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Cancer resistance to PI3K inhibitor therapy can be in part mediated by increases in the PIM1 kinase. However, the exact mechanism by which PIM kinase promotes tumor cell resistance is unknown. Our study unveils the pivotal control of redox signaling by PIM kinases as a driver of this resistance mechanism. PIM1 kinase functions to decrease cellular ROS levels by enhancing nuclear factor erythroid 2-related factor 2 (NRF2)/antioxidant response element activity. PIM prevents cell death induced by PI3K-AKT–inhibitory drugs through a noncanonical mechanism of NRF2 ubiquitination and degradation and translational control of NRF2 protein levels through modulation of eIF4B and mTORC1 activity. Importantly, PIM also controls NAD(P)H production by increasing glucose flux through the pentose phosphate shunt decreasing ROS production, and thereby diminishing the cytotoxicity of PI3K-AKT inhibitors. Treatment with PIM kinase inhibitors reverses this resistance phenotype, making tumors increasingly susceptible to small-molecule therapeutics, which block the PI3K-AKT pathway.

Supplementary Methods Supplementary References Figure S1. PIM and PI3K/AKT inhibitors synergize to suppress PCa cell growth and survival. Figure S2. PIM1 induces expression of ROS scavengers by NRF2 transcription activity. Figure S3. PIM1 promotes glucose carbon flux in various metabolic pathways. Figure S4. PIM and PI3K/AKT inhibitors downregulate NRF2 antioxidant response leading to oxidative DNA damages.

Supplementary Figures S1-S5.

Supplemental Fig. 1. Hypoxia sensitizes breast and colon cancer cells to PIM inhibitors. Supplemental Fig. 2. PIM inhibitors kill hypoxic cancer cells in a HIF-1 independent manner. Supplemental Fig. 3. Chemical structure of pan-PIM kinase inhibitors.

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The document contains supplementary methods and figure legends for supplementary figure 1, 2 and 3.

Supplementary Figure 1: Differential cytolytic potential and activation of Th17IL-1β cells in culture. Supplementary figure 2: Ectonucleotidase expression and persistence of different Th17 cells. Supplementary figure 3: Schematic summary highlighting the effect of different Th17 programming conditions on anti-tumor function.

Pim serine/threonine kinases contribute to prostate tumorigenesis and therapeutic resistance, yet Pim kinase inhibitors seem to have only limited effects on prostate cancer cell survival. Because overexpression of Bcl-2 family members are implicated in chemotherapeutic resistance in prostate cancer, we investigated the cooperative effects of Pim kinase inhibition with ABT-737, a small molecule antagonist of Bcl-2 family members. Strikingly, the addition of ABT-737 to Pim inhibitors triggered a robust apoptosis of prostate cancer cells in vitro and in vivo. Pim inhibitors decreased levels of the Bcl-2 family member Mcl-1, both by blocking 5′-cap dependent translation and decreasing protein half life. In addition, Pim inhibition transcriptionally increased levels of the BH3 protein Noxa by activating the unfolded protein response (UPR), lead to eIF-2α phosphorylation and increased expression of CHOP. Increased levels of Noxa also inactivated the remaining levels of Mcl-1 protein activity. Notably, these specific protein changes were essential to the apoptotic process because ABT-737 did not inhibit Mcl-1 protein activity and Mcl-1 overexpression blocked the apoptotic activity of ABT-737. Our results therefore suggest that this combination treatment could be developed as a potential therapy for human prostate cancer where overexpression of Pim kinases and antiapoptotic Bcl-2 family members drives tumor cell resistance to current anticancer therapies. Cancer Res; 72(1); 294–303. ©2011 AACR.

Supplementary Methods, Figures 1-8, Table 1 from The BH3 Mimetic ABT-737 Induces Cancer Cell Senescence

