Inhibition of c-Myc Overcomes Cytotoxic Drug Resistance in Acute Myeloid Leukemia Cells by Promoting Differentiation

Nowadays, drug resistance still represents a major obstacle to successful acute myeloid leukemia (AML) treatment and the underlying mechanism is not fully elucidated. Here, we found that high expression of c-Myc was one of the cytogenetic characteristics in the drug-resistant leukemic cells. c-Myc over-expression in leukemic cells induced resistance to chemotherapeutic drugs, enhanced colony formation capacity and inhibited cell differentiation induced by all-trans retinoic acid (ATRA). Meanwhile, inhibition of c-Myc by shRNA or specific c-Myc inhibitor 10058-F4 rescued the sensitivity to cytotoxic drugs, restrained the colony formation ability and promoted differentiation. RT-PCR and western blotting analysis showed that down-regulation of C/EBPβ contributed to the poor differentiation state of leukemic cells induced by c-Myc over-expression. Importantly, over-expression of C/EBPβ could reverse c-Myc induced drug resistance. In primary AML cells, the c-Myc expression was negatively correlated with C/EBPβ. 10058-F4, displayed anti-proliferative activity and increased cellular differentiation with up-regulation of C/EBPβ in primary AML cells. Thus, our study indicated that c-Myc could be a novel target to overcome drug resistance, providing a new approach in AML therapy.     

Up-regulation of Translation Eukaryotic Initiation Factor 4E in Nucleophosmin 1 Haploinsufficient Cells Results in Changes in CCAAT Enhancer-binding Protein α Activity: IMPLICATIONS IN MYELODYSPLASTIC SYNDROME AND ACUTE MYELOID LEUKEMIA*

NPM1 is a ubiquitously expressed nucleolar phosphoprotein, the gene for which maps to chromosome 5q35 in close proximity to a commonly deleted region associated with (del)5q, a type of myelodysplastic syndrome (MDS). This region is also a frequent target of deletions in de novo and therapy-related MDS/acute myeloid leukemia. Previous studies have shown that Npm1^+/− mice develop an MDS-like disease that transforms to acute myeloid leukemia over time. To better understand the mechanism by which NPM1 haploinsufficiency causes an MDS phenotype, we generated factor-dependent myeloid cell lines from the bone marrow of Npm1^+/+ and Npm1^+/− mice and demonstrated compromised neutrophil-specific gene expression in the MNPM1^+/− cells. We attribute these observations to increased levels of the shorter, dominant negative leukemogenic isoform (p30) of CCAAT enhancer-binding protein α (C/EBPα). We show that this increase is caused, in part, by elevated levels of the activated translation initiation factor eIF4E, overexpression of which also increases translation of C/EBPαp30 in HEK293 cells. In a positive feedback loop, eIF4E expression is further elevated both at the mRNA and protein levels by C/EBPαp30 but not by the full-length C/EBPαp42. Re-expression of C/EBPαp42 or NPM1 but not C/EBPαp30 in MNPM1^+/− cells partially rescues the myeloid phenotype. Our observations suggest that the aberrant feed-forward pathway that keeps eIF4E and C/EBPαp30 elevated in NPM1^+/− cells contributes to the MDS phenotype associated with NPM1 deficiency.     

Radotinib Induces Apoptosis of CD11b+ Cells Differentiated from Acute Myeloid Leukemia Cells

Radotinib, developed as a BCR/ABL tyrosine kinase inhibitor (TKI), is approved for the second-line treatment of chronic myeloid leukemia (CML) in South Korea. However, therapeutic effects of radotinib in acute myeloid leukemia (AML) are unknown. In the present study, we demonstrate that radotinib significantly decreases the viability of AML cells in a dose-dependent manner. Kasumi-1 cells were more sensitive to radotinib than NB4, HL60, or THP-1 cell lines. Furthermore, radotinib induced CD11b expression in NB4, THP-1, and Kasumi-1 cells either in presence or absence of all trans-retinoic acid (ATRA). We found that radotinib promoted differentiation and induced CD11b expression in AML cells by downregulating LYN. However, CD11b expression induced by ATRA in HL60 cells was decreased by radotinib through upregulation of LYN. Furthermore, radotinib mainly induced apoptosis of CD11b⁺ cells in the total population of AML cells. Radotinib also increased apoptosis of CD11b⁺ HL60 cells when they were differentiated by ATRA/dasatinib treatment. We show that radotinib induced apoptosis via caspase-3 activation and the loss of mitochondrial membrane potential (ΔΨ_m) in CD11b⁺ cells differentiated from AML cells. Our results suggest that radotinib may be used as a candidate drug in AML or a chemosensitizer for treatment of AML by other therapeutics.     

Engagement of SIRPα Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

Background

Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRPα) on macrophages. Although AML cells express SIRPα, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRPα in acute myeloid leukemia.

Design and Methods

We analyzed the expression of SIRPα, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRPα on two low SIRPα expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRPα expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.

