A targetable LIFR-NF-κB-LCN2 axis controls liver tumorigenesis and vulnerability to ferroptosis.

The growing knowledge of ferroptosis has suggested the role and therapeutic potential of ferroptosis in cancer, but has not been translated into effective therapy. Liver cancer, primarily hepatocellular carcinoma (HCC), is highly lethal with limited treatment options. LIFR is frequently downregulated in HCC. Here, by studying hepatocyte-specific and inducible Lifr-knockout mice, we show that loss of Lifr promotes liver tumorigenesis and confers resistance to drug-induced ferroptosis. Mechanistically, loss of LIFR activates NF-κB signaling through SHP1, leading to upregulation of the iron-sequestering cytokine LCN2, which depletes iron and renders insensitivity to ferroptosis inducers. Notably, an LCN2-neutralizing antibody enhances the ferroptosis-inducing and anticancer effects of sorafenib on HCC patient-derived xenograft tumors with low LIFR expression and high LCN2 expression. Thus, anti-LCN2 therapy is a promising way to improve liver cancer treatment by targeting ferroptosis.

A comprehensive overview of metaplastic breast cancer: clinical features and molecular aberrations.

Metaplastic breast cancer (MpBC) is an exceedingly rare breast cancer variant that is therapeutically challenging and aggressive. MpBC is defined by the histological presence of at least two cellular types, typically epithelial and mesenchymal components. This variant harbors a triple-negative breast cancer (TNBC) phenotype, yet has a worse prognosis and decreased survival compared to TNBC. There are currently no standardized treatment guidelines specifically for MpBC. However, prior studies have found that MpBC typically has molecular alterations in epithelial-to-mesenchymal transition, amplification of epidermal growth factor receptor, PI3K/Akt signaling, nitric oxide signaling, Wnt/ß-catenin signaling, altered immune response, and cell cycle dysregulation. Some of these molecular alterations have been studied as therapeutic targets, in both the preclinical and clinical setting. This current review discusses the histological organization and cellular origins of MpBC, molecular alterations, the role of radiation therapy, and current clinical trials for MpBC.

Carbapenems drive the collateral resistance to ceftaroline in cystic fibrosis patients with MRSA.

Chronic airways infection with methicillin-resistant Staphylococcus aureus (MRSA) is associated with worse respiratory disease cystic fibrosis (CF) patients. Ceftaroline is a cephalosporin that inhibits the penicillin-binding protein (PBP2a) uniquely produced by MRSA. We analyzed 335 S. aureus isolates from CF sputum samples collected at three US centers between 2015-2018. Molecular relationships demonstrated that high-level resistance of preceding isolates to carbapenems were associated with subsequent isolation of ceftaroline resistant CF MRSA. In vitro evolution experiments showed that pre-exposure of CF MRSA to meropenem with further selection with ceftaroline implied mutations in mecA and additional mutations in pbp1 and pbp2, targets of carbapenems; no effects were achieved by other ß-lactams. An in vivo pneumonia mouse model showed the potential therapeutic efficacy of ceftaroline/meropenem combination against ceftaroline-resistant CF MRSA infections. Thus, the present findings highlight risk factors and potential therapeutic strategies offering an opportunity to both prevent and address antibiotic resistance in this patient population.

Correction to: A window-of-opportunity trial of the CXCR1/2 inhibitor reparixin in operable HER-2-negative breast cancer.

An amendment to this paper has been published and can be accessed via the original article.

Simultaneous targeting of HER family pro-survival signaling with Pan-HER antibody mixture is highly effective in TNBC: a preclinical trial with PDXs.

