mTOR inhibition by everolimus in childhood acute lymphoblastic leukemia induces caspase-independent cell death.

Increasingly, anti-cancer medications are being reported to induce cell death mechanisms other than apoptosis. Activating alternate death mechanisms introduces the potential to kill cells that have defects in their apoptotic machinery, as is commonly observed in cancer cells, including in hematological malignancies. We, and others, have previously reported that the mTOR inhibitor everolimus has pre-clinical efficacy and induces caspase-independent cell death in acute lymphoblastic leukemia cells. Furthermore, everolimus is currently in clinical trial for acute lymphoblastic leukemia. Here we characterize the death mechanism activated by everolimus in acute lymphoblastic leukemia cells. We find that cell death is caspase-independent and lacks the morphology associated with apoptosis. Although mitochondrial depolarization is an early event, permeabilization of the outer mitochondrial membrane only occurs after cell death has occurred. While morphological and biochemical evidence shows that autophagy is clearly present it is not responsible for the observed cell death. There are a number of features consistent with paraptosis including morphology, caspase-independence, and the requirement for new protein synthesis. However in contrast to some reports of paraptosis, the activation of JNK signaling was not required for everolimus-induced cell death. Overall in acute lymphoblastic leukemia cells everolimus induces a cell death that resembles paraptosis

Silencer of death domains controls cell death through tumour necrosis factor-receptor 1 and caspase-10 in acute lymphoblastic leukemia

Resistance to apoptosis remains a significant problem in drug resistance and treatment failure in malignant disease. NO-aspirin is a novel drug that has efficacy against a number of solid tumours, and can inhibit Wnt signaling, and although we have shown Wnt signaling to be important for acute lymphoblastic leukemia (ALL) cell proliferation and survival inhibition of Wnt signaling does not appear to be involved in the induction of ALL cell death. Treatment of B lineage ALL cell lines and patient ALL cells with NO-aspirin induced rapid apoptotic cell death mediated via the extrinsic death pathway. Apoptosis was dependent on caspase-10 in association with the formation of the death-inducing signaling complex (DISC) incorporating pro-caspase-10 and tumor necrosis factor receptor 1 (TNF-R1). There was no measurable increase in TNF-R1 or TNF-α in response to NO-aspirin, suggesting that the process was ligand-independent. Consistent with this, expression of silencer of death domain (SODD) was reduced following NO-aspirin exposure and lentiviral mediated shRNA knockdown of SODD suppressed expansion of transduced cells confirming the importance of SODD for ALL cell survival. Considering that SODD and caspase-10 are frequently over-expressed in ALL, interfering with these proteins may provide a new strategy for the treatment of this and potentially other cancers.10 page(s

Everolimus-Induced Autophagy is Not Required for Cell Death.

<p>(<b>A–G</b>) NALM6 cells were treated with vehicle or 16 µM everolimus overnight and ultrastructure examined by electron microscopy. Fine arrows indicate autophagic vacuoles containing degrading organelles. Thick arrows indicate stretches of double membrane walling off regions of cytoplasm. Scale bars indicate 100 nm. (<b>H</b>) Cell lysates prepared from NALM6 cells cultured for the indicated time periods with the specified concentration of everolimus were analyzed for LC3-I and LC3-II by Western blotting and sequential blotting for β-actin used as a loading control. (<b>I</b>) The viability of NALM6 cells cultured for 24 h with the indicated concentration of everolimus was assessed with and without the addition of 3MA. The mean ± SD of 3 independent experiments is shown and analyzed using a paired t-test. (<b>J</b>) Analysis of LC3 by Western blotting following a 24 h incubation of NALM6 cells with indicated concentration of everolimus with or without that addition of 3MA. The ratio of LC3-II to LC3-I is indicated below the blot.</p

<i>para</i>-NO-ASA results in activation of executioner caspases and mitochondrial depolarization.

<p>(A) Cleavage of caspase-3 in NALM6 cells by intracellular flow cytometry following 6 hour exposure to 10 µM <i>para</i>-NO-ASA. (B) Caspase-3 activation as measured by flow cytometry, in NALM6 cells untreated or treated with 5 µM or 10 µM <i>para</i>-NO-ASA for the given time. Bars indicate the mean and s.d. of 3 independent experiments. (C) Western blot analysis of PARP activation in NALM6 cells stimulated with the indicated concentrations of <i>para</i>-NO-ASA for 12 hours. The cleaved (89 kD) and uncleaved (116 kD) forms of PARP are indicated. Data is representative of 2 independent experiments. (D) NALM6 cells were treated with 5 µM <i>para</i>-NO-ASA or vehicle for 6 hours and assessed for ΔΨ_m using TMRM to label cells with polarized mitochondria, and apoptosis using annexin V and 7AAD. Representative plots are shown and the mean percentage of cells from 2 experiments in each quadrant indicated. (E) Representative histograms showing cytochrome c release following exposure of NALM6 cells to 5 µM para-NO-ASA for 6 hours. The thin line represents isotype control (Iso) staining and the heavy line cytochrome c (Cyto c) staining. (F) Quantitation of cytochrome c release at the indicated time points following addition of 5 µM <i>para</i>-NO-ASA. The mean and s.d. of replicates is shown. *p = 0.02. (G) NALM6 cells were pre-treated with 100 µM Z-VAD or 2.5 mM NAC for 1 hour prior to exposure to 5 µM <i>para</i>-NO-ASA for 6 h the mean ± s.e. is shown (n≥5). *p<0.05 compared to NO-ASA alone.</p

Multiple caspases are activated by <i>para</i>-NO-ASA.

