BACKGROUND/AIM:The REarranged during Transfection (RET) proto-oncogene fusion is a typical cancer driver gene frequently observed in thyroid and lung cancers. This study characterized the novel dynactin subunit 1 (DCTN1)-RET fusion gene and evaluated the efficacy of RET inhibitors against this fusion. MATERIALS AND METHODS:Thyroid cancer tissue DNA samples were sequenced to identify fusion genes, and an expression vector was generated using extracted RNA. Cell lines stably expressing DCTN1-RET variants, including those lacking the coiled-coil (CC) domain, were established. The functionality of these variants and therapeutic efficacy of RET inhibitors were examined both in vitro and in vivo. RESULTS:The DCTN1-RET fusion gene contains the CC domain from DCTN1 and the kinase domain from RET. Deletion of the CC domain abrogated dimer formation and reduced RET and extracellular signal-regulated kinase phosphorylation. Cells expressing DCTN1-RET exhibited enhanced proliferation and tumorigenesis in vivo. The RET inhibitor TAS0286 effectively suppressed DCTN1-RET-mediated RET autophosphorylation and tumor growth in a mouse subcutaneous tumor model. CONCLUSION:DCTN1-RET is a novel oncogenic fusion gene in thyroid cancer that promotes tumorigenesis through CC domain-mediated dimerization. It represents a potential therapeutic target for RET-specific inhibitors.

Supplementary Table S2 shows the case reports of clinical activity and AUC0-24h of TAS-121.

Futibatinib is a covalent FGFR1-4 inhibitor. In clinical trial, futibatinib demonstrated activity across a broad spectrum of FGFR-aberrant tumors, including FGFR2 fusion/rearrangement-positive cholangiocarcinoma. Some patients with acquired resistance to a prior FGFR inhibitor also experienced responses with futibatinib (Goyal et. al. Cancer Discovery 2022). In fact, futibatinib was active against multiple FGFR mutations conferring resistance to other FGFR inhibitors (Goyal et.al. Cancer Discovery 2019).

Abstract Background. Drug resistance and central nervous system (CNS) metastasis often hampers response to precision therapy for RET-rearranged solid tumors. Vepafestinib (Vepa) is a clinical-stage, potent and selective RET tyrosine-kinase inhibitor (TKI) with proven activity against on-target resistance mutations that arise post first-generation RET TKI. Here, we utilized an array of structural and pharmacokinetic methods to determine blood-brain barrier (BBB) permeability of vepa compared with other RET TKIs. Furthermore, we correlate BBB penetrability of RET inhibitors with in vivo efficacy in preclinical models of RET-rearranged intracranial solid tumors. Methods. RET inhibitors were generated by rational chemical design to identify agents which penetrate BBB more effectively