Novel triterpenoid CDDO-Me is a potent inducer of apoptosis and differentiation in acute myelogenous leukemia.

It has been shown that the novel synthetic triterpenoid CDDO inhibits proliferation and induces differentiation and apoptosis in myeloid leukemia cells. In the current study the effects of the C-28 methyl ester of CDDO, CDDO-Me, were analyzed on cell growth and apoptosis of leukemic cell lines and primary acute myelogenous leukemia (AML). CDDO-Me decreased the viability of leukemic cell lines, including multidrug resistant (MDR)-1-overexpressing, p53(null) HL-60-Dox and of primary AML cells, and it was 3- to 5-fold more active than CDDO. CDDO-Me induced a loss of mitochondrial membrane potential, induction of caspase-3 cleavage, increase in annexin V binding and DNA fragmentation, suggesting the induction of apoptosis. CDDO-Me induced pro-apoptotic Bax protein that preceded caspase activation. Furthermore, CDDO-Me inhibited the activation of ERK1/2, as determined by the inhibition of mitochondrial ERK1/2 phosphorylation, and it blocked Bcl-2 phosphorylation, rendering Bcl-2 less anti-apoptotic. CDDO-Me induced granulo-monocytic differentiation in HL-60 cells and monocytic differentiation in primary cells. Of significance, colony formation of AML progenitors was significantly inhibited in a dose-dependent fashion, whereas normal CD34(+) progenitor cells were less affected. Combinations with ATRA or the RXR-specific ligand LG100268 enhanced the effects of CDDO-Me on cell viability and terminal differentiation of myeloid leukemic cell lines. In conclusion, CDDO-Me is an MDR-1- and a p53-independent compound that exerts strong antiproliferative, apoptotic, and differentiating effects in myeloid leukemic cell lines and in primary AML samples when given in submicromolar concentrations. Differential effects of CDDO-Me on leukemic and normal progenitor cells suggest that CDDO-Me has potential as a novel compound in the treatment of hematologic malignancies.     

Ligation of the CD44 adhesion molecule inhibits drug-induced apoptosis in human myeloid leukemia cells

Adhesion molecules can improve hematopoietic cell survival; however, their role in leukemic cell resistance to drug-induced apoptosis is poorly documented. The CD44 adhesion molecule is strongly expressed on acute myeloid leukemia (AML) blasts. Using 2 myeloid cell lines, HL60 and NB4, evidence is presented that prior incubation with the CD44-specific monoclonal antibody (mAb) A3D8, reported to induce differentiation of AML blasts, significantly decreases apoptosis induced by 3 drugs used in AML chemotherapy: daunorubicin (DNR), mitoxantrone, and etoposide. In addition, in HL60 cells, CD44 ligation with A3D8 mAb fully abrogates the DNR-triggered generation of ceramide, a lipid second messenger involved in the DNR apoptotic signaling pathway. Moreover, results show that the A3D8 mAb and Bcl-2 additively inhibit DNR-induced apoptosis in HL60 cells overexpressing Bcl-2. These results suggest that, to eradicate AML blasts, the differentiation-inducing anti-CD44 mAb A3D8 should not be administered prior to apoptosis-inducing drugs.     

Prognostic impact, concurrent genetic mutations, and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients: further evidence for CEBPA double mutant AML as a distinctive disease entity

We evaluated concurrent gene mutations, clinical outcome, and gene expression signatures of CCAAT/enhancer binding protein alpha (CEBPA) double mutations (CEBPA(dm)) versus single mutations (CEBPA(sm)) in 1182 cytogenetically normal acute myeloid leukemia (AML) patients (16-60 years of age). We identified 151 (12.8%) patients with CEBPA mutations (91 CEBPA(dm) and 60 CEBPA(sm)). The incidence of germline mutations was 7% (5 of 71), including 3 C-terminal mutations. CEBPA(dm) patients had a lower frequency of concurrent mutations than CEBPA(sm) patients (P < .0001). Both, groups were associated with a favorable outcome compared with CEBPA(wt) (5-year overall survival [OS] 63% and 56% vs 39%; P < .0001 and P = .05, respectively). However, in multivariable analysis only CEBPA(dm) was a prognostic factor for favorable OS outcome (hazard ratio [HR] 0.36, P < .0001; event-free survival, HR 0.41, P < .0001; relapse-free survival, HR 0.55, P = .001). Outcome in CEBPA(sm) is dominated by concurrent NPM1 and/or FLT3 internal tandem duplication mutations. Unsupervised and supervised GEP analyses showed that CEBPA(dm) AML (n = 42), but not CEBPA(sm) AML (n = 18), expressed a unique gene signature. A 25-probe set prediction signature for CEBPA(dm) AML showed 100% sensitivity and specificity. Based on these findings, we propose that CEBPA(dm) should be clearly defined from CEBPA(sm) AML and considered as a separate entity in the classification of AML.     

