(P 173) IN VITRO PROFILING OF IDRX-42 AGAINST SECONDARY MUTATIONS FOUND IN TKI-RESISTANT GIST

Objective: Approximately 70-75% of Gastrointestinal stromal tumor (GIST) harbor a KIT mutation and patients with KIT-mutant GIST have durable responses to front-line imatinib (IM). However, clinical resistance emerges in almost all treated patients due to secondary resistance mutations. These secondary mutations are restricted to two regions of the KIT protein: the drug/ATP binding pocket (AP, encoded by exons 13 and 14) and the activation loop (AL, encoded by exons 17 and 18). Although additional TKI drugs has been approved for the second-line (2L, sunitinib), 3L (regorafenib), and 4L/4L+ (ripretinib, RIP), these agents lack activity against the entire spectrum of known resistance mutations. For example, sunitinib is potent against AP mutations (V654A, T670I), but has minimal activity against AL mutations. Conversely, RIP has minimal activity against AP mutations. Currently, IDRX-42 is being tested in a phase 1/1b study of patients with TKI-resistant GIST. However, the cellular potency of IDRX-42 against various primary, secondary, and compound tertiary KIT mutations has not been comprehensively described.

Methods: We used a panel of GIST cell lines with various secondary or compound mutations derived from the parental GIST T1 cell line (primary KIT exon 11 (K11) deletion mutation). We also cloned and transiently expressed additional mutations in CHO cells. Densitometry and curve fitting was used to determine the biochemical IC50 for inhibition of KIT phosphorylation, an accepted surrogate for KIT kinase activity. To assess relative potency, we also profiled IM and RIP using the same cell line models.

Results: Our results are summarized in Table 1. IM was active against primary K11 mutations (K11 exon 11 deletion and K11 V560D) but was much less active against K11 + secondary mutations, KIT exon 9 (K9) primary mutations, and K9 + secondary mutations. In addition, IM was inactive against AP/AL mutations (primary K11 + AP +AL mutations all three mutations in cis). RIP potently inhibited K11 primary mutations, K11 + AL mutations, and K9 + AL mutations. However, RIP was less active against K11 + AP, K9 primary, and K11 + AP/AL mutations. IDRX-42 was active against K11, K11 + V654A, K11 + AL, and K11 + V654A + A829 P (AP/AL), K9 primary, K9 + AP, K9 +AL mutations. However, IDRX-42 lacked potency against the gatekeeper T670I (AP) mutation and AP/AL mutations with T670I (K11+T670I + N822Y, K11 + T670I + N822K). We developed resistant cell lines using GIST T1 cell line subjected to chemical mutagenesis and long-term drug selection with IDRX-42. All IDRX-42 resistant clones had an acquired T670I mutation, but no other secondary mutations were identified. Structural analysis of IDRX-42 bound to wild-type KIT supported the broader spectrum of activity of IDRX-42 against E13 AP and E17 AL mutations due to the considerable ligand flexibility of this compound. However, consistent with our biochemical results, K14 mutations caused steric hindrance to drug binding and thereby decreased drug potency.

Conclusion: IDRX-42 has superior biochemical potency against a panel of primary and secondary KIT mutations compared with IM or RIP. Additionally, IDRX-42 has activity against all tested K11 + secondary/tertiary mutations with the exception of K14 T670I. It should be noted that T670I is an infrequently reported secondary mutation compared to V654A or AL secondary mutations, possibly due to diminished enzymatic activity compared with the parental K11 mutant kinase. Our results indicate that IDRX-42 has the most complete KIT mutation inhibitory profile reported to date.