The three Rs along the TRAIL: Resistance,re-sensitization and reactive oxygen species (ROS)

Abstract

Ligation of the Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors has been associated with cancer specific apoptotic execution in a number of model systems. This has generated tremendous interest in the use of TRAIL as a potential therapeutic modality. However, recent evidence indicates that resistance to TRAIL might present with a therapeutic challenge. In this short report, we review the basic biology of TRAIL signalling in cancer cells, highlight the mechanisms underlying resistance to TRAIL and the ability of small molecule compounds to re-sensitize cells to TRAIL-mediated apoptosis. In particular, we provide evidence that intracellular reactive oxygen species could be critical in regulating the response of cancer cells to TRAIL.     

ALS2CR7 (CDK15) attenuates TRAIL induced apoptosis by inducing phosphorylation of survivin Thr34

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered a promising agent for medical applications because it induces apoptosis selectively in a variety of cancer cells without toxicity to normal human cells. However, its therapeutic potential has been limited by the existence of several cancer cells with TRAIL resistance. TRAIL resistance results from a variety of mechanisms, which occur at various points in the cellular signaling pathways. In this study, we demonstrate that ALS2CR7 (CDK15) can mediate resistance to TRAIL. We also demonstrate that cell viability of TRAIL sensitive HCT116 and MDA-MB-231 cells increased after TRAIL treatment in ALS2CR7 transfected cancer cells compared with vector transfected cancer cells. Furthermore, cell viability was decreased by TRAIL treatment after knockdown with ALS2CR7 siRNA in TRAIL resistant HT29 and MCF-7 cells. We also show that the activated form of apoptotic proteins such as caspase-3, -8 and -9 and PARP increased after TRAIL treatment in the control group, but decreased in the ALS2CR7 transfected group. The expression of survival proteins such as bcl2 and survivin in TRAIL sensitive cancer cells increased in the ALS2CR7 transfected group, but decreased in TRAIL resistant cancer cells treated with ALS2CR7 siRNA. Other survival proteins such as FLIP and XIAP were not affected. ALS2CR7 appears to bind with only survivin, and not bcl2. The phospho-survivin (Thr34) critical in drug resistance was increased by transfection with ALS2CR7, but the expression of death receptors such as DR4 and DR5 was not affected. ALS2CR7 did not bind with any of the death receptors in our study. In summary, our results suggest that ALS2CR7 confers TRAIL resistance to cancer cells via phosphorylation of survivin.     

Knockdown of MTDH sensitizes endometrial cancer cells to cell death induction by death receptor ligand TRAIL and HDAC inhibitor LBH589 co-treatment

Understanding the molecular underpinnings of chemoresistance is vital to design therapies to restore chemosensitivity. In particular, metadherin (MTDH) has been demonstrated to have a critical role in chemoresistance. Over-expression of MTDH correlates with poor clinical outcome in breast cancer, neuroblastoma, hepatocellular carcinoma and prostate cancer. MTDH is also highly expressed in advanced endometrial cancers, a disease for which new therapies are urgently needed. In this present study, we focused on the therapeutic benefit of MTDH depletion in endometrial cancer cells to restore sensitivity to cell death. Cells were treated with a combination of tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL), which promotes death of malignant cells of the human reproductive tract, and histone deacetylase (HDAC) inhibitors, which have been shown to increase the sensitivity of cancer cells to TRAIL-induced apoptosis. Our data indicate that depletion of MTDH in endometrial cancer cells resulted in sensitization of cells that were previously resistant in response to combinatorial treatment with TRAIL and the HDAC inhibitor LBH589. MTDH knockdown reduced the proportion of cells in S and increased cell arrest in G2/M in cells treated with LBH589 alone or LBH589 in combination with TRAIL, suggesting that MTDH functions at the cell cycle checkpoint to accomplish resistance. Using microarray technology, we identified 57 downstream target genes of MTDH, including calbindin 1 and galectin-1, which may contribute to MTDH-mediated therapeutic resistance. On the other hand, in MTDH depleted cells, inhibition of PDK1 and AKT phosphorylation along with increased Bim expression and XIAP degradation correlated with enhanced sensitivity to cell death in response to TRAIL and LBH589. These findings indicate that targeting or depleting MTDH is a potentially novel avenue for reversing therapeutic resistance in patients with endometrial cancer.     

