 283 kinases. In cellular assay, TAS0286/HM05 strongly suppressed phosphorylation of RET expressed in cells with various RET fusions and activating mutations, and inhibited cell proliferation at around 10 nM. At this concentration, apoptosis was also observed. The potency was higher than other multikinase inhibitors with RET inhibitory activity. Furthermore, in in-vivo efficacy studies, TAS0286/HM05 significantly inhibited the growth of tumors harboring various RET fusions and activating mutations at a range of 20 to 100 mg/kg/day without any body weight loss. The antitumor efficacy of TAS0286/HM05 was more potent than pre-existing multikinase inhibitors at their maximum tolerated dose. In particular, TAS0286/HM05 dramatically induced tumor regression of 40% within 15 days in animals implanted with LC-2/ad cells, a human lung adenocarcinoma cell line with CCDC6-RET fusion gene. TAS0286/HM05 is a novel and highly selective RET inhibitor with prominent tolerability. The potency of TAS0286/HM05 against tumors with RET abnormalities was stronger than currently marketed multikinase inhibitors being tested in NSCLC patients with RET fusions. TAS0286/HM05 is a promising agent for future clinical development in patients with RET gene abnormalities. Citation Format: Hidenori Fujita, Isao Miyazaki, Masanori Kato, Yukari Yamada, Keiji Ishida, Tomonori Haruma, Haruka Nagasaki, Kenjiro Ito, Akihiro Hashimoto, Yasuo Kodama, Kaoru Funabashi, Emanuela Lovati, Kazutaka Miyadera, Kenichi Matsuo, Yoshikazu Iwasawa. TAS0286/HM05, a novel highly selective RET inhibitor, prominently inhibits various RET defective tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4784.

The rearranged during transfection (RET) tyrosine kinase is mostly altered by fusion and it makes RET as a attractive therapeutic target for medullary thyroid cancer and non-small cell lung cancer (NSCLC). Multi-kinase inhibitors (mKIs) such as cabozantinib and vandetanib expressed poor tolerability and limited efficacy due to off-target kinase inhibitory activity in academia and clinical cases. As for selective RET inhibitor, compounds such as BLU-667 and LOXO-292 are under development, currently in clinical trials. We are currently developing potent and selective RET inhibitors to address unmet medical needs for RET-driven cancers. Our team has validated its high potency and selectivity via kinase profiling and in-vitro assay results. We also anticipate our compound to overcome certain mutation, such as V804M. Our compounds strongly suppress phosphorylation of RET in tumor cells with various RET fusions and mutations by inhibiting proliferation at below 10 nM. This potency result is higher than those of other mKIs with RET inhibitory activity. The inhibitory concentration of our compounds to VEGFR2 is more than dozens of fold compared with RET. In addition, our compounds reveal low cytotoxicity in Ba/F3 naïve cells in the 1 µM concentration. These findings lead us to conclude that our compounds can be potent and highly selective inhibitors of RET kinase, a potential novel therapeutic agents for RET-driven cancers. Citation Format: Hua Li, SeoHyun Jo, Woomi Do, Hyunkyung Kim, Hwan Kim, Kyung-ah Seo, Ji-hoon Oh, Jieun Choi, Jaeyoung Ahn, Jung Beom Son, Nam Doo Kim. Novel therapeutic agents for RET-driven cancers, which is highly potent and selective inhibitor to RET kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2209.

Pralsetinib (PRL) is a selective Rearranged during Transfection (RET) inhibitor, developed by Blueprint Medicines Corporation for the treatment of RET fusion non-small-cell lung cancer (NSCLC), papillary thyroid cancer (PTC), and medullary thyroid carcinoma (MTC). RET is a known proto-oncogene found in NSCLC, PTC, and MTC. PRL was recently granted accelerated USFDA approval with the brand name GAVRETO™ on 4 September 2020 to treat metastatic RET fusion-positive NSCLC and was updated on 1 December 2020 with the addition of advanced and metastatic RET-altered MTC and PTC in the USA. On 19 November 2021, the European Commission granted conditional marketing authorization to PRL for use as a single agent in adult patients with RET fusion-positive advanced NSCLC. They were not previously treated with an RET inhibitor. This review article summarizes the milestones in the development of PRL, chemistry, chemical (synthesis) research and development, characterization and identification of PRL-resistant RET mutants, the structural basis of resistance to PRL, mechanism of action, pharmacokinetics, pharmacodynamic, adverse effects, and regulatory status, including ongoing clinical trial of PRL and other potential drug candidates, leading to this first approval of PRL for the treatment of various solid tumors (RET fusion NSCLC, MTC, and PTC).

