Lovastatin inhibits erythroleukemia progression through KLF2-mediated suppression of MAPK/ERK signaling

Figures

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  Fig. 1. Lovastatin inhibits leukemia progression independently of FLI1. (a) BALB/c mice were infected with F-MuLV to induce erythroleukemia. At 5 weeks post-viral infection, mice were treated with lovastatin (3 mg/KG) every other day for 2 weeks Overall survival was used to plot a Kaplan–Meier survival curve and significance was calculated by student t-test. (b,c) Spleen weight (b) and hematocrit (c) of leukemic mice at the onset of morbidity. P value by two-tailed student t-test. (d) CB3 cells were treated with 20 µM of lovastatin for 24 h and analyzed by western blot for FLI1 expression. The full-length blots/gels are presented in Supplementary Fig. 5

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  Fig. 2. Lovastatin suppresses cell cycle and induces apoptosis in leukemic cells. (a) Lovastatin inhibits cell proliferation in a dose dependent manner. (b) HEL cells were treated with 20 µM of lovastatin for 24 and 48 h and used for cell cycle analysis. (c) HEL cells were treated with 20 µM of lovastatin for 24 and 48 h and subjected to apoptosis analysis

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  Fig. 3. Lovastatin induces cholesterol biosynthesis gene expression in HEL cells. HEL cells were treated with 20 µM of lovastatin and used for RT-qPCR analysis for the indicated genes. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), by two-tailed student t-test

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  Fig. 4. Lovastatin suppresses the expression of the FAM83A and DDIT4 oncogenes in leukemic cells. (a,b) HEL cells were treated with lovastatin (20 µM) and subjected to RT-qPCR and western blot analysis for expression of FAM83A. (c) HEL cells were treated with lovastatin for 24 h and analyzed by western blotting for expression of ERK and phospho-ERK (P-ERK). GAPDH was used as a loading control. (d) HEL cells were treated with lovastatin (20 µM) and subjected to RT-qPCR for expression of DDIT4. (e) Growth rate of shDDIT4/3 and control cells treated with or without lovastatin (20 µM) for 24 h. (f) Expression of DDIT4 and GAPDH protein in scrambled and shDDIT4/3 cells treated for 24 h with or without lovastatin (20 µM). (g) Expression of ERK, phosphor-ERK and GAPDH proteins in scrambled and shDDIT4/3 cells treated for 24 h with lovastatin (20 µM). The full-length blots/gels are presented in Supplementary Fig. 6

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  Fig. 5. Induction of KLF2 by lovastatin suppresses MAPK/ERK through downregulation of FAM83A. (a,b) HEL cells were treated with 20 µM of lovastatin for 24 h and subjected to RT-qPCR (a) and western blot (b) analysis for expression of KLF2. (c,d) Expression of KLF2 in shKLF2/2, shKLF2/3 and scrambled control cells by RT-qPCR (c) and western blot (d). (e) Growth rate of shKLF2/2 cells versus scrambled control cells for the indicated time points. The significant value between the groups is shown by brackets. (f,g) Expression of FAM83A (f) and DDIT4 (g) in the indicated cells after treatment for 24 h with lovastatin (20 µM). (h) Expression of ERK and phospho-ERK in indicated cells after treatment with lovastatin (20 µM). The full-length blots/gels are presented in Supplementary Fig. 7

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  Fig. 6. KLF2 controls the expression of a subset of cholesterol biosynthesis genes. (a-l) Scrambled and shKLF2/2 cells were treated for 24 h with 20 µM of lovastatin and subjected to RT-qPCR analysis for the expression of (a) MSMO1, (b) MVD, (c) HMGCR, (d) MVK, (e) TM7SF2, (f) HMGCS1, (g) CYP51A1, (h) LSS, (i) EBP, (j) DHCR24, (k) DHCR7, (l) SQLE genes.

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  Fig. 7. Suggested model for the effect of lovastatin on ERK1/2 via KLF2. (a) During leukemia proliferation, expression of FAM83A and DDIT4 promote cell proliferation by activation of ERK. (b) Lovastatin induces the tumor suppressor gene KLF2 leading to downregulation of FAM83A and DDIT4, and suppression of ERK. In addition, KLF2 controls the expression of certain cholesterol biosynthesis genes, which may also attenuate leukemia progression