ABT-737, a small molecule cell-permeable Bcl-2 antagonist that acts by mimicking BH3 proteins, induces apoptotic cell death in multiple cancer types. However, when incubated with this agent many solid tumor cell lines do not undergo apoptosis. The current study reveals a novel mechanism whereby ABT-737 when added to apoptosis-resistant cancer cells has profound biologic effects. In PV-10 cells, a renal cell carcinoma that does not die after ABT-737 treatment, this agent induces a two-fold change in the transcription of nearly 430 genes. Many of these induced mRNA changes are in secreted proteins, IL-6, IL-8, and IL-11 and chemokines CXCL2 and CXCL5, or genes associated with an “inflammatory” phenotype. Strikingly, these gene changes are highly similar to those changes previously identified in cellular senescence. Brief exposure of apoptosis-resistant renal, lung and prostate cancer cell lines to ABT-737, although not capable of inducing cell death, causes the induction of senescence-associated β-galactosidase and inhibition of cell growth consistent with the induction of cellular senescence. Evidence indicates that the induction of senescence occurs as a result of reactive oxygen species elevation followed by low-level activation of the caspase cascade, insufficient to induce apoptosis, but sufficient to lead to minor DNA damage and increases in p53, p21, IL-6 and 8 proteins. By overexpression of a dominant-negative p53 protein, we show that ABT-737-induced cellular senescence is p53-dependent. Thus, in multiple cancer types in which ABT-737 is incapable of causing cell death, ABT-737 may have additional cellular activities that make its use as an anticancer agent highly attractive. Cancer Res; 71(2); 506–15. ©2010 AACR.

The paucity of therapeutic strategies to reduce the severity of radiation-induced lung fibrosis (RILF), a life-threatening complication of intended or accidental ionizing radiation exposure, is a serious unmet need. We evaluated the contribution of eNAMPT (extracellular nicotinamide phosphoribosyltransferase), a damage-associated molecular pattern (DAMP) protein and TLR4 (Toll-like receptor 4) ligand, to the severity of whole-thorax lung irradiation (WTLI)-induced RILF. Wild-type (WT) and

Background/Aims: Increase in vascular permeability is a cardinal feature of all inflammatory diseases and represents an imbalance in vascular contractile forces and barrier-restorative forces, both of which are highly dependent on actin cytoskeletal dynamics. In addition to the involvement of key vascular barrier-regulatory, actin-binding proteins, such as nmMLCK and cortactin, we recently demonstrated a role for a member of the Ena-VASP family known as Ena-VASP-like (EVL) in promoting vascular focal adhesion (FA) remodeling and endothelial cell (EC) barrier restoration/preservation. Methods: To further understand the role of EVL in EC barrier-regulatory processes, we examined EVL-cytoskeletal protein interactions in FA dynamics in vitro utilizing lung EC and in vivo murine models of acute inflammatory lung injury. Deletion mapping studies and immunoprecipitation assays were performed to detail the interaction between EVL and cortactin, and further evaluated by assessment of changes in vascular EC permeability following disruption of EVL-cortactin interaction. Results: Initial studies focusing on the actin-binding proteins, nmMLCK and cortactin, utilized deletion mapping of the cortactin gene (CTTN ) to identify cortactin domains critical for EVL-cortactin interaction and verified the role of actin in promoting EVL-cortactin interaction. A role for profilins, actin-binding proteins that regulate actin polymerization, was established in facilitating EVL-FA binding. Conclusion: In summary, these studies further substantiate EVL participation in regulation of vascular barrier integrity and in the highly choreographed cytoskeletal interactions between key FA and cytoskeletal partners.

The loss of vascular integrity is a cardinal feature of acute inflammatory responses evoked by activation of the TLR4 inflammatory cascade. Utilizing in vitro and in vivo models of inflammatory lung injury, we explored TLR4-mediated dysregulated signaling that results in the loss of endothelial cell (EC) barrier integrity and vascular permeability, focusing on Dock1 and Elmo1 complexes that are intimately involved in regulation of Rac1 GTPase activity, a well recognized modulator of vascular integrity. Marked reductions in Dock1 and Elmo1 expression was observed in lung tissues (porcine, rat, mouse) exposed to TLR4 ligand-mediated acute inflammatory lung injury (LPS, eNAMPT) in combination with injurious mechanical ventilation. Lung tissue levels of Dock1 and Elmo1 were preserved in animals receiving an eNAMPT-neutralizing mAb in conjunction with highly significant decreases in alveolar edema and lung injury severity, consistent with Dock1/Elmo1 as pathologic TLR4 targets directly involved in inflammation-mediated loss of vascular barrier integrity. In vitro studies determined that pharmacologic inhibition of Dock1-mediated activation of Rac1 (TBOPP) significantly exacerbated TLR4 agonist-induced EC barrier dysfunction (LPS, eNAMPT) and attenuated increases in EC barrier integrity elicited by barrier-enhancing ligands of the S1P1 receptor (sphingosine-1-phosphate, Tysiponate). The EC barrier-disrupting influence of Dock1 inhibition on S1PR1 barrier regulation occurred in concert with: 1) suppressed formation of EC barrier-enhancing lamellipodia, 2) altered nmMLCK-mediated MLC2 phosphorylation, and 3) upregulation of NOX4 expression and increased ROS. These studies indicate that Dock1 is essential for maintaining EC junctional integrity and is a critical target in TLR4-mediated inflammatory lung injury.

