Growth Inhibitory Function Of a Novel T-LAK Cell-Originated Protein Kinase Inhibitor On FLT3-ITD Positive Acute Myeloid Leukemia (AML) Cells

T-LAK cell-originated protein kinase (TOPK/PBK), a serine-threonine mitogen-activated protein kinase kinase is highly expressed and correlated with more aggressive disease in several types of cancer, but is undetectable in normal tissues except the testis and fetal tissues. TOPK is up-regulated in a variety of hematologic malignancies including acute myeloid leukemia (AML) and may be involved in disease pathogenesis. To investigate the role of TOPK in AML and to develop the rationale for therapeutic targeting, we initially examined the expression level of TOPK protein in AML cell lines by western blot analysis. TOPK expression was detected at high levels in 8 out of 11 AML cell lines. Importantly, TOPK was not detected in mobilized, peripheral blood-derived stem cells from healthy donors. To determine the role of TOPK in AML, we utilized a loss of function approach in two AML cell lines (MV4-11 and U937 cells) and assessed cell viability and apoptosis by MTS and Annexin/PI staining assays, respectively. Cells transfected with TOPK-siRNA showed significant decrease in cell viability (∼70%, P<0.001, both cell lines) and significant increase in apoptosis 48 hours following transfection compared to cells transfected with control-siRNA.

A small molecule compound that inhibits TOPK kinase is currently undergoing pharmaceutical development for cancer treatment. We treated nine AML cell lines with the compound for 48 hours and assessed cells viability by MTS assay. Decrease in cell viability following treatment with increasing doses of the compound was noted with variable sensitivities among AML cell lines. In addition, the compound showed enhancement of cell differentiation assessed by CD11b staining in U937 cells. Cell lines with FLT3 mutations (MV4-11, MOLM13 and KOCL-48) were significantly more sensitive to the treatment compared to cell lines with unmutated FLT3. We next treated cells with increasing doses of the compound and assessed cells viability and apoptosis by MTS and flow cytometry assays. Following the treatment with the compound, IC₅₀ was <10nM for MV4-11 and MOLM13 cells (FLT3-ITD positive cell lines), and >20nM for U937 and KG1 (FLT3-ITD negative cell lines). 40 nM of the compound induced apoptosis by ∼80% in MV4-11 and MOLM13 cells compared to 49% in U937 and 9% in KG1 cells, at 48 hours following treatment. We also validated the anti-leukemia activity in primary blasts from patients with AML (n=2). FLT3-ITD positive blasts had an IC₅₀ of ∼15nM and showed 40% increase in apoptosis following treatment with 20nM of the compound. To gain a mechanistic insight into why FLT3-ITD mutant cells are preferentially sensitive to this TOPK inhibitor, we performed a gene expression profile microarray analysis on MV4-11 cells treated with 20nM of the compound or transfected with TOPK siRNA in comparison with untreated cells and cells transfected with control-siRNA. We observed significant downregulation in genes involved in cell cycle control pathways in the signatures associated with the compound-treated and TOPK siRNA-transfected cells. Interestingly, FLT3 was among the significantly downregulated genes in the compound treated cells (∼80%) and in TOPK-siRNA transfected cells (∼30%). We then examined the activity and the expression levels of FLT3 protein following the treatment. Consistently, we found that both the phospho-FLT3 and the total FLT3 protein levels were completely depleted in cells treated with 10, 20 and 40 nM of the compound as early as 16 hours following treatment.

In conclusion, TOPK is highly expressed in AML and may act as novel therapeutic target. A novel TOPK kinase inhibitor exhibits preferential cytotoxicity to FLT3-ITD mutated AML cells, possibly through inhibition of FLT3 protein expression. Although further research is needed to determine the mechanism by which the compound inhibits FLT3 protein expression, this novel compound may represent a new targeted therapy for this adverse risk subset of patients with AML.