T-LAK cell-originated protein kinase presents a novel therapeutic target in FLT3-ITD mutated acute myeloid leukemia

Abstract

Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML.

Keywords: AML; CEBPA; FLT3-ITD; TOPK; kinase inhibitor.

Figure 1. TOPK Knock-down decreases cell viability and induces apoptosis

MV4-11, U937 and KG1 cells were transfected with TOPK siRNA or control siRNA; A. western blot was performed to measure TOPK protein level. B. Viability assay was performed 48 hours following transfection. C. Apoptosis assay was performed using annexin and PI staining in MV4-11 and U937 cells 48 hours following transfection. Data are presented as Mean ± SEM, P values were calculated using Student's t-test (*P < 0.05).

Figure 2. TOPK inhibitor inhibits colony formation in leukemia but not normal CD34+ cells

AML blasts were treated with TOPK inhibitor OTS514. A. Viability assay was performed in AML blasts obtained from three AML patients 48 hours following treatment with increasing concentration of OTS514. B. Colony forming assay was performed in sorted CD34+ cells obtained from AML patient and treated with 10 nM of OTS514. C. Colony forming assay was performed in CD34+ cells obtained from healthy donor and treated with 20 and 40 nM of OTS514. Data are presented as Mean ± SEM, P values were calculated using Student's t-test (*P < 0.05).

Figure 3. TOPK inhibitor exhibits preferential anti-leukemia activity in FLT3 mutated AML

A. AML cell lines (n = 10) were treated with increasing concentration of TOPK inhibitor OTS514, and viability assay was performed 48 hours post-treatment, calculated IC50 were compared between FLT3-mut cell lines and FLT3-wt cell lines. B. MV4-11, MOLM13, U937 and KG1 cells were treated with 20 and 40 nM of OTS514 and assessed for apoptotic cells by annexin and PI staining. C. MV4-11, THP-1 and KG1 cells were treated with 20 nM OTS514 and cleaved caspase 3 levels were assessed by western blot and D. cell cycle analysis was performed using BrdU and 7AAD staining. Data are presented as Mean ± SEM, P values were calculated using Mann-Whitney U test (*P < 0.05).

Figure 4. TOPK inhibitor exhibits anti-leukemia activity in FLT3 mutated AML blasts and in MV4-11 murine model

A. Blasts obtained from three AML patients with FLT3-ITD mutations and relapsed following AC220 clinical trial, cells were treated with increasing concentration of OTS514 and viability assay was performed 48 hours later. B. Apoptosis was assessed by annexin and PI staining in blasts obtained from three AML patients with FLT3-ITD mutation C. Blasts from AML patient with FLT3-ITD mutation were treated with 20 and 40 nM of OTS514 and apoptotic cells were assessed by annexin and PI staining 5 days later. D. Image of spleens obtained from vehicle and OTS514 treated mice. E. Quantitative analysis of spleen weights (Mean ± SEM). F. Mean relative mouse body weight ± SD (N = 6 mice per group) in comparison with the body weight just before the administration. G. Survival analysis of OTS514-treated leukemic mice (N = 6) compared with the vehicle-treated controls (N = 6) (P < 0.001).

Figure 5. Targeting TOPK downregulates FLT3 expression, decreases CEBPA phosphorylation and induces myeloid differentiation in AML cells

A. MV4-11 and THP-1 cells were transfected with TOPK siRNA or control siRNA and FLT3 mRNA level was measured 48 hours later by qRT-PCR, and B. FLT3 protein levels were measured by western blot. C. MV4-11, MOLM13, U937 and KG1 cells were treated with 10 nM of OTS514 and FLT3 mRNA levels were measured by qRT-PCR 18 hours later. D. MV4-11 cells were treated with 10, 20 and 40 nM of OTS514 and P-FLT3 and total FLT3 protein level was assessed by western blot. E. MV4-11 and MOLM13 cells were treated with 20 nM of OTS514, P-STAT5 and totalSTAT5 were assessed by western blot. F. MV4-11 and MOLM13 cells were treated with 20 nM of OTS514, P-CEBPA and total CEBPA were assessed by western blot 18 hours later. G. CD11b expression was assessed by flow cytometry in blasts obtained from three AML patients with FLT3-ITD mutation following treatment with 20 and 40 nM of OTS514 (data were normalized to mode fluorescence intensity). Data are presented as Mean ± SEM, P values were calculated using Student's t-test (*p < 0.05).

Figure 6. TOPK is activated in FLT3-ITD positive in AML cells

A. MV4-11 cells were treated with 50 nM of FLT3 inhibitor MLN518 for 18 hours and P-FLT3, P-TOPK and total TOPK protein levels were assessed by western blot. B. THP-1 cells were treated with 100 ng/ml of FLT3 ligand and P-FLT3, P-TOPK and total TOPK were assessed by western blot. U937, MV4-11 and MOLM13 cells were treated with 50 and 100 nM of FLT3 inhibitor MLN518 C. cell viability was assessed by viability assay 48 hours later and D. TOPK protein levels were assessed by western blot 18 hours later. MV4-11 cells were transfected with FLT3 siRNA or control siRNA and E. TOPK mRNA and F. protein levels, G. MYC mRNA and H. protein levels were assessed. MV4-11 and U937 cells were transfected with MYC siRNA or control siRNA and I. TOPK mRNA and J. protein levels were assessed 48 hours later. Data are presented as Mean ± SEM, P values were calculated using Student's t-test (*p < 0.05).

Figure 7. CEBPA P30 contributes to TOPK upregulation in AML

THP-1 cells were transfected with CEBPA siRNA; A. FLT3 and TOPK mRNA and B. protein levels were assessed 48 hours later. THP-1 cells transfected with either empty vector: EV, P30 or P42; C. TOPK and FLT3 mRNA expression and D. protein levels were assessed 48 hours later. E. A schematic figure showing the mechanism by which targeting TOPK could affect FLT3-ITD positive AML cells. Data are presented as Mean ± SEM, P values were calculated using Student's t-test (*p < 0.05).