BACKGROUND: The human epidermal growth factor receptor (HER) family, notably EGFR, is overexpressed in most triple-negative breast cancer (TNBC) cases and provides cancer cells with compensatory signals that greatly contribute to the survival and development of resistance in response to therapy. This study investigated the effects of Pan-HER (Symphogen, Ballerup, Denmark), a novel mixture of six monoclonal antibodies directed against members of the HER family EGFR, HER2, and HER3, in a preclinical trial of TNBC patient-derived xenografts (PDXs). METHODS: Fifteen low passage TNBC PDX tumor samples were transferred into the right mammary fat pad of mice for engraftment. When tumors reached an average size of 100-200 mm3, mice were randomized (n ≥ 6 per group) and treated following three 1-week cycles consisting of three times/week intraperitoneal (IP) injection of either formulation buffer (vehicle control) or Pan-HER (50 mg/kg). At the end of treatment, tumors were collected for Western blot, RNA, and immunohistochemistry analyses. RESULTS: All 15 TNBC PDXs were responsive to Pan-HER treatment, showing significant reductions in tumor growth consistent with Pan-HER-mediated tumor downmodulation of EGFR and HER3 protein levels and significantly decreased activation of associated HER family signaling pathways AKT and ERK. Tumor regression was observed in five of the models, which corresponded to those PDX tumor models with the highest level of HER family activation. CONCLUSIONS: The marked effect of Pan-HER in numerous HER family-dependent TNBC PDX models justifies further studies of Pan-HER in TNBC clinical trials as a potential therapeutic option.

A window-of-opportunity trial of the CXCR1/2 inhibitor reparixin in operable HER-2-negative breast cancer.

BACKGROUND: Cancer stem cells (CSCs) are purported to be responsible for tumor initiation, treatment resistance, disease recurrence, and metastasis. CXCR1, one of the receptors for CXCL8, was identified on breast cancer (BC) CSCs. Reparixin, an investigational allosteric inhibitor of CXCR1, reduced the CSC content of human BC xenograft in mice. METHODS: In this multicenter, single-arm trial, women with HER-2-negative operable BC received reparixin oral tablets 1000 mg three times daily for 21 days before surgery. Primary objectives evaluated the safety of reparixin and the effects of reparixin on CSC and tumor microenvironment in core biopsies taken at baseline and at treatment completion. Signal of activity was defined as a reduction of ≥ 20% in ALDH+ or CD24-/CD44+ CSC by flow cytometry, with consistent reduction by immunohistochemistry. RESULTS: Twenty patients were enrolled and completed the study. There were no serious adverse reactions. CSC markers ALDH+ and CD24-/CD44+ measured by flow cytometry decreased by ≥ 20% in 4/17 and 9/17 evaluable patients, respectively. However, these results could not be confirmed by immunofluorescence due to the very low number of CSC. CONCLUSIONS: Reparixin appeared safe and well-tolerated. CSCs were reduced in several patients as measured by flow cytometry, suggesting targeting of CXCR1 on CSC. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov, NCT01861054. Registered on April 18, 2013.

A randomized, controlled phase II trial of neoadjuvant ado-trastuzumab emtansine, lapatinib, and nab-paclitaxel versus trastuzumab, pertuzumab, and paclitaxel in HER2-positive breast cancer (TEAL study).

BACKGROUND: Neoadjuvant dual human epidermal growth factor receptor (HER2) blockade with trastuzumab and pertuzumab plus paclitaxel leads to an overall pathologic complete response (pCR) rate of 46%. Dual HER2 blockade with ado-trastuzumab emtansine (T-DM1) and lapatinib plus nab-paclitaxel has shown efficacy in patients with metastatic HER2-positive breast cancer. To test neoadjuvant effectiveness of this regimen, an open-label, multicenter, randomized, phase II trial was conducted comparing T-DM1, lapatinib, and nab-paclitaxel with trastuzumab, pertuzumab, and paclitaxel in patients with early-stage HER2-positive breast cancer. METHODS: Stratification by estrogen receptor (ER) status occurred prior to randomization. Patients in the experimental arm received 6 weeks of targeted therapies (T-DM1 and lapatinib) followed by T-DM1 every 3 weeks, lapatinib daily, and nab-paclitaxel weekly for 12 weeks. In the standard arm, patients received 6 weeks of trastuzumab and pertuzumab followed by trastuzumab weekly, pertuzumab every 3 weeks, and paclitaxel weekly for 12 weeks. The primary objective was to evaluate the proportion of patients with residual cancer burden (RCB) 0 or I. Key secondary objectives included pCR rate, safety, and change in tumor size at 6 weeks. Hypothesis-generating correlative assessments were also performed. RESULTS: The 30 evaluable patients were well-balanced in patient and tumor characteristics. The proportion of patients with RCB 0 or I was higher in the experimental arm (100% vs. 62.5% in the standard arm, p = 0.0035). In the ER-positive subset, all patients in the experimental arm achieved RCB 0-I versus 25% in the standard arm (p = 0.0035). Adverse events were similar between the two arms. CONCLUSION: In early-stage HER2-positive breast cancer, the neoadjuvant treatment with T-DM1, lapatinib, and nab-paclitaxel was more effective than the standard treatment, particularly in the ER-positive cohort. TRIAL REGISTRATION: Clinicaltrials.gov NCT02073487 , February 27, 2014.