than currently approved RET TKIs, and with reduced efflux transporter susceptibility. Transcellular transport assays were conducted in LLC-PK1 and MDCK II cells expressing P-gp and BCRP, respectively. Brain penetrability was assessed by total (Kp) and unbound (Kp,uu) brain:plasma concentration ratios in male Balb/c mice and microdialysis studies in freely-moving male Wistar rats. Models of CNS metastasis were generated by implanting cells labelled with luciferase into the cerebellum of mice. Studies were conducted in comparison with selpercatinib (Selp), pralsetinib (Pral) and TPX-0046. Results. Assessment of Kp and Kp,uu parameters of analogs revealed an excellent structure-activity relationship where modification at the 6-position of the pyrrolopyrimidine core resulted in improved BBB penetrability. The analog with the most favorable physiochemical and biological properties was Vepa. Vepa showed poor susceptibility to P-gp and BCRP-mediated efflux and better CNS penetrability (Kp,uu: 1.3) and retention than Sel, Pral and TPX-0046. The ratio of the observed concentrations of Vepa in microdialysates from the prefrontal cortex, cerebrospinal fluid (CSF) and plasma free fraction was approximately 1:1:1; these ratios were maintained from 2h to 6.5h after Vepaadministration (up to 8h for CSF). Importantly, Vepa was more effective than Selp at causing regression of intracranial xenograft tumors of RET fusion-driven malignancies (lung adenocarcinoma and sarcoma), thus extending survival of tumor-bearing animals. Common first-generation RET TKI resistance mutations at the solvent front (G810A/C/S/R), gatekeeper (V804L/M), hinge (Y806C) and roof (L730Q/R) regions remained sensitive to Vepa. Vepa was more selective than Selp, Pral and TPX-0046, inhibiting only RET when profiled against a panel of 256 kinases. In contrast, Selp, Pral and TPX-0045 also inhibited VEGFR2, and 4, 11 and 39 additional kinases, respectively. Conclusions. Vepafestinib is a structurally distinct and specific RET inhibitor, with superior brain penetration and retention kinetics. It is more effective than selpercatinib in controlling CNS disease. Vepafestinib is currently undergoing phase 1 and 2 clinical trial for patients with advanced solid tumors with RET alterations (margaRET, NCT04683250). Citation Format: Igor Odintsov, Kentaro Wakayama, Tom Zhang, Satoru Iguchi, Masanori Kato, Allan JW Lui, Inna Khodos, Morana Vojnic, Claudio Giuliano, Annalisa Bonifacio, Monika A Davare, Elisa de Stanchina, Emanuela Lovati, Marc Ladanyi, Isao Miyazaki, Romel Somwar. Preclinical evaluation of the blood-brain barrier permeability and intracranial efficacy of the next-generation RET inhibitor Vepafestinib [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B157.

