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

Statins suppresses several types of cancers [32], yet the underlying mechanism remains unknown. Using a mouse model of erythroleukemia induced by Friend Murine Leukemia virus (F-MuLV), we investigated the effect of lovastatin on leukemia progression. Erythroleukemia is indeed an aggressive form of leukemia as medium survival is 3–9 month from the time of diagnosis and new drugs desperately needed for treatment of this cancer [29, 30]. Leukemias induced by F-MuLV usually appears in the spleen of infected mice around 3–5 weeks post-viral infection [30]. At 5 weeks post-viral infection, leukemic mice were treated with lovastatin (3 mg/kg), every other day for 2 weeks. Lovastatin strongly inhibited leukemia progression in these mice (Fig. 1a). At the time of death, no difference in the size of tumorigenic spleens was detected between lovastatin treated and control mice (Fig. 1b). The hematocrit values were much higher in the lovastatin treated compared to control mice, but slightly below significance (Fig. 1c), suggesting a positive effect of lovastatin on red blood cell suppression during leukemia progression, which extended survival.

As Fli-1 activation is critical for murine leukemia induction by F-MuLV [33], we tested for Fli-1 levels. Lovastatin had marginal effect on Fli-1 expression (Fig. 1d) on murine erythroleukemia cell line CB3 induced by this retrovirus [29], suggesting that lovastatin inhibits leukemia progression independently of Fli-1.

As lovastatin inhibits erythroleukemia progression in vivo (Fig. 1a), we examined the effect of this statin on erythroleukemia cell survival in culture. Lovastatin strongly inhibited the growth of human erythroleukemia cell line HEL in culture in a dose dependent manner (Fig. 2a), with an IC₅₀ of 18.2 µM [18]. Similar growth inhibition is also seen in the erythroleukemia cell lines K562 and CB3, with IC₅₀ of 30.2 and 35.3 µM, respectively. At 20 µM, lovastatin induced a G₁ cell cycle arrest of HEL cells at both 24 and 48 h post-drug treatment (Fig. 2b). At this concentration, lovastatin also significantly induced apoptosis of HEL cells in culture (Fig. 2c). The above results demonstrate anti-leukemic effects of lovastatin in both tissue culture and mice.

While lovastatin is known to reduce serum cholesterol by inhibiting the HMGCR (3-hydroxy-3-methylglutaryl-Coenzyme A reductase) enzyme in liver [2], the mechanism of its anti-leukemia activity is unknown. Interestingly, treatment of the human erythroleukemia cell line HEL with lovastatin (20 µM) resulted in significant transcriptional upregulation of 16 of 18 cholesterol biosynthesis genes including HMGCR (Fig. 3). Induction of HMGCR and LSS by lovastatin is also tested by western blot (Supplementary Fig. 1a,b; The full-length blots/gels are presented in Supplementary Fig. 8). Expression of FDFT1 and IDI1 was not induced by lovastatin. Interestingly, we previously demonstrated that upregulation of cholesterol biosynthesis genes by liminoid compound A1542 responsible for some of its growth inhibition activity in HEL cells line [18]. As lovastatin also inhibits cell proliferation in culture (Fig. 2a) and in vivo (Fig. 1a), higher cholesterol gene expression by this statin may be in part responsible for leukemia suppression.

Cholesterol biosynthesis gene transcription is regulated by the transcription factors SREBP1 and SREBP2 [34]. Interestingly, lovastatin significantly upregulated SREBP2 (Supplementary Fig. 2a,b). Lovastatin also upregulated expression of apolipoprotein A1 (APOA1), A component of high-density lipoprotein (HDL) involved in cholesterol biosynthesis (Supplementary Fig. 2c) [35].