Inhibition of RIP and c-FLIP enhances TRAIL-induced apoptosis in pancreatic cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently emerged as a cancer therapeutic agent because it is capable of preferentially inducing apoptosis in human cancer over normal cells. The majority of human pancreatic cancers, unfortunately, are resistant to TRAIL treatment. Here, we show that the inhibition of caspase-8 cleavage is the most upstream event in TRAIL resistance in pancreatic cancers. TRAIL treatment led to the cleavage of caspase-8 and downstream caspase-9, caspase-3, and DNA fragmentation factor 45 (DFF45) in TRAIL-sensitive pancreatic cancer cell lines (BXPC-3, PACA-2). This caspase-8-initiated caspase cascade, however, was inhibited in TRAIL-resistant pancreatic cancer cell lines (PANC-1, ASPC-1, CAPAN-1, CAPAN-2). The long and short forms of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP(L), c-FLIP(S)) were highly expressed in the TRAIL-resistant as compared to the sensitive cells; knockdown of c-FLIP(L) and c-FLIP(S) by a short hairpin RNA (shRNA) rendered the resistant cells sensitive to TRAIL-induced apoptosis through the cleavage of caspase-8 and activation of the mitochondrial pathway. Receptor-interacting protein (RIP) has been reported in TRAIL-induced activation of NF-kappaB and we show here that knockdown of RIP sensitized the resistant cells to TRAIL-induced apoptosis. These results indicate the role of c-FLIP and RIP in caspase-8 inhibition and thus TRAIL resistance. Treatment of the resistant cells with camptothecin, celecoxib and cisplatin resulted in the downregulation of c-FLIP and caused a synergistic apoptotic effect with TRAIL. These studies therefore suggest that combination treatment with chemotherapy can overcome TRAIL resistance and enhance TRAIL therapeutic efficacy in treating pancreatic cancers.     

The Transcription Factor Wilms Tumor 1 Confers Resistance in Myeloid Leukemia Cells against the Proapoptotic Therapeutic Agent TRAIL (Tumor Necrosis Factor α-related Apoptosis-inducing Ligand) by Regulating the Antiapoptotic Protein Bcl-xL

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias.     

TRADD is critical for resistance to TRAIL-induced cell death through NF-κB activation

One major obstacle in the clinical application of TRAIL as a cancer therapeutic agent is the acquisition of TRAIL resistance. We found that deficiency of TRADD sensitizes cells to TRAIL-induced apoptosis. Enhanced cell death in TRADD(-/-) MEFs is associated with defective NF-κB activation, indicating that the pro-survival function of TRADD in TRAIL signaling is mediated at least in part via NF-κB activation. Moreover, siRNA knock-down of TRADD in cancer cells sensitizes them to TRAIL-induced apoptosis. Thus, TRADD has a survival role in TRAIL signaling and may be one potential target for overcoming TRAIL resistance in cancer therapy.     

Proteasome inhibitors sensitize ovarian cancer cells to TRAIL induced apoptosis

In the present study we have explored the sensitivity of ovarian cancer cells to TRAIL and proteasome inhibitors. Particularly, we have explored the capacity of proteasome inhibitors to bypass TRAIL resistance of ovarian cancer cells. For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/DDP and A2780/ADR, providing evidence that: (i) the three cell lines are either scarcely sensitive (A2780 and A2780/ADR) or moderately sensitive (A2780/DDP) to the cytotoxic effects of TRAIL; (ii) the elevated c-FLIP expression observed in ovarian cancer cells is a major determinant of TRAIL resistance of these cells; (iii) proteasome inhibitors (PS-341 or MG132) are able to exert a significant pro-apoptotic effect and to greatly enhance the sensitivity of both chemosensitive and chemoresistant A2780 cells to TRAIL; (iv) proteasome inhibitors damage mitochondria through stabilization of BH3-only proteins, Bax and caspase activation and significantly enhance TRAIL-R2 expression; (v) TRAIL-R2, but not TRAIL-R1, mediates the apoptotic effects of TRAIL on ovarian cancer cells. Importantly, studies on primary ovarian cancer cells have shown that these cells are completely resistant to TRAIL and proteasome inhibitors markedly enhance the sensitivity of these cells to TRAIL. Given the high susceptibility of ovarian cancer cells to proteasome inhibitors, our results further support the experimental use of these compounds in the treatment of ovarian cancer.     