Pharmacologic interventions to halt/reverse the vascular remodeling and right ventricular dysfunction in pulmonary arterial hypertension (PAH) remains an unmet need. We previously demonstrated extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a DAMP (damage-associated molecular pattern protein) contributing to PAH pathobiology via TLR4 ligation. We examined the role of endothelial cell (EC)-specific eNAMPT in experimental PH and an eNAMPT-neutralizing mAb as a therapeutic strategy to reverse established PH. Hemodynamic/echocardiographic measurements and tissue analyses were performed in Sprague Dawley rats exposed to 10% hypoxia/Sugen (three weeks) followed by return to normoxia and weekly intraperitoneal delivery of the eNAMPT mAb (1 mg/kg). WT C57BL/6J mice and conditional EC-cNAMPT ec−/− mice were exposed to 10% hypoxia (three weeks). Biochemical and RNA sequencing studies were performed on rat PH lung tissues and human PAH PBMCs. Hypoxia/Sugen-exposed rats exhibited multiple indices of severe PH (right ventricular systolic pressure, Fulton index), including severe vascular remodeling, compared to control rats. PH severity indices and plasma levels of eNAMPT, IL-6, and TNF- α were all significantly attenuated by eNAMPT mAb neutralization. Compared to hypoxia-exposed WT mice, cNAMPT ec−/− KO mice exhibited significantly reduced PH severity and evidence of EC to mesenchymal transition (EndMT). Finally, biochemical and RNAseq analyses revealed eNAMPT mAb-mediated rectification of dysregulated inflammatory signaling pathways (TLR/NF-κB, MAP kinase, Akt/mTOR) and EndMT in rat PH lung tissues and human PAH PBMCs. These studies underscore EC-derived eNAMPT as a key contributor to PAH pathobiology and support the eNAMPT/TLR4 inflammatory pathway as a highly druggable therapeutic target to reduce PH severity and reverse PAH.

Therapeutic strategies to prevent or reduce the severity of radiation pneumonitis are a serious unmet need. We evaluated extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a damage-associated molecular pattern protein (DAMP) and Toll-Like Receptor 4 (TLR4) ligand, as a therapeutic target in murine radiation pneumonitis. Radiation-induced murine and human NAMPT expression was assessed in vitro, in tissues (IHC, biochemistry, imaging), and in plasma. Wild type C57Bl6 mice (WT) and Nampt(+/−) heterozygous mice were exposed to 20Gy whole thoracic lung irradiation (WTLI) with or without weekly IP injection of IgG(1) (control) or an eN-AMPT-neutralizing polyclonal (pAb) or monoclonal antibody (mAb). BAL protein/cells and H&E staining were used to generate a WTLI severity score. Differentially-expressed genes (DEGs)/pathways were identified by RNA sequencing and bioinformatic analyses. Radiation exposure increases in vitro NAMPT expression in lung epithelium (NAMPT promoter activity) and NAMPT lung tissue expression in WTLI-exposed mice. Nampt(+/−) mice and eNAMPT pAb/mAb-treated mice exhibited significant histologic attenuation of WTLI-mediated lung injury with reduced levels of BAL protein and cells, and plasma levels of eNAMPT, IL-6, and IL-1 β. Genomic and biochemical studies from WTLI-exposed lung tissues highlighted dysregulation of NFkB/cytokine and MAP kinase signaling pathways which were rectified by eNAMPT mAb treatment. The eNAMPT/TLR4 pathway is essentially involved in radiation pathobiology with eNAMPT neutralization an effective therapeutic strategy to reduce the severity of radiation pneumonitis.

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P‐bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P‐body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P‐body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P‐bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin β1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer‐relevant functions and suggest that modulation of P‐body activity may represent a new paradigm for cancer treatment.

