Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia.

BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.

Targeting PKC-mediated signal transduction pathways using enzastaurin to promote apoptosis in acute myeloid leukemia-derived cell lines and blast cells.

Recent studies in acute myeloid leukemia (AML) suggest activation of pro-proliferative signaling cascades including those mediated by protein kinase C (PKC) represent a poor prognostic factor for patients. The classical PKC isoforms α and ß generally support survival signaling and have emerged as important targets for anti-cancer therapy. Enzastaurin is a PKC ß inhibitor and is in clinical trials for lymphomas, gliomas, and lung cancer. Presently, it is not known if enzastaurin could be effective against AML. In the current study, we found that high dose enzastaurin was found to promote apoptosis in the AML-derived cell lines and in blast cells from AML patients. The mechanism of cell death, however, likely does not involve PKC ß as another PKC ß inhibitor was not toxic to AML cell lines and did not promote enzastaurin-induced cell killing. While enzastaurin is fairly specific for PKC ß, the agent can inhibit other PKC isoforms at higher concentrations. Enzastaurin was effective at inhibiting PKC α phosphorylation and membrane localization in the AML cell lines and suppressed phosphorylation of BCL2. Furthermore, enzastaurin suppressed activation of ERK (which can be activated by PKC α). Analysis of the serine/threonine phosphorylation profile in HL60 cells after enzastaurin treatment revealed that the drug inhibits the phosphorylation of a distinct set of proteins while promoting phosphorylation of another set of proteins. This suggests the drug may regulate multiple signaling pathways. Taken together, these findings suggest that enzastaurin could be effective in the therapy of AML.

TGF-beta receptor kinase inhibitor LY2109761 reverses the anti-apoptotic effects of TGF-beta1 in myelo-monocytic leukaemic cells co-cultured with stromal cells.

Transforming growth factor beta1 (TGF-beta1) is an essential regulator of cell proliferation, survival and apoptosis, depending on the cellular context. TGF-beta1 is also known to affect cell-to-cell interactions between tumour cells and stromal cells. We investigated the role of TGF-beta1 in the survival of myelo-monocytic leukaemia cell lines co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSC). Treatment with recombinant human (rh)TGF-beta1 inhibited spontaneous and cytarabine-induced apoptosis in U937 cells, most prominently in U937 cells directly attached to MSCs. Conversely, the pro-survival effects of TGF-beta1 were inhibited by LY2109761 or TGF-beta1 neutralizing antibody. rhTGF-beta1 increased pro-survival phosphorylation of Akt, which was inhibited by LY2109761. The combination of rhTGF-beta1 and MSC co-culture induced significant upregulation of C/EBPbeta gene (CEBPB) and protein expression along with increased C/EBPbeta liver-enriched activating protein: liver-enriched inhibitory protein ratio, suggesting the novel role of C/EBPbeta in TGF-beta1-mediated U937 cell survival in the context of stromal cell support. In summary, these results indicate that TGF-beta1 produced by BM stromal cells promotes the survival and chemoresistance of leukaemia cells under the direct cell-to-cell interactions. The blockade of TGF-beta signalling by LY2109761, which effectively inhibited the pro-survival signalling, may enhance the efficacy of chemotherapy against myelo-monocytic leukaemic cells in the BM microenvironment.

Cellular transformation by the HTLV-I Tax protein, a jack-of-all-trades.

The human T-cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that is responsible for adult T-cell leukemia and a neurological disease, HTLV-I-associated myelopathy/tropical spastic paraparesis. HTLV-I encodes an oncogenic protein, Tax, which affects a variety of cellular functions prompting it to be referred to as a jack-of-all trades. The ability of Tax to both transcriptionally regulate cellular gene expression and to functionally inactivate proteins involved in cell-cycle progression and DNA repair provide the basis for Tax-mediated transformation and leukemogenesis. This review will concentrate on the effects of Tax on the dysregulation of the G(1)/S and G(2)/M checkpoints as well as the suppression of DNA damage repair leading to cellular transformation.

Cooperativity of p19ARF, Mdm2, and p53 in murine tumorigenesis.

The p19ARF gene product responds to oncogenic stresses by interfering with the inhibitory effects of Mdm2 on p53, thus enhancing p53 activity and its antiproliferative functions. The absence of p19ARF in the mouse leads to early tumor susceptibility, presumably in part due to decreased p53 activity. To examine the tumorigenic cooperativity of p19ARF, Mdm2, and p53 in vivo, p19ARF-deficient mice were crossed first to p53-deficient mice and then to Mdm2 transgenic mice. The progeny were monitored for tumors. Cooperativity between p19ARF and p53 deficiencies in accelerating tumor formation was observed for most genotypes except p53-/- p19ARF-/- mice. p53-/- p19ARF-/- mice had a tumor incidence similar to p53-/- mice. In this context, tumor suppression by ARF appears to be primarily p53 dependent. The majority of the p19ARF+/- tumors deleted the wildtype p19ARF allele, in agreement with the previous studies, suggesting that p19ARF is a classic 'two hit' tumor suppressor. In a p53+/- background, however, all p19ARF+/- tumors retained a wildtype ARF allele and most also retained wildtype p53. In the second cross between p19ARF-deficient and Mdm2 transgenic mice, cooperativity in tumor incidence between Mdm2 overexpression and ARF deficiency was observed, consistent with the role of p19ARF in negatively regulating Mdm2 activity. These experiments further demonstrate in vivo the inter-relationships of the p19ARF-Mdm2-p53 signaling axis in tumor suppression.

