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.

Key Points VCAM-1/VLA-4 triggers reciprocal NF-κB activation in leukemia and stromal cells and mediates cross-talk between leukemia and stromal cells. VCAM-1/VLA-4 and NF-κB signaling plays a pivotal role in the development of leukemia chemoresistance.

Triptolide, a diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook.f, has shown antitumor activities in a broad range of solid tumors. Here, we examined its effects on leukemic cells and found that, at 100 nM or less, it potently induced apoptosis in various leukemic cell lines and primary acute myeloid leukemia (AML) blasts. We then attempted to identify its mechanisms of action. Triptolide induced caspasedependent cell death accompanied by a significant decrease in XIAP levels. IntroductionTripterygium wilfordii Hook.f, a member of the Celastraceae family of plants, has been used in Chinese medicine for centuries. Triptolide, a diterpenoid, was first isolated from the plant and structurally characterized in 1972 1 and has been used for the treatment of a variety of autoimmune diseases and as an immunosuppressant in patients with organ and tissue transplantations. [1][2][3][4] Recently, triptolide was shown to have antitumor properties by suppressing the growth and inducing apoptosis of a broad range of human tumor cells. [5][6][7][8][9] Triptolide was also shown to sensitize cells to death induced by various agents, such as Apo2/Trail, TNF-␣, and different chemotherapeutic agents. [10][11][12] However, despite the recognized potent antitumor activity of triptolide, our knowledge regarding its mechanism of action is still limited. So far it is known only that triptolide blocks TNF-␣-mediated induction of c-IAP1 and c-IAP2, and the activation of NFB, 10,13,14 and induces caspase activation. 15,16 Acute myeloid leukemia (AML) is an aggressive hematologic malignancy. Despite major efforts during the past 30 years, limited progress has been made in the treatment of AML. The current primary treatment for AML is chemotherapy, which induces cell death mainly by apoptosis mediated by either the intrinsic mitochondrial or the extrinsic death receptor pathway, both of which lead to caspase activation and cell disintegration. The development of innovative therapies and identification of more effective drugs, therefore, remain high priorities for leukemia research. Because of its antitumor properties, triptolide holds promise as a treatment for leukemia. However, other than recent reports that triptolide induced apoptosis in U937 cells by activating caspase-3 and downregulating XIAP, 16 and that it down-regulated Bcr-Abl expression and induced apoptosis in K562 cells, 17 virtually no work has been done to elucidate the activity and mechanisms of triptolide in leukemia. Through an understanding of the molecular mechanisms that mediate the proapoptotic activity of triptolide, it will be possible to better understand its antileukemic effects and to determine whether it is a candidate for clinical use. In addition, knowing the molecular targets of triptolide and its mechanism of action will enable us to design rational combination therapies that more efficiently eradicate leukemic cells. In the study described here, we examined the effects of triptolide on various leukemic cell lines and primary AML blasts a...

