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

BACKGROUND: Lovastatin, an HMG-CoA inhibitor and an effective cholesterol lowering drug, exhibits anti-neoplastic activity towards several types of cancer, although the underlying mechanism is still not fully understood. Herein, we investigated mechanism of growth inhibition of leukemic cells by lovastatin. METHODS: RNAseq analysis was used to explore the effect of lovastatin on gene expression in leukemic cells. An animal model of leukemia was used to test the effect of this statin in vivo. FAM83A and DDIT4 expression was knocked-downed in leukemia cells via lentivirus-shRNA. Western blotting, RT-qPCR, cell cycle analysis and apoptosis assays were used to determine the effect of lovastatin-induced growth suppression in leukemic cells in vitro. RESULTS: Lovastatin treatment strongly inhibited cancer progression in a mouse model of erythroleukemia induced by Friend virus. In tissue culture, lovastatin inhibited cell proliferation through induction of G1 phase cell cycle arrest and apoptosis. Interestingly, lovastatin induced most known genes associated with cholesterol biosynthesis in leukemic cells. Moreover, it suppressed ERK1/2 phosphorylation by downregulating FAM83A and DDIT4, two mediators of MAP-Kinase signaling. RNAseq analysis of lovastatin treated leukemic cells revealed a strong induction of the tumor suppressor gene KLF2. Accordingly, lentivirus-mediated knockdown of KLF2 antagonized leukemia cell suppression induced by lovastatin, associated with higher ERK1/2 phosphorylation compared to control. We further show that KLF2 induction by lovastatin is responsible for lower expression of the FAM83A and DDIT4 oncogenes, involved in the activation of ERK1/2. KLF2 activation by lovastatin also activated a subset of cholesterol biosynthesis genes that may further contribute to leukemia suppression. CONCLUSIONS: These results implicate KLF2-mediated FAM83A/DDIT4/MAPK suppression and activation of cholesterol biosynthesis as the mechanism of leukemia cell growth inhibition by lovastatin.

4SC-202 induces apoptosis in myelodysplastic syndromes and the underlying mechanism.

Epigenetic modifications play crucial roles in regulating the self-renewal and differentiation of hematopoiesis. 4SC-202, a novel inhibitor of histone lysine-specific demethylase 1 (LSD1) and class I histone deacetylases (HDACs), is a potential therapeutic agent to treat myelodysplastic syndrome (MDS). However, it remains unclarified of the mechanism of 4SC-202. In the study, we found that 4SC-202 treatment could inhibit cell viability, induce apoptosis and cause G2/M cell cycle arrest in MDS cell line SKM-1. Heme oxygenase-1 (HO-1) was correlated with disease progression and chemotherapy resistance. Here, we reported that 4SC-202 could down-regulate the expression of HO-1, and up-regulation of HO-1 could significantly attenuate the 4SC-202-induced apoptosis in SKM-1 cells. In addition, the activation of NF-κB pathway was suppressed by 4SC-202, while up-regulation of HO-1 significantly weakened the 4SC-202-induced suppression of the NF-κB pathway, thereby attenuating the efficacy of 4SC-202. However, down-regulation of HO-1 enhanced the sensitivity of 4SC-202 against SKM-1 cells. Moreover, SKM-1 cells were transfected with HO-1 overexpression lentivirus, subsequently injected into the tail vein of NOD/SCID mice, followed by administration of 4SC-202 in mice. As a result, up-regulation HO-1 could partially attenuate 4SC-202-suppressed MDS cells growth in NOD/SCID mice. In conclusion, 4SC-202 could induce apoptosis via the NF-κB pathway, and our present finding may provide a novel therapeutic strategy for MDS.

Deletion of HO-1 blocks development of B lymphocytes in mice.

B lymphocytes, a key cluster of cells composing the immune system, can protect against abnormal biological factors. Heme oxygenase-1 (HO-1) plays important roles in cell proliferation and immune regulation, but its effects on the development and growth of B lymphocytes are still unknown. Herein, the count of B lymphocytes in HO-1 gene knockout (HO-1+/-) mice was significantly lower than that of the HO-1 gene wild-type (HO-1WT) mice. Meanwhile, the cell count of HO-1+/- mice did not recover after irradiation for one week, due to the G0/G1 phase arrest of Pro-B cells and the augmented apoptosis of Pre-B cells. Up-regulation of HO-1 by lentivirus attenuated the Pro-B cell cycle arrest and Pre-B cell apoptosis. To understand the molecular mechanism by which HO-1 knockout blocked B lymphocyte development, protein-to-protein interaction network and Western blot were used. The PI3K/AKT signaling pathway mediated the regulatory effects of HO-1 on B lymphocytes. In conclusion, HO-1 is a crucial transcriptional repressor for B cell development.

