Relationship between KIT inhibition by bezuclastinib and effects on disease burden in mouse models of systemic mastocytosis

Abstract Background: Nonadvanced Systemic Mastocytosis (NonAdvSM) is primarily driven by activating mutations in the KIT proto-oncogene, with up to 95% of SM patients positive for a point mutation at codon 816 (D816V). Other mutations at this same residue and activating alterations in exons 11 and 17 have been identified but occur with lower incidence. This leads to ligand-independent activation of KIT receptor tyrosine kinase, neoplastic mast cell infiltration of tissues, and inappropriate release of mediators causing severe inflammation. The approval of the first KIT-targeted therapy, avapritinib (AVA), demonstrates proof-of-concept clinical efficacy in these patients, but effects on mast cell burden in blood and bone marrow are limited. The level of KIT inhibition that will lead to optimal loss of mutant clones has not previously been delineated, and it is unclear if higher levels of target engagement will result in greater molecular remission. Bezuclastinib (BEZU) is an orally bioavailable, small molecule inhibitor targeting mutated KIT kinase that is under development for the treatment of patients with NonAdvSM. Extensive biochemical and cell-based selectivity profiling demonstrate that BEZU has a unique selectivity profile that may allow higher target engagement in NonAdvSM patients, consistent with the top line results from the Summit clinical trial (NCT05186753) reported in July of 2025. The purpose of the work herein was to utilize nonclinical models to delineate in vivo target engagement and translate this to target engagement observed in clinical trials in this indication. Methods: To establish an in vivo IC50 for pKIT inhibition, tumor-bearing animals were administered a single dose of BEZU (1-300 mg/kg) or AVA (0.2-100 mg/kg), and pKIT and drug concentrations measured 4 hours later. For a functional target engagement readout, Ba/F3-KIT-D816V cells were injected intravenously into nude mice, and spleen weights and drug exposure (AUC) analyzed after 9 days drug administration of BEZU (0.1-200 mg/kg), AVA (0.3-100 mg/kg), or the AVA analog, elenestinib (ELE, 1-200 mg/kg). The ROSA-KIT-D816V model of human SM was utilized to relate target engagement to effects on mutant mast cell burden. In this model, NSG mice were sub-lethally irradiated and transplanted with ROSA-KIT-D816V cells by intravenous injection, then BEZU (100 mg/kg, QD) or AVA (0.6 mg/kg, QD) administration was initiated 7 days later. Efficacy at study end (Study Day 64) was reported as mutant mast cell burden in peripheral blood, bone marrow, and spleen, as well as human tryptase levels in plasma. Results: Data from multiple independent studies were compiled to determine in vivo IC50 for inhibition of mutant KIT phosphorylation and the half maximal effective AUC to inhibit splenomegaly (EAUC50). In these studies, BEZU in vivo activity was 325 ng/mL and 3333ng∙hr/mL for pKIT IC50 and splenomegaly EAUC50, respectively. BEZU was approximately 2x more potent than AVA in these models (535 ng/mL and 6915 ng∙hr/mL for IC50 and EAUC50, respectively). AVA was similar to ELE which had a splenomegaly EAUC50 of 6070 ng∙hr/mL. Efficacy in the ROSA model was evaluated using mouse dose levels selected to match human exposures reported in NonAdvSM clinical studies, specifically 100 mg in Summit for BEZU and 25 mg in PIONEER for AVA. Drug treatments were generally well tolerated for both drugs and were within 2x reported clinical exposures at study end for both Cmax and AUC corrected for species differences in protein binding. BEZU treatment compared to vehicle resulted in statistically significant inhibition of mutant mast cell burden in peripheral blood, bone marrow, and spleen (-93%, -84%, and -89% decrease, respectively), and reduced human plasma tryptase (-72%). The drug concentrations resulting in these effects reached target engagement levels of IC88 for pKIT at Cmax and EAUC84 for total drug exposure. In contrast, AVA at the NonAdvSM clinical exposure match showed no statistically significant impact on efficacy endpoints, with substantially lower target engagement limited to IC25 for pKit and EAUC27 for splenomegaly. Conclusions: Overall, these analyses suggest that while partial inhibition of KIT achieved clinically may yield a measurable clinical effect, substantially higher target coverage is likely needed to observe significant impact on clonal mast cell expansion.