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]

Purpose: Disseminated melanoma is highly therapy resistant. The finding that 66% of melanomas harbor the activating BRAF V600E mutation has raised expectations for targeting the Ras/RAF/mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway in melanoma. This study addresses the anti-melanoma activity of the MEK inhibitor AZD6244 (ARRY-142886). Experimental Design: We recently have shown that growing melanoma cells as threedimensional collagen-implanted spheroids enhances resistance to the MEK inhibitor U0126. Here, we investigated the anti-melanoma activity of AZD6244 in two-dimensional cell culture, the three-dimensional spheroid model, and an in vivo model. Results: In two-dimensional cell culture, AZD6244 was cytostatic and reduced the growth of melanoma cells in a concentration-dependent fashion through the induction of G 1 -phase cell cycle arrest. In our three-dimensional spheroid model, the effects of AZD6244 were largely cytostatic and reversible, with drug washout leading to spheroid regrowth. Finally, 1205Lu cells were grown as tumor xenografts in severe combined immunodeficient mice. After tumor establishment, mice were dosed twice daily with 0, 10, or 30 mg/kg AZD6244 p.o. AZD6244 treatment decreased phospho-ERK in the tumors and significantly suppressed tumor growth. The original tumors remained viable, suggesting that AZD6244 monotherapy was largely cytostatic, and not proapoptotic in this model. Further studies showed that co-administration of AZD6244 (30 mg/kg) with docetaxel (15 mg/kg) led to tumor regression, indicating the potential for MEK inhibitor/chemotherapy drug combinations. Conclusions: Inhibition of MEK is cytostatic as a monotherapy in melanoma, but cytotoxic when combined with docetaxel.

Unlike other tumors, melanomas harbor wild-type (WT) p53 but exhibit impaired p53-dependent apoptosis. The mechanisms for the impaired p53 activation are poorly understood but may be linked to the high expression of the p53 suppressor Mdm2, which is found in >50% of melanoma lesions. Here, we describe an organometallic glycogen synthase kinase 3B (GSK3B) inhibitor (DW1/2) as a potent activator of p53 and inducer of cell death in otherwise highly chemoresistant melanoma cells. Using RNA interference and pharmacologic approaches, we show that p53 is required for the cytotoxic effects of this organometallic inhibitor. The DW1/2 compound was barely able to induce cell death in melanoma cells with p53 mutations, further confirming the requirement for p53-WT in the cytotoxic effects of the GSK3B inhibition. Mechanistic analysis of the p53-dependent cell death indicated an apoptotic mechanism involving depolarization of mitochondrial membrane potential, caspase cleavage, and elevated NOXA expression. The effect of p53 was not simply due to passive up-regulation of protein expression as adenoviralmediated overexpression of p53 was not able to induce cell death. Treatment of melanoma cells with DW1/2 was instead found to decrease levels of Mdm2 and Mdm4. The importance of Mdm2 down-regulation in DW1/2-induced apoptosis was confirmed by treating the p53-WT cells with the p53/Mdm2 antagonist Nutlin-3. Taken together, our data provide a new strategy for the pharmacologic activation of p53 in melanoma, which may be a viable approach for overcoming apoptotic resistance in melanoma and offer new hope for rational melanoma therapy.

The loss of tumour phospho-extracellular responsive kinase (pERK) positivity is the major treatment biomarker for mitogen-activated protein kinase/extracellular responsive kinase (MEK) inhibitors. Here, we demonstrate that there is a poor correlation between pERK inhibition and the anti-proliferative effects of MEK inhibitors in melanoma cells. We suggest that Ki67 is a better biomarker for future clinical studies.