Downregulation of Bid is associated with PKCepsilon-mediated TRAIL resistance

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent as it selectively kills tumor cells but spares normal cells. Resistance to TRAIL by tumor cells limits its therapeutic use. We have previously shown that protein kinase C-epsilon (PKCepsilon) acts as an antiapoptotic protein in MCF-7 breast cancer cells. In the present study, we have investigated the mechanism(s) by which PKCepsilon contributes to TRAIL resistance. Overexpression of PKCepsilon inhibited caspase-8 and -9 activation, release of mitochondrial cytochrome c and cell death induced by TRAIL, but did not interfere with the recruitment of caspase-8 to the death-inducing signaling complex. Knockdown/inhibition of PKCepsilon resulted in enhanced sensitivity to TRAIL. The level of Bcl-2 was increased and Bid was decreased by PKCepsilon at both the protein and mRNA level but PKCepsilon had no effect on Bax. Knockdown of Bcl-2 by siRNA reversed TRAIL resistance in PKCepsilon-overexpressing cells, whereas depletion of Bid contributed to TRAIL resistance in MCF-7 cells. A decrease in Bid content was also associated with inhibition of TRAIL-induced caspase-8 activation. Furthermore, PKCepsilon depletion or overexpression of DN-PKCepsilon was associated with a decrease in Bcl-2 protein level. Thus, our results suggest that PKCepsilon acts upstream of mitochondria and mediates TRAIL resistance via both Bcl-2 and Bid in MCF-7 cells.     

Triptolide sensitizes TRAIL-induced apoptosis in prostate cancer cells via p53-mediated DR5 up-regulation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy. However, a number of prostate cancer cells exhibit high resistance to TRAIL effect. In this study, we found that Triptolide, a Chinese medicine, significantly sensitizes prostate cancer cells to TRAIL-mediated cellular apoptosis by up-regulating DR5 expression. Triptolide treatment can suppress Akt/Hdm2 signaling pathway, and lead to p53 accumulation, thereby up-regulating DR5 expression. Taken together, all evidences indicate that Triptolide may become a promising therapeutic agent that prevents the progression of prostate cancer.     

Getting TRAIL back on track for cancer therapy

Unlike other members of the TNF superfamily, the TNF-related apoptosis-inducing ligand (TRAIL, also known as Apo2L) possesses the unique capacity to induce apoptosis selectively in cancer cells in vitro and in vivo. This exciting discovery provided the basis for the development of TRAIL-receptor agonists (TRAs), which have demonstrated robust anticancer activity in a number of preclinical studies. Subsequently initiated clinical trials testing TRAs demonstrated, on the one hand, broad tolerability but revealed, on the other, that therapeutic benefit was rather limited. Several factors that are likely to account for TRAs'' sobering clinical performance have since been identified. First, because of initial concerns over potential hepatotoxicity, TRAs with relatively weak agonistic activity were selected to enter clinical trials. Second, although TRAIL can induce apoptosis in several cancer cell lines, it has now emerged that many others, and importantly, most primary cancer cells are resistant to TRAIL monotherapy. Third, so far patients enrolled in TRA-employing clinical trials were not selected for likelihood of benefitting from a TRA-comprising therapy on the basis of a valid(ated) biomarker. This review summarizes and discusses the results achieved so far in TRA-employing clinical trials in the light of these three shortcomings. By integrating recent insight on apoptotic and non-apoptotic TRAIL signaling in cancer cells, we propose approaches to introduce novel, revised TRAIL-based therapeutic concepts into the cancer clinic. These include (i) the use of recently developed highly active TRAs, (ii) the addition of efficient, but cancer-cell-selective TRAIL-sensitizing agents to overcome TRAIL resistance and (iii) employing proteomic profiling to uncover resistance mechanisms. We envisage that this shall enable the design of effective TRA-comprising therapeutic concepts for individual cancer patients in the future.     

