Novel Small Molecule FLT3 Inhibitors for the Treatment of FLT3-ITD AML

FMS-like Tyrosine Kinase 3 (FLT3) is a receptor tyrosine kinase that promotes growth and survival of hematopoietic stem and progenitor cells. Mutations in the gene encoding FLT3 are found in 20-30% of acute myeloid leukemias (AML), with approximately 25% of all AMLs containing an internal tandem duplication (ITD) in the juxtamembranal region. FLT3-ITD AML is associated with poor prognosis, with a 5 year survival rate of approximately 15% as compared to 40% for wild-type FLT3 AML. As such, FLT3 inhibitors have been developed to treat FLT-ITD AML. Although the inhibitors show considerable efficacy in vitro and in animal models, clinical trials using FLT3 inhibitors as single agents have been underwhelming. A Phase II study of quizartinib (AC220) showed few complete remissions in AML patients treated with AC220 as a single agent. Additionally, patients who relapse after FLT3 inhibitor treatment can develop FLT3-inhibitor resistant clones, many of which acquire a D835Y substitution. Thus, there is a need for a FLT3 inhibitor that increases complete remission rates and reduces relapse for FLT3-ITD AML patients.

Through chemical and structure-activity relationship studies, we have identified a novel class of FLT3 tyrosine kinase inhibitors. After initial biochemical and functional analysis, NCGC-2327 emerged as our lead compound, and further optimized with improved solubility, stability, and permeability properties suitable for in vivo utility (NCGC-1481). Both compounds have excellent selectivity against the kinase activity of FLT3-ITD and FLT-ITD D835Y at a subnanomolar concentration (IC50 <5.08 x 10^-10 M). AC220, NCGC-2327, and NCGC-1481 treatment of primary human FLT3-ITD-mutant AML cells show EC50 at subnanomolar concentrations (0.5 nM, 0.4 nM, and 0.1 nM respectively) as determined by CellTiter Glo proliferation assays. To ensure that our compounds were selectively effective against FLT3-ITD-mutant AML, primary human NRas-mutant AML cells treated with the inhibitors revealed an EC50 outside of the tested range (>30 µM). To assess the ability of the compounds to induce apoptosis, we treated FLT3-ITD-mutant AML cells with AC220, NCGC-2327, NCGC-1481, or Crenolanib (a FLT3 inhibitor that can inhibit FLT3-ITD-D835Y) for 72 hours. NCGC-148-treated cells showed the greatest levels of apoptosis (AnnexinV+) as compared to Crenolanib, AC220, and NCGC-2327 (P< 0.01). Consistent with the viability assays, NCGC-1481 treatment showed the greatest inhibition of leukemic progenitor function in methylcellulose (Vehicle: 189 ± 39, Crenolanib: 151 ± 32, AC220: 36 ± 3, NCGC-2327: 22 ± 3, and NCGC-1481: 3 ± 2) (P<0.01).

Relapse and resistance is a primary concern for patients treated with AC220, therefore we investigated leukemic subclonal resistance in vitro after treatment with Crenolanib, AC220, NCGC-2327, or NCGC-1481. FLT3-ITD-mutant AML cells were treated for 72 hours, washed and then allowed to recover in the absence of the compounds for one week. Subclonal recovery was assessed by measuring cell viability (AnnexinV+) and leukemic progenitor function (methylcellulose) for up to 1 week post treatment. NCGC-2327 and NCGC-1481 delayed, and in some instances prevented, subclonal recovery as compared to AC220 or Crenolanib treatment.

NCGC-2327 and NCGC-1481 show comparable potency to current FLT3 inhibitors (i.e., AC220 and Crenolanib) in regards to inhibition of FLT3 signaling, proliferation, and induction of apoptosis in FLT3-ITD-mutant AML. However, NCGC-2327 and NCGC-1481 are exquisitely effective at preventing subclonal recovery of FLT3-ITD-mutant AML as compared to both AC220 and Crenolanib. Taken together, these findings suggest our novel FLT3 inhibitors show promise for the treatment of FLT3-ITD positive AML, and particularly for patients that have intrinsic and/or acquired resistance to FLT3 tyrosine kinase inhibitors.