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Despite being highly effective for newly diagnosed chronic myeloid leukemia (CML), imatinib not only is inactive against quiescent CML stem cells, but also has limited activity against blast crisis (BC) CML. The relative activity of Bcr-Abl and the expression levels of antiapoptotic proteins in proliferating and quiescent CD34(+) BC CML progenitor cells and the effects of targeting antiapoptotic proteins in these cells are unknown. Here we report higher levels of p-CrkL in quiescent than in proliferating CD34(+) progenitor cells and comparable expression levels of Bcl-2, Bcl-xL, Mcl-1 and XIAP in the two populations in BC CML. Inhibition of Bcl-2/Bcl-xL by ABT-737 in cells from patients with tyrosine kinase inhibitor (TKI)-resistant BC CML promoted apoptosis in quiescent CD34(+) progenitor cells with an efficacy similar to that in proliferating cells. Combination of ABT-737 with imatinib (which decreases Mcl-1 levels) or triptolide (which decreases Mcl-1 and XIAP) synergistically induced death of both proliferating and quiescent CD34(+) progenitor cells obtained from TKI-resistant BC CML patients. These results suggest that antiapoptotic proteins are critical targets in BC CML and that activation of apoptosis signaling can eliminate both proliferating and quiescent CD34(+) progenitor cells in BC CML, independent of response to TKIs. [ABSTRACT FROM AUTHOR]

Checkpoint immunotherapy has yielded meaningful responses across many cancers but has shown modest efficacy in advanced prostate cancer. B7 homolog 3 protein (B7-H3/ CD276 ) is an immune checkpoint molecule and has emerged as a promising therapeutic target. However, much remains to be understood regarding B7-H3's role in cancer progression, predictive biomarkers for B7-H3-targeted therapy, and combinatorial strategies. Our multi-omics analyses identified B7-H3 as one of the most abundant immune checkpoints in prostate tumors containing PTEN and TP53 genetic inactivation. Here, we sought in vivo genetic evidence for, and mechanistic understanding of, the role of B7-H3 in PTEN/TP53- deficient prostate cancer. We found that loss of PTEN and TP53 induced B7-H3 expression by activating transcriptional factor Sp1. Prostate-specific deletion of Cd276 resulted in delayed tumor progression and reversed the suppression of tumor-infiltrating T cells and NK cells in Pten/Trp53 genetically engineered mouse models. Furthermore, we tested the efficacy of the B7-H3 inhibitor in preclinical models of castration-resistant prostate cancer (CRPC). We demonstrated that enriched regulatory T cells and elevated programmed cell death ligand 1 (PD-L1) in myeloid cells hinder the therapeutic efficacy of B7-H3 inhibition in prostate tumors. Last, we showed that B7-H3 inhibition combined with blockade of PD-L1 or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) achieved durable antitumor effects and had curative potential in a PTEN/TP53 -deficient CRPC model. Given that B7-H3-targeted therapies have been evaluated in early clinical trials, our studies provide insights into the potential of biomarker-driven combinatorial immunotherapy targeting B7-H3 in prostate cancer, among other malignancies.

MCL-1 is known to play a major role in resistance to BCL-2 inhibition, but the contribution of other BCL-2 family proteins has not been fully explored. We, here, demonstrate the ineffectiveness of MCL-1 inhibitor AMG176 in venetoclax-resistant, and conversely, of venetoclax in AMG176-resistant acute myelogenous leukemia (AML). Like cells with acquired resistance to venetoclax, cells with acquired resistance to AMG176 express increased MCL-1. Both cells with acquired resistance to venetoclax and to AMG176 express increased levels of BCL-2 and BCL-2A1, decreased BAX, and/or altered levels of other BCL-2 proteins. Cotargeting BCL-2 and MCL-1 was highly synergistic in AML cell lines with intrinsic or acquired resistance to BH3 mimetics or engineered to genetically overexpress BCL-2 or BCL-2A1 or downregulate BAX. The combination effectively eliminated primary AML blasts and stem/progenitor cells resistant to or relapsed after venetoclax-based therapy irrespective of mutations and cytogenetic abnormalities. Venetoclax and AMG176 combination markedly suppressed antiapoptotic BCL-2 proteins and AML stem/progenitor cells and dramatically extended mouse survival (median 336 vs. control 126 days; P < 0.0001) in a patient-derived xenograft (PDX) model developed from a venetoclax/hypomethylating agent therapy-resistant patient with AML. However, decreased BAX levels in the bone marrow residual leukemia cells after 4-week combination treatment may represent a resistance mechanism that contributed to their survival. Enhanced antileukemia activity was also observed in a PDX model of monocytic AML, known to be resistant to venetoclax therapy. Our results support codependence on multiple antiapoptotic BCL-2 proteins and suppression of BAX as mechanisms of AML resistance to individual BH3 mimetics. Cotargeting of MCL-1 and BCL-2 eliminates otherwise apoptosis-resistant cells., (©2022 American Association for Cancer Research.)

Although highly effective, BCR-ABL1 tyrosine kinase inhibitors do not target chronic myeloid leukemia (CML) stem cells. Most patients relapse upon tyrosine kinase inhibitor therapy cessation. We reported previously that combined BCR-ABL1 and BCL-2 inhibition synergistically targets CML stem/progenitor cells. p53 induces apoptosis mainly by modulating BCL-2 family proteins. Although infrequently mutated in CML, p53 is antagonized by MDM2, which is regulated by BCR-ABL1 signaling. We hypothesized that MDM2 inhibition could sensitize CML cells to tyrosine kinase inhibitors. Using an inducible transgenic Scl-tTa- BCR-ABL1 murine CML model, we found, by RT-PCR and CyTOF proteomics increased p53 signaling in CML bone marrow (BM) cells compared with controls in CD45 + and linage-SCA-1 + C-KIT + populations. CML BM cells were more sensitive to exogenous BH3 peptides than controls. Combined inhibition of BCR-ABL1 with imatinib and MDM2 with DS-5272 increased NOXA level, markedly reduced leukemic linage-SCA-1 + C-KIT + cells and hematopoiesis, decreased leukemia burden, significantly prolonged the survival of mice engrafted with BM cells from Scl-tTa- BCR-ABL1 mice, and significantly decreased CML stem cell frequency in secondary transplantations. Our results suggest that CML stem/progenitor cells have increased p53 signaling and a propensity for apoptosis. Combined MDM2 and BCR-ABL1 inhibition targets CML stem/progenitor cells and has the potential to improve cure rates for CML., (Copyright© 2020 Ferrata Storti Foundation.)

