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Acute Myeloid Leukemia (AML) is one of the most common leukemias in adults and if not treated is rapidly fatal.1 FLT3 kinase plays a critical role in the differentiation and survival of hematopoietic stem cells in bone marrow.2 The internal tandem duplication of FLT3 kinase (FLT3-ITD) as a driving oncogenic mutation has been found in approximately 30% of AML patients and has been actively pursued as a drug discovery target for AML.3 A number of small molecule inhibitors of FLT3 kinase exist that are undergoing clinical investigation, such as crenolanib,4 AC220 (quizartinib),5 and PKC412 (midostaurin).6, 7 Recently PKC412 has received FDA’s break through therapy designation for the FLT3-ITD+ AML. The preclinical studies demonstrated that myelosuppression toxicity of PKC412 and AC220 might be due to off-target effects, such as inhibition of c-KIT.8 Recently we discovered that the BTK kinase inhibitor, ibrutinib (PCI-32765), displays a sub-micromolar GI50 against FLT3-ITD-positive AML cancer cell lines, such as MOLM13, MOLM14, and MV4-11,9 however exhibits no apparent activity against c-KIT. An effort to improve the efficacy of ibrutinib led to the development of the novel and highly potent FLT3 kinase inhibitor, CHMFL-FLT3-165, which displays high selectivity toward BTK and c-KIT kinases, and exhibits impressive inhibitory efficacy against FLT3-ITD-positive AML cancer cell lines and mutant FLT3-expressing primary patient cells, and reduces leukemia growth in preclinical in vivo xenograft and engraftment models.
Through a structure-guided drug design approach, we discovered the lead compound, CHMFL-FLT3-165 (abbreviated "compound 165"), (Fig. 1A) which displays an IC50 of 12 nM and 4 nM against FLT3 wild type (wt) and FLT3-ITD kinases, respectively, in the Z’LYTE biochemical assay. (Fig. 1B) A kinetic study demonstrated that compound 165 is an ATP competitive inhibitor. (Fig. 1C) A molecule modeling study showed that 165 adopted a typical type I binding conformation in the FLT3 kinase. (Supplemental Fig. 1) We tested compound 165 against a panel of engineered FLT3 wt/mutant-expressing BaF3 cell lines and found that it was highly potent against the FLT3-ITD mutant (GI50: <0.3 nM) and exhibited great selectivity compared to the parental BaF3 cell line (GI50: 2.2 µM, around 7000-fold selectivity). (Fig. 1D and supplemental Table 1) Compound 165 also demonstrated great inhibitory activity against FLT3 wt (GI50: 8 nM) and other oncogenic mutations of FLT3, such as FLT3-D835Y (GI50: 21 nM), FLT3-D835H (GI50: 3 nM) and FLT3-D835V (GI50: 12 nM). However, it was slightly less potent against drug resistant mutations FLT3-ITD-D835Y (GI50: 73 nM) and FLT3-ITD-F691L (GI50: 67 nM), and displayed low activity against FLT3-K663Q (GI50: 840 nM). Drug-induced inhibition of cell growth could be rescued by the addition of IL-3, which suggests that the anti-proliferation effect observed with the isogenic BaF3 cells is due to an on-target effect. (Supplemental Table 2) Compound 165 displayed single digit nanomolar activity against the human FLT3-ITD AML cancer cell lines, MOLM13 (GI50: 3 nM), MOLM14 (GI50: 8 nM), and MV4-11 (GI50: 2 nM) (Fig. 1E and Supplemental Table 3). In contrast, compound 165 was comparatively inactive against FLT3 wt- expressing cell lines, such as SKM-1, NB4, SU-DHL-2, U2932, JVM-2 and Namalwa (GI50s: all over 1600 nM). The potent inhibitory activities displayed against FLT3-ITD-positive AML cells were recapitulated in colony formation assays. (Supplemental Fig. 2) Kinome-wide selectivity profiling with DiscoveRx’s KinomScan™ technology10 at a concentration of 1000 nM showed that compound 165 exhibited good selectivity (S score (0.1)=0.03) and also showed binding affinity against kinases, such as BLK, HER2, LATS2, MEK5, PDGFRβ, RET, SRMS and YES. (Fig. 1F and Supplemental Table 4) As most of the FLT3 inhibitors in clinical trials are multi-targeted compounds, and simultaneous inhibition of c-KIT and FLT3 may result in myelosuppression as has been reported for AC220,8 we then tested compound 165 against c-KIT kinase and FLT3 kinase in the TEL transfused BaF3 cells. Compound 165 inhibited FLT3 wt and FLT-ITD auto-phosphorylation (Y589/Y591) with EC50 of 22 nM and 13 nM respectively. While it inhibited c-KIT kinase auto-phosphorylation site Y823 with an EC50 of 289 nM, it did not exhibit apparent activity against two other important phosphorylation sites(Y703 and Y719, EC50: >1000 nM), which suggests that compound 165 may exhibit a better safety profile. (Supplemental Fig. 3) In addition, compound 165 also displayed good selectivity against BTK kinase (IC50: 190 nM, around 10 fold selectivity). (Supplemental Fig. 4)