Up-regulation of HO-1 promotes resistance of B-cell acute lymphocytic leukemia cells to HDAC4/5 inhibitor LMK-235 via the Smad7 pathway.

PURPOSE: HDAC4/5 and Smad7 are potential therapeutic targets for the onset and progression of B-cell acute lymphocytic leukemia (B-ALL) and indices for clinical prognosis. In contrast, HO-1 (heat shock protein 32) plays a key role in protecting tumor cells from apoptosis. METHODS: HDAC4/5, HO-1 and Smad7 expressions in 34 newly diagnosed B-ALL cases were detected by real-time PCR and Western blot. Lentivirus and small interference RNA were used to transfect B-ALL cells. The expression of Smad7 was detected after treatment with LMK-235 or Hemin and ZnPP. Apoptosis and proliferation were evaluated by flow cytometry, CCK-8 assay and Western blot. RESULTS: HDAC4/5 was overexpressed in B-ALL patients with high HO-1 levels. Increasing the concentration of HDAC4/5 inhibitor LMK-235 induced the decrease of Smad7 and HO-1 expressions and the apoptosis of B-ALL cells by suppressing the phosphorylation of AKT (Protein kinase B). Up-regulating HO-1 alleviated the decrease of Smad7 expression and enhanced B-ALL resistance to LMK-235 by activating p-AKT which reduced the apoptosis of B-ALL cells and influenced the survival of leukemia patients. Silencing Smad7 also augmented the apoptosis rate of B-ALL cells by suppressing p-AKT. CONCLUSION: HO-1 played a key role in protecting tumor cells from apoptosis, and HDAC4/5 were related with the apoptosis of B-ALL cells. LMK-235 may be able to improve the poor survival of leukemia patients.

Crucial role of HO-1/IRF4-dependent apoptosis induced by panobinostat and lenalidomide in multiple myeloma.

Inhibition of histone deacetylase (HDAC) is a promising therapeutic strategy for various hematologic cancers. Panobinostat has been approved for treating patients with multiple myeloma (MM) by the FDA. Since the mechanism for the resistance of panobinostat to MM remains elusive, we aimed to clarify this mechanism and the synergism of panobinostat with lenalidomide. The mRNA and protein of transcription factor IRF4 were overexpressed in CD138+ mononuclear cells from MM patients compared with in those from healthy donors. Given that direct IRF4 inhibitors are clinically unavailable, we intended to explore the mechanism by which IRF4 expression was regulated in MM. Heme oxygenase-1 (HO-1) promotes the growth and drug resistance of various malignant tumors, and its expression is positively correlated with IRF4 mRNA and protein expression levels. Herein, panobinostat induced acetylation of histone H3K9 and activation of caspase-3 in MM cells, being inversely correlated with the reduction of HO-1/IRF4/MYC protein levels. Adding Z-DEVD-FMK, a caspase-3 inhibitor, abolished the HO-1/IRF4 reduction by panobinostat alone or in combination with lenalidomide, suggesting that caspase-3-mediated HO-1/IRF4/MYC degradation occurred. Given that lenalidomide stabilized cereblon and facilitated IRF4 degradation in MM cells, we combined it with LBH589, an HDAC inhibitor. LBH589 and lenalidomide exerted synergistic effects, and LBH589 reversed the efficacy of lenalidomide on the resistance of CD138+ primary MM cells, in part due to simultaneous suppression of HO-1, IRF4 and MYC. The results provide an eligible therapeutic strategy for targeting MM depending on the IRF4 network and clinical testing of this drug combination in MM patients.

Low-dose staurosporine selectively reverses BCR-ABL-independent IM resistance through PKC-α-mediated G2/M phase arrest in chronic myeloid leukaemia.

Imatinib (IM) resistance has become a critical problem for the treatment of patients with relapsed chronic myeloid leukaemia (CML), so novel therapies are in need. Various isotypes of protein kinases C (PKCs) are up-regulated in CML and related with BCR-ABL regulating several signalling pathways that are crucial to malignant cellular transformation. However, it is still unknown whether PKC isotypes play crucial roles in IM resistance. Therefore, we herein used a PKC pan-inhibitor staurosporine (St). To protect normal cells from damage, a proper dose of St was used, at which IM-resistant CML cells were selectively killed in combination with IM but normal cells survived. The IM resistance of CML cells was best reversed by 4 nM St alone, mainly depending on the G2/M phase arrest. Cell cycle-related proteins p21, CDK2, cyclin A and cyclin B were down-regulated. Meanwhile, PKC-α was more significantly decreased than other PKC isotypes at this concentration. The PKC-α-dependent G2/M phase arrest was induced by down-regulation of CDC23, an important regulator of mitotic progression. Low-dose St also reversed IM resistance in vivo. In conclusion, low-dose St selectively increased the sensitivity of IM-resistant CML to IM by arresting cell cycle in the G2/M phase through PKC-α-dependent CDC23 inhibition.