Erlotinib exhibits antineoplastic off-target effects in AML and MDS: a preclinical study

Erlotinib, an inhibitor of the epidermal growth factor receptor (EGFR), induces differentiation, cell-cycle arrest, and apoptosis of EGFR-negative myeloblasts of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), as well as in EGFR-negative cell lines representing these diseases (P39, KG-1, and HL 60). This off-target effect can be explained by inhibitory effects on JAK2. Apoptosis induction coupled to mitochondrial membrane permeabilization occurred independently from phenotypic differentiation. In apoptosis-sensitive AML cells, erlotinib caused a rapid (within less than 1 hour) nucleocytoplasmic translocation of nucleophosmin-1 (NPM-1) and p14(ARF). Apoptosis-insensitive myeloblasts failed to manifest this translocation yet became sensitive to apoptosis induction by erlotinib when NPM-1 was depleted by RNA interference. Moreover, erlotinib reduced the growth of xenografted human AML cells in vivo. Erlotinib also killed CD34(+) bone marrow blasts from MDS and AML patients while sparing normal CD34(+) progenitors. This ex vivo therapeutic effect was once more associated with the nucleocytoplasmic translocation of NPM-1 and p14(ARF). One patient afflicted with both MDS and non-small cell lung cancer manifested hematologic improvement in response to erlotinib. In summary, we here provide novel evidence in vitro, ex vivo, and in vivo for the potential therapeutic efficacy of erlotinib in the treatment of high-risk MDS and AML.     

Ellagic acid,a natural polyphenolic compound,induces apoptosis and potentiates retinoic acid-induced differentiation of human leukemia HL-60 cells

All-trans retinoic acid (ATRA) is a standard drug used for differentiation therapy in acute promyelocytic leukemia. To potentiate this therapy, we examined the effect of ellagic acid (EA), a natural polyphenolic compound with antiproliferative and antioxidant properties, on the growth and differentiation of HL-60 acute myeloid leukemia cells. EA was found to induce apoptosis, which was blocked by pan-caspase inhibitor, Z-VAD-FMK. EA activated the caspase-3 pathway and enhanced the expressions of myeloid differentiation markers (CD11b, MRP-14 protein, granulocytic morphology) induced by ATRA treatment. These results indicate that EA is a potent apoptosis inducer and also effectively potentiates ATRA-induced myeloid differentiation of HL-60 cells.     

Retinoic acid inhibits sodium butyrate-induced monocytic differentiation of HL60 cells while synergistically inducing granulocytoid differentiation

The human myeloid leukemia cell line HL60 is an in vitro model to study myeloid differentiation. HL60 cells differentiate along different cell type lineages in response to a variety of compounds. The direction of differentiation is usually inducer-specific. However, the response of HL60 cells to sodium n-butyrate (NaB) is pleiotropic. NaB induces HL60 along the monocytic, neutrophilic, eosinophilic, and basophilic pathways. In this study we saw that physiologic concentrations of all-trans-retinoic acid (RA) switched the direction of NaB-induced differentiation from monocytic to granulocytic. We showed previously (Breitman & He, Cancer Res 1990: 50: 6268-6273) that combinations of RA and NaB synergistically induce HL60 to cells that reduce nitroblue tetrazolium. The present study shows that this synergy was even greater if the parameter measured was mature granulocytes. Our results raise the possibility that the endogenous RA in the serum used to grow cells in culture may affect the direction of differentiation of HL60 cells induced by NaB. Furthermore, our results may provide additional rationale for the use of combinations of RA and NaB in the treatment of some leukemias.     

Induction of differentiation of human myeloid leukemias: surface changes probed with monoclonal antibodies

The surface changes occurring in three acute myeloid leukemia cell lines (HL60, ML3, and KG1) induced to differentiate by a variety of agents (dimethylsulfoxide, retinoic acid, 12-O-tetradecanoylphorbol-13- acetate, and factors present in lymphocyte conditioned medium) were probed using monoclonal antibodies that are differentiation stage- and lineage-specific. In all cases, the differentiated phenotype was defective and varied with the inducing agent and the cell line used. HL60 proved to be the most sensitive to the effect of the inducers. Retinoic acid was better than DMSO, and TPA was better than the medium factors in the ability to induce granulocytic and monocytic differentiation, respectively, in HL60 cells. These findings indicate that the differentiation block in acute myeloid leukemias is heterogeneous and that each cell line has different phenotypic characteristics that are responsible for the extent of differentiation obtained with a given inducer. These results also suggest that the extent of the differentiation response in vitro may be improved by the use of more suitable inducers for each specific leukemic line.     

