D898_E901 RET Deletion Is Oncogenic, Responds to Selpercatinib, and Treatment Resistance Can Arise Via RET-Independent Mechanisms.

PURPOSE: We analyzed the oncogenic potential of RET Δ898-901 mutant and its response to selpercatinib, vandetanib, and cabozantinib in vitro and in a clinical case. MATERIALS AND METHODS: A 35-year-old man with a medullary thyroid cancer (MTC) harboring a somatic D898_E901 RET deletion was sequentially treated with vandetanib, selpercatinib, cabozantinib, and fluorouracil (5-FU)-dacarbazine. Functional study of RET Δ898-901 mutant was performed in HEK-293T, NIH-3T3, and Ba/F3 cells. RET C634R and wild-type cells served as positive and negative controls, respectively. RESULTS: The patient showed primary resistance to vandetanib and secondary resistance to selpercatinib after 12 months. Comprehensive next-generation sequencing of a progressing lesion during selpercatinib showed no additional RET mutation but an acquired complete genetic loss of CDKN2A, CDKN2B, and MTAP genes. Subsequent treatment with cabozantinib and 5-FU-dacarbazine had poor efficacy. In vitro, RET Δ898-901 showed higher ligand-independent RET autophosphorylation compared with RET C634R and similar proliferation rates in cell models. Subcutaneous injection of Δ898-901 NIH 3T3 cells in nude mice produced tumors of around 500 mm3 in 2 weeks, similarly to RET C634R cells. Selpercatinib inhibited cell growth of Ba/F3 RET Δ898-901 and RET C634R with a similar half maximal inhibitory concentration (IC50) of approximately 3 nM. Vandetanib was five-fold less effective at inhibiting cell growth promoted by RET Δ898-901 mutant (IC50, 564 nM) compared with RET C634R one (IC50, 91 nM). Cabozantinib efficiently inhibited Ba/F3 RET C634 proliferation (IC50, 25.9 nM), but was scarcely active in Ba/F3 RET 898-901 (IC50 > 1,350 nM). CONCLUSION: D898_E901 RET deletion is a gain-of-function mutation and responds to tyrosine kinase inhibitors in MTC. RET Δ898-901 mutant is sensitive to selpercatinib and vandetanib, and acquired resistance to selpercatinib may develop via RET-independent mechanisms.

Discovery of N-Trisubstituted Pyrimidine Derivatives as Type I RET and RET Gatekeeper Mutant Inhibitors with a Novel Kinase Binding Pose.

Mutations of the rearranged during transfection (RET) kinase are frequently reported in cancer, which make it as an attractive therapeutic target. Herein, we discovered a series of N-trisubstituted pyrimidine derivatives as potent inhibitors for both wild-type (wt) RET and RETV804M, which is a resistant mutant for several FDA-approved inhibitors. The X-ray structure of a representative inhibitor with RET revealed that the compound binds in a unique pose that bifurcates beneath the P-loop and confirmed the compound as a type I inhibitor. Through the structure-activity relationship (SAR) study, compound 20 was identified as a lead compound, showing potent inhibition of both RET and RETV804M. Additionally, compound 20 displayed potent antiproliferative activity of CCDC6-RET-driven LC-2/ad cells. Analysis of RET phosphorylation indicated that biological activity was mediated by RET inhibition. Collectively, N-trisubstituted pyrimidine derivatives could serve as scaffolds for the discovery and development of potent inhibitors of type I RET and its gatekeeper mutant for the treatment of RET-driven cancers.

Targeted activity of the small molecule kinase inhibitor Pz-1 towards RET and TRK kinases.

We have recently described Pz-1, a benzimidazole-based type-2 RET and VEGFR2 inhibitor. Based on a kinome scan, here we show that Pz-1 is also a potent (IC50 < 1 nM) TRKA/B/C inhibitor. Pz-1 potently inhibited proliferation of human cancer cells carrying either RET- or TRKA oncoproteins (IC50 ~ 1 nM), with a negligible effect against RET- and TRKA-negative cells. By testing mutations, known to mediate resistance to other compounds, RET G810R/S, but not L730I/V, E732K, V738A and Y806N, showed some degree of resistance to Pz-1. In the case of TRKA, G595R and F589L, but not G667C, showed some degree of resistance. In xenograft models, orally administered Pz-1 almost completely inhibited RET- and TRKA-mutant tumours at 1-3 mg/kg/day but showed a reduced effect on RET/TRKA-negative cancer models. The activity, albeit reduced, on RET/TRKA-negative tumours may be justified by VEGFR2 inhibition. Tumours induced by NIH3T3 cells transfected by RET G810R and TRKA G595R featured resistance to Pz-1, demonstrating that RET or TRKA inhibition is critical for its anti-tumourigenic effect. In conclusion, Pz-1 represents a new powerful kinase inhibitor with distinct activity towards cancers induced by oncogenic RET and TRKA variants, including some mutants displaying resistance to other drugs.