The pathogenesis of acute myeloid leukemia (AML) involves serial acquisition of mutations controlling several cellular processes, requiring combination therapies affecting key downstream survival nodes in order to treat the disease effectively. The BCL2 selective inhibitor venetoclax has potent anti-leukemia efficacy; however, resistance can occur due to its inability to inhibit MCL1, which is stabilized by the MAPK pathway. In this study, we aimed to determine the anti-leukemia efficacy of concomitant targeting of the BCL2 and MAPK pathways by venetoclax and the MEK1/2 inhibitor cobimetinib, respectively. The combination demonstrated synergy in seven of 11 AML cell lines, including those resistant to single agents, and showed growth-inhibitory activity in over 60% of primary samples from patients with diverse genetic alterations. The combination markedly impaired leukemia progenitor functions, while maintaining normal progenitors. Mass cytometry data revealed that BCL2 protein is enriched in leukemia stem/progenitor cells, primarily in venetoclax-sensitive samples, and that cobimetinib suppressed cytokine-induced pERK and pS6 signaling pathways. Through proteomic profiling studies, we identified several pathways inhibited downstream of MAPK that contribute to the synergy of the combination. In OCI-AML3 cells, the combination downregulated MCL1 protein levels and disrupted both BCL2:BIM and MCL1:BIM complexes, releasing BIM to induce cell death. RNA sequencing identified several enriched pathways, including MYC, mTORC1, and p53 in cells sensitive to the drug combination. In vivo , the venetoclax-cobimetinib combination reduced leukemia burden in xenograft models using genetically engineered OCI-AML3 and MOLM13 cells. Our data thus provide a rationale for combinatorial blockade of MEK and BCL2 pathways in AML., (Copyright© 2020 Ferrata Storti Foundation.)

In B-cell acute lymphoblastic leukemia (B-ALL), activation of Notch signaling leads to cell-cycle arrest and apoptosis. We aimed to harness knowledge acquired by understanding a mechanism of Notch-induced cell death to elucidate a therapeutically viable target in B-ALL. To this end, we identified that Notch activation suppresses Polo-like kinase 1 (PLK1) in a B-ALL-specific manner. We identified that PLK1 is expressed in all subsets of B-ALL and is highest in Philadelphia-like (Ph-like) ALL, a high-risk subtype of disease. We biochemically delineated a mechanism of Notch-induced PLK1 downregulation that elucidated stark regulation of p53 in this setting. Our findings identified a novel posttranslational cascade initiated by Notch in which CHFR was activated via PARP1-mediated PARylation, resulting in ubiquitination and degradation of PLK1. This led to hypophosphorylation of MDM2 Ser260 , culminating in p53 stabilization and upregulation of BAX. shRNA knockdown or pharmacologic inhibition of PLK1 using BI2536 or BI6727 (volasertib) in B-ALL cell lines and patient samples led to p53 stabilization and cell death. These effects were seen in primary human B-ALL samples in vitro and in patient-derived xenograft models in vivo These results highlight PLK1 as a viable therapeutic target in B-ALL. Efficacy of clinically relevant PLK1 inhibitors in B-ALL patient-derived xenograft mouse models suggests that use of these agents may be tailored as an additional therapeutic strategy in future clinical studies., (©2019 American Association for Cancer Research.)

Purpose: Wnt/β-catenin signaling is required for leukemic stem cell function. FLT3 mutations are frequently observed in acute myeloid leukemia (AML). Anomalous FLT3 signaling increases β-catenin nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKI) are used clinically to treat FLT3 -mutated AML patients, but with limited efficacy. We investigated the antileukemia activity of combined Wnt/β-catenin and FLT3 inhibition in FLT3 -mutant AML. Experimental Design: Wnt/β-catenin signaling was inhibited by the β-catenin/CBP antagonist C-82/PRI-724 or siRNAs, and FLT3 signaling by sorafenib or quizartinib. Treatments on apoptosis, cell growth, and cell signaling were assessed in cell lines, patient samples, and in vivo in immunodeficient mice by flow cytometry, Western blot, RT-PCR, and CyTOF. Results: We found significantly higher β-catenin expression in cytogenetically unfavorable and relapsed AML patient samples and in the bone marrow-resident leukemic cells compared with circulating blasts. Disrupting Wnt/β-catenin signaling suppressed AML cell growth, induced apoptosis, abrogated stromal protection, and synergized with TKIs in FLT3 -mutated AML cells and stem/progenitor cells in vitro The aforementioned combinatorial treatment improved survival of AML-xenografted mice in two in vivo models and impaired leukemia cell engraftment. Mechanistically, the combined inhibition of Wnt/β-catenin and FLT3 cooperatively decreased nuclear β-catenin and the levels of c-Myc and other Wnt/β-catenin and FLT3 signaling proteins. Importantly, β-catenin inhibition abrogated the microenvironmental protection afforded the leukemic stem/progenitor cells. Conclusions: Disrupting Wnt/β-catenin signaling exerts potent activities against AML stem/progenitor cells and synergizes with FLT3 inhibition in FLT3 -mutant AML. These findings provide a rationale for clinical development of this strategy for treating FLT3 -mutated AML patients. Clin Cancer Res; 24(10); 2417-29. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Tyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC), both related to persistent β-catenin activation, remain formidable challenges for chronic myeloid leukemia (CML). We observed overexpression of β-catenin in BC-CML stem/progenitor cells, particularly in granulocyte-macrophage progenitors, and highest among a novel CD34 + CD38 + CD123 hi Tim-3 hi subset as determined by CyTOF analysis. Co-culture with mesenchymal stromal cells (MSCs) induced the expression of β-catenin and its target CD44 in CML cells. A novel Wnt/β-catenin signaling modulator, C82, and nilotinib synergistically killed KBM5 T315I and TKI-resistant primary BC-CML cells with or without BCR-ABL kinase mutations even under leukemia/MSC co-culture conditions. Silencing of β-catenin by short interfering RNA restored sensitivity of primary BCR-ABL T315I/E255V BC-CML cells to nilotinib. Combining the C82 pro-drug, PRI-724, with nilotinib significantly prolonged the survival of NOD/SCID/IL2Rγ null mice injected with primary BCR-ABL T315I/E255V BC-CML cells. The combined treatment selectively targeted CML progenitors and inhibited CD44, c-Myc, survivin, p-CRKL and p-STAT5 expression. In addition, pretreating primary BC-CML cells with C82, or the combination, but not with nilotinib alone, significantly impaired their engraftment potential in NOD/SCID/IL2Rγ-null-3/GM/SF mice and significantly prolonged survival. Our data suggest potential benefit of concomitant β-catenin and Bcr-Abl inhibition to prevent or overcome Bcr-Abl kinase-dependent or -independent TKI resistance in BC-CML.

