Oncogenic mutations of the receptor tyrosine kinase KIT drive cancers such as gastrointestinal stroma tumors (GIST) and systemic mastocytosis (SM). The mutations in the juxtamembrane domain (Exon 11, E11) and extracellular domain (E9) are most commonly found in GIST, and the A-Loop D816V mutation (E17) in over 90% SM cases. Currently approved KIT inhibitors either lacked efficacy against certain mutants, like imatinib, or lacked selectivity such as the multi-kinase targeting sunitinib and regorafenib. Primary or acquired resistance or intolerance is common and poses a great clinical challenge. Non-kinase scaffold functions of oncoproteins are being revealed, as exemplified by EGFR, BTK, and BCR-ABL1. Since all the currently approved TKIs only inhibit the catalytic activity, the scaffold functions may constitute residual oncogenic signaling and eventually lead to acquired resistance and disease progression. We reasoned that a degrader, rather than an inhibitor, may potentially provide more benefit for patients with oncogenic KIT.

TGRX-3544 is a heterobifunctional degrader which promotes ubiquitination and proteasomal degradation of oncogenic KIT. In GIST T1 cells, TGRX-3544 potently induced degradation of Del560-578 (E11) mutant KIT (DC50 = 1.6 nM at 24h). The degradation activity was also robustly observed in Ba/F3 isogenic cells expressing KIT Del557-558 (E11), InsAY502-503 (E9), D816V (E17), and Del557-558/D816V (E11/E17) mutants, but not wildtype KIT. TGRX-3544 potently inhibited proliferation of GIST T1 (KIT E11) cells and Kasumi-1 (KIT N822K, E17) cells. It also inhibited proliferation of Ba/F3-KIT cells expressing KIT E11, E17, and E11/E17 mutants (IC50 0.9 to 3.9 nM), as well as the E9 mutant. The cellular activity of TGRX-3544 against the E11, E17 and E11/E17 mutants was superior to that of all the currently approved KIT inhibitors imatinib, sunitinib, regorafenib, avapritinib and ripretinib. Unlike other KIT inhibitors, TGRX-3544 had little or no activity against the closely related PDGFRA or FLT3, nor did it show cytotoxicity in a panel of normal cell lines (IC50 > 10000 nM), suggesting its high selectivity. In vivo, TGRX-3544 led to dose-dependent tumor regression in xenograft models expressing E11 or E11/E17 mutant KIT. In the E11/E17 model, TGRX-3544 was much more efficacious than the corresponding warhead. TGRX-3544 rapidly reduced the protein levels of E11 or E11/E17 mutant KIT by over 50% 4h after oral dosing. It also diminished the downstream signaling pathways of tumor samples as indicated by reduced phosphorylation of KIT, STAT5, AKT and ERK1/2.

In summary, we have developed a highly mutant-selective, event-driven, scaffold-eliminating KIT degrader with superior cellular potency and in vivo oral activity. The balanced preclinical profile of TGRX-3544 supports its further clinical investigation in KIT-driven malignancies such as GIST and SM.