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2024 Background: Patients with RET fusion-positive NSCLC have an estimated 25% incidence of CNS metastasis at diagnosis, and up to 40% during disease progression. Effective anti-RET therapy that penetrates the blood-brain barrier is essential to extending survival. TAS0953/HM06 is a structurally distinct RET-specific inhibitor that exhibits a distinct binding mode to RET and is effective against RET solvent front (G810) and gatekeeper (V804) mutations. TAS0953/HM06 also inhibits growth of xenograft tumors established from RET fusion-driven tumors of multiple histologies. TAS0953/HM06, therefore, represents a potentially effective strategy to overcome the emergence of acquired resistance to first generation RET-selective inhibitors. Here, we compared the brain penetration and efficacy of TAS0953/HM06 to selpercatinib (FDA-approved RET inhibitor) in models of intracranial RET fusion-positive cancers, specifically NSCLC and sarcoma. Methods: We compared the brain: plasma ratio of unbound TAS0953/HM06 and selpercatinib in mice to determine the unbound partition coefficient, Kpuu, brain. We injected ECLC5 (NSCLC cell line, TRIM33-RET) and HMSC-RET (immortalized human mesenchymal stem cells in which SPECCL1-RET was introduced by CRISPR-Cas9 genomic engineering, sarcoma model) cells expressing luciferase into the cerebellum of mice. Tumor-bearing mice were treated with TAS0953/HM06 (50 mg/kg BID), selpercatinib (10 mg/kg BID) or vandetanib (multi-kinase RET inhibitor, 50 mg/kg QD), and assessed weekly for tumor growth via bioluminescence imaging. Results: Kpuu, brain, of TAS0953/HM06 and selpercatinib were 1.3 and 0.20, respectively. Substances with brain Kpuu > 0.3 in mice are regarded as brain-penetrable. TAS0953/HM06 was superior to selpercatinib at inhibiting growth of ECLC5 (p < 0.0001) and HMSC-RET (p = 0.0005) brain xenograft tumors, and increasing survival of tumor-bearing animals (ECLC5: TAS0953/HM06 139±0.5 days, selpercatinib 95+2.3 days, p = 0.002; HMSC-RET: TAS0953/HM06 41± 2.2 days, selpercatinib 20±3 days, p = 0.0001). Vandetanib, which is highly brain-penetrant, did not cause a significant decrease in growth of either brain tumor xenograft models. At the doses used, the 3 RET inhibitors induced similar regression in several peripheral subcutaneous xenograft tumor models. Conclusions: Our data in animal models suggest that TAS0953/HM06 penetrates the CNS more effectively than selpercatinib, and is superior at decreasing CNS disease and extending survival. TAS0953/HM06 represents a promising new therapeutic option for patients with RET fusions with acquired resistance mutations, including those with brain metastasis and those resistant to first-generation selective RET inhibitors. TAS0953/HM06 is currently undergoing a biomarker-driven phase 1/ 2 clinical trial for patients with solid tumors driven by RET alterations (NCT04683250).

INTRODUCTION Brain metastases are the most common brain tumors and occur in 10-30% of cancer patients, whereas leptomeningeal disease (LMD) occurs in approximately 5% of adults with systemic malignancies. Tractable preclinical disease models that faithfully represent metastasis to the brain and recapitulate LMD are needed to improve our understanding of the biological basis of CNS disease as well developing effective therapeutic strategies. Our goal in this study was to generate representative preclinical disease models using two methods. METHODS We isolated tumor cells from CSF of 16 patients with cytologically proven LMD (9 NSCLC, 1 melanoma, 1 ovarian cancer, 1 endometrial cancer, and 4 breast cancer) and implanted the cells subcutaneously into the flank of immunocompromised mice. Cell lines were also generated from PDX tissues. Models were characterized by next-generation sequencing (NGS). We also generated a model of CNS metastasis of kinase-driven sarcoma by intracardiac (IC) injection of human mesenchymal stem cells (HMSC) expressing a SPECC1L::RET fusion (CRISPR-Cas9 gene editing). The cells (HMSC-RET) were also labeled with a luciferase construct to allow non-invasive bioluminescence imaging. RESULTS We established three PDX models (2 lung, 1 ovarian) from CSF (19% success rate compared to approximately 33% for solid tumors) and matched cell lines from the resulting PDX tissues. Intracardiac injection of HMSC-RET cells resulted in tumors establishing in several peripheral organs and the brain. SUMMARY AND CONCLUSIONS We have established disease models of CNS metastasis and LMD. Translational studies where patients with clinical suspicion of LMD undergo CSF sampling, NGS/ctDNA analysis, and PDX modeling are crucial in improving our understanding of this metastatic compartment and investigating novel treatment paradigms. Future studies will be focused on examining the biochemical and genetic nature of these tumors as well as developing effective therapeutic strategies.