Anti-angiogenesis therapy has shown clinical benefit in patients with high-grade serous ovarian cancer (HGSC), but adaptive resistance rapidly emerges. Thus, approaches to overcome such resistance are needed. We developed the setting of adaptive resistance to anti-VEGF therapy, and performed a series of in vivo experiments in both immune competent and nude mouse models. Given the pro-angiogenic properties of tumor-associated macrophages (TAMs) and the dominant role of CSF1R in macrophage function, we added CSF1R inhibitors following emergence of adaptive resistance to anti-VEGF antibody. Mice treated with a CSF1R inhibitor (AC708) after anti-VEGF antibody resistance had little to no measurable tumor burden upon completion of the experiment while those that did not receive a CSF1R inhibitor still had abundant tumor. To mimic clinically used regimens, mice were also treated with anti-VEGF antibody and paclitaxel until resistance emerged, and then a CSF1R inhibitor was added. The addition of a CSF1R inhibitor restored response to anti-angiogenesis therapy, resulting in 83% lower tumor burden compared to treatment with anti-VEGF antibody and paclitaxel alone. Collectively, our data demonstrate that the addition of a CSF1R inhibitor to anti-VEGF therapy and taxane chemotherapy results in robust anti-tumor effects., Competing Interests: CONFLICTS OF INTEREST RLC serves on the advisory board and as a consultant for Genentech/Roche.

Mass cytometry presents an exceptional opportunity to interrogate the biology of highly heterogeneous cell populations, owing to the ability to collect highly parametric proteomic data at a single cell level. However, sample-to-sample variability, due to antibody staining and/or instrument sensitivity, can introduce substantial artifacts into the data, which can in turn lead to erroneous conclusions. This variability can be eliminated by sample barcoding which enables samples to be pooled, stained and run simultaneously. Existing mass cytometry barcoding approaches require time intensive labeling, reduce the number of biologically meaningful parameters and/or rely on expensive reagents. We present an approach utilizing monoisotopic cisplatin to perform cell barcoding that does not require cell permeabilization, can be completed in 10 minutes and can be utilized in combination with existing barcoding techniques to greatly increase the number of samples which can be multiplexed to improve throughput and consistency.

Although overexpression/activation of focal adhesion kinase (FAK) is widely known in solid tumors to control cell growth, survival, invasion, metastasis, gene expression, and stem cell self-renewal, its expression and function in myeloid leukemia are not well investigated. Using reverse-phase protein arrays in large cohorts of newly diagnosed acute myeloid leukemia (AML) and myeloid dysplastic syndrome (MDS) samples, we found that high FAK expression was associated with unfavorable cytogenetics ( P = 2 × 10 -4 ) and relapse ( P = 0.02) in AML. FAK expression was significantly lower in patients with FLT3 -ITD ( P = 0.0024) or RAS ( P = 0.05) mutations and strongly correlated with p-SRC and integrinβ3 levels. FAK protein levels were significantly higher in CD34 + ( P = 5.42 × 10 -20 ) and CD34 + CD38 - MDS ( P = 7.62 × 10 -9 ) cells compared with normal CD34 + cells. MDS patients with higher FAK in CD34 + cells tended to have better overall survival ( P = 0.05). FAK expression was significantly higher in MDS patients who later transformed to compared with those who did not transform to AML and in AML patients who transformed from MDS compared with those with de novo AML. Coculture with mesenchymal stromal cells (MSC) increased FAK expression in AML cells. Inhibition of FAK decreased MSC-mediated adhesion/migration and viability of AML cells and prolonged survival in an AML xenograft murine model. Our results suggest that FAK regulates leukemia-stromal interactions and supports leukemia cell survival; hence, FAK is a potential therapeutic target in myeloid leukemia. Mol Cancer Ther; 16(6); 1133-44. ©2017 AACR ., (©2017 American Association for Cancer Research.)

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., Competing Interests: Joel Leverson is an employee of AbbVie Inc, which developed ABT-199. The study was in part supported by research funding from AbbVie Inc., (Copyright © 2016, American Association for the Advancement of Science.)

mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The active-site mTOR inhibitors (asTORi) represent a promising new approach to targeting mTOR in AKT/mTOR signaling. MLN0128 is an orally-administered, second-generation asTORi, currently in clinical development. We examined the anti-leukemic effects and the mechanisms of action of MLN0128 in AML cell lines and primary samples, with a particular focus on its effect in AML stem/progenitor cells. MLN0128 inhibited cell proliferation and induced apoptosis in AML by attenuating the activity of mTOR complex 1 and 2. Using time-of-flight mass cytometry, we demonstrated that MLN0128 selectively targeted and functionally inhibited AML stem/progenitor cells with high AKT/mTOR signaling activity. Using the reverse-phase protein array technique, we measured expression and phosphorylation changes in response to MLN0128 in 151 proteins from 24 primary AML samples and identified several pro-survival pathways that antagonize MLN0128-induced cellular stress. A combined blockade of AKT/mTOR signaling and these pro-survival pathways facilitated AML cell killing. Our findings provide a rationale for the clinical use of MLN0128 to target AML and AML stem/progenitor cells, and support the use of combinatorial multi-targeted approaches in AML therapy.

To better understand how the apoptosis repressor with caspase recruitment domain (ARC) protein confers drug resistance in acute myeloid leukemia (AML), we investigated the role of ARC in regulating leukemia-mesenchymal stromal cell (MSC) interactions. In addition to the previously reported effect on AML apoptosis, we have demonstrated that ARC enhances migration and adhesion of leukemia cells to MSCs both in vitro and in a novel human extramedullary bone/bone marrow mouse model. Mechanistic studies revealed that ARC induces IL1β expression in AML cells and increases CCL2, CCL4, and CXCL12 expression in MSCs, both through ARC-mediated activation of NFκB. Expression of these chemokines in MSCs increased by AML cells in an ARC/IL1β-dependent manner; likewise, IL1β expression was elevated when leukemia cells were co-cultured with MSCs. Further, cells from AML patients expressed the receptors for and migrated toward CCL2, CCL4, and CXCL12. Inhibition of IL1β suppressed AML cell migration and sensitized the cells co-cultured with MSCs to chemotherapy. Our results suggest the existence of a complex ARC-regulated circuit that maintains intimate connection of AML with the tumor microenvironment through NFκB/IL1β-regulated chemokine receptor/ligand axes and reciprocal crosstalk resulting in cytoprotection. The data implicate ARC as a promising drug target to potentially sensitize AML cells to chemotherapy.

The Bcr-Abl tyrosine kinase regulates several Bcl-2 family proteins that confer resistance to apoptosis in chronic myeloid leukemia (CML) cells. Given p53's ability to modulate the expression and activity of Bcl-2 family members, we hypothesized that targeting Bcr-Abl, Bcl-2, and p53 concomitantly could have therapeutic benefits in blast crisis (BC) CML and in quiescent CML CD34+ cells that are insensitive to tyrosine kinase inhibitors (TKI). We examined the effects of the MDM2 inhibitor nutlin3a and its combination with the dual Bcl-2 and Bcl-xL inhibitor ABT-737, and the Bcr-Abl inhibitor nilotinib on BC CML patient samples. We found that in quiescent CD34+ progenitors, p53 expression is significantly lower, and MDM2 is higher, compared to their proliferating counterparts. Treatment with nutlin3a induced apoptosis in bulk and CD34+CD38- cells, and in both proliferating and quiescent CD34+ progenitor CML cells. Nutlin3a synergized with ABT-737 and nilotinib, in part by inducing pro-apoptotic, and suppressing anti-apoptotic, Bcl-2 proteins. Nilotinib inhibited the expression of Bcl-xL and Mcl-1 in BC CML cells. These results demonstrate that p53 activation by MDM2 blockade can sensitize BC CML cells, including quiescent CD34+ cells, to Bcl-2 inhibitor- and TKI-induced apoptosis. This novel strategy could be useful in the therapy of BC CML.

Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which allows measurements of up to 120 parameters in single cells without correction for spectral overlap. The aim of this study was the evaluation of intracellular signaling in antigen-defined stem/progenitor cell subsets in primary AML. CyTOF and conventional FCM yielded comparable results on LSC phenotypes defined by CD45, CD34, CD38, CD123, and CD99. Intracellular phosphoprotein responses to ex vivo cell signaling inhibitors and cytokine stimulation were assessed in myeloid leukemia cell lines and one primary AML sample. CyTOF and conventional FCM results were confirmed by western blotting. In the primary AML sample, we investigated the cell responses to ex vivo stimulation with stem cell factor and BEZ235-induced inhibition of PI3K and identified activation patterns in multiple PI3K downstream signaling pathways including p-4EBP1, p-AKT, and p-S6, particularly in CD34(+) subsets. We evaluated multiple signaling pathways in antigen-defined subpopulations in primary AML cells with FLT3-ITD mutations. The data demonstrated the heterogeneity of cell phenotype distribution and distinct patterns of signaling activation across AML samples and between AML and normal samples. The mTOR targets p-4EBP1 and p-S6 were exclusively found in FLT3-ITD stem/progenitor cells, but not in their normal counterparts, suggesting both as novel targets in FLT3 mutated AML. Our data suggest that CyTOF can identify functional signaling pathways in antigen-defined subpopulations in primary AML, which may provide a rationale for designing therapeutics targeting LSC-enriched cell populations., (© 2015 International Society for Advancement of Cytometry.)

Overexpression of the apoptosis repressor with caspase recruitment domain (ARC, also termed NOL3) protein predicts adverse outcome in patients with acute myeloid leukaemia (AML) and confers drug resistance to AML cells. The second mitochondrial-derived activator of caspases (SMAC, also termed DIABLO) mimetic, birinapant, promotes extrinsic apoptosis in AML cells. SMAC mimetics induce cleavage of cellular inhibitor of apoptosis (cIAP) proteins, leading to stabilization of the nuclear factor-κB (NF-κB)-inducing kinase (MAP3K14, also termed NIK) and activation of non-canonical NF-κB signalling. To enhance the therapeutic potential of SMAC mimetics in AML, we investigated the regulation and role of ARC in birinapant-induced apoptosis. We showed that birinapant increases ARC in AML and bone marrow-derived mesenchymal stromal cells (MSCs). Downregulation of MAP3K14 by siRNA decreased ARC levels and suppressed birinapant-induced ARC increase. Reverse-phase protein array analysis of 511 samples from newly diagnosed AML patients showed that BIRC2 (also termed cIAP1) and ARC were inversely correlated. Knockdown of ARC sensitized, while overexpression attenuated, birinapant-induced apoptosis. Furthermore, ARC knockdown in MSCs sensitized co-cultured AML cells to birinapant-induced apoptosis. Our data demonstrate that ARC is regulated via BIRC2/MAP3K14 signalling and its overexpression in AML or MSCs can function as a resistant factor to birinapant-induced leukaemia cell death, suggesting that strategies to inhibit ARC will improve the therapeutic potential of SMAC mimetics., (© 2014 John Wiley & Sons Ltd.)

The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress both intrinsic and extrinsic apoptosis. We previously reported that ARC expression is a strong, independent adverse prognostic factor in acute myeloid leukemia (AML). Here, we investigated the regulation and role of ARC in AML. ARC expression is upregulated in AML cells co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and suppressed by inhibition of MAPK and PI3K signaling. AML patient samples with RAS mutations (N = 64) expressed significantly higher levels of ARC than samples without RAS mutations (N = 371) (P = 0.016). ARC overexpression protected and ARC knockdown sensitized AML cells to cytarabine and to agents that selectively induce intrinsic (ABT-737) or extrinsic (TNF-related apoptosis inducing ligand) apoptosis. NOD-SCID mice harboring ARC-overexpressing KG-1 cells had significantly shorter survival than mice injected with control cells (median 84 vs 111 days) and significantly fewer leukemia cells were present when NOD/SCID IL2Rγ null mice were injected with ARC knockdown as compared to control Molm13 cells (P = 0.005 and 0.03 at 2 and 3 weeks, respectively). Together, these findings demonstrate that MSCs regulate ARC in AML through activation of MAPK and PI3K signaling pathways. ARC confers drug resistance and survival advantage to AML in vitro and in vivo, suggesting ARC as a novel target in AML therapy.

Background: Acute myeloid leukemia (AML) therapy has limited long-term efficacy because patients frequently develop disease relapse because of the inability of standard chemotherapeutic agents to target AML stem/progenitor cells. Here, we identify deregulated apoptotic components in AML stem/progenitor cells and investigate the individual and combinatorial effects of the novel inhibitor of apoptosis (IAP) protein antagonist and second mitochondrial-derived activator of caspases (SMAC) mimetic birinapant and demethylating epigenetic modulators., Methods: Protein expression was measured by reversed-phase protein array in AML patient (n = 511) and normal (n = 21) samples and by western blot in drug-treated cells. The antileukemic activity of birinapant and demethylating agents was assessed in vitro and in an in vivo AML mouse xenograft model (n = 10 mice per group). All statistical tests were two-sided., Results: Compared with bulk AML cells, CD34(+)38(-) AML stem/progenitors expressed increased cIAP1 and caspase-8 levels and decreased SMAC levels (one-way analysis of variance followed by Tukey's multiple comparison test, P < .001). Birinapant induced death receptor-/caspase-8-mediated apoptosis in AML cells, including in AML stem/progenitor cells, but not in normal CD34(+) cells. Demethylating agents modulated extrinsic apoptosis pathway components and, when combined with birinapant, were highly synergistic in vitro (combination index < 1), and also more effective in vivo (P < .001, by Student t test, for the median survival of birinapant plus 5-azacytadine vs birinapant alone or vs controls)., Conclusions: cIAP1, SMAC, and caspase-8 appear to play a role in AML stem cell survival, and synergistic targeting of these cells with birinapant and demethylating agents shows potential utility in leukemia therapy.