PI3 Kinase inhibition on TRAIL-induced apoptosis correlates with androgen-sensitivity and p21 expression in prostate cancer cells

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many types of cancer cells. TRAIL is considered a therapeutic target, therefore, it was of interest to examine molecular mechanisms that may modulate sensitivity to TRAIL signaling in prostate cancer cells. LNCaP cells were found to be relatively resistant to TRAIL induced cell death while PC3 cells were sensitive. PI3-kinase (PI3 K) inhibitors were able to render LNCaP cells sensitive to TRAIL but conferred resistance to PC3 cells. PI3 K inhibitors were associated with an increase in p21^{waf1, cip1} expression in PC3 cells where as p21 decreases in LNCaP cells suggesting that p21 may impart TRAIL resistance. Since androgen receptor (AR) signaling can be modulated by AKT, and p21 is an AR responsive gene, the impact of PI3 K inhibition on TRAIL sensitivity was evaluated in AR transfected PC3 cells (PC3AR). The expression of AR was significantly downregulated by PI3 K inhibition in LNCaP cells, which have an intact AR signaling axis. PC3AR cells expressed higher levels of p21 protein and were relatively resistant to TRAIL compared to control cells. Finally, using adenoviral p21 gene transfer we directly demonstrated that p21 can confer resistance to TRAIL-induced cell death. These results suggest that TRAIL resistance is not regulated simply by a PI3 K/AKT survival pathway associated with inactivating PTEN mutations but may also be modulated by downstream AR responsive targets such as p21. These findings may have significant clinical implications for the utility of TRAIL in the management of prostate cancer.     

Thiourea compound AW00178 sensitizes human H1299 lung carcinoma cells to TRAIL-mediated apoptosis

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a wide variety of cancer cells. Recently, cancer cell resistance to TRAIL-mediated apoptosis has become a challenging issue in the development of TRAIL-based anti-cancer therapies. In this study, we found that 1-(5-chloro-2-methyl-phenyl)-3-[4-(5-trifluoromethyl-pyrazol-1-yl)-phenyl]-thiourea (AW00178) was able to sensitize TRAIL-resistant human lung cancer H1299 cells to TRAIL-mediated apoptosis. Treatment with AW00178, either alone or in combination with TRAIL, induced the expression of CHOP, a protein related to TRAIL sensitivity, and reduced the expression of survivin, an anti-apoptotic protein involved in TRAIL resistance. Additionally, AW00178, alone or in combination with TRAIL, induced the activation of c-Jun and inactivation of Akt. A pharmacologic inhibition study revealed that c-Jun activation and Akt inactivation were strongly related to CHOP induction and survivin down-regulation, respectively. In summary, these results suggested that AW00178 mediated sensitization to TRAIL-mediated apoptosis in H1299 cells by increasing sensitivity and decreasing resistance to TRAIL via the induction of c-Jun-dependent CHOP expression and the reduction of Akt-dependent survivin expression, respectively.     

Neobavaisoflavone sensitizes apoptosis via the inhibition of metastasis in TRAIL-resistant human glioma U373MG cells

Aims

Neobavaisoflavone (NBIF), an isoflavone isolated from Psoralea corylifolia (Leguminosae), has striking anti-inflammatory and anti-cancer effects. NBIF inhibits the proliferation of prostate cancer in vitro and in vivo.

Main methods

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a key endogenous molecule that selectively induces apoptosis in cancer cells with little or no toxicity in normal cells. However, some cancer cells, including U373MG cells, are resistant to TRAIL-mediated apoptosis. We demonstrated that the cell viability, migration and invasion assay were used in U373MG glioma cells.

