Cytarabine-Resistant FLT3-ITD Leukemia Cells are Associated with TP53 Mutation and Multiple Pathway Alterations-Possible Therapeutic Efficacy of Cabozantinib

Abstract

Internal tandem duplication of FLT3 juxtamembrane domain (FLT3-ITD)-positive acute myeloid leukemia (AML) leads to poor clinical outcomes after chemotherapy. We aimed to establish a cytarabine-resistant line from FLT3-ITD-positive MV4-11 (MV4-11-P) cells and examine the development of resistance. The FLT3-ITD mutation was retained in MV4-11-R; however, the protein was underglycosylated and less phosphorylated in these cells. Moreover, the phosphorylation of ERK1/2, Akt, MEK1/2 and p53 increased in MV4-11-R. The levels of Mcl-1 and p53 proteins were also elevated in MV4-11-R. A p53 D281G mutant emerged in MV4-11-R, in addition to the pre-existing R248W mutation. MV4-11-P and MV4-11-R showed similar sensitivity to cabozantinib, sorafenib, and MK2206, whereas MV4-11-R showed resistance to CI-1040 and idarubicin. MV4-11-R resistance may be associated with inhibition of Akt phosphorylation, but not ERK phosphorylation, after exposure to these drugs. The multi-kinase inhibitor cabozantinib inhibited FLT3-ITD signaling in MV4-11-R cells and MV4-11-R-derived tumors in mice. Cabozantinib effectively inhibited tumor growth and prolonged survival time in mice bearing MV4-11-R-derived tumors. Together, our findings suggest that Mcl-1 and Akt phosphorylation are potential therapeutic targets for p53 mutants and that cabozantinib is an effective treatment in cytarabine-resistant FLT3-ITD-positive AML.

Keywords: Cytarabine; FLT3-ITD; acute myeloid leukemia; drug-resistance.

Figure 1

FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation and protein expression were detected in MV4-11-P and MV4-11-R cells. (A) GeneScan analysis showed the presence of the FLT3-ITD mutation (162bps, red frame) instead of wildtype FLT3 (130bps) in both MV4-11-P and MV4-11-R cells. (B) FLT3-ITD mRNA expression was revealed by qPCR. Data are representative of two independent experiments each performed in triplicate. (C) Total and phosphorylated FLT3 protein was observed in mature (160 kDa) and immature (130 kDa) forms. Representative Western blots of three independent experiments are shown.

Figure 2

Differential kinase phosphorylation was observed between MV4-11-P and MV4-11-R cells. (A) Human phospho-kinase array blots showed increased (shown in red) and decreased (shown in blue) phosphorylation of multiple kinases in MV4-11-R compared with MV4-11-P. Spots for each phospho-kinase antibody are in duplicate. (B) Average pixel densities of duplicate spots on the human phospho-kinase array blots were analyzed using ImageJ. (C) Cell lysates of MV4-11-P and MV4-11-R were analyzed for selected total/phosphorylated kinases. Representative Western blots of three independent experiments are shown.

Figure 3

Apoptosis-related proteins in MV4-11-P and MV4-11-R were analyzed by human apoptosis array. (A) Blots of the array show increased p53 phosphorylation at serine 15, 46, and 392 in MV4-11-R compared to MV4-11-P. (B) Average pixel densities for duplicate spots of phosphorylated p53 were analyzed using ImageJ.

Figure 4

Differential Mcl-1 protein expression was observed between MV4-11-P and MV4-11-R. (A) Mcl-1 mRNA levels show no significant difference between MV4-11-P and MV4-11-R by qPCR. Quantitative data are representative of three independent experiments. (B) Western blot analysis shows that Mcl-1 protein expression is increased in MV4-11-R. Representative Western blots from three independent experiments are shown.

Figure 5

Sequencing analyses of the TP53 gene reveal the emergence of a new TP53 mutation, D281G, in MV4-11-R. (A) The R248W (CGG → TGG, red frame) TP53 mutation was detected in MV4-11-P, while both R248W and D281G (GAC → GGC, red frame) mutations were observed in MV4-11-R using Sanger sequencing analysis. (B) The percentage of mutant antisense-alleles for D281G and R248W mutations in MV4-11-P and MV4-11-R was determined by pyrosequencing.

Figure 6

(A–E) Cytotoxicity curves and IC₅₀ values of anti-cancer drugs for MV4-11-P and MV4-11-R were evaluated by MTS assays. Data are representative of at least two independent experiments each performed in triplicate. Cells were drug-treated for 72 h before performing the MTS assay. (F) Phosphorylation of Akt and ERK in response to drug treatment in MV4-11-P and MV4-11-R was detected by Western blot analysis. Cells were treated with cytarabine, MK2206, or CI-1040 for 6 h before collection of cell lysates. DMSO (0.01%) served as a control. Representative Western blots of two independent experiments with similar results were shown.

Figure 7

Cabozantinib inhibits FLT3-ITD signaling in MV4-11-R cells and growth of MV4-11-R-derived tumors. Kinase phosphorylation of the FLT3-ITD signaling pathway in cultured MV4-11-R cells was evaluated by Western blot analysis (A). MV4-11-R tumor xenografts were grown in nude mice; when tumor sizes reached 100–200 mm³, mice were then given 30 mg/kg of cabozantinib-malate in a single dose. Mice were sacrificed after 4 h or 24 h, and protein extracts were prepared from the tumor tissues and analyzed by Western blotting (B). To assess tumor growth, mice with MV4-11-R xenograft tumors reaching 100–200 mm³ were randomly given vehicle, 10 mg/kg, or 30 mg/kg cabozantinib-malate daily in four five-day courses (days 1–5, 7–11, 13–17, and 19–23). Tumor volumes were measured every 1–2 days (C), and Kaplan–Meier survival curves of the different mice groups were plotted against the number of days after treatment (D). Multiple tumors from each treatment group were obtained, weighed (E), stained with hematoxylin and eosin (H&E) or Ki-67 antibodies, and quantified for the number of Ki-67-positive cells (F). Quantification data represents the mean ± SD from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bars = 200 μM.