To further understand the role of XIAP in acute myeloid leukemia (AML), we suppressed XIAP expression by antisense oligonucleotides and determined the effect on gene expression profiles and biological pathways. XIAP inhibition upregulated expression of proteasome genes in a manner similar to the proteasome inhibitor bortezomib or MG132; decreased 20S proteasome activity, an effect which was diminished in the presence of a pan-caspase inhibitor; and increased IκBα, Mcl-1, and HSP70 in AML cells. In addition to multiple functions already described, XIAP contributes to increased proteasome activity in AML cells, and the antitumor effect of XIAP inhibition may be mediated in part through caspase-dependent proteasome inhibition., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Survivin, a member of the inhibitors of apoptosis protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignancies, including leukemias. To better understand its role in acute myeloid leukemia (AML), we profiled survivin expression in samples obtained from 511 newly diagnosed AML patients and in CD34(+)38(-) AML stem/progenitor cells using a validated reverse-phase protein array; we correlated its levels with clinical outcomes and with levels of other proteins in the same sample set. We found that survivin levels were higher in bone marrow than in paired peripheral blood leukemic cells (n = 140, P = .0001) and that higher survivin levels significantly predicted shorter overall (P = .016) and event-free (P = .023) survival in multivariate Cox model analysis. Importantly, survivin levels were significantly higher in CD34(+)38(-) AML stem/progenitor cells than in bulk blasts and total CD34(+) AML cells (P < .05). Survivin expression correlated with the expressions of multiple proteins involved with cell proliferation and survival. Particularly, its expression strongly correlated with HIF1α in the stem/progenitor cell compartment. These results suggest that survivin is a prognostic biomarker in AML and that survivin, which is overexpressed in AML stem/progenitor cells, remains a potentially important target for leukemia therapy.

Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-X(L), ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

Triptolide, isolated from the herb Tripterygium wilfordii, has been shown to potently induce apoptosis in various malignant cells by inhibiting RNA synthesis and nuclear factor-κB activity. Previously, we showed that triptolide promotes apoptosis in acute myeloid leukemia (AML) cells via the mitochondria-mediated pathway, in part, by decreasing levels of the anti-apoptotic proteins XIAP and Mcl-1. MRx102 is a triptolide derivative, currently in preclinical development. Here we show that MRx102 potently promoted apoptosis in AML cell lines, with EC(50) values of 14.5±0.6 nM and 37.0±0.9 nM at 48 h for OCI-AML3 and MV4-11 cells, respectively. MRx102, at low nanomolar concentrations, also induced apoptosis in bulk, CD34(+) progenitor, and more importantly, CD34(+)CD38(-) stem/progenitor cells from AML patients, even when they were protected by coculture with bone marrow derived mesenchymal stromal cells. MRx102 decreased XIAP and Mcl-1 protein levels and inhibited RNA synthesis in OCI-AML3 cells. In vivo, MRx102 greatly decreased leukemia burden and increased survival time in non-obese diabetic/severe combined immunodeficiency mice harboring Ba/F3-ITD cells. Collectively, we demonstrated that MRx102 has potent antileukemic activity both in vitro and in vivo, has the potential to eliminate AML stem/progenitor cells and overcome microenvironmental protection of leukemic cells, and warrants clinical investigation.

Regulators of apoptosis in acute myeloid leukemia (AML) have been extensively studied and are considered excellent therapeutic targets. Apoptosis repressor with caspase recruitment domain (ARC), an antiapoptotic protein originally found to be involved in apoptosis of cardiac cells, was recently demonstrated to be overexpressed in several solid tumors. To assess its importance in AML, we profiled ARC expression in 511 newly diagnosed AML patients using a validated robust reverse-phase protein array and correlated ARC levels with clinical outcomes. ARC was variably expressed in samples from patients with AML. ARC level was not associated with cytogenetic groups or with FLT-3 mutation status. However, patients with low or medium ARC protein levels had significantly better outcomes than those with high ARC levels: longer overall survival (median, 53.9 or 61.6 vs 38.9 weeks, P = .0015) and longer remission duration (median, 97.6 or 44.7 vs 31.1 weeks, P = .0007). Multivariate analysis indicated that ARC was a statistically significant independent predictor of survival in AML (P = .00013). Inhibition of ARC promoted apoptosis and sensitized cytosine arabinoside-induced apoptosis in OCI-AML3 cells. These results suggest that ARC expression levels are highly prognostic in AML and that ARC is a potential therapeutic target in AML.

XIAP, a potent caspase inhibitor, is highly expressed in acute myeloid leukemia (AML) cells and contributes to chemoresistance. A multi-center phase 1/2 trial of XIAP antisense oligonucleotide AEG35156 in combination with idarubicin/cytarabine was conducted in 56 patients with relapsed/refractory AML. Herein we report the pharmacodynamic studies of the patients enrolled at M. D. Anderson Cancer Center. A total of 13 patients were enrolled in our institution: five in phase 1 (12-350 mg/m² AEG35156) and eight in phase 2 (350 mg/m² AEG35156) of the protocol. AEG35156 was administered on 3 consecutive days and then weekly up to a maximum of 35 days. Blood samples were collected from patients on days 1 through 5 and on day 28-35 post-chemotherapy for detection of XIAP levels and apoptosis. AEG35156 treatment led to dose-dependent decreases of XIAP mRNA levels (42-100% reduction in phase 2 patients). XIAP protein levels were reduced in all five samples measured. Apoptosis induction was detected in 1/4 phase 1 and 4/5 phase 2 patients. Importantly, apoptosis was most pronounced in CD34+38- AML stem cells and all phase 2 patients showing apoptosis induction in CD34+38- cells achieved response. We conclude that at 350 mg/m², AEG35156 is effective in knocking down XIAP in circulating blasts accompanied by the preferential induction of apoptosis in CD34+38- AML stem cells.

Chronic myeloid leukemia (CML) is a clonal multistep myeloproliferative disease originating from and ultimately sustained by a rare population of BCR-ABL(+) cells with multilineage stem cell properties. Imatinib, the most successful of molecular targeted therapies, has revolutionized treatment of patients with CML. Despite this achievement, CML is often not curable, largely due to the innate insensitivity of CML stem cells, particularly when in a quiescent state. This failure of not only imatinib but also the second-generation tyrosine kinase inhibitors (TKIs) frequently leads to relapse upon drug discontinuation. Thus, any curative therapy must eliminate CML stem cells. A comprehensive understanding of the biological properties of CML stem cells and an elucidation of the molecular mechanisms and signaling pathways enabling these CML stem cells to self-renew, combined with insight into the regulation of apoptosis signaling and the mechanisms governing the interaction of CML stem cells with their bone marrow microenvironment, will facilitate the development of therapies for targeting these cells. Here, we discuss the biological properties of CML stem cells and potential strategies to eliminate them., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Activation of p53 by murine double minute (MDM2) antagonist nutlin-3a or inhibition of X-linked inhibitor of apoptosis (XIAP) induces apoptosis in acute myeloid leukemia (AML) cells. We demonstrate that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and down-regulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a-induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. Because both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic.