Key findings

In this study, we found that NBIF sensitizes human U373MG glioma cells to TRAIL-mediated apoptosis. Co-treatment of TRAIL and NBIF effectively induced Bid cleavage and activated caspases 3, 8, and 9. Importantly, DR5 expression was upregulated by NBIF. We also observed that the combination NBIF and TRAIL increased expression of BAX. We further demonstrate that NBIF induced TRAIL-mediated apoptosis in human glioma cells by suppressing migration and invasion, and by inhibiting anoikis resistance.

Significance

Taken together, our results suggest that NBIF reduces the resistance of cancer cells to TRAIL and that the combination of NBIF and TRAIL may be a new therapeutic strategy for treating TRAIL-resistant glioma cells.     

The flavonolignan silibinin potentiates TRAIL-induced apoptosis in human colon adenocarcinoma and in derived TRAIL-resistant metastatic cells

Silibinin, a flavonolignan, is the major active component of the milk thistle plant (Silybum marianum) and has been shown to possess anti-neoplastic properties. TNF-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent which selectively induces apoptosis in cancer cells. However, resistance to TRAIL-induced apoptosis is an important and frequent problem in cancer treatment. In this study, we investigated the effect of silibinin and TRAIL in an in vitro model of human colon cancer progression, consisting of primary colon tumor cells (SW480) and their derived TRAIL-resistant metastatic cells (SW620). We showed by flow cytometry that silibinin and TRAIL synergistically induced cell death in the two cell lines. Up-regulation of death receptor 4 (DR4) and DR5 by silibinin was shown by RT-PCR and by flow cytometry. Human recombinant DR5/Fc chimera protein that has a dominant-negative effect by competing with the endogenous receptors abrogated cell death induced by silibinin and TRAIL, demonstrating the activation of the death receptor pathway. Synergistic activation of caspase-3, -8, and -9 by silibinin and TRAIL was shown by colorimetric assays. When caspase inhibitors were used, cell death was blocked. Furthermore, silibinin and TRAIL potentiated activation of the mitochondrial apoptotic pathway and down-regulated the anti-apoptotic proteins Mcl-1 and XIAP. The involvement of XIAP in sensitization of the two cell lines to TRAIL was demonstrated using the XIAP inhibitor embelin. These findings demonstrate the synergistic action of silibinin and TRAIL, suggesting chemopreventive and therapeutic potential which should be further explored.     

Salinomycin Potentiates the Cytotoxic Effects of TRAIL on Glioblastoma Cell Lines

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to exhibit therapeutic activity in cancer. However, many tumors remain resistant to treatment with TRAIL. Therefore, small molecules that potentiate the cytotoxic effects of TRAIL could be used for combinatorial therapy. Here we found that the ionophore antibiotic salinomycin acts in synergism with TRAIL, enhancing TRAIL-induced apoptosis in glioma cells. Treatment with low doses of salinomycin in combination with TRAIL augmented the activation of caspase-3 and increased TRAIL-R2 cell surface expression. TRAIL-R2 upmodulation was required for mediating the stimulatory effect of salinomycin on TRAIL-mediated apoptosis, since it was abrogated by siRNA-mediated TRAIL-R2 knockdown. Salinomycin in synergism with TRAIL exerts a marked anti-tumor effect in nude mice xenografted with human glioblastoma cells. Our results suggest that the combination of TRAIL and salinomycin may be a useful tool to overcome TRAIL resistance in glioma cells and may represent a potential drug for treatment of these tumors. Importantly, salinomycin+TRAIL were able to induce cell death of well-defined glioblastoma stem-like lines.     