Novel Human-Derived RET Fusion NSCLC Cell Lines Have Heterogeneous Responses to RET Inhibitors and Differential Regulation of Downstream Signaling

Rearranged during transfection (RET) receptor tyrosine kinase was first identified over thirty years ago as a novel transforming gene. Since its discovery and subsequent pathway characterization, RET alterations have been identified in numerous cancer types and are most prevalent in thyroid carcinomas and non-small cell lung cancer (NSCLC). In other tumor types such as breast cancer and salivary gland carcinomas, RET alterations can be found at lower frequencies. Aberrant RET activity is associated with poor prognosis of thyroid and lung carcinoma patients, and is strongly correlated with increased risk of distant metastases. RET aberrations encompass a variety of genomic or proteomic alterations, most of which confer constitutive activation of RET. Activating RET alterations, such as point mutations or gene fusions, enhance activity of signaling pathways downstream of RET, namely PI3K/AKT, RAS/RAF, MAPK, and PLCγ pathways, to promote cell proliferation, growth, and survival. Given the important role that mutant RET plays in metastatic cancers, significant efforts have been made in developing inhibitors against RET kinase activity. These efforts have led to FDA approval of Selpercatinib and Pralsetinib for NSCLC, as well as, additional selective RET inhibitors in preclinical and clinical testing. This review covers the current biological understanding of RET signaling, the impact of RET hyperactivity on tumor progression in multiple tumor types, and RET inhibitors with promising preclinical and clinical efficacy.

e21693 Background: Fusions involving the REarranged during Transfection ( RET) gene are known oncogenic drivers in non-small cell lung cancer (NSCLC). The patient profile is distinct from a genomically-unselected population of NSCLC patients and may lead to the potentially confounded conclusion that RET fusion-positive patients have a better prognosis. This study characterized clinical outcomes in patients with RET fusion-positive NSCLC versus those without when correcting for known demographic differences. Methods: The Flatiron ClinicoGenomics Database, electronic medical record data linked to Foundation Medicine, Inc genomic test results, was used to identify individuals diagnosed with advanced or metastatic NSCLC who initiated anti-cancer systemic therapy between JAN2011 and JUN2019 in the US. Follow up data were available through JUN2019. Patients were considered RET+ if a fusion was reported and RET- if no fusion was reported. Baseline characteristics and tumor response were compared using Fisher’s exact, chi-squared or t-tests; unadjusted and adjusted COX models were used to compare progression-free survival (PFS) and overall survival (OS). Results: There were 5,807 eligible patients with NSCLC; 46 (0.8%) and 5,761 (99.2%) were RET+ and RET-, respectively. Patients with RET+ tumors were significantly younger (63 vs 67 yrs), better performance status, less likely to have smoking history (37% vs 82%), more likely to have non-squamous tumors (98% vs 76%) (all p < 0.05), and less likely to have ALK, ROS1, KRAS or BRAF alterations, which were each 0% in the RET+ group. There were no differences in total lines of therapy received; treatment regimens were comparable. In unadjusted analyses, patients with RET+ tumors had no significant differences in tumor response (p = 0.17) or PFS (p = 0.06) but significantly better OS outcomes (p = 0.005) from the start of first-line therapy. After adjusting for all available baseline covariates, survival outcomes were not significantly different between RET- and RET+ groups (PFS hazard ratio [HR] = 1.24, 95% confidence interval [CI] = 0.86, 1.78, p = 0.26; OS HR = 1.53, 95% CI = 0.95, 2.44, p = 0.08). Conclusions: Patients with RET fusion-positive NSCLC have different baseline characteristics than patients without these fusions. The unadjusted differences in OS between groups appear to be driven by baseline demographic features and not the presence of the RET fusion, however further research is needed. Conclusions are limited by the rarity of RET fusions in NSCLC and subsequent small sample size.

Rearranged during transfection (RET), a receptor tyrosine kinase, is activated by glial cell line-derived neurotrophic factor family ligands. Chromosomal rearrangement or point mutations in RET are observed in patients with papillary thyroid and medullary thyroid carcinomas. Oncogenic alteration of RET results in constitutive activation of RET activity. Therefore, inhibiting RET activity has become a target in thyroid cancer therapy. Here, the anti-tumor activity of a novel RET inhibitor was characterized in medullary thyroid carcinoma cells. The indirubin derivative LDD-2633 was tested for RET kinase inhibitory activity. In vitro, LDD-2633 showed potent inhibition of RET kinase activity, with an IC50 of 4.42 nM. The growth of TT thyroid carcinoma cells harboring an RET mutation was suppressed by LDD-2633 treatment via the proliferation suppression and the induction of apoptosis. The effects of LDD-2633 on the RET signaling pathway were examined; LDD-2633 inhibited the phosphorylation of the RET protein and the downstream molecules Shc and ERK1/2. Oral administration of 20 or 40 mg/kg of LDD-2633 induced dose-dependent suppression of TT cell xenograft tumor growth. The in vivo and in vitro experimental results supported the potential use of LDD-2633 as an anticancer drug for thyroid cancers.

