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.

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 F 1.23 Mmol/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. [Cancer Res 2008;68(9):3413-20]

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IntroductionThe triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivatives, CDDO-methylester (CDDO-Me) and CDDO-imidazole (CDDO-Im), induce growth arrest and apoptosis of a variety of solid tumor and leukemic cell lines in vitro and in vivo. 1,2 Different signaling pathways account for the proapoptotic and antiproliferative effects of CDDO. CDDO induces apoptosis through both caspase-independent and -dependent mechanisms, the latter involving caspase-8 activation, Bid cleavage, cytochrome c release, and caspase-3 activation. [3][4][5][6] Furthermore, JNK, p38, and ERK pathways are involved in CDDO-induced apoptosis of tumor cell lines 7-9 mediated by disrupted intracellular redox balance and involving decreased glutathione and increased reactive oxygen species [9][10][11][12] CDDO-induced growth arrest of breast cancer cell lines correlates with transactivated PPARgamma and leads to up-regulation of p21 cip1waf1 , GADD153, CCAAT enhancer-binding proteins (CEBPs), and proteasome-regulatory factors, and to down-regulation of cyclin D1, PCNA, and IRS1. 13 CDDO and CDDO-Im activate the TGF␤ pathway through activation of Smad2/3,14,15 which is required for the repression of inflammatory molecules by CDDO. 16 Interestingly, CDDO and its derivatives also induce differentiation of leukemic cells. 1,2,7,17 Differentiation of normal hematopoietic stem cells into their mature progeny critically depends on a fine-tuned interplay of hematopoietic transcription factors. 18 Among these, we have recently shown that increased CEBP beta (CEBPB) expression is critical for CDDO-Im-induced monocytic differentiation, and this was partially dependent upon ERK activation and TGF␤-mediated Smad activation. 17 Granulocytic differentiation requires the presence of functional CEBPA, since mice with a targeted disruption of the CEBPA gene demonstrate a selective lack of granulocytes and an accumulation of immature myeloid cells. 19 The expression and/or function of CEBPA is severely altered in a significant fraction of acute myeloid leukemia (AML) subtypes. 20,21 CEBPA is mutated in 7% of all AML cases with normal cytogenetics, and this results in a balance shift from the transcriptionally active full-length isoform (p42) toward the dominantnegatively acting p30 isoform. 22 The fusion protein AML1-ETO suppresses CEBPA transcription, 23 and AML1-MDS1-EVI1 and CBFB-SMMHC oncogenes inhibit CEBPA translation through activation of the RNA-binding protein calreticulin. 24,25 In these AML subtypes, re-expression of functional CEBPA restores granulocytic differentiation, suggesting that suppression of CEBPA is essential for the phenotype of AML blasts. Likewise, differentiation of myeloid progenitors in chronic myelogenous leukemia The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use...

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 p19 ARF in the mouse leads to early tumor susceptibility, presumably in part due to decreased p53 activity. To examine the tumorigenic cooperativity of p19 ARF , Mdm2, and p53 in vivo, p19 ARF -deficient mice were crossed first to p53-deficient mice and then to Mdm2 transgenic mice. The progeny were monitored for tumors. Cooperativity between p19 ARF and p53 deficiencies in accelerating tumor formation was observed for most genotypes except p53À/À p19 ARF À/À mice. p53À/À p19 ARF À/À mice had a tumor incidence similar to p53À/À mice. In this context, tumor suppression by ARF appears to be primarily p53 dependent. The majority of the p19 ARF þ /À tumors deleted the wildtype p19 ARF allele, in agreement with the previous studies, suggesting that p19 ARF is a classic 'two hit' tumor suppressor. In a p53 þ /À background, however, all p19 ARF þ /À tumors retained a wildtype ARF allele and most also retained wildtype p53. In the second cross between p19 ARF -deficient and Mdm2 transgenic mice, cooperativity in tumor incidence between Mdm2 overexpression and ARF deficiency was observed, consistent with the role of p19 ARF in negatively regulating Mdm2 activity. These experiments further demonstrate in vivo the inter-relationships of the p19 ARF -Mdm2-p53 signaling axis in tumor suppression.