RationaleThe severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target.MethodsWild-type C57BL/6J or endothelial cell (EC)-cNAMPT−/−knockout mice (targeted ECNAMPTdeletion) were exposed to either a lipopolysaccharide (LPS)-induced (“one-hit”) or a combined LPS/ventilator (“two-hit”)-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were usedin vitroandin vivo.ResultsImmunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models.In vitrohuman lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption.In vivostudies in wild-type and EC-cNAMPT−/−mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models.ConclusionsThese findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker andNAMPTgenotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.

The Pim and AKT serine/threonine protein kinases are implicated as drivers of cancer. Their regulation of tumor growth is closely tied to the ability of these enzymes to mainly stimulate protein synthesis by activating mTORC1 (mammalian target of rapamycin complex 1) signaling, although the exact mechanism is not completely understood. mTORC1 activity is normally suppressed by amino acid starvation through a cascade of multiple regulatory protein complexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activity of Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEP domain containing protein 5), a component of GATOR1 complex, contains Pim and AKT protein kinase phosphorylation consensus sequences. DEPDC5 phosphorylation by Pim and AKT kinases was confirmed in cancer cells through the use of phospho-specific antibodies and transfection of phospho-inactive DEPDC5 mutants. Consistent with these findings, during amino acid starvation the elevated expression of Pim1 overcame the amino acid inhibitory protein cascade and activated mTORC1. In contrast, the knockout of DEPDC5 partially blocked the ability of small molecule inhibitors against Pim and AKT kinases both singly and in combination to suppress tumor growth and mTORC1 activity in vitro and in vivo. In animal experiments knocking in a glutamic acid (S1530E) in DEPDC5, a phospho mimic, in tumor cells induced a significant level of resistance to Pim and the combination of Pim and AKT inhibitors. Our results indicate a phosphorylation-dependent regulatory mechanism targeting DEPDC5 through which Pim1 and AKT act as upstream effectors of mTORC1 to facilitate proliferation and survival of cancer cells.

The proviral integration site for Moloney murine leukemia virus (PIM) serine/threonine kinases have an oncogenic and prosurvival role in hematological and solid cancers. However, the mechanism by which these kinases drive tumor growth has not been completely elucidated. To determine the genes controlled by these protein kinases, we carried out a microarray analysis in T‐cell acute lymphoblastic leukemia (T‐ALL) comparing early progenitor (ETP‐ALL) cell lines whose growth is driven by PIM kinases to more mature T‐ALL cells that have low PIM levels. This analysis demonstrated that the long noncoding RNA (lncRNA) H19 was associated with increased PIM levels in ETP‐ALL. Overexpression or knockdown of PIM in these T‐ALL cell lines controlled the level of H19 and regulated the methylation of the H19 promoter, suggesting a mechanism by which PIM controls H19 transcription. In these T‐ALL cells, the expression of PIM1 induced stem cell gene expression (SOX2, OCT‐4, and NANOG) through H19. Identical results were found in prostate cancer (PCa) cell lines where PIM kinases drive cancer growth, and both H19 and stem cell gene levels. Small molecule pan‐PIM inhibitors (PIM‐i) currently in clinical trials reduced H19 expression in both of these tumor types. Importantly, the knockdown of H19 blocked the ability of PIM to induce stem cell genes in T‐ALL cells, suggesting a novel signal transduction cascade. In PCa, increases in SOX2 levels have been shown to cause both resistance to the androgen deprivation therapy (ADT) and the induction of neuroendocrine PCa, a highly metastatic form of this disease. Treatment of PCa cells with a small molecule pan‐PIM‐i reduced stem cell gene transcription and enhanced ADT, while overexpression of H19 suppressed the ability of pan‐PIM‐i to regulate hormone blockade. Together, these results demonstrate that the PIM kinases control the level of lncRNA H19, which in turn modifies stem cell gene transcription regulating tumor growth., The present study demonstrates that in T‐ALL and prostate cancer, proviral integration site for Moloney murine leukemia virus (PIM) kinase induction increases long noncoding RNA‐H19 that in turn regulates stem cell genes. In resistant T‐ALL, PIM1/H19 signaling restores partial sensitivity to pan‐PIM inhibitors (PIM‐i). In prostate cancer, elevated PIM1 contributes to androgen deprivation therapy resistance reversed by a combination with PIM‐i. These data suggest a novel and clinically relevant pathway controlled by PIM kinases.