Kinesin spindle protein (KSP), a microtubule-associated motor protein essential for cell cycle progression, is overexpressed in many cancers and is a potential anti-tumor target. We found that inhibition of KSP by a selective inhibitor, ARRY-520, blocked cell cycle progression, leading to apoptosis in acute myeloid leukemia cell lines that express high levels of KSP. Knockdown of p53, overexpression of XIAP and mutation in caspase-8 did not significantly affect sensitivity to ARRY-520, suggesting that the response is independent of p53, XIAP and the extrinsic apoptotic pathway. Although ARRY-520 induced mitotic arrest in both HL-60 and Bcl-2-overexpressing HL-60Bcl-2 cells, cell death was blunted in HL-60Bcl-2 cells, suggesting that the apoptotic program is executed through the mitochondrial pathway. Accordingly, inhibition of Bcl-2 by ABT-737 was synergistic with ARRY-520 in HL-60Bcl-2 cells. Furthermore, ARRY-520 increased Bim protein levels prior to caspase activation in HL-60 cells. ARRY-520 significantly inhibited tumor growth of xenografts in SCID mice and inhibited AML blast but not normal colony formation, supporting a critical role for KSP in proliferation of leukemic progenitor cells. These results demonstrate that ARRY-520 potently induces cell cycle block and subsequent death in leukemic cells via the mitochondrial pathway and has the potential to eradicate AML progenitor cells.

The advent of Bcr-Abl tyrosine kinase inhibitors (TKI) has revolutionized the treatment of chronic myelogenous leukemia (CML). However, resistance evolves due to BCR-ABL mutations and other mechanisms. Furthermore, patients with blast crisis CML are less responsive and quiescent CML stem cells are insensitive to these inhibitors. We found that triptolide, a diterpenoid, at nanomolar concentrations, promoted equally significant death of KBM5 cells, a cell line derived from a Bcr-Abl-bearing blast crisis CML patient and KBM5STI571 cells, an imatinib-resistant KBM5 subline bearing the T315I mutation. Similarly, Ba/F3 cells harboring mutated BCR-ABL were as sensitive as Ba/F3Bcr-Abl(p210wt) cells to triptolide. Importantly, triptolide induced apoptosis in primary samples from blast crisis CML patients, who showed resistance to Bcr-Abl TKIs in vivo, with less toxicity to normal cells. Triptolide decreased X-linked inhibitor of apoptosis protein, Mcl-1, and Bcr-Abl protein levels in K562, KBM5, and KBM5STI571 cells and in cells from blast crisis CML patients. It sensitized KBM5, but not KBM5STI571, cells to imatinib. More importantly, triptolide also induced death of quiescent CD34(+) CML progenitor cells, a major problem in the therapy of CML with TKIs. Collectively, these results suggest that triptolide potently induces blast crisis CML cell death independent of the cellular responses to Bcr-Abl TKIs, suggesting that triptolide could eradicate residual quiescent CML progenitor cells in TKI-treated patients and benefit TKI-resistant blast crisis CML patients.

Acute myeloid leukemia (AML) cells are relatively resistant to tumor necrosis factor alpha-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 TRAIL-induced 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.

Tetra-O-methyl nordihydroguaiaretic acid (M4N) was shown to induce G2 arrest and suppress human xenograft tumor growth by inhibiting Cdc2 and survivin. We examined the effect of M4N on leukemia and found that M4N inhibited growth and induced cell death in leukemic cell lines and blasts from AML patients. However, no significant changes in Cdc2 and survivin levels and G2 arrest were observed. Cell death and growth inhibition were dependent neither on XIAP, Bcl-2, and Bcl-X(L) levels nor on caspase-8. M4N did not promote cell differentiation in HL-60 cells. Interestingly, significant inhibition of AKT phosphorylation was observed in M4N treated OCI-AML3 cells. Collectively, our data showed that M4N inhibited cell growth and induced cell death in both leukemic cell lines and AML patient sample via a mechanism not mediated by Cdc2 and survivin inhibition and suggested that the extrinsic and the mitochondrial apoptotic pathways are not essential.

In order to investigate the pharmacological basis of 'Yang-invigorating' action, the effect of oral treatment with the methanolic extract of 'Yang-invigorating' herbs on ATP-generation capacity was examined, using heart homogenates prepared from herb-pretreated mice. Tonifying (i.e., health-promoting) herbs of other functional categories were also included for comparison. The results indicated that 'Yang-invigorating' Chinese tonifying herbs could invariably enhance myocardial ATP-generation capacity, with the extent of stimulation varying among the herbs. In contrast, 'Yin-nourishing' herbs either did not stimulate or even decreased myocardial ATP-generation capacity. While 'Qi-invigorating' herbs produced variable effects on myocardial ATP-generation capacity, most of the 'blood-enriching' herbs did not cause any significant changes. The results obtained from studies using myocardial mitochondrial fractions isolated from herb-pretreated mice suggest that 'Yang-invigorating' herbs might speed up ATP generation by increasing mitochondrial electron transport. The ensemble of results has provided evidence for the first time to support the pharmacological basis of 'Yang invigoration' in Chinese medicine.

Patients with blast crisis (BC) CML frequently become resistant to Imatinib, a Bcr-Abl tyrosine kinase-targeting agent. Eg5, a microtubule-associated motor protein has been described to be highly expressed in BC CML by microarray analysis (Nowicki et al., Oncogene 2003; 22:3952-63). We investigated the regulation of Eg5 by Bcr-Abl tyrosine kinase and its potential as a therapeutic target in BC CML. Eg5 was highly expressed in all Philadelphia chromosome positive (Ph(+)) cell lines and BC CML patient samples. Inhibition of Bcr-Abl by Imatinib downregulated Eg5 expression in Imatinib-sensitive KBM5 and HL-60p185 cells, but not in Imatinib-resistant KBM5-STI571, harboring a T315I mutation, and Bcr-Abl-negative HL-60 cells. Blocking Eg5 expression with antisense oligonucleotide (Eg5-ASO) or inhibiting its activity with the small-molecule Eg5 inhibitor, S-trityl-L-cysteine induced G(2)/M cell cycle block and subsequent cell death in both Imatinib-sensitive and -resistant cells. Further, Eg5-ASO treatment of SCID mice harboring KBM5 cell xenografts significantly prolonged the median survival of the animals (p = 0.03). Our findings suggest that Eg5 is downstream of and regulated by Bcr-Abl tyrosine kinase in Philadelphia chromosome positive cells. Inhibition of Eg5 expression or its activity blocks cell cycle progression and induces cell death independent of the cellular response to Imatinib. Therefore, Eg5 could be a potential therapeutic target for the treatment of BC CML, in particular Imatinib-resistant BC CML.