The rearranged during transfection (RET) is a receptor protein tyrosine kinase. Oncogenic RET fusions or mutations are found most often in non-small cell lung cancer (NSCLC) and in thyroid cancer, but also increasingly in various types of cancers at low rates. In the last few years, two potent and selective RET protein tyrosine kinase inhibitors (TKIs), pralsetinib (BLU-667) and selpercatinib (LOXO-292, LY3527723) were developed and received regulatory approval. Although pralsetinib and selpercatinib gave high overall response rates (ORRs), < 10% of patients achieved a complete response (CR). The RET TKI-tolerated residual tumors inevitably develop resistance by secondary target mutations, acquired alternative oncogenes, or MET amplification. RET G810 mutations located at the kinase solvent front site were identified as the major on-target mechanism of acquired resistance to both selpercatinib and pralsetinib. Several next-generation of RET TKIs capable of inhibiting the selpercatinib/pralsetinib-resistant RET mutants have progressed to clinical trials. However, it is likely that new TKI-adapted RET mutations will emerge to cause resistance to these next-generation of RET TKIs. Solving the problem requires a better understanding of the multiple mechanisms that support the RET TKI-tolerated persisters to identify a converging point of vulnerability to devise an effective co-treatment to eliminate the residual tumors.

9065 Background: Rearranged during transfection ( RET) gene alterations are the oncogenic driver in diverse tumor types, including RET fusions identified in 1-2% of non-small cell lung cancers (NSCLC). While RET-selective tyrosine kinase inhibitors (TKIs) selpercatinib and pralsetinib are effective, acquired drug resistance remains a challenge. Here, we report the initial results from the RETgistry, an international consortium aimed at elucidating mechanisms of resistance to RET TKIs across RET-altered solid tumors. Methods: This was a retrospective analysis performed across 16 institutions. Patients (pts) were eligible if they had advanced solid tumor harboring an oncogenic RET alteration, received ≥1 RET TKI with disease progression, and underwent resistant tumor or liquid biopsy for next-generation sequencing (NGS). Results: A total of 103 time-distinct biopsies (62 tissue, 30 plasma, 11 paired tissue/plasma) were included in analysis, obtained from 88 pts with progression on a RET-selective TKI [selpercatinib (n = 70), pralsetinib (n = 14), selpercatinib and pralsetinib sequentially (n = 4)]. Pts had the following tumor types: 72 NSCLC (69% KIF5B-RET, 21% CCDC6-RET, 10% other RET fusion), 13 medullary thyroid cancer (TC) (54% RET M918T, 46% other RET mutation), and 2 papillary and 1 anaplastic TC (all, RET fusion). Median age was 58 (range, 21-86); 48% were male with 89% never/light smokers. Median duration of RET TKI preceding biopsies [first-line, n = 32 (31%); second-line, n = 42 (41%); third-/greater-line, n = 29 (28%)] was 16.5 months (mos) (95% CI, 14.0-19.6); median PFS was 14.1 mos (95% CI, 9.3-17.0). Resistant biopsies were obtained at median 15.0 mos from TKI initiation (range, 1.8-58.8). Acquired RET mutations were detected in 14 (14%), most common being G810 substitutions in 12 (12%). Potential off-target resistance gene alterations identified in 43 cases (42%) included MET amplification (14%), BRAF V600E or fusion (2%), KRAS gain or mutation (5%), ERBB2 amplification (2%), EGFR amplification (3%), ROS1 fusion (1%), and activating PIK3CA mutation or PTEN loss (4%). No resistant lung cancer biopsy demonstrated small cell transformation. The duration of TKI therapy (HR 0.64, p = 0.11) or PFS (HR 0.75, p = 0.33) did not differ according to the presence of on-target vs off-target resistance. NGS analyses of pre-RET-selective TKI tumors (n = 92) revealed frequent co-occurring alterations in TP53 (29%) and CDKN2A/B (12%). Conclusions: On-target resistance to RET inhibition due to acquired RET mutations was less common than off-target resistance, identified in 14%. The majority of RET TKI resistance is mediated by off-target mechanisms such as bypass receptor tyrosine kinase activation. Further studies are warranted to enable the development of therapeutic strategies to address resistance in pts with RET-altered tumors. The RETgistry accrual and data analyses continue.