Supplementary methods shows "Synthesis of TAS-121" and the metohds that is relevant to the experiments shown in Supplementary datas.

PDF - 243KB, Supplementary Figure S1. Growth inhibition of TAS-115 against MET amplified cancer cell lines. Supplementary Figure S2. Kinase inhibitor-induced cell damage in rat cardiomyocytes after 96 h of treatment. Supplementary Figure S3. Gene expression changes in mice bearing human gastric cancer SC-9 after TAS-115 or sunitinib treatment. Supplementary Figure S4. Anti-tumor efficacy of TAS-115 against athymic mice transplanted with MET-amplified human gastric cancer Hs746T (A), NUGC-4 (B). Supplementary Figure S5. The inhibitory activity of sunitinib against adenosine monophosphate (AMP)-activated protein kinase (AMPK). Supplementary Table S1. Kinase inhibitory activity of TAS-115 and sunitinib against 192 kinases. Supplementary Table S2. Cell growth inhibition of TAS-115, sunitinib, sorafenib and crizotinib against MET-amplified or MET-inactivated cancer cell lines. Supplementary Table S3. Median survival times (MST) in the NUGC-4 peritoneal dissemination model after TAS-115 or sunitinib treatment.

Background: Futibatinib is a next-generation covalent FGFR1-4 inhibitor. Kinase inhibitors are subject to drug resistance due to acquired mutations of their target kinase, which limits the duration of response in patients. Here we compared the time to emergence of drug resistance caused by FGFR2 kinase mutations between futibatinib and reversible ATP-competitive FGFR inhibitors. Methods: A library with random amino acid mutations in two regions (#1 and #2) of the kinase domain of FGFR2 was transfected as TEL-FGFR2 fusion into a Ba/F3 cell line, resulting in FGFR2-dependent cell growth. Using this as a model system for clonal selection, cells were exposed to futibatinib, pemigatinib and erdafitinib, and cell count measured over time. Surviving drug resistant clones were isolated and sequenced to determine the kinase domain mutations. Results: The emergence of resistant clones due to mutations in FGFR2 kinase domain occurred most rapidly in the pemigatinib (100 nM)-treated cells with 8 days from cell seeding to >500-fold increase in resistant cells compared to 2 days in the control group. In erdafitinib (100 nM) treated cells, the T500 (time to reach 500-fold cell count) was 17 days. In contrast, no resistant cells to futibatinib (40 & 100 nM) appeared during the measurement period leading to a T500 of greater than 35 days. At the concentration that completely inhibits the proliferation of BaF/3 cells expressing wild-type FGFR, treatment of the library-transfected cells with pemigatinib (12 nM), erdafitinib (12 nM), and futibatinib (3 nM) resulted in an average (n = 2) of 93, 27, and 13 resistant clones in region #1, respectively, and 28, 21 and 1 clones in region #2, respectively. Sequencing of the resistant clones for these three FGFR inhibitors identified mutations at the following key amino acid residues: N550, E566 and K642 (regulatory triad), V565 (gatekeeper region), and K660 (activation loop); these were also identified previously in patients who developed resistance to pemigatinib, infigratinib, and Debio1347. For further analysis, 10 FGFR2 mutations (N550D/K, V563L, V565I/L, E566A/G, K642I/ R and K660M) were selected those with ≥5 clones identified as resistant to any of the tested drugs. Among them, futibatinib inhibited 8 mutants and erdafitinib inhibited 5 mutants at half-maximal effective concentrations (IC50’s) comparable to that with wild-type FGFR; pemigatinib activity against all FGFR mutants was attenuated by ≥5-fold vs wild-type. Conclusions: Futibatinib, a covalent FGFR1-4 inhibitor, exhibited sustained antitumor effects compared to ATP-competitive inhibitors with fewer and later onset of acquired FGFR2 kinase resistance mutations. These data may potentially explain the numerically favorable differences in duration of response for futibatinib compared with ATP-competitive FGFR inhibitors for treatment of cholangiocarcinoma patients. No conflict of interest.

RET receptor tyrosine kinase is activated in various cancers (lung, thyroid, colon and pancreatic, among others) through oncogenic fusions or gain-of-function single-nucleotide variants. Small-molecule RET kinase inhibitors became standard-of-care therapy for advanced malignancies driven by RET. The therapeutic benefit of RET inhibitors is limited, however, by acquired mutations in the drug target as well as brain metastasis, presumably due to inadequate brain penetration. Here, we perform preclinical characterization of vepafestinib (TAS0953/HM06), a next-generation RET inhibitor with a unique binding mode. We demonstrate that vepafestinib has best-in-class selectivity against RET, while exerting activity against commonly reported on-target resistance mutations (variants in RETL730, RETV804 and RETG810), and shows superior pharmacokinetic properties in the brain when compared to currently approved RET drugs. We further show that these properties translate into improved tumor control in an intracranial model of RET-driven cancer. Our results underscore the clinical potential of vepafestinib in treating RET-driven cancers.

Supplementary Fig. S4 shows dose-dependency of Cmax (A) and AUC0-24h (B) in nude mice.

Supplementary Fig. S3 shows cytotoxicity of EGFR-TKIs for normal human epidermal keratinocytes.

Supplementary Fig. S5 shows anti-tumor effect of TAS-121 in HCC827 xenograft mouse models.

Supplementary Fig. S1 shows chemical structure and binding mode of TAS-121.

Supplementary Fig. S2 shows inhibitory activity of TAS-121 for ERBB family kinases.