Does hybridoma technology still have a place in transfusion medicine?

Hybridoma technology has contributed to virtually all areas of biology and medicine and set the scene for important advances in cell biology and immunodiagnostics. Blood transfusion and transfusion medicine have benefited significantly from development in hybridoma technology, and this has resulted in the generation of a wide range of monoclonal antibodies to human blood group antigens. These antibodies have also had an impact on diagnostic techniques such as enzyme-linked immunosorbent assay and flow cytometry as well as on the application of therapeutic agents in the clinical environment. However, rapid advances in the development of knockout and transgenic mice as well as in nucleic acid-based immunization may well lead hybridoma technology into a new era.

Activation of p53 signaling by MI-63 induces apoptosis in acute myeloid leukemia cells.

Non-mutational inactivation of p53 is frequent in acute myeloid leukemia (AML) via overexpression of MDM2. We report that treatment with MI-63, a novel inhibitor of MDM2, activates p53 signaling to induce apoptosis in AML cell lines and primary samples. Cell lines naturally devoid of p53 or expressing shRNA targeting p53 are refractory to apoptosis induction by MI-63, indicating that the effects of MI-63 require p53 expression. MI-63 induced G1 phase arrest and increased p21 expression. MI-63 induced pronounced apoptosis in all primary AML samples tested, and most important, was effective in inducing cell death of leukemia 'stem' cells. In addition, MI-63 showed synergy with both doxorubicin and AraC. Interestingly, treatment with MI-63 also led to a reduction in levels of MDM4 protein, a repressor of p53 mediated transcription, in AML cells. Our results warrant investigation of MI-63 or its analogs as anti-leukemic agents, alone or in combination with traditional chemotherapeutic agents.

Role of peroxisome proliferator-activated receptor-gamma and its coactivator DRIP205 in cellular responses to CDDO (RTA-401) in acute myelogenous leukemia.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor (NR) family of transcription factors with important regulatory roles in cellular growth, differentiation, and apoptosis. Using proteomic analysis, we showed expression of PPARgamma protein in a series of 260 newly diagnosed primary acute myelogenous leukemia (AML) samples. Forced expression of PPARgamma enhanced the sensitivity of myeloid leukemic cells to apoptosis induced by PPARgamma agonists 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and 15-deoxy-(12,14)-15DPGJ(2), through preferential cleavage of caspase-8. No effects on cell cycle distribution or differentiation were noted, despite prominent induction of p21 in PPARgamma-transfected cells. In turn, antagonizing PPARgamma function by small interfering RNA or pharmacologic PPARgamma inhibitor significantly diminished apoptosis induction by CDDO. Overexpression of coactivator protein DRIP205 resulted in enhanced differentiation induction by CDDO in AML cells through PPARgamma activation. Studies with DRIP205 deletion constructs showed that the NR boxes of DRIP205 are not required for this coactivation. In a phase I clinical trial of CDDO (RTA-401) in leukemia, CDDO induced an increase in PPARgamma mRNA expression in six of nine patient samples; of those, induction of differentiation was documented in four patients and that of p21 in three patients, all expressing DRIP205 protein. In summary, these findings suggest that cellular levels of PPARgamma regulate induction of apoptosis via caspase-8 activation, whereas the coactivator DRIP205 is a determinant of induction of differentiation, in response to PPARgamma agonists in leukemic cells.

Mechanisms of antileukemic activity of the novel Bcl-2 homology domain-3 mimetic GX15-070 (obatoclax).

In this study, we investigated the mechanism of apoptosis induction of obatoclax (GX15-070), a novel Bcl-2 homology domain-3 (BH3) mimetic, in acute myeloid leukemia (AML) cell lines and primary AML samples. Obatoclax inhibited cell growth of HL-60, U937, OCI-AML3, and KG-1 cell lines. Apoptosis induction contributed to the observed antiproliferative effects at concentrations of this agent that mirror its affinity for antiapoptotic Bcl-2 proteins. We show that obatoclax can promote the release of cytochrome c from isolated leukemia cell mitochondria and that apoptosis induced by this agent is preceded by the release of Bak from Mcl-1, liberation of Bim from both Bcl-2 and Mcl-1, and the formation of an active Bak/Bax complex. Notably, apoptosis was diminished, but not fully prevented, in the absence of Bak/Bax or Bim, suggesting that obatoclax has additional targets that contribute to its cytotoxicity. At growth inhibitory doses that did not induce apoptosis or decrease viability, obatoclax induced an S-G(2) cell-cycle block. Obatoclax induced apoptosis in AML CD34+ progenitor cells with an average IC(50) of 3.59 +/- 1.23 micromol/L although clonogenicity was inhibited at concentrations of 75 to 100 nmol/L. Obatoclax synergized with the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells and synergistically induced apoptosis in combination with AraC in leukemic cell lines and in primary AML samples. In conclusion, we show that obatoclax potently induces apoptosis and decreases leukemia cell proliferation and may be used in a novel therapeutic strategy for AML alone and in combination with other targeted agents and chemotherapeutics.