Effects of schisandrin B enantiomers ((+)Sch B and (-)Sch B) treatment on cellular reduced glutathione (GSH) level and heat shock protein (Hsp)25/70 production were investigated in H9c2 cardiomyocytes. (+)Sch B and (-)Sch B at 6.25 muM produced a time-dependent and biphasic change in cellular GSH level and Hsp25/70 production, with the stimulatory effect of (-)Sch B being more potent. The GSH- and Hsp-enhancing effects were accompanied by a parallel cytoprotection against xanthine oxidase/xanthine-induced toxicity, with the biphasic time course of (+)Sch B- or (-)Sch B-induced protection being superimposed with that of the increase in GSH level but not Hsp25/70 production. The results indicate that (-)Sch B produces more potent enhancing effects on cellular GSH and Hsp production as well as protection against oxidative injury than (+)Sch B in cardiomyocytes.

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 caspase-dependent cell death accompanied by a significant decrease in XIAP levels. Forced XIAP overexpression attenuated triptolide-induced cell death. Triptolide also decreased Mcl-1 but not Bcl-2 and Bcl-X(L) levels. Bcl-2 overexpression suppressed triptolide-induced apoptosis. Further, triptolide induced loss of the mitochondrial membrane potential and cytochrome C release. Caspase-9 knock-out cells were resistant, while caspase-8-deficient cells were sensitive to triptolide, suggesting criticality of the mitochondrial but not the death receptor pathway for triptolide-induced apoptosis. Triptolide also enhanced cell death induced by other anticancer agents. Collectively, our results demonstrate that triptolide decreases XIAP and potently induces caspase-dependent apoptosis in leukemic cells mediated through the mitochondrial pathway at low nanomolar concentrations. The potent antileukemic activity of triptolide in vitro warrants further investigation of this compound for the treatment of leukemias and other malignancies.

The effects of pretreatment with Dang-Gui Buxue Tang (DBT, a decoction of Astragali and Angelica roots) and its component herb extracts on myocardial ischaemia-reperfusion (IR) injury were examined in rats ex vivo. DBT and its component herb extracts could protect against myocardial IR injury in a dose-dependent manner. The more potent cardioprotection afforded by DBT pretreatment than that of a mixture of Astragali and Angelica root extracts was associated with a much higher extraction yield of active ingredients from Angelica root in the herbal decoction. The high level of active ingredients might increase their bioavailability after oral administration. DBT pretreatment could enhance myocardial mitochondrial as well as red blood cell (RBC) glutathione status, thereby increasing their resistance to oxidative stress-induced injury in rats. The measurement of RBC glutathione status may serve as a useful index for the antioxidant effect produced by DBT treatment in human subjects.

KBM5 cells, derived from a patient with blast crisis Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML), and imatinib-resistant KBM5 (KBM5-STI571) cells were found to express high levels of survivin. Inhibition of Bcr-Abl by imatinib significantly decreased survivin expression and cell viability in KBM5, but much less so in KBM5-STI571 cells. Inhibition of MEK, downstream of the Bcr-Abl signaling cascade decreased survivin expression and cell viability in both KBM5 and KBM5-STI571 cells. In addition, down-regulation of survivin by a survivin antisense oligonucleotide (Sur-AS-ODN) inhibited cell growth and induced maximal G2M block at 48 hours, whereas cell death was observed only at 72 hours in both KBM5 and KBM5-STI571 cells as shown by annexin V staining. Further, the combination of Sur-AS-ODN and imatinib induced more cell death in KBM5 cells than did either treatment alone. Down-regulating survivin also decreased colony-forming units (CFUs) in blast crisis CML patient samples. Our data therefore suggest that survivin is regulated by the Bcr-Abl/MAPK cascade in Ph+ CML. The facts that down-regulating survivin expression induced cell-growth arrest and subsequent cell death regardless of the cell response to imatinib and enhanced the sensitivity to imatinib suggest the potential therapeutic utility of this strategy in patients with CML, both imatinib sensitive and resistant.

Effects of schisandrin B enantiomer ((+)Sch B and (-)Sch B) treatment on the reduced cellular glutathione (GSH) level and susceptibility to menadione-induced toxicity were investigated and compared in AML12 hepatocytes. (+)Sch B or (-)Sch B treatment at 6.25 micromol/l produced a time-dependent change in cellular GSH level, with the maximal stimulation occurring 16 h after dosing. (+)Sch B/(-)Sch B pretreatment for 16 h dose-dependently protected against menadione toxicity, with the maximum degree of protection observable at 6.25 micromol/l and the extent of protection afforded by (-)Sch B being larger than that of (+)Sch B. The cytoprotection was associated with a parallel enhancement in cellular GSH level in both non-menadione (control) and menadione-intoxicated cells. While the GSH depletion produced by buthionine sulfoximine/phorone treatment largely abrogated the cytoprotective action of (+)Sch B/(-)Sch B, it almost completely abolished the GSH-enhancing effect of (+)Sch B and (-)Sch B in both control and menadione-treated cells. Both (+)Sch B and (-)Sch B treatments increased the GSH reductase activity in control and menadione-treated cells, with the stimulatory action of (-)Sch B being more potent than that of (+)Sch B in the control condition. (+)Sch B and (-)Sch B also enhanced the gamma-glutamate cysteine ligase activity in menadione-intoxicated cells. The results indicate that (-)Sch B is more effective than (+)Sch B in enhancing cellular GSH and protecting against oxidant injury in hepatocytes., (Copyright 2006 S. Karger AG, Basel.)

In order to explore the role of cytochrome P-450 (P-450) in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effect of 1-aminobenzotriazole (ABT, a broad spectrum inhibitor of P-450) on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression was examined in Sch B-treated mice. The non-specific and partial inhibition of cytochrome P-450 (P-450) by ABT pretreatment significantly caused a protraction in the time-course of Sch B-induced enhancement in hepatic mitGAS and Hsp25/70 expression in mice. Using mouse liver microsomes as a source of P-450, Sch B, but not dimethyl diphenyl bicarboxylate (a non-hepatoprotective analog of Sch B), was found to serve as a co-substrate for the P-450-catalyzed NADPH oxidation reaction, with a concomitant production of oxidant species. Taken together, the results suggest that oxidant species generated from P-450-catalyzed reaction with Sch B may trigger the antioxidant and heat shock responses in mouse liver.

Cordyceps sinensis (Berk.) Sacc. (Cordyceps), a popular Chinese tonifying herb, was revered for being both 'Yin-nourishing' and 'Yang-invigorating' in Chinese medicine. In order to establish the pharmacological basis for the 'Yin-nourishing' and 'Yang-invigorating' action of Cordyceps, the effects of wild and cultured Cordyceps on concanavalin A (Con A)-stimulated splenocytes, an in vitro bioassay for 'Yin-nourishment', and myocardial ATP generation capacity, an ex vivo bioassay for 'Yang-invigoration', were investigated in mice. The results indicated that methanolic extracts of wild and cultured Cordyceps enhanced both the Con A-stimulated splenocyte proliferation in vitro and myocardial mitochondrial ATP generation ex vivo in mice, with no significant difference in potency of action between the two types of Cordyceps. While the immuno-potentiating effect was associated with the increase in interleukin II production, the stimulation of myocardial ATP generation was paralleled by an enhancement in mitochondrial electron transport. When compared with typical 'Yin' and 'Yang' tonifying Chinese herbs, Cordyceps was found to possess both 'Yin-nourishing' and 'Yang-invigorating' activities, with a lower potency in both modes of action. The pharmacological characterization of Cordyceps by means of contemporary bioassays is consistent with the time-honored clinical observation from Chinese herbalists.