To further understand the mechanism of lovastatin growth inhibition, HEL cells were treated with lovastatin (20 µM) for 24 h and subjected to RNAseq analysis. Among genes whose expression was significantly up- or down-regulated at least 2.5 fold by lovastatin (Supplementary Table 1), we detected FAM83A, a known regulator of the RAS pathway, whose level was reduced by 2 folds in lovastatin-treated cells [20, 21]. Reduced expression of FAM83A by lovastatin was confirmed by RT-qPCR and western blot (Fig. 4a,b). As FAM83A regulates MAPK activity, we tested for ERK1/2 phosphorylation on Thr202/Tyr204 in lovastatin (20 µM)-treated HEL cells, and observed a strong suppression of P-ERK (Fig. 4c). Interestingly, RNAseq data also revealed strong reduction in the level of DNA damage-inducible transcript 4 (DDIT4), also known as DNA damage response 1 (REDD1). DDIT4 was shown to accelerate growth of gastric cancer cells through upregulation of MAPK [27]. Herein, we showed that lovastatin strongly suppressed DDIT4 transcription in HEL cells (Fig. 4d). To further confirm this observation, we knockdown DDIT4 using lentivirus shRNA. Two cell lines shDDIT4/2 and shDDIT4/3 were successfully generated, in which shDDIT4/3 revealed significant downregulation (Fig. 4e). By western blot, while the level of DDIT4 reduced following lovastatin (20 µM) treatment, this downregulation was further increased in shDDIT4/3 cells (Fig. 4f). Cell proliferation was significantly reduced in shDDIT4/3 cells versus control, while this level further decreased when treated with lovastatin (Fig. 4g). Moreover, as the level of phosphor-ERK is significantly reduced in shDDIT4/3 cells, this level further decreased after treatment with lovastatin (Fig. 4h). These results suggest that lovastatin may suppress cell proliferation by inhibiting MAPK activity through FAM83A and DDIT4.

RNAseq analysis also identified higher expression (above 400 times) of Kruppel Like Factor 2 (KLF2) in lovastatin treated HEL cells (Supplementary Table 1). This upregulation by lovastatin was confirmed by RT-qPCR (Fig. 5a) and Western blotting (Fig. 5b). Interestingly, KLF2 expression was significantly higher in enlarged spleens isolated from three leukemic mice treated with lovastatin versus three DMSO treated controls (Supplementary Fig. 3; The full-length blots/gels are presented in Supplementary Fig. 9). Since lovastatin suppresses cell proliferation (Fig. 2a), we examined whether this inhibition is mediated through KLF2 upregulation, as this transcription factor acts as a tumor suppressor gene [28]. Three lentivirus shRNA KLF2s (shKLF2-1, shKLF2-2 or shKLF2-3) were introduced into HEL cells. While knockdown with shKLF2-1 was not effective (data not shown), significant downregulation of KLF2 transcription was seen with shKLF2-2 and shKLF2-3 lentiviruses (Fig. 5c). As expected, KLF2 induction was significantly lower following lovastatin (20 µM) treatment in shKLF2-2 and shKLF2-3 cells by both RT-qPCR (Fig. 5c) and western blot (Fig. 5d), relative to control scrambled cells.

To detect if knockdown of KLF2 influenced cell proliferation in culture, the growth of shKLF2-2 and scrambled control cells was compared by MTT assay. Knockdown of KLF2 marginally affected the proliferation of shKLF2-2 cells compared to control cells (Fig. 5e). However, when these cells were treated with lovastatin, the growth suppression was lower in shKLF2-2 than in scrambled control cells (Fig. 5e). These results suggests that KLF2 in part is involved in growth suppression by lovastatin. Interestingly, while the level of FAM83A and DDIT4 are significantly downregulated by lovastatin in scrambled control cells, in shKLF2-2 cells this level slightly increased, although not statistically significant (Fig. 5f,g). In addition, the level of phospho-ERK is much higher in KLF2-2 than in scrambled control cells after treatment with lovastatin (Fig. 5h). These results suggest that KLF2 may suppress cell proliferation through the FAM83A/DDIT4/ERK pathway.

Since cholesterol biosynthesis genes were regulated by lovastatin in leukemia cells, we examined whether this regulation is mediated through KLF2. Indeed, by examining 12 cholesterol biosynthesis genes, we showed that induction of MSMO1, MVD, HMGCR, MVK, TM7SF2 and HMGCS1 gene expression by lovastatin is much lower in shKLF2-2 cells compared to control (Fig. 6a-f). However, this induction is similar for CYP51A1, LSS, EBP, DHCR24, DHCR7 and even higher for SQLE in shKLF2-2 cells (Fig. 6g-l). In shKLF2-2 cells, while the level of SREBP1 was constant, higher expression of SREBP2 was observed, further demonstrating a partial role for KLF2 in cholesterol biosynthesis (Supplementary Fig. 4). In the proposed model depicted in Fig. 7a, FAM83A and DDIT4 expression promotes leukemia cell proliferation through activation of ERK. Lovastatin inhibits leukemia cell survival and proliferation in part through suppression of the FAM83A/DDIT4/ERK1/2 pathway, mediated by the induction of the tumor suppressor gene KLF2, as well as cholesterol biosynthesis genes (Fig. 7b).