Discovery of a Proteolysis Targeting Chimera for TRKA and RET-derived oncoproteins

We designed Proteolysis Targeting Chimeras (PROTACs) to target Tropomyosin Receptor Kinase A (TrkA) and Rearranged during Transfection (RET) oncogenic proteins. We analyzed a series of 22 PROTACs, based on the RET and TrkA small molecule inhibitor Pz-1 and ligands of E3 Ligase complex component Cereblon (CRBN). The compounds were tested in TPC-1 and KM12 cells, derived from papillary thyroid carcinoma and colorectal carcinoma, and harboring the CCDC6-RET and TPM3-TRKA oncoproteins, respectively. We identified several RET and TPM3-TRKA PROTACs, able to induce their degradation. Consistently, one of the most active degraders, compound 9, exhibited a strong anti-proliferative effect in several Cancer cell lines derived from human medullary and papillary thyroid, lung and colon cancers, displaying either RET or TRKA-derived oncoproteins, with an IC₅₀ dose of one digit nM. Mechanistically, TPM3-TRKA degradation by compound 9 was dependent on CRBN-mediated polyubiquitination and proteasomal degradation; accordingly, it was hindered by inhibitors of the Proteasome (MG132) or Cullins (MLN4924), by dominant negative Cullin 4A mutant, and by free pomalidomide. Saturating amounts of compound 9 featured loss of activity, consistently with the bivalent binding of a PROTAC ("hook effect"). Finally, a compound 9 derivative, compound 20, induced in vivo degradation of TMP3-TRKA in KM12 cells mouse xenografts. In conclusion, our study indicated that PROTAC-mediated degradation is an efficient strategy to intercept RET and TrkA oncogenic signaling.