Clear cell sarcoma (CCS) is an uncommon malignant mesenchymal neoplasm of young adults with a predilection for tendons and aponeuroses of distal extremities, a distinctive nested growth pattern, melanocytic differentiation, and usually an EWSR1::ATF1 fusion. Distinction from melanoma can be challenging but is critical for clinical management. Rare cases of primary bone CCS have been reported. The purpose of this study was to evaluate the clinicopathologic features of a series of primary bone CCS. Three cases of primary bone CCS were identified out of 140 CCS diagnosed between 2010 and 2021. Two patients were female, and 1 patient was male; ages were 19, 47, and 61 years. All tumors arose in the long bones of the extremities (femur, humerus, fibula). Two tumors also involved regional lymph nodes at presentation. Two showed characteristic histologic features, in the form of nests and fascicles of uniform epithelioid to spindle cells with prominent nucleoli and pale eosinophilic to clear cytoplasm; 1 tumor showed sheet-like growth, unusual focal pleomorphism, and more notable nuclear atypia. By immunohistochemistry, S100 protein was positive in 2/3 cases, SOX10 in 3/3, HMB-45 in 2/3, MiTF in 2/2, and melan A in 1/3. All cases were confirmed to harbor EWSR1 rearrangement and EWSR1::ATF1 fusion or t(12;22). On follow-up, all 3 patients developed metastases and died of disease, 5, 18, and 21 months after diagnosis. In summary, CCS rarely presents in the skeleton. At such locations, distinction from metastatic melanoma is particularly challenging. Clinical and pathologic features are similar to conventional CCS of soft tissue. Primary bone CCS may pursue an aggressive clinical course.

Background: Vepafestinib (TAS0953/HM06, Vepa) is a 2nd generation RET-selective inhibitor that effectively penetrates the brain, and inhibits the wildtype RET kinase domain (KD) and RET KD mutants (G810, V804, Y806, L730) (presented at AACR-NCI-EROTC 2021 meeting). RET rearrangements are found in an increasing number of soft tissue sarcomas, including infantile fibrosarcoma (IFS). Here we investigated the efficacy of Vepa in comparison to other RET-selective inhibitors in preclinical models of pediatric sarcomas harboring RET fusions. Methods: Multiple preclinical models of SPECC1L::RET-driven sarcomas were established: 1) Paired patient-derived xenograft (PDX) and cell line models from a brain metastasis (BM) of an IFS tumor (SR-Sarc-0001); 2) A human mesenchymal stem cell line with RET fusion introduced with CRISPR-Cas9 (HMSC-RET); 3) A murine BM model produced by injection of luciferase-expressing HMSC-RET into the cerebellum. CNS penetration of Vepa was assessed by pharmacokinetic profiling in the prefrontal cortex (PFC), cerebrospinal fluid (CSF), and plasma in freely-moving male Han Wistar rats after oral administration of 3, 10, or 50 mg/kg single doses. Results: Exposure of SR-Sarc-0001 and HMSC-RET cells to Vepa resulted in dose- and time-dependent decreases in phosphorylation of RET, ERK1/2, AKT, STAT3 and S6, expression changes in cell cycle regulators (p27 up, cyclin D1 down), induction of pro-apoptosis proteins (c-PARP, BIM), and loss of MYC expression. Growth of SR-Sarc-0001 (IC50: 0.09 µM, 95% CI: 0.03-0.2) and HMSC-RET cells (IC50: 0.2 µM, 95% CI: 0.09-0.5), but not parental HMSC cells (IC50 &gt; 1 µM), was suppressed by Vepa, with concomitant elevation of caspase 3/7 activity. Vepa was more effective than vandetanib and similar to the FDA-approved RET inhibitors, selpercatinib (Selp) and pralsetinib (Pral), in all in vitro assays. Significant regression of SR-Sarc-0001 PDX tumors was seen after Vepa treatment (64.8 ± 0.5%). Notably, no regrowth was observed up to 46 days after cessation of Vepa treatment, whereas 25 days after stopping Selp (10 mg/kg BID) and Pral (15 mg/kg BID) treatment, 1/5 and 3/5 tumors started to regrow, respectively. Similar efficacy was observed in the HMSC-RET xenograft model. Vepa was more effective than Selp at blocking HMSC-RET brain xenograft tumor growth (p=0.001) and increasing survival (p=0.0001). CNS penetration of Vepa was excellent, with near-equivalent concentrations detected in the PFC, CSF, and plasma-free fraction after equilibration between body fluid compartments. Conclusions: Our preclinical results suggest that vepafestinib has the potential to more effectively manage CNS metastasis compared to selpercatinib, representing a promising new therapeutic option for patients with RET-driven sarcomas. Vepafestinib is currently in a phase 1/2 trial for adult patients with advanced solid tumors harboring RET alterations (margaRET, NCT04683250). Citation Format: Igor Odintsov, Ryan C. Cheng, Allan J. Lui, Tom Zhang, Yue C. Lu, Renate I. Kurth, Morana Vojnic, Inna Khodos, Qing Chang, Kevin Chen, Claudio Giuliano, Annalisa Bonifacio, Isao Miyazaki, Elisa de Stanchina, Emanuela Lovati, Marc Ladanyi, Romel Somwar. Efficacy of vepafestinib in preclinical models of RET fusion-driven sarcoma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4007.