Down-regulation of Mcl-1 potentiates HDACi-mediated apoptosis in leukemic cells.

Mcl-1 is an anti-apoptotic Bcl-2 family member, whose degradation is supposedly required for induction of apoptosis. However, histone deacetylase inhibitors (HDACi) induce apoptosis primarily through the Bak/Mcl-1/Noxa and Bim pathways without decreasing Mcl-1. To investigate this discrepancy, we examined the role of Mcl-1 on HDACi-mediated apoptosis. Inhibition of either Class I or Class II HDAC by selective HDACi caused an upregulation of Mcl-1 mRNA and protein. Down-regulation of Mcl-1 by three structurally unrelated cyclin dependent kinase inhibitors potentiated HDACi-mediated apoptosis in primary chronic lymphocytic leukemic (CLL) cells and K562 cells. Sensitivity to HDACi-induced apoptosis was increased ~10-fold by the cyclin dependent kinase inhibitors. Nanomolar concentrations of HDACi, ~300-fold lower than required to induce apoptosis alone, sensitized cells to TRAIL, emphasizing that the mechanism(s) whereby HDACi induce apoptosis is clearly distinct from those by which they sensitize to TRAIL. Furthermore knockdown of Mcl-1 potentiated HDACi-mediated apoptosis in K562 cells. Thus HDACi-mediated Mcl-1 upregulation plays an important anti-apoptotic regulatory role in limiting the efficacy of HDACi-induced apoptosis, which can be overcome by combination with an agent that down-regulates Mcl-1. Thus a clinical trial in some cancers is warranted using a combination of an HDACi with agents that down-regulate Mcl-1

Apoptosis induced by histone deacetylase inhibitors in leukemic cells is mediated by Bim and Noxa

Several histone deacetylase inhibitors (HDACi), which have recently entered early clinical trials, exert their anti-cancer activity in part through the induction of apoptosis although the precise mechanism of this induction is not known. Induction of apoptosis by structurally diverse HDACi in primary cells from patients with chronic lymphocytic leukemia (CLL) and different leukemic cell lines was mediated by the Bcl-2 regulated intrinsic pathway and demonstrated a requirement for de novo protein synthesis. A marked time dependent induction of the pro-apoptotic BH3-only proteins, Bim, Noxa and Bmf was observed, which preceded the induction of apoptosis. A key role for both Bim and Noxa was proposed in HDACi-mediated apoptosis based on our findings that siRNA for Bim and Noxa but not Bmf largely prevented the HDACi induced loss in mitochondrial membrane potential, caspase processing and phosphatidylserine externalization. Noxa, induced by HDACi, in CLL cells and tumor cell lines, bound extensively to Mcl-1, a major anti-apoptotic Bcl-2 family member present in CLL cells. Our data strongly suggests that HDACi induce apoptosis primarily through inactivation of anti-apoptotic Bcl-2 family members by increases in Bim and Noxa and highlights these increases as a potential clinical target for CLL/lymphoma therapy

Receptor-mediated endocytosis is not required for TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared to normal cells. Other members of the TNF family of death ligands (Tumor necrosis factor, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signalling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but rather amplified the apoptotic signalling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization are required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin- dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors are required for transmission of its apoptotic signal, recruitment of FADD and procaspase-8 to form the TRAIL-associated DISC occurred at 4 oC, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL-R1/R2, unlike TNFTNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8 or transmission of an apoptotic signal in BJAB type I cells

Receptor-selective TRAIL Mutants Target Lymphoid Tumor cells for Apoptosis via TRAIL-R1: Implications for Therapy.

Receptor-selective TRAIL Mutants Target Lymphoid Tumor cells for Apoptosis via TRAIL-R1: Implications for Therapy

The COP9 signalosome-mediated deneddylation is stimulated by caspases during Apoptosis.

The COP9 signalosome-mediated deneddylation is stimulated by caspases during Apoptosis

Diminished Sensitivity of Chronic Lymphocytic Leukemia Cells to ABT-737 and ABT-263 Due to Albumin Binding in Blood

Purpose: Inhibition of the antiapoptotic BCL2 family is one of the most promising areas of anticancer drug development. However, ABT-737, a specific BCL2 inhibitor, is neither orally bioavailable nor metabolically stable. To overcome these problems, the structurally related molecule ABT-263 was synthesized and recently entered clinical trials in hematologic malignancies, including chronic lymphocytic leukemia (CLL). Almost all laboratory studies have been carried out with ABT-737 rather than ABT-263, the drug being used in clinical trials. Currently there are no published data on the comparative effects of these inhibitors. To gain insight into the potential value or limitations of ABT-263 in the clinic, we assessed its ability to induce apoptosis in clinically relevant cellular models of CLL. Experimental Design: The susceptibility of freshly isolated primary CLL cells to these inhibitors was compared in standard culture conditions and in conditions that more closely mimic in vivo conditions in a whole blood assay system. Results: ABT-737 was more potent than ABT-263 at inducing apoptosis in CLL cells. In whole blood, ∼100-fold higher concentrations of both drugs were required to induce apoptosis. We found that ABT-263 was highly bound by albumin and that an increased albumin binding of ABT-263 as compared with ABT-737 accounted for the differential sensitivity of CLL cells. Conclusions: Our data indicate that the exquisite in vitro sensitivity of CLL cells to BCL2 inhibitors may be lost in vivo due to high cell densities and the albumin binding of ABT-263. Modification of ABT-263 may yield a BCL2 inhibitor with greater bioavailability and more favorable pharmacokinetics

Evidence for the involvement of carbon-centred radicals in the induction of apoptotic cell death by artemisinin compounds

Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these compounds towards rapidly dividing human carcinoma cells and cell lines has been reported and it is hypothesised that activation of the endoperoxide bridge, by an iron (II) species, to form C-centred radicals, is essential for cytotoxicity. The studies described here have utilised artemisinin derivatives ; dihydroartemisinin, 10β-(p-bromophenoxy) dihydroartemisinin and 10β-(p-fluorophenoxy) dihydroartemisinin to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death, and to elucidate the molecular mechanism of cell death. In vitro studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukaemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells (PBMC). Deoxy-10β-(p-fluorophenoxy) dihydroartemisinin, which lacks the peroxide bridge, was 50- to 130-fold less active in the same cells confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity using LC-MS analysis to monitor endoperoxide bridge activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not insensitive PBMC. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death; characterized by concentration- and time-dependent mitochondrial membrane depolarisation, activation of caspases-3 and –7, sub-G0/G1 DNA formation and attenuation by z-VAD.fmk, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of metabolic activation of the peroxide bridge to radical species, which trigger caspase-dependent apoptosis

Concurrent upregulation of BCL-XL and BCL2A1 induces ~1000-fold resistance to ABT-737 in chronic lymphocytic leukemia.

ABT-737 and its orally active analog, ABT-263, are rationally designed inhibitors of BCL2 and BCL-XL. ABT-263 shows promising activity in early phase 1 clinical trials in B-cell malignancies, particularly chronic lymphocytic leukemia (CLL). In vitro, peripheral blood CLL cells are extremely sensitive to ABT-737 (EC50 7 nM), with rapid induction of apoptosis in all 60 patients tested, independent of parameters associated with disease progression and chemotherapy resistance. In contrast to data from cell lines, ABT-737–induced apoptosis in CLL cells was largely MCL1-independent. Because CLL cells within lymph nodes are more resistant to apoptosis than those in peripheral blood, CLL cells were cultured on CD154-expressing fibroblasts in the presence of interleukin-4 (IL-4) to mimic the lymph node microenvironment. CLL cells thus cultured developed an approximately 1000-fold resistance to ABT-737 within 24 hours. Investigations of the underlying mechanism revealed that this resistance occurred upstream of mitochondrial perturbation and involved de novo synthesis of the antiapoptotic proteins BCL-XL and BCL2A1, which were responsible for resistance to low and high ABT-737 concentrations, respectively. Our data indicate that after therapy with ABT-737–related inhibitors, resistant CLL cells might develop in lymph nodes in vivo and that treatment strategies targeting multiple BCL2 antiapoptotic members simultaneously may have synergistic activity

Endoplasmic reticulum membrane reorganization is regulated by ionic homeostasis.

Recently we described a new, evolutionarily conserved cellular stress response characterized by a reversible reorganization of endoplasmic reticulum (ER) membranes that is distinct from canonical ER stress and the unfolded protein response (UPR). Apogossypol, a putative broad spectrum BCL-2 family antagonist, was the prototype compound used to induce this ER membrane reorganization. Following microarray analysis of cells treated with apogossypol, we used connectivity mapping to identify a wide range of structurally diverse chemicals from different pharmacological classes and established their ability to induce ER membrane reorganization. Such structural diversity suggests that the mechanisms initiating ER membrane reorganization are also diverse and a major objective of the present study was to identify potentially common features of these mechanisms. In order to explore this, we used hierarchical clustering of transcription profiles for a number of chemicals that induce membrane reorganization and discovered two distinct clusters. One cluster contained chemicals with known effects on Ca(2+) homeostasis. Support for this was provided by the findings that ER membrane reorganization was induced by agents that either deplete ER Ca(2+) (thapsigargin) or cause an alteration in cellular Ca(2+) handling (calmodulin antagonists). Furthermore, overexpression of the ER luminal Ca(2+) sensor, STIM1, also evoked ER membrane reorganization. Although perturbation of Ca(2+) homeostasis was clearly one mechanism by which some agents induced ER membrane reorganization, influx of extracellular Na(+) but not Ca(2+) was required for ER membrane reorganization induced by apogossypol and the related BCL-2 family antagonist, TW37, in both human and yeast cells. Not only is this novel, non-canonical ER stress response evolutionary conserved but so also are aspects of the mechanism of formation of ER membrane aggregates. Thus perturbation of ionic homeostasis is important in the regulation of ER membrane reorganization

TRAIL signals to apoptosis in CLL cells primarily through TRAIL R-1 whereas cross-linked agonistic TRAIL R-2 antibodies facilitate signalling via TRAIL R-2

TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, which is being developed as an anti-tumour agent due to its selective toxicity to tumour cells, induces apoptosis by binding to two membrane-bound receptors, TRAIL-R1 and TRAIL-R2. Clinical trials have been initiated with various preparations of TRAIL as well as agonistic mAbs to TRAIL-R1 and TRAILR2. Previously we reported that prior treatment of primary chronic lymphocytic leukaemia (CLL) cells with histone deacetylase inhibitors (HDACi) was required to sensitize CLL cells to TRAIL and using various receptor-selective TRAIL mutant ligands we demonstrated that CLL cells signalled to apoptosis primarily through TRAIL-R1. Some, but not all, agonistic TRAIL-receptor antibodies require cross-linking in order to induce apoptosis. We now demonstrate that CLL cells can signal to apoptosis through the TRAIL-R2 receptor, but only after cross-linking of the agonistic TRAIL-R2 antibodies, LBY135 and lexatumumab (HGS-ETR2). In contrast, signalling through TRAIL-R1 by receptor-selective ligands or certain agonistic antibodies, such as mapatumumab (HGS-ETR1) occurs in the absence of cross-linking. These results further highlight important differences in apoptotic signalling triggered through TRAIL-R1 and TRAIL-R2 in primary tumour cells. Such information is clearly important for the rational optimisation of TRAIL therapy in primary lymphoid malignancies, such as CLL