Activity of the multikinase inhibitor TP‐0903 in RAS mutant acute myeloid leukemia
Authors: Jeon Jae Yoon , Eisenmann Eric , Niu Mingshan , Garrison Dominique , Buelow Daelynn , Thomas Megan Zavorka , Whatcott Clifford , Warner Steven , Orwick Shelley , Hertlein Erin , Byrd John , Bhatnagar Bhavana , Baker Sharyn D. 
Published: April 2020

Clonal heterogeneity and co‐occurring mutations contribute to drug resistance in AML. Leukemia samples from patients progressing on gilteritinib, a FLT3 inhibitor, were selected for RAS mutant clones, which are known to contribute preclinically to resistance (McMahon CM, Cancer Discov 2019). Previously, we demonstrated that TP‐0930, a multi‐kinase inhibitor, targets FLT3‐ITD and resistance‐conferring tyrosine kinase domain mutations in preclinical models. Here, we present the activity of TP‐0903 in RAS pathway mutant models of AML.KiNativ assay was performed in OCI‐AML3 cells (NRAS Q61L) treated with TP‐0903 (100 nM, 2h). Inhibition of cell viability was evaluated in AML cell lines (MTT) and primary samples (CellTiter Glo). Inhibition of AURKA/B, AKT, ERK, MCL‐1, and BCL‐2 were determined by western blot. Apoptosis, cell cycle, and differentiation assays were performed in OCI‐AML3 using annexin V, DAPI and CD11b‐APC staining. In vivo activity of TP‐0903 was evaluated in a systemic OCI‐AML3‐Luc+ xenograft model; 5e5 luciferase expressing cells were tail vein injected to NSG mice and TP‐0903 50 mg/kg or vehicle was given orally once dailyx5/week. Progression of leukemia was monitored via bioluminescence imaging and Kaplan Meier was used for survival analysis.In Kinativ assay, inhibition of native kinases were similar to those observed in a previous binding assay. We observed that TP‐0903 inhibited ACK1 (activated CDC42 kinase 1 or TNK2) and GCK (germinal center kinase or MAP4K2) by 87 and 46%, respectively. Inhibition of ACK1 (Kd = 7.3 nM, IC50 = 30.7 nM) and GCK (Kd = 1.8 nM, IC50 = 2.5 nM) was confirmed in binding and kinase assays. In RAS mutant AML cell lines (OCI‐AML3, THP1, HL60, and U937), TP‐0903 inhibited viability with IC50 values of 26 – 99 nM. In OCI‐AML3, TP‐0903 at 100 nM inhibited pAURKA/B, pAKT and MCL‐1; caused a G2‐M arrest and polyploidy (20 nM, 24h); and induced apoptosis (20–50 nM, 72h). TP‐0903, compared to gilteritinib, was more potent in inhibiting 4 ex vivo human primary AML samples with N/KRAS mutations co‐occurring with wt FLT3 or FLT3‐ITD, along with other mutations (IC50 range, 21 – 60 vs 260 – 1370 nM). In a 14‐day CFU assay, TP‐0903 inhibited colony formation of a FLT3‐ITD and NRAS mutant primary sample compared to DMSO (mean, 65 vs 188 CFUs). In an OCI‐AML3‐Luc+ xenograft study, TP‐0903 suppressed the outgrowth of leukemia at day 21 (P<0.0001) and prolonged median survival by 9 days compared to veh‐treated mice (P<0.0001). At study endpoint, spleen weight was higher in veh‐treated mice than in TP‐0903‐treated mice (mean ± SD, 333 ± 23 vs 181 ± 21 mg, P=0.0029).TP‐0903, a novel multi‐kinase inhibitor, shows promising in vitro and in vivo activity in RAS mutant AML. In a previous report (Nonami A, Blood 2015), an integrated approach involving cell‐based pharmacologic screening combined with KiNativ, gene expression profiling and mechanism studies, identified 2 kinase signaling pathways (ACK1/AKT and GCK) that synergistically contributed to the growth of NRAS mutant leukemia cells, including OCI‐AML3. Based on our data, TP‐0903 may exert its activity in RAS mutant AML via inhibiting ACK1 and GCK.