We then tested the effect of compound 165 on FLT3-ITD-mediated signaling in the FLT3-ITD positive AML cancer cell lines, MOLM13, MOLM14 and MV4-11. At a concentration of 100 nM, compound 165 almost completely suppressed FLT3 auto-phosphorylation at the Tyr589/591 site in all three cell lines. (Fig. 2A) Phosphorylation of the FLT3 kinase downstream mediator STAT5 Try694 was almost completely inhibited at 30 nM. In addition, phosphorylation of ERK and AKT and expression of c-MYC were also significantly suppressed at 100 nM, which further validates compound 165 as a highly potent FLT3 kinase inhibitor and the observed anti-proliferation effects as on target. In the FLT3-ITD+ patient primary cells, 500 nM concentration of compound 165 could completely block FLT3-ITD Y589/591 auto-phosphorylation. (Fig. 2B) In addition, in the purified CD34+ bone marrow cells, upon FLT3 ligand stimulation, compound 165 could also effectively inhibit the FLT3 wt Y589/591’s auto-phosphorylation. (Fig. 2B, Supplemental Fig. 5) Flow cytometry analysis demonstrated that at 30 nM, compound 165 strongly arrests cell cycle progression in the G0/G1 phase. (Fig. 2C) At 24 h, 30 nM compound 165 significantly induced apoptosis in MOLM13 cells, as evidenced by an increase in PARP and Caspase-3 cleavage. (Fig. 2D) This strong apoptotic induction was also observed in MOLM14 and MV4-11 cells. In FLT3-ITD-positive primary AML patient cells, compound 165 exhibited dose-dependent anti-proliferation effects with less inhibition of normal bone marrow cells. (Fig. 2E and Supplemental Table 5) Comparison to the genetically pure FLT3-ITD+ AML cell lines, the anti-proliferative efficacy of compound 165 against primary cells is relatively weak, which might be due to the heterogeneous genetic background of patient primary cells. (Supplemental Table 5) In addition, although compound 165 could effectively inhibit FLT3 wt’s phosphorylation in the bone marrow cells at 1µM (Fig.2B), it did not affect bone marrow’s proliferation at this concentration (Fig. 2E), indicating that there is a safety window when only FLT3 kinase was inhibited. In an MV4-11 cell inoculated xenograft mouse model, compound 165 displayed dose-dependent tumor inhibition activity, and at 50mg/kg/day dosage it almost completely suppressed tumor progression. (Fig. 2F) Immunohistochemistry staining demonstrated that the proliferation of tumor cells was greatly inhibited and apoptosis was induced. (Supplemental Fig. 6) Additionally, no apparent toxicity was observed. (Supplemental Fig. 7) In the MV4-11 cell inoculated engraftment model, 25mg/kg/day dosage of compound 165 could significantly reduce the MV4-11 cells in the PBMC, bone marrow, spleen and lymphoma nodes. (Fig. 2G)
In summary, we have discovered a highly potent FLT3 kinase inhibitor, CHMFL-FLT3-165, which exhibits strong biochemical inhibition against FLT3 wt/ITD kinases, potent anti-proliferation effects against FLT3-ITD-positive leukemia cell lines and patient primary cells, as well as significant in vivo tumor suppression. Compared to the FLT3 inhibitors currently received FDA approval (PKC412) and inhibitor in advanced-stage clinical development (such as AC220), compound 165 may be associated with less adverse effects, such as myelosuppression, in that it exhibited over 20-fold selectivity toward FLT3 versus c-KIT kinase. Potent suppression of the drug-resistant FLT3-ITD mutations (FLT3-ITD-D835Y, F691L) combined with tyrosine kinase domain (TKD) point mutants (D835Y/E/H) is also potentially clinically advantageous. Compound 165 displays GI50s of less than 100 nM against these compound mutations in the TEL transfused BaF3 cells, which is comparable potency to that exhibited by crenolanib.4 While our studies show that compound 165 is selective for FLT3 as an oncogenic target, it should be noted that contribution of other drug targets of compound 165, such as Her2 and PDGFRβ, to the observed anti-leukemia effects cannot be ruled out. Taken together, we believe CHMFL-FLT3-165 might be a valuable potential novel therapeutic agent for FLT3-ITD positive AML. CHMFL-FLT3 is currently undergoing extensive preclinical safety evaluation.
J. Liu, Q. Liu and W. Wang are supported by the grant of “Cross-disciplinary Collaborative Teams Program for Science, Technology and Innovation (2014-2016)” from Chinese Academy of Sciences. Z. Zhao is supported by Anhui Province Natural Science Foundation Annual Key Program (grant number: 1301023011). We want to thank China “Thousand Talents Program” support for Prof. Q. Liu and “Hundred Talents Program” of The Chinese Academy of Sciences support for Prof. J. Liu, and W. Wang. Z. Zhai is supported by Key Projects of Provincial Natural Science Research in Collages and Universities of Anhui Province. (Grant number: KJ2014Z017)
CONFLICT OF INTEREST
Dr. Shanchun Zhang is shareholder of Hefei Cosource medicine Technology Co. LTD.
Supplementary information is available at Leukemia’s website