Fig. 4: Pharmacological inhibition of WNK1 reduces AML growth.
a WNK1 inhibitors elicit a dose–dependent decrease in human OXSR1 and STK39 phosphorylation. Western blot analysis of phospho-OXSR1(S325)/phospho-STK39(S373) and total OXSR1 and STK39 in human THP1 lysates treated with WNK463 or Compound 12 at the indicated concentrations. Ponceau S staining served as a loading control. Blots are from one representative experiment (n = 1). The samples were derived from the same experiment, but different gels for phospho-OXSR1(S325)/phospho-STK39(S373), another for OXSR1/STK39 (total) were processed in parallel. b–d Growth curve of MA9 (b), human THP1 (c) and human MOLM13 (d) leukaemia cells treated with Compound 12 at the indicated concentrations. Data are presented as mean ± SD of three independent experiments (n = 3). e IC50 values of WNK1 inhibitors for a panel of indicated cancer lines. f Schematic of WNK inhibitor treatment strategy. Sublethally irradiated B6.SJL recipient mice were transplanted with MA9 leukaemia cells, and the mice were intraperitoneally injected with Compound 12 (72 mg/kg) on day 4 after transplantation. The treatment with Compound12 was sustained for a period of 14 days. g Kaplan–Meier survival curves of recipient mice transplanted with MA9 leukaemia cells receiving oil or Compound12 treatment. Statistical significance was calculated using a log-rank test. h Western blot analysis of p-OXSR1, p-STK39 and p-S6K in AML patient samples treated with WNK463. AML #1 and AML #2 are MLL-rearranged, while AML #3, AML #4, and AML #5 are MLL-non-rearranged. Patient samples were treated with WNK463 at 1, 3, or 10 µM for 2 h. Blots for each patient sample are from one representative experiment (n = 1). The samples were derived from the same experiment, but different gels for p-STK39(S373)/p-OXSR1(S325) and Vinculin, another for p-P70S6K (T389) were processed in parallel. Vinculin served as a loading control. i WNK463 dose–response curves for a panel of primary AML samples. Data are presented as mean ± S.D. (n = 3). The IC50 for each sample is shown in brackets. j Percentage of Annexin V-positive cells among human hCD45-positive cells in a panel of human primary AML samples following treatment with WNK463 at the indicated concentrations. Analysis of AML#1, AML#2, and AML#3 was performed on day 3 after treatment with WNK463, whereas AML#4 and AML#5 were analysed on day 7. Data are presented as mean ± s.d. (n = 3). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post-hoc test to compare each WNK463 concentration to the untreated control.