<p>(A) Western blot of cell lysates from cells treated with <i>para</i>-NO-ASA for the indicated times. Western Blot of caspases-8 (cleaved forms only), -9 (full length, p47 and cleaved forms), caspase-10 (full length only), Bid (full length and cleaved forms) and full length Noxa and Puma are shown. Note that the caspase-8 antibody (#9496) only recognised the cleaved forms and not the intact protein, while the caspase-10 antibody only recognised the full-length form of the protein. (B) NALM6 cells were treated with 5 µM NO-ASA for 6 h and cell lysates analysed for caspase-8 or caspase-9 activity. The mean±SD of 3 experiments is shown. *p<0.05 compared to control. (C) Representative histograms showing <i>para</i>-NO-ASA mediated activation of caspase 10 as determined by flow cytometry. Time of exposure to <i>para</i>-NO-ASA is indicated on the histograms. The mean and s.d. from one of two independent experiments is shown. (D) Gene expression was assessed using quantitative RT-PCR in NALM6 cells treated with 10 µM <i>para</i>-NO-ASA for 12 hours. *p<0.05.</p

SODD is over-expressed in ALL cells and expression is required for ALL cell growth.

<p>(A) SODD expression was determined by semi-quantitative RT-PCR in NALM6 and REH cells treated with 10 µM <i>para</i>-NO-ASA for 12 h. (B) SODD expression was determined by western blotting following treatment with 10 µM <i>para</i>-NO-ASA for the indicated times. (C) Western blot analysis of SODD in normal peripheral blood mononuclear cells (PBMC), indicated cell lines (upper blots) or patient samples (lower blots). Patient samples had been expanded in NOD/SCID mice to obtain sufficient cells for Western blotting. (D) NALM6 transduced with lentiviral constructs expressing GFP alone (Control) or containing one of two shRNA specific for SODD (SODD1 and SODD2). The level of SODD protein (D) was determined in LK63 cells after 44 days in culture while mRNA levels were assessed in NALM6 cells on day 6 of culture by qRT-PCR and was normalised to the levels of GAPDH (E). The mean and s.e.m. of three independent experiments is shown. (F) The total cell number (left panels), and the percentage (centre panels) and total number of GFP+ transduced cells (right panels) was monitored over time. The mean and s.d. of duplicate cultures is shown.</p

Schematic diagram illustrating the proposed mechanism of action of <i>para</i>-NO-ASA.

<p><i>para</i>-NO-ASA down regulates SODD allowing self-aggregation, or enhancing TNF-α-induced, activation of signalling through TNF-R1. TNF-R1 signalling triggers the extrinsic apoptosis cascade including cleavage of pro-caspase-10 and Bid, mitochondrial depolarization, outer membrane permeabilization and release of cytochrome c. This is followed by cleavage of pro-caspase-9 and the executioner caspases-3 and -7. While signalling through TNF-R1 can activate NF-κB this may be suppressed by caspase-mediated cleavage of TRAF. In contrast, TNF-R2 signal induces cell survival and proliferation pathways, predominantly through NF-κB but also MAPK signalling. In ALL cells where SODD is over expressed, exogenous TNF-α is likely to predominantly signal through TNF-R2 resulting in increased survival and proliferation. Activating TNF-R1 by down regulation of SODD provides a mechanism for inducing cell death without increasing proliferation and survival signals. FADD, Fas-associated death domain; IKK, inhibitor of κB kinase; I-κB, inhibitor of κB; TRADD, TNF-R1-associated death domain; TRAF, TNF receptor-associated factor.</p

<i>para</i>-NO-ASA inhibit the proliferation and induces cell death in ALL cell lines and patient samples.

<p>(A) The proliferation of the indicated ALL cell lines as determined by ³H-thymidine incorporation following 24 hours incubation with <i>para</i>-NO-ASA. Vertical bars represent standard deviation of quadruplicate determinations from one of 2 independent experiments. Curves were generated using the line of best fit in Excel software. IC₅₀ values are indicated. (B) Cell cycle analysis of ALL cell lines treated with indicated concentrations of <i>para</i>-NO-ASA for 24 hours. Representative plots including the mean from duplicate assessments are shown. (C) The viability of pre-B ALL patient samples or patient derived stromal dependent cell lines was assessed after 24 hours of culture in the presence of DMSO (Cont), 5 or 10 µM <i>para</i>-NO-ASA by annexin V and 7AAD staining with dual negative cells being considered viable. Vertical bars represent standard deviation of quadruplicate determinations.</p