Ceramide promotes apoptosis in chronic myelogenous leukemia-derived K562 cells by a mechanism involving caspase-8 and JNK.

Ceramide is a sphingolipid that activates stress kinases such as p38 and c-JUN N-Terminal Kinase (JNK). Though Chronic Myelogenous Leukemia (CML) derived K562 cells resist killing by short chain C2-ceramide, we report here that longer chain C6-ceramide promotes apoptosis in these cells. C6-ceramide induces cleavage of Caspase-8 and Caspase-9, but only Caspase-8 is required for apoptosis. The sphingolipid killed CML derived KBM5 cells and, to a lesser extent, imatinib-resistant KBM5-STI cells suggesting that BCR-ABL can not completely block C6-ceramide-induced apoptosis but the kinase may regulate the process. BCR-ABL is known to suppress Protein Phosphatase 2A (PP2A) in CML cells. While C6-ceramide can activate PP2A in acute leukemia cells, the sphingolipid did not activate the phosphatase in K562 cells. C6-ceramide did not activate p38 kinase but did promote JNK activation and phosphorylation of JUN. Inhibition of JNK by pharmacological agent protected K562 cells from C6-ceramide suggesting that JNK plays an essential role in C6-ceramide mediated apoptosis. Furthermore, the sphingolipid promoted MCL-1 phosphorylation by a mechanism that, at least in part, involves JNK. The findings presented here suggest that Caspase-8, JNK, and perhaps MCL-1 may play important roles in regulating cell death and may represent new targets for therapeutic strategies for CML.

PPARgamma-active triterpenoid CDDO enhances ATRA-induced differentiation in APL.

Acute promyelocytic leukemia (APL) is associated with oncogenic PML-RARalpha that acts as a dominant negative transcriptional repressor of retinoic acid (RA) receptor target genes by recruiting histone deacetylase (HDAC). The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor family that forms heterodimers with retinoid X receptor (RXR). In addition to RAR targets, PML-RARalpha silence a wide range of nuclear receptor target genes including PPARgamma targets. All-trans-retinoic acid (ATRA), a ligand for the RA receptor (RAR), restores normal retinoid signaling and induces terminal differentiation of APL cells; however, APL cells can develop resistance to ATRA. Using ATRA sensitive NB4 and ATRA-resistant derivative MR2 cell lines, we demonstrate that PPARgamma ligand 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO) enhances pro-apoptotic and differentiating effects of ATRA in ATRA-sensitive NB4 cells and partially reverses ATRA resistance in MR2 cells. The CDDO/ATRA combination synergistically induces RARbeta2 expression both in ATRA-sensitive and -resistant APL cells. RARbeta2 MrNA induction by CDDO/ATRA was mediated in part by enhanced H3-Lys9 acetylation in the RARbeta2 promoter which in turn increased the affinity of RARbeta for betaRARE. PPARgamma specific inhibitor T007 and silencing of PPARgamma by siRNA diminished CDDO-induced maturation and RARbeta2 mRNA along with PPARgamma induction indicating that PPARgamma activation is at least partially responsible for the RARbeta2 transcription and maturation induction. In an in vivo mouse model of APL, CDDO derivative CDDO-methyl ester markedly enhanced ATRA-induced maturation and extended the survival of mice. In summary, these results provide rationale for the combined targeting of RAR and PPARgamma nuclear receptors in the therapy of APL.

CDDO induces granulocytic differentiation of myeloid leukemic blasts through translational up-regulation of p42 CCAAT enhancer binding protein alpha.

2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of tumor cells in vitro and in vivo. Here we assessed the effects of CDDO on CCAAT enhancer-binding protein alpha (CEBPA), a transcription factor critical for granulocytic differentiation. In HL60 acute myeloid leukemia (AML) cells, CDDO (0.01 to 2 muM) induces apoptosis in a dose-dependent manner. Conversely, subapoptotic doses of CDDO promote phagocytic activity and granulocytic-monocytic differentiation of HL60 cells through increased de novo synthesis of p42 CEBPA protein. CEBPA translational up-regulation is required for CDDO-induced granulocytic differentiation and depends on the integrity of the CEBPA upstream open reading frame (uORF). Moreover, CDDO increases the ratio of transcriptionally active p42 and the inactive p30 CEBPA isoform, which, in turn, leads to transcriptional activation of CEBPA-regulated genes (eg, GSCFR) and is associated with dephosphorylation of eIF2alpha and phosphorylation of eIF4E. In concordance with these results, CDDO induces a CEBPA ratio change and differentiation of primary blasts from patients with acute myeloid leukemia (AML). Because AML is characterized by arrested differentiation, our data suggest the inclusion of CDDO in the therapy of AML characterized by dysfunctional CEBPA expression.