Intratumoral hypoxia is a significant obstacle to the successful treatment of solid tumors, and it is highly correlated with metastasis, therapeutic resistance, and disease recurrence in cancer patients. As a result, there is an urgent need to develop effective therapies that target hypoxic cells within the tumor microenvironment. The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases represent a prosurvival pathway that is upregulated in response to hypoxia, in a HIF-1–independent manner. We demonstrate that pharmacologic or genetic inhibition of PIM kinases is significantly more toxic toward cancer cells in hypoxia as compared with normoxia. Xenograft studies confirm that PIM kinase inhibitors impede tumor growth and selectively kill hypoxic tumor cells in vivo. Experiments show that PIM kinases enhance the ability of tumor cells to adapt to hypoxia-induced oxidative stress by increasing the nuclear localization and activity of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), which functions to increase the expression of antioxidant genes. Small molecule PIM kinase inhibitors prevent Nrf2 from accumulating in the nucleus, reducing the transcription of cytoprotective genes and leading to the build-up of intracellular reactive oxygen species (ROS) to toxic levels in hypoxic tumor cells. This toxic effect of PIM inhibitors can be successfully blocked by ROS scavengers, including N-acetyl cystine and superoxide dismutase. Thus, inhibition of PIM kinases has the potential to oppose hypoxia-mediated therapeutic resistance and induce cell death in the hypoxic tumor microenvironment. Mol Cancer Ther; 15(7); 1637–47. ©2016 AACR.

The Pim family of serine/threonine protein kinases (Pim 1, 2, and 3) contribute to cellular transformation by regulating glucose metabolism, protein synthesis, and mitochondrial oxidative phosphorylation. Drugs targeting the Pim protein kinases are being tested in phase I/II clinical trials for the treatment of hematopoietic malignancies. The goal of these studies was to identify Pim substrate(s) that could help define the pathway regulated by these enzymes and potentially serve as a biomarker of Pim activity. To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 and 2 (IRS1/2) as potential Pim substrates. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation. Our study demonstrates in both malignant and normal cells using either genetic or pharmacological inhibition of the Pim kinases or overexpression of this family of enzymes that human IRS1S1101 and IRS2S1149 are Pim substrates. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. These results demonstrate that IRS1S1101 is a novel substrate for the Pim kinases and provide a novel marker for evaluation of Pim inhibitor therapy.

Cancer resistance to PI3K inhibitor therapy can be in part mediated by increases in the PIM1 kinase. However, the exact mechanism by which PIM kinase promotes tumor cell resistance is unknown. Our study unveils the pivotal control of redox signaling by PIM kinases as a driver of this resistance mechanism. PIM1 kinase functions to decrease cellular ROS levels by enhancing nuclear factor erythroid 2-related factor 2 (NRF2)/antioxidant response element activity. PIM prevents cell death induced by PI3K-AKT–inhibitory drugs through a noncanonical mechanism of NRF2 ubiquitination and degradation and translational control of NRF2 protein levels through modulation of eIF4B and mTORC1 activity. Importantly, PIM also controls NAD(P)H production by increasing glucose flux through the pentose phosphate shunt decreasing ROS production, and thereby diminishing the cytotoxicity of PI3K-AKT inhibitors. Treatment with PIM kinase inhibitors reverses this resistance phenotype, making tumors increasingly susceptible to small-molecule therapeutics, which block the PI3K-AKT pathway.

Summary Increased telomerase activity is associated with malignancy and poor prognosis in human cancer, but the development of targeted agents has not yet provided clinical benefit. Here we report that, instead of targeting the telomerase enzyme directly, small molecules that bind to the G-hairpin of the hTERT G-quadruplex-forming sequence kill selectively malignant cells without altering the function of normal cells. RG260 targets the hTERT G-quadruplex stem-loop folding but not tetrad DNAs, leading to downregulation of hTERT expression. To improve physicochemical and pharmacokinetic properties, we derived a small-molecule analog, RG1603, from the parent compound. RG1603 induces mitochondrial defects including PGC1α and NRF2 inhibition and increases oxidative stress, followed by DNA damage and apoptosis. RG1603 injected as a single agent has tolerable toxicity while achieving strong anticancer efficacy in a tumor xenograft mouse model. These results demonstrate a unique approach to inhibiting the hTERT that functions by impairing mitochondrial activity, inducing cell death.