Synergistic anti-cancer effects via co-delivery of TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) and doxorubicin using micellar nanoparticles

The use of small molecule drugs in cancer chemotherapy has mostly been limited by dose-dependent toxicity and development of drug resistance resulting from repeated administrations. To overcome such problems, efforts have been made to develop drug delivery systems that can bear multiple therapeutic agents in one system. The purpose of this study is to deliver human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) and doxorubicin (Dox, an anti-cancer drug) with micellar nanoparticles self-assembled from a biodegradable cationic copolymer P(MDS-co-CES) to achieve synergistic cytotoxic effects in cancer cells. Exogenously expressed TRAIL using recombinant methods shows great potential in cancer therapy as it induces cell death selectively in cancer cells with limited toxicity to normal tissues. Dox-loaded nanoparticles and TRAIL formed stable nanocomplexes with a size of ～ 225 nm and zeta potential of ～ 70 mV. Effects of nanocomplexes on both wild type and TRAIL-resistant SW480 colorectal carcinoma cells were investigated. The assemblies of Dox and TRAIL with P(MDS-co-CES) nanoparticles were efficiently delivered to cancer cells. Receptor-blocking studies showed that the nanocomplexes entered cells via death receptor-mediated endocytosis. Synergism in cell death induction was analysed by the isobologram method to study drug interactions. Cytotoxicity of the nanocomplexes to non-cancerous cells was significantly lower than cancerous cells. Anti-proliferative effects of nanocomplexes were retained in remaining cancer cells in long-term cultures after treatment with the nanocomplexes. In summary, this Dox and TRAIL co-delivery system can be a promising candidate for cancer treatment.     

Effect of UV irradiation on colorectal cancer cells with acquired TRAIL resistance

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily. TRAIL shows strong cytotoxicity to many cancer cells but minimal cytotoxicity to most normal cells. Interestingly, our recent studies have demonstrated that pretreatment with TRAIL induces acquired resistance to TRAIL (Song et al. 2007 J Biol Chem 282: 319). Acquired TRAIL resistance develops within 1 day and gradually decays within 5 days after TRAIL treatment. In our current study, we examined whether human colorectal carcinoma CX-1 cells with acquired TRAIL resistance are resistant to UV irradiation as well. CX-1 cells were treated with 200 ng/ml TRAIL for 6 h and incubated various times (0.25-5 days) and then challenged to UV irradiation. Unexpectedly, we observed an increase in apoptosis in acquired TRAIL resistant cells after UVC as well as UVB exposure. This was due to an increase in caspase activation which was mediated through cytochrome c release. These results suggest that cells with acquired TRAIL resistance are sensitive to UV irradiation.     

Selective CDK9 inhibition overcomes TRAIL resistance by concomitant suppression of cFlip and Mcl-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many cancer cells without causing toxicity in vivo. However, to date, TRAIL-receptor agonists have only shown limited therapeutic benefit in clinical trials. This can, most likely, be attributed to the fact that 50% of all cancer cell lines and most primary human cancers are TRAIL resistant. Consequently, future TRAIL-based therapies will require the addition of sensitizing agents that remove crucial blocks in the TRAIL apoptosis pathway. Here, we identify PIK-75, a small molecule inhibitor of the p110α isoform of phosphoinositide-3 kinase (PI3K), as an exceptionally potent TRAIL apoptosis sensitizer. Surprisingly, PI3K inhibition was not responsible for this activity. A kinome-wide in vitro screen revealed that PIK-75 strongly inhibits a panel of 27 kinases in addition to p110α. Within this panel, we identified cyclin-dependent kinase 9 (CDK9) as responsible for TRAIL resistance of cancer cells. Combination of CDK9 inhibition with TRAIL effectively induced apoptosis even in highly TRAIL-resistant cancer cells. Mechanistically, CDK9 inhibition resulted in downregulation of cellular FLICE-like inhibitory protein (cFlip) and Mcl-1 at both the mRNA and protein levels. Concomitant cFlip and Mcl-1 downregulation was required and sufficient for TRAIL sensitization by CDK9 inhibition. When evaluating cancer selectivity of TRAIL combined with SNS-032, the most selective and clinically used inhibitor of CDK9, we found that a panel of mostly TRAIL-resistant non-small cell lung cancer cell lines was readily killed, even at low concentrations of TRAIL. Primary human hepatocytes did not succumb to the same treatment regime, defining a therapeutic window. Importantly, TRAIL in combination with SNS-032 eradicated established, orthotopic lung cancer xenografts in vivo. Based on the high potency of CDK9 inhibition as a cancer cell-selective TRAIL-sensitizing strategy, we envisage the development of new, highly effective cancer therapies.