In the 16-week pilot study, the effect of a Yang-promoting Chinese herbal suppository preparation (VI-28) on the red cell antioxidant status was examined in 31 healthy male subjects aged 41-66 years old. VI-28 treatment for 12 weeks (one suppository (0.3 g) daily for week 1-4; one every 2 days for week 5-8; one every 3 days for week 9-12) produced a time/dose-dependent alteration in red cell antioxidant status. The VI-28-induced change is characterized by a slight depletion in cellular reduced glutathione (GSH) level and a decrease in susceptibility to peroxide-induced lipid peroxidation as well as increases in catalase (CAT) and Cu-Zn-superoxide dismutase (SOD) activities. While a reversal trend of change was observed in cellular GSH level, the susceptibility to lipid peroxidation as well as the CAT activity after the cessation of treatment for 4 weeks, the SOD activity exhibited a protracted increase. The results indicate that VI-28 treatment enhances red cell antioxidant status in male subjects. The beneficial effect of VI-28 treatment on red cells may re fl ect a corresponding change in antioxidant status of peripheral tissues.

The 'Yin-Yang' theory is an ancient Chinese philosophy that underlies the practice of traditional Chinese medicine. Although Yang-tonic herbs tend to boost body function possibly through enhancing the mitochondrial oxidative processes, the Yin property (i.e. antioxidant potential) of these herbs can also play a role in safeguarding mitochondrial ATP generation. The pharmacological basis of 'Yang-invigoration' by Chinese tonic herbs might be due primarily to the enhancement of mitochondrial ATP generation.

In the present study, the hepatoprotective effect of metformin (Met), a dimethylbiguanide anti-hyperglycemic, was examined in a mouse model of liver damage induced by chronic repeated administration of carbon tetrachloride (CCl(4)) (5 microl/kg, twice a week for 12 weeks). Met, when given orally in drinking water at an estimated daily dose of 25 or 50 mg/kg for 10 weeks starting 2 weeks after CCl(4) challenge, protected against CCl(4) hepatotoxicity. The results indicate that the hepatoprotection afforded by Met treatment at a dose of 25 mg/kg against CCl(4) toxicity may at least in part be mediated by the enhancement of mitochondrial glutathione redox status.

In this study, the time course of schisandrin B- (Sch B-) induced changes in hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (HSP) 25/70 induction was examined to study their differential roles in the hepatoprotection afforded by Sch B pretreatment against carbon tetrachloride (CCl(4)) toxicity in mice. Dimethyl diphenyl bicarboxylate (DDB), a nonhepatoprotective analog of Sch B, was also included for comparison. The results indicate that Sch B treatment (2 mmol/kg) produced maximum enhancement in hepatic mtGAS and increases in both hepatic HSP 25 and HSP 70 levels at 24 h after dosing. While the extent of hepatoprotection afforded by Sch B pretreatment against CCl(4) was found to correlate inversely with the elapsed time postdosing, the protective effect was associated with the ability to sustain mtGAS and/or HSP 70 levels in a CCl(4)-intoxicated condition. On the other hand, DDB (2 mmol/kg) treatment, which did not sustain mtGAS and HSP 70 level, could not protect against CCl(4) toxicity. Abolition of the Sch B-mediated enhancement of mtGAS by buthionine sulfoximine/phorone did not completely abrogate the hepatoprotective action of Sch B. The results indicate that Sch B pretreatment independently enhances mtGAS and induces HSP 25/70 production, particularly under conditions of oxidative stress, thereby protecting against CCl(4) hepatotoxicity.

The in vivo antioxidant action of a lignan-enriched extract of the fruit of Schisandra chinensis (FS) and an anthraquinone-containing extract of the root of Polygonum multiflorum (PME) was compared with their respective active constituents schisandrin B (Sch B) and emodin by examining their effect on hepatic mitochondrial glutathione antioxidant status in control and carbon tetrachloride (CCl 4 )-intoxicated mice. FS and PME pretreatments produced a dose-dependent protection against CCl 4 hepatotoxicity, with the effect of FS being more potent. Pretreatment with Sch B, emodin or alpha-tocopherol (alpha-Toc) also protected against CCl 4 hepatotoxicity, with the effect of Sch B being more potent. The extent of hepatoprotection afforded by FS/Sch B and PME/emodin pretreatment against CCl 4 toxicity was found to correlate well with the degree of enhancement in hepatic mitochondrial glutathione antioxidant status, as evidenced by increases in reduced glutathione level and activities of glutathione reductase, glutathione peroxidase as well as glutathione S-transferases, in both control and CCl 4 -intoxicated mice. alpha-Toc, which did not enhance mitochondrial glutathione antioxidant status, seemed to be less potent in protecting against CCl 4 hepatotoxicity. The ensemble of results indicates that FS/PME produced a more potent in vivo antioxidant action than alpha-Toc by virtue of their ability to enhance hepatic mitochondrial glutathione antioxidant status and that the differential potency of FS and PME can be attributed to the difference in in vivo antioxidant potential between Sch B and emodin. Abbreviations. ALT:alanine aminotransferases CCl 4 :carbon tetrachloride FS:lignan-enriched extract of Schisandra fruit GRD:glutathione reductase GSH:reduced glutathione GSH-Px: Se-glutathione peroxidase GST:glutathione S-transferases mt:mitochondrial MDA:malondialdehyde PME:anthraquinone-containing fraction of Polygonum root Sch B:schisandrin B SDH:sorbitol dehydrogenase alpha-Toc:alpha-tocopherol

Intragastric administration (100-200 micromol/kg) of tacrine (THA) or bis(7)-THA could cause an acute and dose-dependent increase in plasma alanine aminotransferases activity in mice at 6 h after the drug administration. The increase in plasma enzyme activity was associated with an increase in hepatic malondialdehyde level, an indirect index of oxidative tissue damage. Pretreating mice with schisandrin B (Sch B), an active dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, at a daily dose of 0.125-0.5 mmol/kg for 3 days protected against the THA/bis(7)-THA induced hepatic oxidative damage in a dose-dependent manner. Sch B treatment (0.025-0.5 mmol/kg/day x 5) also enhanced the passive avoidance-response in mice as assessed by the step-through task experiment. The ensemble of results suggests that Sch B may be useful for reducing the potential hepatotoxicity of THA/bis(7)-THA in anti-Alzheimer's therapy.