Discovery of pyrazolo-thieno[3,2-d]pyrimidinylamino-phenyl acetamides as type-II pan-tropomyosin receptor kinase (TRK) inhibitors: Design, synthesis, and biological evaluation.

Tropomyosin receptor kinase (TRK) represents an attractive oncology target for cancer therapy related to its critical role in cancer formation and progression. NTRK fusions are found to occur in 3.3% of lung cancers, 2.2% of colorectal cancers, 16.7% of thyroid cancers, 2.5% of glioblastomas, and 7.1% of pediatric gliomas. In this paper, we described the discovery of the type-II pan-TRK inhibitor 4c through the structure-based drug design strategy from the original hits 1b and 2b. Compound 4c exhibited excellent in vitro TRKA, TRKB, and TRKC kinase inhibitory activity and anti-proliferative activity against human colorectal carcinoma derived cell line KM12. In the NCI-60 human cancer cell lines screen, compound 4g demonstrated nearly 80% of growth inhibition for KM12, while only minimal inhibitory activity was observed for the remaining 59 cancer cell lines. Western blot analysis demonstrated that 4c and its urea cousin 4k suppressed the TPM3-TRKA autophosphorylation at the concentrations of 100 nM and 10 nM, respectively. The work presented that 2-(4-(thieno[3,2-d]pyrimidin-4-ylamino)phenyl)acetamides could serve as a novel scaffold for the discovery and development of type-II pan-TRK inhibitors for the treatment of TRK driven cancers.

Bioisosteric Discovery of NPA101.3, a Second-Generation RET/VEGFR2 Inhibitor Optimized for Single-Agent Polypharmacology.

RET receptor tyrosine kinase is a driver oncogene in human cancer. We recently identified the clinical drug candidate Pz-1, which targets RET and VEGFR2. A key in vivo metabolite of Pz-1 is its less active demethylated pyrazole analogue. Using bioisosteric substitution methods, here, we report the identification of NPA101.3, lacking the structural liability for demethylation. NPA101.3 showed a selective inhibitory profile and an inhibitory concentration 50 (IC50) of <0.003 µM for both RET and VEGFR2. NPA101.3 inhibited phosphorylation of all tested RET oncoproteins as well as VEGFR2 and proliferation of cells transformed by RET. Oral administration of NPA101.3 (10 mg/kg/day) completely prevented formation of tumors induced by RET/C634Y-transformed cells, while it weakened, but did not abrogate, formation of tumors induced by a control oncogene (HRAS/G12V). The balanced synchronous inhibition of both RET and VEGFR2, as well the resistance to demethylation, renders NPA101.3 a potential clinical candidate for RET-driven cancers.

RET Gene Fusions in Malignancies of the Thyroid and Other Tissues.

Following the identification of the BCR-ABL1 (Breakpoint Cluster Region-ABelson murine Leukemia) fusion in chronic myelogenous leukemia, gene fusions generating chimeric oncoproteins have been recognized as common genomic structural variations in human malignancies. This is, in particular, a frequent mechanism in the oncogenic conversion of protein kinases. Gene fusion was the first mechanism identified for the oncogenic activation of the receptor tyrosine kinase RET (REarranged during Transfection), initially discovered in papillary thyroid carcinoma (PTC). More recently, the advent of highly sensitive massive parallel (next generation sequencing, NGS) sequencing of tumor DNA or cell-free (cfDNA) circulating tumor DNA, allowed for the detection of RET fusions in many other solid and hematopoietic malignancies. This review summarizes the role of RET fusions in the pathogenesis of human cancer.

Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase.

Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the 'DFG-out' inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the 'gatekeeper' V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.

Identification of two novel RET kinase inhibitors through MCR-based drug discovery: design, synthesis and evaluation.

From an MCR fragment library, two novel chemical series have been developed as inhibitors of RET, which is a kinase involved in the pathology of medullary thyroid cancer (MTC). Structure activity relationship studies (SAR) identified two sub-micromolar tractable leads, 6g and 13g. 6g was confirmed to be a Type-II RET inhibitor. 13g and 6g inhibited RET in cells transformed by RET/C634. A RET DFG-out homology model was established and utilized to predict Type-II inhibitor binding modes.