IntroductionApoptosis is genetically programmed cell death regulated by multiple proteins. Inhibitors of apoptosis proteins (IAPs) are a family of related proteins that suppress cell death by inhibiting upstream and terminal caspases. [1][2][3][4][5][6][7][8] These proteins suppress apoptosis induced by a variety of stimuli, including tumor necrosis factor, Fas, menadione, staurosporine, etoposide, paclitaxel, and growth factor withdrawal. 1,4,5,9 The expression of at least some IAPs is up-regulated by various growth factors [10][11][12] supporting their roles in survival. Among them, Survivin is considered important because it links cell death and cell proliferation. 13,14 Survivin is not expressed in adult terminal differentiated tissues, but it is expressed in most human cancers and cancer cell lines. 1,13 Disruption of Survivin expression causes cell death and cell division defects, which result in polyploidy and multinucleated cells. [15][16][17] Studies from different groups have demonstrated that the elevated expression of Survivin is associated with poor prognosis and increased tumor recurrence in many cancers [18][19][20][21] and in acute myelogenous leukemia (AML). 22 Our previous studies showed that Survivin is overexpressed in the blasts of AML patient samples analyzed. 10,23 XIAP is the most widely expressed IAP. It is also the most potent inhibitor of caspases, with an inhibition constant (K i ) in the low nanomolar range. 2,3,24 Elevated expression of XIAP has been reported in certain cancers and cancer cell lines. 25,26 Downregulation of XIAP has been shown to induce apoptosis in chemoresistant human ovarian cancer cells 27 and to sensitize non-small cell lung carcinoma to low-dose ␥-irradiation. 28 We recently reported variable expression levels of XIAP in primary AML samples and found its expression inversely correlated with the survival of patients with AML. 29 Most current anticancer therapies, including chemotherapy, trigger tumor cell death by inducing apoptosis. Caspases are believed to function as the central mediators of apoptosis. On activation, they selectively cleave a variety of key structural proteins of the cytoskeleton and nucleus and numerous proteins involved in DNA repair and signaling pathways, resulting in DNA fragmentation. 30 Inhibiting caspase activation has been shown to prevent apoptosis. 30,31 This is supported by the fact that the overexpression of IAPs inhibits apoptotic cell death. [1][2][3][4][5] However, recent studies suggest that caspase activation is not the only pathway by which anticancer drugs induce cell death. Anticancer drugs can also induce caspase-independent cell death 32 and necrosis. 33 Recently, a number of caspase-independent cell death factors, which are released from mitochondria along with cytochrome c, have been described, among them apoptosis-inducing factor (AIF), 34-36 endonuclease, EndoG, 37 and HtrA2 (omi), which may be able to trigger caspase-independent cell death through its intrinsic serine protease activity. [38][39][40][41] Caspase-in...

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein level in bone marrow cells, particularly in Lin−Sca-1+cKit+ cells of inducible CML in mice as determined by CyTOF mass cytometry. Further, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly decreased leukemic Lin−Sca-1+cKit+ cell numbers and long-term stem cell frequency, and prolonged survival in a murine CML model. Additionally, this combination effectively eradicated CD34+CD38−, CD34+CD38+, and quiescent stem/progenitor CD34+ cells from BC CML patient samples. Our results suggest that BCL-2 is a key survival factor for CML stem/progenitor cells and that combined inhibition of BCL-2 and BCR-ABL tyrosine kinase has the potential to significantly improve depth of response and cure rates of chronic phase and BC CML.

XIAP is a member of the inhibitors-of-apoptosis family of proteins, which inhibit caspases and block cell death, with prognostic importance in AML. Here we demonstrate that cytokines regulate the expression of XIAP in leukemic cell lines and primary AML blasts. Inhibition of phosphatidylinositol-3 kinase (PI3K) with LY294002 and of the mitogen-activated protein kinase (MAPK) cascade by PD98059 resulted in decreased XIAP levels (3478.7 and 2375.7%, respectively). We then generated OCI-AML3 cells with constitutively phosphorylated Akt (p473-Akt) by retroviral gene transfer. Neither these nor Akt inhibitor-treated OCI-AML3 cells showed changes in XIAP levels, suggesting that XIAP expression is regulated by PI3K downstream effectors other than Akt. The induction of XIAP expression by cytokines through PI3K/MAPK pathways is consistent with its role in cell survival. Exposure of leukemic cells to chemotherapeutic agents decreased XIAP protein levels by caspase-dependent XIAP cleavage. Targeting XIAP by XIAP antisense oligonucleotide resulted in downregulation of XIAP, activation of caspases and cell death, and sensitized HL-60 cells to Ara-C. Our results suggest that XIAP is regulated by cytokines through PI3K, and to a lesser degree through MAPK pathways. Selective downregulation of XIAP expression might be of therapeutic benefit to leukemic patients.