Targeted inhibition of VEGF-modulated survival and arsenic sensitivity in acute myeloid leukemia (AML)

Vascular endothelial growth factor (VEGF) is a specific growth factor for tumor-associated angiogenesis, and is also involved in leukemogenesis; however, its exact role in leukemia development remains elusive. In this study we used antisense oligonucleotide (AS) to manipulate VEGF function in acute myeloid leukemia (AML). HL60 and primary AML cells were transfected with VEGF AS (0.3 μmol/l). Cell proliferation and survival were assessed using the trypan blue exclusion assay. The viability of cells was determined using MTT. The IC50 values of arsenic trioxide (ATO) in HL60 cell were calculated by ICp software. The results showed that VEGF AS effectively inhibited AML cell proliferation and survival 72 hours post-transfection and exhibited time dependence. The IC50 value of ATO was significantly down-regulated by VEGF AS in HL60. Meanwhile, VEGF AS alone induced cell apoptosis, and promoted ATO-induced apoptosis. There is synergistic inhibitory effects between AS and ATO. VEGF AS down-regulated VEGF protein level in the supernatants and cellular VEGF mRNA level by western blot and real-time PCR. Therefore, targeted inhibition of VEGF suppressed survival and increased arsenic sensitivity in AML.     

Effects of anti-CD44 monoclonal antibodies on differentiation and apoptosis of human myeloid leukemia cell lines.

Acute myeloid leukemia (AML) is a heterogeneous leukemia characterized by the blockage of myeloid differentiation at different stages, which define distinct AML subtypes. We have recently reported that the ligation of CD44 with 2 activating monoclonal antibodies (mAbs), A3D8 and H90, triggers terminal differentiation of leukemic blasts in AML-M1/2 to AML-M5 subtypes, which are the most frequent ones. However, fresh AML blasts have short in vitro lifespans. Therefore, to find relevant in vitro cellular models for further studying the mechanisms involved in CD44-induced differentiation, we investigated whether CD44 ligation with A3D8 and H90 mAbs can induce terminal differentiation of THP-1, NB4, and HL60 cells, each interesting models of AML-M5 (monoblastic subtype), AML-M3 (promyelocytic subtype), and AML-M2 (myeloblastic subtype), respectively. We also study whether CD44 ligation induces a loss of proliferative capacity, an important feature of late-stage myeloid differentiation. In the second part of our study, we investigated whether A3D8 and H90 anti-CD44 mAbs can induce the differentiation and inhibit the proliferation of KG1a cells, which are very immature AML-M0 blasts. Using functional, antigenic, and cytologic criteria, we presently show that A3D8 and/or H90 induce terminal differentiation of THP-1, HL60, and NB4 cell lines and strongly inhibit their proliferation. Interestingly, cell-specific effects of H90 and A3D8 are observed. We also observe that incubation with A3D8 for 3 to 6 days induces an apoptotic cell death that is moderate in the case of THP-1 and HL60 cells and massive in the case of NB4 cells. Finally, our results demonstrate for the first time that it is possible to reverse the leukemic blockage of KG1a cells by using both an anti-CD44 mAb and retinoic acid. This result may provide a new experimental basis for a differentiative therapy in AML-M0 patients.     

Disulfiram/copper complex activated JNK/c-jun pathway and sensitized cytotoxicity of doxorubicin in doxorubicin resistant leukemia HL60 cells

Resistance to chemotherapy and non-specific cytotoxicity are the major challenges to the treatment of acute myeloid leukemia (AML). In this study, we demonstrated that the disulfiram/copper (DS/Cu) complex alone exhibited cytotoxicity to doxorubicin (Dox) resistant leukemia HL60 cells (HL60/Dox) and enhanced cytotoxicity of Dox to HL60/Dox cells. DS/Cu inhibited Bcl-2 expression and enhanced Dox-induced apoptosis. DS/Cu/Dox in combination significantly induced c-Jun expression and JNK and c-Jun phosphorylation. JNK inhibitor Sp600125 attenuated DS/Cu/Dox-induced apoptosis and suppressed DS/Cu/Dox-induced protein expression in JNK/c-jun pathway. This study suggested that DS/Cu complex may re-sensitize HL60/Dox cells to Dox through activating JNK/c-jun as well as inhibiting anti-apoptotic bcl-2 expression.     

The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated

Impairment of CCAAT Enhancer Binding Protein alpha (CEBPA) function is a common finding in acute myeloid leukemia; nevertheless, its relevance for acute promyelocytic leukemia pathogenesis is unclear. We analyzed the expression and assessed the methylation status of the core and upstream promoters of CEBPA in acute promyelocytic leukemia at diagnosis. Patients with acute promyelocytic leukemia (n = 18) presented lower levels of CEBPA expression compared to healthy controls (n = 5), but higher levels than those in acute myeloid leukemia with t(8;21) (n = 9) and with inv(16) (n = 5). Regarding the core promoter, we detected no methylation in 39 acute promyelocytic leukemia samples or in 8 samples from controls. In contrast, analysis of the upstream promoter showed methylation in 37 of 39 samples, with 17 patients showing methylation levels over 30%. Our results corroborate data obtained in animal models showing that CEBPA is down-regulated in acute promyelocytic leukemia stem cells and suggest that epigenetic mechanisms may be involved.