VraSR and Virulence Trait Modulation during Daptomycin Resistance in Methicillin-Resistant Staphylococcus aureus Infection.

Methicillin-resistant Staphylococcus aureus (MRSA) threatens human health in hospital and community settings. The lipopeptide antibiotic daptomycin (DAP) is a frequently used treatment option for MRSA infection. DAP exposure can cause bacterial resistance because mutations are induced in genes implicated in cell membrane and cell wall metabolism. Adaptations aimed at surviving antimicrobial pressure can affect bacterial physiology and modify in vivo aptitude and pathogenesis. In this study, clinical DAP-susceptible (DAPs) and DAP-resistant (DAPr) MRSA isolates were used to investigate associations between DAP resistance and staphylococcal virulence. We previously found that VraSR is a critical sensor of cell membrane/wall homeostasis associated with DAP acquisition during MRSA infection. The present study found that DAPr CB1634 and CB5014 MRSA strains with vraSR upregulation were less virulent than their susceptible counterparts, CB1631 and CB5013. Differential gene-transcription profile analysis revealed that DAPr CB1634 had decreased agr two-component system expression, virulence factors, and highly suppressed hemolysis activity. Functional genetic analysis performed in DAPr CB1634 strains using vraSR inactivation followed by gene complementation found that vraSR acted as a transcriptional agrA regulator. These results indicated that VraSR has a broad range of regulatory functions. VraSR also appeared to affect DAPr adherence to epithelial cells, which would affect DAPr strain colonization and survival in the host. The correlation between DAP resistance and decreased virulence was also found in the CB5013 (DAPs) and CB5014 (DAPr) pair. Taken together, these findings are the first evidence that DAP resistance and MRSA virulence are tightly connected and involve compromised expression of regulatory and virulence determinants.IMPORTANCE Methicillin-resistant S. aureus continues to develop resistance to antimicrobials, including those in current clinical use as daptomycin (DAP). Resistance to DAP arises by mutations in cell membrane and cell wall genes and/or upregulation of the two-component VraSR system. However, less is known about the connection between the pathogen and virulence traits during DAP resistance development. We provide new insights into VraSR and its regulatory role for virulence factors during DAP resistance, highlighting coordinated interactions that favor the higher persistence of MRSA DAP-resistant strains in the infected host.

Evaluation of anti-PD-1-based therapy against triple-negative breast cancer patient-derived xenograft tumors engrafted in humanized mouse models.

