CHMFL-FLT3-165

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 ∼30% of the AML patients and has been actively pursued as a drug discovery target for AML.³ A number of small-mol. inhibitors of FLT3 kinase exist that are undergoing clin. investigation such as crenolanib,⁴ AC220 quizartinib⁵ and PKC412 midostaurin.^6, 7 Recently PKC412 has received Food and Drug Administration FDA's break through therapy designation for the FLT3-ITD⁺ AML.The preclin. 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 growth inhibition of 50% GI₅₀ against FLT3-ITD-pos. 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 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-pos. AML cancer cell lines and mutant FLT3-expressing primary patient cells, and reduces leukemia growth in preclin. in vivo xenograft and engraftment models.Through a structure-guided drug design approach, we discovered the lead compound, CHMFL-FLT3-165 abbreviated 'compound 165' Figure 1a, which displays an the half maximal inhibitory concentration IC₅₀ of 12 and 4 nM against FLT3 wild-type wt and FLT3-ITD kinases, resp., in the Z'LYTE biochem. assay Figure 1b.A kinetic study demonstrated that compound 165 is an ATP competitive inhibitor Figure 1c.A mol. modeling study showed that 165 adopted a typical type I binding conformation in the FLT3 kinase Supplementary Figure 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 with the parental BaF3 cell line GI₅₀: 2.2 nM, around 7000-fold selectivity Figure 1d and Supplementary 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 interleukin-3, which suggests that the anti-proliferation effect observed with the isogenic BaF3 cells is due to an on-target effect Supplementary 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 Figure 1e and Supplementary 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-pos. AML cells were recapitulated in colony formation assays Supplementary Figure 2.Kinome-wide selectivity profiling with DiscoveRx's KinomScan technol.¹⁰ 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 Figure 1f and Supplementary Table 4.As most of the FLT3 inhibitors in clin. 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 and 13 nM, resp.Although 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 Supplementary Figure 3.In addition, compound 165 also displayed good selectivity against BTK kinase IC₅₀: 190 nM, around 10-fold selectivity Supplementary Figure 4.We then tested the effect of compound 165 on FLT3-ITD-mediated signaling in the FLT3-ITD-pos. 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 Figure 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 the 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 Figure 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 Figure 2b, Supplementary Figure 5.Flow cytometry anal. demonstrated that at 30 nM, compound 165 strongly arrests cell cycle progression in the G0/G1 phase Figure 2c.At 24 h, 30 nM compound 165 significantly induced apoptosis in MOLM13 cells, as evidenced by an increase in poly ADP ribose polymerase and caspase-3 cleavage Figure 2d.This strong apoptotic induction was also observed in MOLM14 and MV4-11 cells.In FLT3-ITD-pos. primary AML patient cells, compound 165 exhibited dose-dependent anti-proliferation effects with less inhibition of normal bone marrow cells Figure 2e and Supplementary Table 5.Comparison with 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 Supplementary Table 5.In addition, although compound 165 could effectively inhibit FLT3 wt's phosphorylation in the bone marrow cells at 1 nM Figure 2b, it did not affect bone marrow's proliferation at this concentration Figure 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 50 mg/kg/day dosage it almost completely suppressed tumor progression Figure 2f.Immunohistochem. staining demonstrated that the proliferation of tumor cells was greatly inhibited and apoptosis was induced Supplementary Figure 6.In addition, no apparent toxicity was observed Supplementary Figure 7.In the MV4-11 cell-inoculated engraftment model, 25 mg/kg/day dosage of compound 165 could significantly reduce the MV4-11 cells in the peripheral blood mononuclear cell, bone marrow, spleen and lymphoma nodes Figure 2g.In summary, we have discovered a highly potent FLT3 kinase inhibitor, CHMFL-FLT3-165, which exhibits strong biochem. inhibition against FLT3 wt/ITD kinases, potent anti-proliferation effects against FLT3-ITD-pos. leukemia cell lines and patient primary cells, as well as significant in vivo tumor suppression.Compared with the FLT3 inhibitors currently received FDA approval PKC412 and inhibitor in advanced-stage clin. 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 vs. c-KIT kinase.Potent suppression of the drug-resistant FLT3-ITD mutations FLT3-ITD-D835Y and F691L combined with tyrosine kinase domain point mutants D835Y/E/H is also potentially clin. 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 Although 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-pos. AML.CHMFL-FLT3-165 is currently undergoing extensive preclin. safety evaluation.