The PIM1 oncogene is over-expressed in human prostate cancer epithelial cells. Importantly, we observe that in human hyperplastic and cancerous prostate glands PIM1 is also markedly elevated in prostate fibroblasts, suggesting an important role for this kinase in epithelial/stromal crosstalk. The ability of PIM1 to regulate the biologic activity of stromal cells is demonstrated by the observation that expression of PIM1 kinase in human prostate fibroblasts increases the level and secretion of the extracellular matrix molecule, collagen 1A1 (COL1A1), the pro-inflammatory chemokine CCL5, and the platelet-derived growth factor receptors (PDGFR). PIM1 is found to regulate the transcription of CCL5. In co-cultivation assays where PIM1 over-expressing fibroblasts are grown with BPH1 prostate epithelial cells, PIM1 activity markedly enhances the ability of these fibroblasts to differentiate into myofibroblasts and express known markers of cancer-associated fibroblasts (CAFs). This differentiation can be reversed by the addition of small molecule PIM kinase inhibitors. Western blots demonstrate that PIM1 expression in prostate fibroblasts stimulates the phosphorylation of molecules that regulate 5'Cap driven protein translation, including 4EBP1 and eIF4B. Consistent with the hypothesis that the kinase controls translation of specific mRNAs in prostate fibroblasts, we demonstrate that PIM1 expression markedly increases the level of COL1A1 and PDGFRβ mRNA bound to polysomes. Together these results point on PIM1 as a novel factor in regulation of the phenotype and differentiation of fibroblasts in prostate cancer by controlling both the transcription and translation of specific mRNAs.

To determine the toxicity and oncologic outcome of neoadjuvant chemoradiotherapy (CRT) followed by curative total mesorectal excision (TME) in the elderly (≥70 yrs) and younger (70 yrs) rectal cancer patients.Sufficient data for elderly rectal cancer patients who received definitive trimodality have not been accumulated yet.A total of 1232 rectal cancer patients who received neoadjuvant CRT and TME were enrolled in this study. After propensity-score matching, 310 younger patients and 310 elderly patients were matched with 1:1 manner. Treatment response, toxicity, surgical outcome, recurrence, and survival were assessed and compared between the 2 groups of patients.The median age was 58 years for the younger patient group and 74 years for the elderly group. Pathologic complete response rates were not significantly different between the 2 groups (younger and elderly: 17.1% vs 14.8%, P = 0.443). The 5-year recurrence-free survival (younger and elderly: 67.7% vs 65.5%, P = 0.483) and overall survival (younger and elderly: 82.9% vs. 79.5%, P = 0.271) rates were not significantly different between the 2 groups either. Adjuvant chemotherapy after surgery was less frequently delivered to the elderly than that to younger patients (83.9% vs 69.0%). Grade 3 or higher acute hematologic toxicity was observed more frequently in the elderly than that in the younger group (9.0% vs 16.1%, P = 0.008). Late complication rate was higher in the elderly group compared with that in the younger group without statistical significance (2.6% vs 4.5%, P = 0.193).Although acute hematologic toxicity was observed more frequently in the elderly patients than that in the younger patients, elderly rectal cancer patients with good performance status who received preoperative CRT and TME showed favorable tumor response and recurrence-free survival similar to younger patients.

The Pim protein kinases contribute to transformation by enhancing the activity of oncogenic Myc and Ras, which drives significant metabolic changes during tumorigenesis. In this report, we demonstrate that mouse embryo fibroblasts (MEFs) lacking all three isoforms of Pim protein kinases, triple knockout (TKO), cannot tolerate the expression of activated K-Ras (K-Ras(G12V)) and undergo cell death. Transduction of K-Ras(G12V) into these cells markedly increased the level of cellular reactive oxygen species (ROS). The addition of N-acetyl cysteine attenuated ROS production and reversed the cytotoxic effects of K-Ras(G12V) in the TKO MEFs. The altered cellular redox state caused by the loss of Pim occurred as a result of lower levels of metabolic intermediates in the glycolytic and pentose phosphate pathways as well as abnormal mitochondrial oxidative phosphorylation. TKO MEFs exhibit reduced levels of superoxide dismutase (Sod), glutathione peroxidase 4 (Gpx4) and peroxiredoxin 3 (Prdx3) that render them susceptible to killing by K-Ras(G12V)-mediated ROS production. In contrast, the transduction of c-Myc into TKO cells can overcome the lack of Pim protein kinases by regulating cellular metabolism and Sod2. In the absence of the Pim kinases, c-Myc transduction permitted K-Ras(G12V)-induced cell growth by decreasing Ras-induced cellular ROS levels. These results demonstrate that the Pim protein kinases have an important role in regulating cellular redox, metabolism and K-Ras-stimulated cell growth.