BACKGROUND: Breast cancer has been considered not highly immunogenic, and few patients benefit from current immunotherapies. However, new strategies are aimed at changing this paradigm. In the present study, we examined the in vivo activity of a humanized anti-programmed cell death protein 1 (anti-PD-1) antibody against triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) tumor models. METHODS: To circumvent some of the limitations posed by the lack of appropriate animal models in preclinical studies of immunotherapies, partially human leukocyte antigen-matched TNBC PDX tumor lines from our collection, as well as human melanoma cell lines, were engrafted in humanized nonobese diabetic/severe combined immunodeficiency IL2Rγnull (hNSG) mice obtained by intravenous injection of CD34+ hematopoietic stem cells into nonlethally irradiated 3-4-week-old mice. After both PDXs and melanoma cell xenografts reached ~ 150-200 mm3, animals were treated with humanized anti-PD-1 antibody or anti-CTLA-4 and evaluated for tumor growth, survival, and potential mechanism of action. RESULTS: Human CD45+, CD20+, CD3+, CD8+, CD56+, CD68+, and CD33+ cells were readily identified in blood, spleen, and bone marrow collected from hNSG, as well as human cytokines in blood and engrafted tumors. Engraftment of TNBC PDXs in hNSG was high (~ 85%), although they grew at a slightly slower pace and conserved their ability to generate lung metastasis. Human CD45+ cells were detectable in hNSG-harbored PDXs, and consistent with clinical observations, anti-PD-1 antibody therapy resulted in both a significant reduction in tumor growth and increased survival in some of the hNSG PDX tumor lines, whereas no such effects were observed in the corresponding non-hNSG models. CONCLUSIONS: This study provides evidence associated with anti-PD-1 immunotherapy against TNBC tumors supporting the use of TNBC PDXs in humanized mice as a model to overcome some of the technical difficulties associated with the preclinical investigation of immune-based therapies.

Activating Transcription Factor 4 Modulates TGFß-Induced Aggressiveness in Triple-Negative Breast Cancer via SMAD2/3/4 and mTORC2 Signaling.

Purpose: On the basis of the identified stress-independent cellular functions of activating transcription factor 4 (ATF4), we reported enhanced ATF4 levels in MCF10A cells treated with TGFß1. ATF4 is overexpressed in patients with triple-negative breast cancer (TNBC), but its impact on patient survival and the underlying mechanisms remain unknown. We aimed to determine ATF4 effects on patients with breast cancer survival and TNBC aggressiveness, and the relationships between TGFß and ATF4. Defining the signaling pathways may help us identify a cell signaling-tailored gene signature.Experimental Design: Patient survival data were determined by Kaplan-Meier analysis. Relationship between TGFß and ATF4, their effects on aggressiveness (tumor proliferation, metastasis, and stemness), and the underlying pathways were analyzed in three TNBC cell lines and in vivo using patient-derived xenografts (PDX).Results: ATF4 overexpression correlated with TNBC patient survival decrease and a SMAD-dependent crosstalk between ATF4 and TGFß was identified. ATF4 expression inhibition reduced migration, invasiveness, mammosphere-forming efficiency, proliferation, epithelial-mesenchymal transition, and antiapoptotic and stemness marker levels. In PDX models, ATF4 silencing decreased metastases, tumor growth, and relapse after chemotherapy. ATF4 was shown to be active downstream of SMAD2/3/4 and mTORC2, regulating TGFß/SMAD and mTOR/RAC1-RHOA pathways independently of stress. We defined an eight-gene signature with prognostic potential, altered in 45% of 2,509 patients with breast cancer.Conclusions: ATF4 may represent a valuable prognostic biomarker and therapeutic target in patients with TNBC, and we identified a cell signaling pathway-based gene signature that may contribute to the development of combinatorial targeted therapies for breast cancer. Clin Cancer Res; 24(22); 5697-709. ©2018 AACR.

Pharmacological Inhibition of NOS Activates ASK1/JNK Pathway Augmenting Docetaxel-Mediated Apoptosis in Triple-Negative Breast Cancer.

