We report compounds 5 (CG416) and 6 (CG428) as two first-in-class tropomyosin receptor kinase (TRK) degraders that target the intracellular kinase domain of TRK. Degraders 5 and 6 reduced levels of the tropomyosin 3 (TPM3)-TRKA fusion protein in KM12 colorectal carcinoma cells and inhibited downstream PLCγ1 signaling at sub-nanomolar concentrations. Both degraders also degraded human wild-type TRKA with similar potency. Interestingly, both degraders, especially 6, showed selectivity for the degradation of endogenous TPM3-TRKA over ectopically expressed ATP/GTP binding protein-like 4 (AGBL4)-TRKB or ETS variant transcription factor 6 (ETV6)-TRKC fusion proteins in KM12 cells. Global proteomic profiling assays demonstrated that 5 is highly selective for the intended target. TPM3-TRKA protein degradation induced by 5 and 6 was further confirmed to be mediated through cereblon and the ubiquitin-proteasome system. Compared with the parental TRK kinase inhibitor, both degraders exhibited higher potency for inhibiting growth of KM12 cells. Moreover, both 5 and 6 showed good plasma exposure levels in mice. Therefore, 5 and 6 are valuable chemical tool compounds for investigating the in vivo function of TRK fusion during tumorigenesis. Our study also paves the way for pharmacological degradation of TRK.

BRAF is among the most frequently mutated oncogenes in human cancers. Multiple small molecule BRAF kinase inhibitors have been approved for treating melanoma carrying BRAF-V600 mutations. However, the benefits of BRAF kinase inhibitors are generally short-lived. Small molecule-mediated targeted protein degradation has recently emerged as a novel pharmaceutical strategy to remove disease proteins through hijacking the cellular ubiquitin proteasome system (UPS). In this study, we developed thalidomide-based heterobifunctional compounds that induced selective degradation of BRAF-V600E, but not the wild-type BRAF. Downregulation of BRAF-V600E suppressed the MEK/ERK kinase cascade in melanoma cells and impaired cell growth in culture. Abolishing the interaction between degraders and cereblon or blocking the UPS significantly impaired the activities of these degraders, validating a mechanistic role of UPS in mediating targeted degradation of BRAF-V600E. These findings highlight a new approach to modulate the functions of oncogenic BRAF mutants and provide a framework to treat BRAF-dependent human cancers.

Abstract Small molecule-mediated targeted protein degradation offers a systemic approach to deplete disease-causing proteins. The tropomyosin receptor kinase (TRK) family kinases are receptors for neurotrophic factors and primarily function in the central nervous system. As results of chromosomal rearrangement events, TRK kinases are re-expressed as fusion proteins and implicated in a wide diversity of human malignancies. These fusion proteins universally retain the kinase domain and function as key oncodrivers. Two TRK kinase inhibitors, entrectinib and larotrectinib, have been approved to treat TRK fusion-expressing cancers with compelling clinical evidence, highlighting the significance of TRK fusion kinases as oncology targets. In the current study, we report the development of selective TRK degraders. Lead compounds induced rapid degradation of different TRK fusion variants identified in patients at subnanomolar concentrations. Degradation of the fusion kinases significantly compromised TRK-dependent cancer cell growth with low nanomolar IC50. The lead compound exhibited approximately 16% oral bioavailability in mouse and effectively controlled TRK-driven xenograft tumor growth. Pharmacodynamic analysis demonstrated sustained degradation of the TRK fusion in xenograft tumors. Collectively, these findings provide the framework to develop a novel approach that modulates oncogenic TRK fusion kinases for cancer treatment. Citation Format: Xiaoran Han, Liqun Chen, Chengwei Zhang, Bingyang Jiao, Yanke Chen, Jing Liu, Michael Plewe, Jialiang Wang. Targeted protein degradation for the treatment of TRK fusion-driven cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5331.