Background: The emergence of secondary FGFR kinase domain (KD) mutations with FGFRi9s leads to drug resistance and disease progression. The frequency of resistance mutations with individual FGFRi9s is unknown. Using an unbiased library-based approach, we examined the development of acquired resistance with the irreversible FGFRi futibatinib and the ATP-competitive FGFRi9s erdafitinib (ERD) and pemigatinib (PEM). Methods: Mutant (mt) libraries were generated with random mutagenesis in two FGFR2 KD regions: #1 (amino acids [aa] 540-569), and #2 (aa 640-669). Library were transfected as TEL-fusions into Ba/F3 cells, resulting in FGFR2-dependent cell growth. Transfected cells were treated with FGFRi9s at concentrations suppressing parental cell line growth. Surviving drug-resistant clones were counted, isolated, and sequenced to identify the KD mutation. The inhibitory activity of FGFRi9s against resistant clones was assessed using phospho-FGFR2 ELISA. Results: Treatment of the library-transfected Ba/F3-TEL-FGFR2 KD cells with PEM, ERD, and futibatinib resulted in an average (n=2) of 93, 27, and 13 resistant clones in region #1 and 28, 21 and 1 resistant in region #2, respectively. Sequencing (table) identified, among others, the following aa mutations: N549, E565, and K641 (regulatory triad), V564 (gatekeeper region) and K659 (activation loop), also seen in patients who developed resistance to Debio1347, infigratinib, and PEM. Among FGFR mts shown (table), futibatinib inhibited 8/10, and ERD, 5/10 mts at IC50 values comparable to wild type (wt) FGFR; however, PEM activity against all FGFR mts was attenuated by ≥5-fold vs wt. Conclusions: Resistance mutations identified with this unbiased library-based in vitro approach were consistent with those observed in the clinic. Acquired drug resistance mutations were less frequent with futibatinib than with ATP-competitive FGFRi9s. Futibatinib demonstrated the most robust inhibition of drug resistant mts. Citation Format: Hiroshi Sootome, Suzuko Kato, Masanori Kato, Hiroshi Hirai. Acquired resistance to ATP-competitive and irreversible FGFR inhibitors (FGFRi9s): A library-based approach [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 1117.

Abstract RET gene fusions are oncogenic drivers in multiple tumor types and are estimated to be present in 1% to 2% of non-squamous non-small cell lung cancers (NSCLCs). Although the first-generation selective RET inhibitors selpercatinib and pralsetinib show clinical antitumor activity in NSCLCs, acquired resistances driven by RET solvent front mutation (G810R/S/C) have emerged. In such cases, no other treatment options are currently available for these patients after relapse with selpercatinib or pralsetinib. TAS0953/HM06, a novel adenosine triphosphate-competitive and highly selective RET inhibitor, is a next-generation RET inhibitor that may overcome solvent front mutant resistance. We performed a panel test of 254 kinases and found that 23-nM TAS0953/HM06 displayed only RET inhibition as plotted over 50% inhibited kinases. TAS0953/HM06 potently inhibits recombinant wild-type (WT) RET at sub-nanomolar concentrations that are similar to the IC50 values of selpercatinib and pralsetinib in WT RET. We investigated the cellular potencies against RET WT fusions and RET mutations, including the gatekeeper mutations V804L/M and selpercatinib- or pralsetinib-resistant mutations G810R/S, in engineered BA/F3 cells. TAS0953/HM06 inhibited the growth of the RET fusion overexpressed in Ba/F3 cells, including both the V804M/L and G810R/S RET mutations and WT RET. In addition, we examined the crystal structure of the RET kinase domain complexed with an analogue of TAS0953/HM06. X-ray crystal structures of the complex revealed that the analogue has unique binding mode to RET respect to selpercatinib and pralsetinib: it does not fill the space in the direction of the side chain of G810, suggesting that TAS0953/HM06 effectively circumvents steric hindrance from solvent front substitutions. This feature likely contributes to the ability of TAS0953/HM06 to maintain its biological potency in G810 mutations. Finally, we evaluated the antitumor activity of various doses of TAS0953/HM06 in xenograft tumor model derived from Ba/F3 cells from mice expressing WT or G810R KIF5B-RET (these mice show resistance to selpercatinib and pralsetinib). A Doses as low as of 10 mg/kg twice a day, TAS0953/HM06 were able to significantly inhibit tumor growth and phospho-RET. Taken together, the results show that, in addition to its selective high potency against WT RET, TAS0953/HM06 shows significant potency against G810R solvent front mutation which is highly resistant to selpercatinib and pralsetinib, in vitro and in vivo. Therefore, TAS0953/HM06 might represent a new therapeutic option for patients with RET mutations, including those resistant to 1st generation selective RET inhibitors. The safety and efficacy of TAS0953/HM06 are currently being investigated in a phase 1/2 clinical trials (NCT04683250). Citation Format: Isao Miyazaki, Keiji Ishida, Masanori Kato, Tatsuya Suzuki, Hidenori Fujita, Shuichi Ohkubo, Yoshikazu Iwasawa. Discovery of TAS0953/HM06, a novel next generation RET-specific inhibitor capable of inhibiting RET solvent front mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P06-02.