Purpose: Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress response and by inducible nitric oxide synthase (iNOS). Our aim was to determine the effects of pharmacologic NOS inhibition on TNBC.Experimental Design: TNBC cell lines, SUM-159PT, MDA-MB-436, and MDA-MB-468, were treated with docetaxel and NOS inhibitor (L-NMMA) for 24, 48, and 72 hours. Apoptosis was assessed by flow cytometry using Annexin-V and propidium iodide. Western blot was used to assess ER stress and apoptosis, and rtPCR was used to evaluate s-XBP1. TNBC patient-derived xenografts (PDX) were treated either with vehicle, docetaxel, or combination therapy (NOS inhibition + docetaxel). Mouse weight and tumor volumes were recorded twice weekly. Docetaxel concentration was determined using mass spectrometry. To quantify proliferation and apoptosis, PDX tumor samples were stained using Ki67 and TUNEL assay.Results:In vitro, L-NMMA ameliorated the iNOS upregulation associated with docetaxel. Apoptosis increased when TNBC cells were treated with combination therapy. In TNBC PDXs, combination therapy significantly reduced tumor volume growth and increased survival proportions. In the BCM-5998 PDX model, intratumoral docetaxel concentration was higher in mice receiving combination therapy. Coupling docetaxel with NOS inhibition increased EnR-stress response via coactivation of ATF4 and CHOP, which triggered the pASK1/JNK proapoptotic pathway, promoting cleavage of caspases 3 and 9.Conclusions: iNOS is a critical target for docetaxel resistance in TNBC. Pharmacologic inhibition of NOS enhanced chemotherapy response in TNBC PDX models. Combination therapy may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. Clin Cancer Res; 24(5); 1152-62. ©2018 AACR.

HN1L Promotes Triple-Negative Breast Cancer Stem Cells through LEPR-STAT3 Pathway.

Here, we show that HEMATOLOGICAL AND NEUROLOGICAL EXPRESSED 1-LIKE (HN1L) is a targetable breast cancer stem cell (BCSC) gene that is altered in 25% of whole breast cancer and significantly correlated with shorter overall or relapse-free survival in triple-negative breast cancer (TNBC) patients. HN1L silencing reduced the population of BCSCs, inhibited tumor initiation, resensitized chemoresistant tumors to docetaxel, and hindered cancer progression in multiple TNBC cell line-derived xenografts. Additionally, gene signatures associated with HN1L correlated with shorter disease-free survival of TNBC patients. We defined HN1L as a BCSC transcription regulator for genes involved in the LEPR-STAT3 signaling axis as HN1L binds to a putative consensus upstream sequence of STAT3, LEPTIN RECEPTOR, and MIR-150. Our data reveal that BCSCs in TNBC depend on the transcription regulator HN1L for the sustained activation of the LEPR-STAT3 pathway, which makes it a potentially important target for both prognosis and BCSC therapy.

Oligonucleotide aptamers: new tools for targeted cancer therapy.

Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed "chemical antibodies." In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.

Histone deacetylase inhibitors activate NF-kappaB in human leukemia cells through an ATM/NEMO-related pathway.

Mechanisms underlying histone deacetylase inhibitor (HDACI)-mediated NF-kappaB activation were investigated in human leukemia cells. Exposure of U937 and other leukemia cells to LBH-589 induced reactive oxygen species (ROS) followed by single strand (XRCC1) and double strand (gamma-H2AX) DNA breaks. Notably, LBH-589 lethality was markedly attenuated by small interfering RNA (siRNA) knockdown of the DNA damage-linked histone, H1.2. LBH-589 triggered p65/RelA activation, NF-kappaB-dependent induction of Mn-SOD2, and ROS elimination. Interference with LBH-589-mediated NF-kappaB activation (e.g. in I kappaB alpha super-repressor transfected cells) diminished HDACI-mediated Mn-SOD2 induction and increased ROS accumulation, DNA damage, and apoptosis. The Mn-SOD2 mimetic TBAP (manganese(III)-tetrakis 4-benzoic acid porphyrin) prevented HDACI-induced ROS and NF-kappaB activation while dramatically attenuating DNA damage and cell death. In contrast, TRAF2 siRNA knockdown, targeting receptor-mediated NF-kappaB activation, blocked TNFalpha- but not HDACI-mediated NF-kappaB activation and lethality. Consistent with ROS-mediated DNA damage, LBH-589 exposure activated ATM (on serine 1981) and increased its association with NEMO. Significantly, siRNA NEMO or ATM knockdown blocked HDACI-mediated NF-kappaB activation, resulting in diminished MnSOD2 induction and enhanced oxidative DNA damage and cell death. In accord with the recently described DNA damage/ATM/NEMO pathway, SUMOylation site mutant NEMO (K277A or K309A) cells exposed to LBH-589 displayed diminished ATM/NEMO association, NEMO and p65/RelA nuclear localization/activation, and MnSOD2 up-regulation. These events were accompanied by increased ROS production, gamma-H2AX formation, and cell death. Together, these findings indicate that in human leukemia cells, HDACIs activate the cytoprotective NF-kappaB pathway through an ATM/NEMO/SUMOylation-dependent process involving the induction of ROS and DNA damage and suggest that blocking NF-kappaB activation via the atypical ATM/NEMO nuclear pathway can enhance HDACI antileukemic activity.

