STI-8591, a Novel Tyrosine Kinase Inhibitor, Shows Excellent Antileukemic Activity in FLT3-Mutated (mut) Acute Myeloid Leukemia (AML)

Abstract

FMS-related tyrosine kinase 3 ( FLT3) mutations, including internal tandem duplications ( FLT3-ITD) and tyrosine kinase domain ( FLT3-TKD), are found in approximately 30% of AML patients. FLT3 has emerged as a promising molecular target in therapy of AML. Herein, we report on the activity of STI-8591, a novel kinase inhibitor developed by Hangzhou ACEA Pharmaceutical Research Co, in AML models carrying FLT3 mut, including those associated with TKI resistance [e.g., FLT3-ITD(F691L)]. We first determined the in vitro antileukemic effects of STI-8591 in FLT3 mut human AML cell lines (MV4-11, MOLM-13), a murine cell line Ba/F3 transduced with FLT3-TKD(D835Y)[Ba/F3 FLT3-TKD(D835Y)] or FLT3-ITD-TKD(D835Y) [Ba/F3 FLT3-ITD-TKD(D835Y)] or FLT3-ITD/F691L [Ba/F3 FLT3-ITD(F691L), which has been associated with drug-resistant, and primary blasts from FLT3 mut AML patients. STI-8591 effectively inhibited cell proliferation and induced apoptosis in FLT3-ITD/TKD mut cell lines (IC 50: 0.33 to 9.19nM at 24 hours). Of note, STI-8591 demonstrates 4-7-fold higher antileukemic activity on otherwise resistant Ba/F3 FLT3-ITD(F691L ) cells than the currently available FLT3 inhibitors, Gilteritinib and Quizartinib. Similar antileukemic activity of STI-8591 was observed on primary FLT3 mut AML blasts (median IC 50: 13.67nM, n=5). The antileukemic mechanism of STI-8591 is associated with inhibition of the phosphorylation of the mutated FLT3 protein and its downstream targets (i.e., STAT5, AKT, and ERK) as shown by western blot analysis. To test the in vivo antileukemic activity of STI-8591, we utilized 4 FLT3 mut AML murine and human models, obtained by transplanting: 1. BM mononuclear cells (MNCs) from CD45.2 MLL PTD/WT FLT3 ITDITD AML mice congenic CD45.1 B6 mice; 2. murine cell lines [i.e. Ba/F3 FLT3-TKD(D835Y) , Ba/F3 FLT3-ITD-TKD(D835Y), or Ba/F3 FLT3-ITD(F691L), 3. Luciferase-expressing FLT3 mut MOLM13 cells (MOLM13 Luci) into NCG mice; 4. primary blasts from FLT3 mut AML patients into NSGS mice. We treated these mice for 3-4 weeks [3 to 30mg/kg/day(d), oral gavage]. For the MLL PTD/WT FLT3 ITDITD model (n=10 mice/group), compared with Vehicle(V)-treated mice [median survival: 44 d], we observed significantly lower engraftment and longer overall survival (OS) in mice treated with STI-8591 10mg/kg (52 d, p<0.0001) or 30mg/kg (57 d, p<0.0001) or Gilteritinib 10mg/kg (49 d, p=0.0029). At 10mg/kg dosage, STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.0195). Treatment of STI-8591 also demonstrated significant benefits to mice transplanted with FLT3 mut Ba/F3 cells. For the TKI-resistant Ba/F3 FLT3-ITD(F691L) model (n=10/group), STI-8591 (median:19 and 37 d for 10 and 30 mg/kg) and Gilteritinib (17 and 24 d for 10 and 30 mg/kg) -treated mice had longer OS than V-treated (12 d) mice. At equivalent dosage (10 and 30mg/kg), STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.0013 and p<0.0001). For the MOLM13 Luci+ model (n=5/group), mice receiving STI-8591 or Gilteritinib treatment demonstrated a significant reduction in disease burden as observed by bioluminescence imaging (BLI), and prolonged OS. Compared with V-treated mice (median: 23 d), we observed longer OS in Gilteritinib (10mg/kg, 33 d, p=0.0023) and STI-8591 (3mg/kg, 27 d, p=0.0179; 10mg/kg, 34 d, p=0.0023; and 30mg/kg, 35 d, p=0.0023) -treated mice. At 10 mg/kg dosage, STI-8591- and Gilteritinib- treated mice showed no significant difference in OS (p=0.2411). Similar experiments are ongoing in FLT3 mut primary AML patient-derived xenografts (PDXs) and will be reported at the ASH meeting. To evaluate antileukemic activity of STI-8591 in mice bearing disease in central nervous system (CNS), we implanted 5×10 5 /3 µL MOLM13 cells intracranially into SCID mice to construct the CNS leukemia model (n=9/group). Compared with V-treated mice (median: 11 d), we observed a longer OS in mice treated with Gilteritinib (10 mg/kg, 18 d, p=0.0004) or STI-8591 (10 mg/kg, 23 d, p<0.0001; and 30 mg/kg, 26 d, p<0.0001). At 10 mg/kg dosage, STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.005). In conclusion, STI-8591, a novel TKI exhibited a potent antileukemic activity in various FLT3 mut AML models, including those carrying FLT3 mut associated with drug resistance. These results supported further clinical development of STI-8591 for treatment of AML.