Abstract TAS-121 is a novel orally active selective covalent inhibitor of the mutant EGFR. We performed preclinical characterization of TAS-121 and compared its efficacy and selectivity for common EGFR mutations (Ex19del and L858R), first- and second- generation EGFR-tyrosine kinase inhibitor (EGFR-TKI) resistance mutation (T790M), and uncommon mutations (G719X and L861Q) with those of other EGFR-TKIs. We also commenced investigation of the clinical benefits of TAS-121. The IC50 for intracellular EGFR phosphorylation was determined by using Jump-In GripTite HEK293 cells transiently transfected with EGFR expression vectors. Mouse xenograft models were used to evaluate the antitumor activity of TAS-121. TAS-121 potently inhibited common activating and resistance EGFR mutations to the same extent as another third-generation EGFR-TKI (osimertinib). In addition, TAS-121 showed equivalent inhibitory activity against some uncommon mutations such as G719X and L861Q. Furthermore, TAS-121 demonstrated greater selectivity for mutant EGFRs versus the wild-type EGFR compared with other EGFR-TKIs. Moreover, TAS-121 displayed antitumor activity in SW48 (EGFR G719S) and NCI-H1975 (EGFR L858R/T790M) xenograft models, and achieved an objective response in patients with NSCLC with EGFR mutations including G719A mutation. In conclusion, TAS-121 is a novel third-generation EGFR-TKI and demonstrates antitumor activities in patients with NSCLC expressing either common or uncommon EGFR mutations.

Title: TAS-121, TAS-121 shows effect on EGFR G719X mutation. Abstract TAS-121 is a novel orally active selective covalent inhibitor of the mutant epidermal growth factor receptor (EGFR). We performed preclinical characterization of TAS-121 and compared its efficacy and selectivity for common EGFR mutations (Ex19del and L858R), first- and second- generation EGFR-tyrosine kinase inhibitor (EGFR-TKI) resistance mutation (T790M), and uncommon mutations (G719X and L861Q) with those of other EGFR-TKIs. We also commenced investigation of the clinical benefits of TAS-121. The 50% inhibitory for intracellular EGFR phosphorylation was determined by using Jump-In GripTite HEK293 cells transiently transfected with EGFR expression vectors. Mouse xenograft models were used to evaluate the antitumor activity of TAS-121. TAS-121 potently inhibited common activating and resistance EGFR mutations to the same extent as another third-generation EGFR-TKI (osimertinib). In addition, TAS-121 showed equivalent inhibitory activity against some uncommon mutations such as G719X and L861Q. Furthermore, TAS-121 demonstrated greater selectivity for mutant EGFRs versus the wild-type EGFR compared with other EGFR-TKIs. Moreover, TAS-121 displayed antitumor activity in SW48 (EGFR G719S) and NCI-H1975 (EGFR L858R/T790M) xenograft models, and achieved an objective response in NSCLC patients with EGFR mutations including G719A mutation. In conclusion, TAS-121 is a novel third-generation EGFR-TKI and demonstrates anti-tumor activities in patients with NSCLC expressing either common or uncommon EGFR mutations.

Abstract 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.

This paper proposes a stereo wind-noise suppressor with frequency-domain noise averaging. A directional gain for diffuse wind noise is estimated frame by frame using a null beamformer based on interchannel phase difference which blocks the target signal. The wind-noise gain estimate is commonly multiplied by the input noisy signal to generate channel dependent wind noise estimates in order to cope with interchannel wind-noise imbalance. Interchannel phase agreement by target signal dominance or incidentally equal wind-noise phase, which leads to underestimation, is offset by averaging channel dependent wind-noise estimates along frequency. Evaluation results show that the mean PESQ score by the proposed wind-noise suppressor reaches 2.1 which is 0.2 higher than that by the wind-noise suppressor without averaging and 0.3 higher than that by a conventional monaural-noise suppressor with a statistically significant difference.