Identification of an orally available compound with potent and broad FLT3 inhibition activity

Abstract

FLT3 internal tandem duplication (FLT3-ITD) is an activating mutation found in 20-30% of patients with acute myeloid leukemia (AML), which makes FLT3 an attractive target for the treatment of AML. Although FLT3-mutant patients respond to current FLT3 inhibitors, relapse usually happens because of the acquisition of resistant secondary mutations at the FLT3 catalytic domain, which is mainly on D835. In the search for compounds with broad FLT3 inhibition activities, we screened a kinase inhibitor library by using our unique FLT3 substrate and identified JAK3 inhibitor VI (designated JI6 hereafter) as a novel FLT3 inhibitor, which selectively targets FLT3 D835 mutants as well as FLT3-ITD. JI6 effectively inhibited FLT3-ITD-containing MV4-11 cells and HCD-57 cells transformed with FLT3-ITD and D835 mutants. Furthermore, administration of JI6 effectively targeted FLT3 signaling in vivo and suppressed the myeloproliferative phenotypes in FLT3-ITD knock-in mice, and significantly prolonged the survival of immunodeficient mice implanted with the transformed HCD-57 cells. Therefore, JI6 is a promising candidate for the development of next-generation anti-AML drugs.

Figure 1. JI6 selectively inhibits FLT3 in in vitro kinase assays

(A) Chemical structure of JI6. (B) Tyrosine kinase activities of recombinant proteins containing catalytic domains of FLT3, FLT3-D835Y, FLT3-D835H, JAK3, and c-KIT were analyzed with GST-FLT3S as a substrate in the presence of various concentrations of JI6. Tyrosine phosphorylation of GST-FLT3S was detected by using anti-phosphotyrosine antibody PY20, and its protein level, by Coomassie blue staining. (C) The relative kinase activity was calculated based on the density of the western blot bands normalized to the control group. Error bars denote standard deviation (n = 3).

Figure 2. JI6 selectively inhibits FLT3-ITD-containing MV4-11 cell

(A) MV4-11 and HL60 were cultured in the presence of 50 nM JI6. Viable cells were counted by using the trypan blue exclusion method. (B) MV4-11, HL60, Karpas 299, and Jurkat cells were cultured in the presence of various concentrations of JI6 for 48 hours. Cell viability was assessed by XTT assays. (C) Wright-Giemsa staining of MV4-11 and HL60 cells treated with 0 or 50 nM JI6 for 24 h. Black arrows point to mitotic cells. Error bars denote standard deviation (n = 3).

Figure 3. JI6 selectively inhibits cell viability and FLT3 signaling of HCD-57 cells transformed by FLT3-ITDand FLT3-D835 mutants

(A) Parental and oncogenic tyrosine kinase-transformed HCD-57 cells were cultured in the presence of various concentrations of JI6 or sorafenib for 48 hours. Cell viability was assessed by XTT assays. Error bars denote standard deviation (n = 3). (B) FLT3-ITD- and FLT3-D835Y-transformed HCD-57 cells were treated with the indicated concentrations of JI6 for 3 hours. Cell extracts were subjected to western blot analyses with antibodies against phosphorylated forms of FLT3 (pY591), ERK1/2 (pT202/pY204), and AKT (pS473). Equal protein loading was demonstrated by blotting with house-keeping gene product GAPDH.

Figure 4. JI6 induces apoptosis and cell cycle arrest in both FLT3-ITD- and FLT3-D835Y transfromed HCD-57 cells

(A) Apoptosis of parental and FLT3-ITD- or FLT3-D835Y-transformed HCD-57 cells induced by treatment with 0, 100, and 500 nM JI6 or sorafenib for 24 hours. Cells were stained with Cy5-labeled annexin V and propidium iodide, and data represent the percentages of apoptotic (annexin V-positive and propidium iodide-negative) plus necrotic (annexin V-positive and propidium iodide-positive) cells. (B) and (C) Cell cycle analyses of FLT3-ITD- and FLT3-D835Y-transformed HCD-57 treated with 0, 100, and 500 nM of JI6 or sorafenib for 24 hours. Percentages of cells in G1, S+G2 phases were calculated by using the ModFit software. Error bar denotes standard deviation (n=3).

Figure 5. JI6 effectively inhibits FLT3 signaling in vivo

NSG mice were engrafted with FLT3-D835Y-transformed HCD-57 cells for three weeks. (A) Flow cytometric analyses of bone marrow and spleen cells. Note that both tissues are infiltrated with FLT3-D835Y-transformed HCD-57 cells which are positive for CD71 and GFP. (B) Western blot analyses of bone marrow and spleen cell extracts from mice that were treated with vehicle control or JI6 through intraperitoneal injection at 30 mg/kg for 4 hours. Red blood cells were lyzed prior to analyses of bone marrow and spleen cells. Antibodies used were pFLT3 (pY591), FLT3, pERK1/2 (pT202/pY204), ERK1/2, pAKT (pS473) and AKT. Equal protein loading was demonstrated by blotting with house-keeping gene product GAPDH.

Figure 6. JI6 effectively inhibit the proliferation of FLT3-D835Y-transformed HCD-57 in immunodeficient mice and prolongs the animal survival

NSG mice were engrafted with FLT3-D835Y-transformed HCD-57 cells through intravenous injection. JI6 treatment was started 24 hours later by intraperitoneal injection of JI6 at a single daily dose of 15 mg/kg for 3 weeks. (A) Spleen weights of engrafted mice after 3 weeks of treatment with JI6 or vehicle. Error bars denote standard deviation (n≥7 as indicated). (B) Kaplan-Meier analysis of animal survival. Mice were treated with JI6 or vehicle from day 1 to day 21.

Figure 7. JI6 suppresses myeloproliferative phenotypes in FLT3-ITD knock-in mice

(A) FLT3-ITD knock-in mice were treated with JI6 through oral gavage at a daily dose of 25 mg/kg for 3 weeks. White blood cells were analyzed weekly for selected mice, and spleen was weighted after all the mice were euthanized at the end of the treatment. Error bars denote standard deviation (n≥3 as indicated). (B) Hematoxylin and eosin staining of paraffin-embedded sections of spleen and liver from drug- or vehicle-treated mice. Arrows point to typical myeloid cells, which are essentially eliminated in the drug-treated mice.