HDAC Inhibitors--CHI's Third Annual Conference.

The HDAC Inhibitors meeting, held in Boston, included topics covering new therapeutic developments in the field of HDAC inhibitors. This conference report highlights selected presentations on HDAC inhibitors for the treatment of cancer, inflammation, and neurodegenerative and other CNS diseases. Investigational drugs discussed include several compounds under evaluation by Karus Therapeutics Ltd, OCID-4681 (Orchid Research Laboratories Ltd) and EVP-0334 (EnVivo Pharmaceuticals Inc).

Role of histone deacetylase inhibitor-induced reactive oxygen species and DNA damage in LAQ-824/fludarabine antileukemic interactions.

The role of reactive oxygen species (ROS) production on DNA damage and potentiation of fludarabine lethality by the histone deacetylase inhibitor (HDACI) LAQ-824 was investigated in human leukemia cells. Preexposure (24 h) of U937, HL-60, Jurkat, or K562 cells to LAQ-824 (40 nmol/L) followed by fludarabine (0.4 micromol/L) dramatically potentiated apoptosis (>or=75%). LAQ-824 triggered an early ROS peak (30 min-3 h), which declined by 6 h, following LAQ-824-induced manganese superoxide dismutase 2 (Mn-SOD2) upregulation. LAQ-824/fludarabine lethality was significantly diminished by either ROS scavengers N-acetylcysteine or manganese (III) tetrakis (4-benzoic acid) porphyrin or ectopic Mn-SOD2 expression and conversely increased by Mn-SOD2 antisense knockdown. During this interval, LAQ-824 induced early (4-8 h) increases in gamma-H2AX, which persisted (48 h) secondary to LAQ-824-mediated inhibition of DNA repair (e.g., down-regulation of Ku86 and Rad50, increased Ku70 acetylation, diminished Ku70 and Ku86 DNA-binding activity, and down-regulated DNA repair genes BRCA1, CHEK1, and RAD51). Addition of fludarabine further potentiated DNA damage, which was incompatible with cell survival, and triggered multiple proapoptotic signals including activation of nuclear caspase-2 and release of histone H1.2 into the cytoplasm. The latter event induced activation of Bak and culminated in pronounced mitochondrial injury and apoptosis. These findings provide a mechanistic basis for understanding the role of early HDACI-induced ROS generation and modulation of DNA repair processes in potentiation of nucleoside analogue-mediated DNA damage and lethality in leukemia. Moreover, they show for the first time the link between HDACI-mediated ROS generation and the recently reported DNA damage observed in cells exposed to these agents.

RAC3 down-regulation sensitizes human chronic myeloid leukemia cells to TRAIL-induced apoptosis.

The nuclear receptor coactivator RAC3 plays important roles in many biological processes and tumorigenesis. We found that RAC3 is over-expressed in human chronic myeloid leukemia cells K562, which are normally resistant to TRAIL-induced apoptosis. RAC3 down-regulation by siRNA rendered these cells sensitive to TRAIL-induced cell death. In addition to the up-regulation of TRAIL receptors, the process involves Bid, caspases and PARP activation, loss of mitochondrial membrane potential, and release of AIF, cytochrome c and Smac/DIABLO to the cytoplasm. We conclude that RAC3 is required for TRAIL resistance and that this anti-apoptotic function is independent of its role in hormone receptor signaling.