Although many melanomas harbor either activating mutations in BRAF or NRAS, there remains a substantial, yet little known, group of tumors without either mutation. Here, we used a genomic strategy to define a novel group of melanoma cell lines with co-overexpression of cyclin-dependent kinase 4

The multidrug resistance 1 (MDR1) gene product P-glycoprotein (P-gp) is frequently implicated in cross-resistance of tumors to chemotherapeutic drugs. In contrast, acute promyelocytic leukemia (APL) cells do not express MDR1 and are highly sensitive to anthracyclines. The combination of ATRA and the novel histone deacetylase inhibitor (HDACI) depsipeptide (FK228) induced P-gp expression and prevented growth inhibition and apoptosis in NB4 APL cells subsequently exposed to doxorubicin (DOX). ATRA/ FK228 treatment after exposure to DOX, however, enhanced apoptosis. Both agents, ATRA or FK228, induced MDR1 mRNA. This effect was significantly enhanced by ATRA/FK228 administered in combination, due in part to increased H4 and H3-Lys9 acetylation of the MDR1 promoter and recruitment of the nuclear transcription factor Y alpha (NFYA) transcription activator to the CCAAT box. Cotreatment with specific P-gp inhibitor PSC833 reversed cytoprotective effects of ATRA/FK228. G 1 cell-cycle arrest and p21 mRNA induction were also observed in response to ATRA/FK228, which may IntroductionAcute promyelocytic leukemia (APL) cells are highly sensitive to anthracyclines in part due to the lack of expression of the multidrug resistance 1 (MDR1) protein P-glycoprotein (P-gp). 1,2 In this study, we investigated the effects of ATRA and FK228, alone and in combination, on the cytotoxicity of doxorubicin (DOX). Pretreatment by ATRA combined with FK228 prevented DOX-induced apoptosis in NB4 APL cells. However, when DOX treatment preceded ATRA/FK228, DOXinduced cell death was enhanced.The MDR1 gene product P-gp functions as a transmembrane efflux pump for a variety of chemotherapeutic drugs, including anthracyclines, [3][4][5][6] and overexpression of the MDR1 gene is a negative prognostic factor in acute myelogenous leukemias (AMLs). 7 Numerous studies have reported the successful inhibition of P-gp function in vitro using cyclosporine A, PSC833, and other compounds. [8][9][10][11] MDR1 gene expression can also be silenced, however, by epigenetic mechanisms involving histone deacetylases (HDACs) and DNA methyltransferases. [12][13][14][15][16] For example, the nuclear transcription factor Y (NF-Y) heteromeric complex binds to the CCAAT core sequence in the promoters of a variety of eukaryotic genes, including human MDR1, 12,[16][17][18] and acts as a histone acetylation regulator and transcription activator. 12,19 APL cells, which do not express MDR1, are associated with the oncogenic transcription factor PML-RAR␣ that represses transcription of the genes encoding the RA receptor targets through histone deacetylation. The PML-RAR␣ chimeric protein, moreover, has been suspected to be the factor suppressing MDR1 through chromatin remodeling. 20 A number of HDAC inhibitors (HDACIs) are currently being tested in clinical trials against a variety of cancers. Recently, there has been strong interest in HDACIs as anti-APL agents because of their synergistic activity with ATRA. [21][22][23][24] In vivo data demonstrated that HDACIs can overcome ...

We investigated the antileukemic activity and molecular mechanisms of action of a newly synthesized ring-substituted diindolylmethane derivative, 1,1-bis[3V-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane (DIM #34), in acute myelogenous leukemia (AML) cells. DIM #34 inhibited AML cell growth via the induction of apoptosis and abrogated clonogenic growth of primary AML samples. Exposure to DIM #34 induced loss of mitochondrial inner transmembrane potential, release of cytochrome c into the cytosol, and caspase activation. Bcl-2-overexpressing, Bax knockout, and caspase-9-deficient cells were partially resistant to cell death, suggesting the involvement of the intrinsic apoptotic pathway. Furthermore, DIM #34 transiently inhibited the phosphorylation and activity of the extracellular signalregulated kinase and abrogated Bcl-2 phosphorylation. Because other methylene-substituted diindolylmethane analogues have been shown to transactivate the nuclear receptor peroxisome proliferator-activated receptor ; (PPAR;), we studied the role of PPAR; in apoptosis induction. Cotreatment of cells with a selective PPAR; antagonist or with retinoid X receptor and retinoic acid receptor ligands partially modulated apoptosis when combined with DIM #34, suggesting PPAR; receptor-dependent and receptorindependent cell death. Together, these findings suggest that diindolylmethanes are a new class of compounds that selectively induce apoptosis in AML cells through the modulation of the extracellular signal-regulated kinase and PPARg signaling pathways. (Cancer Res 2005; 65(7): 2890-8)