Acute myeloid leukemia (AML) cells are relatively resistant to tumor necrosis factor ␣-related apoptosis-inducing ligand (TRAIL). We previously reported that triptolide, a potent anticancer agent from a Chinese herb, decreases XIAP in leukemic cells. We evaluated the combination of triptolide and TRAIL and found synergistic promotion of apoptosis in AML cells. XIAP-overexpressing U937 cells (U937XIAP) were more resistant to TRAIL than U937neo cells, and inhibition of XIAP with the small-molecule inhibitor 1396-11 enhanced TRAIL-induced apoptosis, implying XIAP as a resistance factor in AML. Furthermore, triptolide increased DR5 levels in OCI-AML3, while the DR5 increase was blunted in p53-knockdown OCI-AML3 and p53-mutated U937 cells, confirming a role for p53 in the regulation of DR5. In support of this finding, disruption of MDM2-p53 binding with subsequent increase in p53 levels by nutlin3a increased DR5 levels and sensitized OCI-AML3 cells to TRAIL. The combination of 1396-11 plus nutlin3a plus TRAIL was more effective than either the 1396-11 and TRAIL or nutlin3a and TRAIL combinations in OCI-AML3 cells, further supporting the role of triptolide as a sensitizer to TRAILinduced apoptosis in part by independent modulation of XIAP expression and p53 signaling. Thus, the combination of triptolide and TRAIL may provide a novel strategy for treating AML by overcoming critical mechanisms of apoptosis resistance.

IntroductionAt a fundamental level, patients with acute leukemia do not respond to treatment because the malignant blasts are not eradicated by current chemotherapy. In part, this failure is due to defects in apoptosis pathways. 1 Therefore, agents that overcome roadblocks to apoptosis could be therapeutically useful for this disease.Classically, apoptosis is caused by the activation of caspases, a family of intracellular cysteine proteases that cleave substrates at aspartic acid residues. 2,3 Currently, at least 4 pathways for initiation of caspase activation exist: (1) the mitochondrial pathway with cytochrome c; (2) the death receptor pathway with the tumor necrosis factor (TNF) family of death receptors; (3) direct caspase activation by cytolytic T-cell protease Granzyme B; and (4) a pathway connected to the endoplasmic reticulum. These pathways launch a proteolytic cascade, in which upstream (initiator) caspases cleave and activate downstream (effector) caspases.The inhibitor of apoptosis proteins (IAPs) are a family of endogenous caspase inhibitors that share a common baculoviral IAP repeat (BIR) domain. To date, 8 IAP family members exist in humans. Of these, XIAP is probably the best characterized with respect to its structure and biochemical mechanisms. XIAP inhibits caspases 3, 7, and 9, but not caspases 1, 6, 8, or 10. 4 XIAP contains 3 tandem baculovirus IAP repeat (BIR) domains and a really interesting new gene (RING) domain. The second BIR domain of XIAP (BIR2) inhibits caspases 3 and 7, while the third BIR domain (BIR3) inhibits caspase 9. Through their ability to inhibit caspases, IAPs act as antiapoptotic proteins [5][6][7][8][9][10] and are promising therapeutic targets. Inhibition of XIAP by antisense strategies or peptides that bind and inhibit the BIR3 domain of XIAP sensitizes malignant cells to chemotherapy. [11][12][13][14][15][16][17][18] Based on the knowledge that XIAP directly inhibits active caspase 3, we devised an enzymatic derepression assay to screen for molecules that relieve protease inhibition. Using this assay, we screened combinatorial libraries of chemical compounds and identified active agents based on different pharmacophores. The initial report of these XIAP inhibitors described a series of compounds based on the polyphenylurea pharmacophore including the active compound N- -methyl-NЈ-phenylurea (1396-12) and structural analogues. 19 Corresponding to their activity in the enzymatic assay, active polyphenylurea-based inhibitors but not inactive controls, induced rapid apoptosis of several types of tumor cell lines. 19 We determined that active compounds inhibit XIAP by binding its BIR2 domain at a site distinct from the binding pocket of the endogenous XIAP inhibitor second modulator of apoptotic proteases (SMAC). 20 Given the potential therapeutic utility of IAP inhibition, we tested these chemical IAP inhibitors in cultured leukemia cell lines For personal use only. on June 19, 2019. by guest www.bloodjournal.org From and primary acute myelogenous leukemia (AML) patie...