Results

By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRPα is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0–M3. Interestingly, AML patients with high SIRPα expression had a poor prognosis. Our results also showed that SIRPα is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRPα with an agonistic antibody in the cells stably expressing chimeric SIRPα, led to inhibition of growth and induction of programmed cell death. Finally, the SIRPα-derived signaling synergized with the activity of established antileukemic drugs.

Conclusions

Our data indicate that triggering of SIRPα has antileukemic effect and may function as a potential therapeutic target in AML.     

NAD(P)H:quinone Oxidoreductase 1 (NQO1) Competes with 20S Proteasome for Binding with C/EBPα Leading to Its Stabilization and Protection against Radiation-induced Myeloproliferative Disease

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that protects cells against radiation and chemical-induced oxidative stress. Disruption of NQO1 gene in mice leads to increased susceptibility to myeloproliferative disease. In this report, we demonstrate that NQO1 controls the stability of myeloid differentiation factor C/EBPα against 20S proteasomal degradation during radiation exposure stress. Co-immunoprecipitation studies showed that NQO1, C/EBPα, and 20S all interacted with each other. C/EBPα interaction with 20S led to the degradation of C/EBPα. NQO1 in presence of its cofactor NADH protected C/EBPα against 20S degradation. Deletion and site-directed mutagenesis demonstrated that NQO1 and 20S competed for the same binding region ²⁶⁸SGAGAGKAKKSV²⁷⁹ in C/EBPα. Mutagenesis studies also revealed that NQO1Y127/Y129 required for NADH binding is essential for NQO1 stabilization of C/EBPα. Exposure of mice and HL-60 cells to 3 Grays of γ-radiation led to increased NQO1 that stabilized C/EBPα against 20S proteasomal degradation. This mechanism of NQO1 regulation of C/EBPα may provide protection to bone marrow against adverse effects of radiation exposure. The studies have significance for human individuals carrying hetero- or homozygous NQO1P187S mutation and are deficient or lack NQO1 protein.     

NRH:Quinone Oxidoreductase 2 (NQO2) Protein Competes with the 20 S Proteasome to Stabilize Transcription Factor CCAAT Enhancer-binding Protein α (C/EBPα), Leading to Protection against γ Radiation-induced Myeloproliferative Disease

NRH:quinone oxidoreductase 2 (NQO2) is a flavoprotein that protects cells against radiation and chemical-induced oxidative stress. Disruption of the NQO2 gene in mice leads to γ radiation-induced myeloproliferative diseases. In this report, we showed that the 20 S proteasome and NQO2 both interact with myeloid differentiation factor CCAAT-enhancer-binding protein α (C/EBPα). The interaction of the 20 S proteasome with C/EBPα led to the degradation of C/EBPα. NQO2, in the presence of its cofactor NRH, protected C/EBPα against 20 S degradation. Deletion and site-directed mutagenesis demonstrated that NQO2 and 20 S competed for the same binding region of S(268)GAGAGKAKKSV(279) in C/EBPα. Exposure of mice and HL-60 cells to γ radiation enhanced the levels of NQO2, which led to an increased NQO2 interaction with C/EBPα and decreased 20 S interaction with C/EBPα. NQO2 stabilization of C/EBPα was independent of NQO1, even though both interacted with the same C/EBPα domain. NQO2^−/− mice, deficient in NQO2, failed to stabilize C/EBPα. This contributed to the development of γ radiation-induced myeloproliferative disease in NQO2^−/− mice.     

Interleukin (IL)-32β-mediated CCAAT/Enhancer-binding Protein α (C/EBPα) Phosphorylation by Protein Kinase Cδ (PKCδ) Abrogates the Inhibitory Effect of C/EBPα on IL-10 Production

We previously reported that IL-32β promotes IL-10 production in myeloid cells. However, the underlying mechanism remains elusive. In this study, we demonstrated that IL-32β abrogated the inhibitory effect of CCAAT/enhancer-binding protein α (C/EBPα) on IL-10 expression in U937 cells. We observed that the phosphorylation of C/EBPα Ser-21 was inhibited by a PKCδ-specific inhibitor, rottlerin, or IL-32β knockdown by siRNA and that IL-32β shifted to the membrane from the cytosol upon phorbol 12-myristate 13-acetate treatment. We revealed that IL-32β suppressed the binding of C/EBPα to IL-10 promoter by using ChIP assay. These data suggest that PKCδ and IL-32β may modulate the effect of C/EBPα on IL-10 expression. We next demonstrated by immunoprecipitation that IL-32β interacted with PKCδ and C/EBPα, thereby mediating C/EBPα Ser-21 phosphorylation by PKCδ. We showed that IL-32β suppressed the inhibitory effect of C/EBPα on IL-10 promoter activity. However, the IL-10 promoter activity was reduced to the basal level by rottlerin treatment. When C/EBPα serine 21 was mutated to glycine (S21G), the inhibitory effect of C/EBPα S21G on IL-10 promoter activity was not modulated by IL-32β. Taken together, our results show that IL-32β-mediated C/EBPα Ser-21 phosphorylation by PKCδ suppressed C/EBPα binding to IL-10 promoter, which promoted IL-10 production in U937 cells.