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. 2013 Nov 21;122(22):3607-15.
doi: 10.1182/blood-2013-07-513044. Epub 2013 Sep 17.

Crenolanib is active against models of drug-resistant FLT3-ITD-positive acute myeloid leukemia

Eric I Zimmerman  1 , David C Turner, Jassada Buaboonnam, Shuiying Hu, Shelley Orwick, Michael S Roberts, Laura J Janke, Abhijit Ramachandran, Clinton F Stewart, Hiroto Inaba, Sharyn D Baker
Affiliations

Crenolanib is active against models of drug-resistant FLT3-ITD-positive acute myeloid leukemia

Eric I Zimmerman et al. Blood. .

Abstract

FLT3 kinase internal tandem duplication (ITD) mutations are common in acute myeloid leukemia (AML) and are associated with poor clinical outcomes. Although initial responses to FLT3 tyrosine kinase inhibitors (TKIs) are observed in FLT3-ITD-positive patients, subsequent relapse often occurs upon acquisition of secondary FLT3 kinase domain (KD) mutations, primarily at residues D835 and F691. Using biochemical assays, we determined that crenolanib, a novel TKI, demonstrates type I properties and is active against FLT3 containing ITD and/or D835- or F691-activating mutations. Potent activity was observed in FLT3-ITD-positive AML cell lines. Crenolanib delayed the outgrowth of MV4-11 cells in a xenograft mouse model, whereas in combination with the type II TKI sorafenib, a significant decrease in leukemic burden (P < .001) and prolonged survival (P < .01) was observed compared with either type I or II TKI alone. Crenolanib was active against Ba/F3 cells harboring FLT3-ITD and secondary KD mutations and sorafenib-resistant MOLM-13 cells containing FLT3-ITD/D835Y both in vitro and in vivo. In addition, crenolanib inhibited drug-resistant AML primary blasts with FLT3-ITD and D835H/Y mutations. These preclinical data demonstrate that crenolanib is effective against FLT3-ITD containing secondary KD mutations, suggesting that crenolanib may be a useful therapeutic agent for TKI-naive and drug-resistant FLT3-ITD-positive AML.

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Figures

Figure 1
Figure 1
Crenolanib potently inhibits FLT3-ITD AML cell viability and FLT3 signaling. AML cells were treated with DMSO or increasing concentrations of crenolanib (A) for 72 hours and cell viability was measured or (B) for 1 hour and lysed. Western blot analysis was performed on FLT3 immunoprecipitation eluent or whole-cell lysate by use of the indicated antibodies . Cell viability measurements represent the mean ± SEM of 3 experiments with 6 replicates each (n = 18). IP, immunoprecipitation.
Figure 2
Figure 2
Drug combination effects on MV4-11 cell viability and FLT3 signaling. (A) MV4-11 and MOLM-13 cells were treated with DMSO or increasing concentrations of crenolanib (CRE), sorafenib (SOR), or both at a fixed concentration ratio for 72 hours and cell viability was measured. Calcusyn combination index (CI) values were generated for drug combination effects on cell viability. Cell viability experiments were performed in triplicate with 6 replicates each (n = 18; CI value > 1.0: antagonism; CI value = 1.0: additivity; CI value < 1.0: synergism). (B) MV4-11 cells were treated for 1 hour with DMSO or the indicated concentration of crenolanib (CRE), sorafenib (SOR), or both (combo) and lysed. Western blot analysis was performed on whole-cell lysate by use of the indicated antibodies. Densitometry measurements were normalized to total protein amount.
Figure 3
Figure 3
Antileukemic effects of the combination of crenolanib and sorafenib in a MV4-11 xenograft AML mouse model. Female NSG mice engrafted with MV4-11-luc cells were treated with vehicle, crenolanib 15 mg/kg intraperitoneally, sorafenib 15 mg/kg orally, or crenolanib (15 mg/kg, intraperitoneally) plus sorafenib (15 mg/kg, orally) once daily (Monday to Friday) for 3 consecutive weeks beginning on day 17. (A-B) Leukemic cell bone marrow infiltration was monitored by noninvasive luciferase imaging (*P < .05; **P < .01; ***P < .001). (C) Kaplan-Meier analysis of animal survival (**P = .0017 for crenolanib vs vehicle; P = .0017 for sorafenib vs vehicle; P = .0017 for the drug combination versus vehicle; P = .0021 and P = .0026 for the drug combination vs crenolanib or sorafenib alone, respectively). Black bar denotes treatment period.
Figure 4
Figure 4
Crenolanib activity against Ba/F3 cells expressing various FLT3 mutants in vitro and in vivo. (A-B) Ba/F3 cells expressing FLT3 mutants were treated with DMSO or increasing concentrations of crenolanib (A) for 72 hours and viability was measured or (B) for 1 hour and lysed. Western blot analysis was performed on FLT3 immunoprecipitation eluent by use of the indicated antibodies. Cell viability measurements represent the mean ± SEM of 2 to 4 experiments with 6 replicates each (n = 12-24). (C-D) Male NSG mice engrafted with Ba/F3 FLT3-ITD/D835H-luc cells were treated with vehicle, crenolanib 15 mg/kg intraperitoneally twice daily, or sorafenib 15 mg/kg orally once daily for 10 consecutive days beginning on day 1 (5 mice per treatment group). (C) Ba/F3 FLT3-ITD/D835H-luc cell bone marrow infiltration was monitored by noninvasive luciferase imaging. Black bar denotes treatment period (*P = .0157; **P = .0013). (D) Representative whole-body luciferase images from each treatment group are shown.
Figure 5
Figure 5
Crenolanib activity against MOLM-13-RES cells in vitro and in vivo. (A-B) MOLM-13-luc and MOLM-13-RES-luc cells were treated with DMSO or increasing concentrations of crenolanib or sorafenib (A) for 72 hours and viability was measured or (B) for 1 hour and lysed. Western blot analysis was performed on FLT3 immunoprecipitation eluent using the indicated antibodies. Cell viability measurements represent the mean ± SEM of 2 to 3 experiments with 6 replicates each (n = 12-18). (C-D) Female NSG mice engrafted with MOLM-13-RES-luc cells were treated with vehicle, sorafenib 15 mg/kg orally once daily, or crenolanib 15 mg/kg intraperitoneally once or twice daily (Monday to Friday) for 3 consecutive weeks beginning on day 3. (C) Leukemic cell bone marrow infiltration was monitored by noninvasive luciferase imaging (**P < .01; ***P < .001). (D) Kaplan-Meier analysis of animal survival (***P < .0001 for crenolanib once daily vs vehicle; ***P < .0001 for crenolanib twice daily vs vehicle). Black bar denotes treatment period.
Figure 6
Figure 6
Crenolanib activity against TKI-resistant pediatric AML primary blasts ex vivo. Primary blast samples were treated with DMSO or increasing concentrations of crenolanib or sorafenib for 72 hours. Cell viability measurements represent the mean ± SEM of 4 replicates.
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