The multikinase inhibitor sorafenib potentiates TRAIL lethality in human leukemia cells in association with Mcl-1 and cFLIPL down-regulation.

Interactions between the multikinase inhibitor sorafenib and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were examined in malignant hematopoietic cells. Pretreatment (24 h) of U937 leukemia cells with 7.5 micromol/L sorafenib dramatically increased apoptosis induced by sublethal concentrations of TRAIL/Apo2L (75 ng/mL). Similar interactions were observed in Raji, Jurkat, Karpas, K562, U266 cells, primary acute myelogenous leukemia blasts, but not in normal CD34+ bone marrow cells. Sorafenib/TRAIL-induced cell death was accompanied by mitochondrial injury and release of cytochrome c, Smac, and AIF into the cytosol and caspase-9, caspase-3, caspase-7, and caspase-8 activation. Sorafenib pretreatment down-regulated Bcl-xL and abrogated Mcl-1 expression, whereas addition of TRAIL sharply increased Bid activation, conformational change of Bak (ccBak) and Bax (ccBax), and Bax translocation. Ectopic Mcl-1 expression significantly attenuated sorafenib/TRAIL-mediated lethality and dramatically reduced ccBak while minimally affecting levels of ccBax. Similarly, inhibition of the receptor-mediated apoptotic cascade with a caspase-8 dominant-negative mutant significantly blocked sorafenib/TRAIL-induced lethality but not Mcl-1 down-regulation or Bak/Bax conformational change, indicating that TRAIL-mediated receptor pathway activation is required for maximal lethality. Sorafenib/TRAIL did not increase expression of DR4/DR5, or recruitment of procaspase-8 or FADD to the death-inducing signaling complex (DISC), but strikingly increased DISC-associated procaspase-8 activation. Sorafenib also down-regulated cFLIP(L), most likely through a translational mechanism, in association with diminished eIF4E phosphorylation, whereas ectopic expression of cFLIP(L) significantly reduced sorafenib/TRAIL lethality. Together, these results suggest that in human leukemia cells, sorafenib potentiates TRAIL-induced lethality by down-regulating Mcl-1 and cFLIP(L), events that cooperate to engage the intrinsic and extrinsic apoptotic cascades, culminating in pronounced mitochondrial injury and apoptosis.

Mechanism and functional role of XIAP and Mcl-1 down-regulation in flavopiridol/vorinostat antileukemic interactions.

The mechanism and functional significance of XIAP and Mcl-1 down-regulation in human leukemia cells exposed to the histone deacetylase inhibitor vorinostat and the cyclin-dependent kinase inhibitor flavopiridol was investigated. Combined exposure of U937 leukemia cells to marginally toxic concentrations of vorinostat and flavopiridol resulted in a marked increase in mitochondrial damage and apoptosis accompanied by pronounced reductions in XIAP and Mcl-1 mRNA and protein. Down-regulation of Mcl-1 and XIAP expression by vorinostat/flavopiridol was associated with enhanced inhibition of phosphorylation of RNA polymerase II and was amplified by caspase-mediated protein degradation. Chromatin immunoprecipitation analysis revealed that XIAP and Mcl-1 down-regulation were also accompanied by both decreased association of nuclear factor-kappaB (XIAP) and increased E2F1 association (Mcl-1) with their promoter regions, respectively. Ectopic expression of Mcl-1 but not XIAP partially protected cells from flavopiridol/vorinostat-mediated mitochondrial injury at 48 h, but both did not significantly restored clonogenic potential. Flavopiridol/vorinostat-mediated transcriptional repression of XIAP, Mcl-1-enhanced apoptosis, and loss of clonogenic potential also occurred in primary acute myelogenous leukemia (AML) blasts. Together, these findings indicate that transcriptional repression of XIAP and Mcl-1 by flavopiridol/vorinostat contributes functionally to apoptosis induction at early exposure intervals and raise the possibility that expression levels may be a useful surrogate marker for activity in current trials.