Sphingolipid metabolism is being increasingly recognized as a key pathway in regulating cancer cell survival and proliferation. However, very little is known about its role in multiple myeloma (MM). We investigated the potential of targeting sphingosine kinase 2 (SK2) for the treatment of MM. We found that SK2 was overexpressed in MM cell lines and in primary human bone marrow (BM) CD1381 myeloma cells. Inhibition of SK2 by SK2- specific short hairpin RNA or ABC294640 (a SK2 specific inhibitor) effectively inhibited myeloma cell proliferation and induced caspase 3–mediated apoptosis. ABC294640 inhibited primary human CD1381 myeloma cells with the same efficacy as with MM cell lines. ABC294640 effectively induced apoptosis of myeloma cells, even in the presence of BM stromal cells. Furthermore, we found that ABC294640 downregulated the expression of pS6 and directed c-Myc and myeloid cell leukemia 1 (Mcl-1) for proteasome degradation. In addition, ABC294640 increased Noxa gene transcription and protein expression. ABC294640, per se, did not affect the expression of B-cell lymphoma 2 (Bcl-2), but acted synergistically with ABT-737 (a Bcl-2 inhibitor) in inducing myeloma cell death. ABC294640 suppressed myeloma tumor growth in vivo in mouse myeloma xenograft models. Our data demonstrated that SK2 provides a novel therapeutic target for the treatment of MM.This trial was registered at www.clinicaltrials.gov as #NCT01410981.

// Sathish K.R. Padi 1 , Libia A. Luevano 1 , Ningfei An 2 , Ritu Pandey 1, 3 , Neha Singh 1 , Jin H. Song 1, 3 , Jon C. Aster 4 , Xue-Zhong Yu 5 , Shikhar Mehrotra 6 , Andrew S. Kraft 1 1 University of Arizona Cancer Center, University of Arizona, Tucson, AZ, USA 2 Department of Pathology, Pediatric Hematology/Oncology Division, University of Chicago, Chicago, IL, USA 3 Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA 4 Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA 5 Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, USA 6 Department of Surgery, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA Correspondence to: Andrew S. Kraft, email: akraft@uacc.arizona.edu Keywords: PIM kinase, T-ALL, ETP-ALL, tyrosine kinase inhibitor, ponatinib Received: October 15, 2016 Accepted: March 08, 2017 Published: March 17, 2017 ABSTRACT New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.

Pim serine/threonine kinases contribute to prostate tumorigenesis and therapeutic resistance, yet Pim kinase inhibitors seem to have only limited effects on prostate cancer cell survival. Because overexpression of Bcl-2 family members are implicated in chemotherapeutic resistance in prostate cancer, we investigated the cooperative effects of Pim kinase inhibition with ABT-737, a small molecule antagonist of Bcl-2 family members. Strikingly, the addition of ABT-737 to Pim inhibitors triggered a robust apoptosis of prostate cancer cells in vitro and in vivo. Pim inhibitors decreased levels of the Bcl-2 family member Mcl-1, both by blocking 5′-cap dependent translation and decreasing protein half life. In addition, Pim inhibition transcriptionally increased levels of the BH3 protein Noxa by activating the unfolded protein response (UPR), lead to eIF-2α phosphorylation and increased expression of CHOP. Increased levels of Noxa also inactivated the remaining levels of Mcl-1 protein activity. Notably, these specific protein changes were essential to the apoptotic process because ABT-737 did not inhibit Mcl-1 protein activity and Mcl-1 overexpression blocked the apoptotic activity of ABT-737. Our results therefore suggest that this combination treatment could be developed as a potential therapy for human prostate cancer where overexpression of Pim kinases and antiapoptotic Bcl-2 family members drives tumor cell resistance to current anticancer therapies. Cancer Res; 72(1); 294–303. ©2011 AACR.

ABT-737, a small molecule cell-permeable Bcl-2 antagonist that acts by mimicking BH3 proteins, induces apoptotic cell death in multiple cancer types. However, when incubated with this agent many solid tumor cell lines do not undergo apoptosis. The current study reveals a novel mechanism whereby ABT-737 when added to apoptosis-resistant cancer cells has profound biologic effects. In PV-10 cells, a renal cell carcinoma that does not die after ABT-737 treatment, this agent induces a two-fold change in the transcription of nearly 430 genes. Many of these induced mRNA changes are in secreted proteins, IL-6, IL-8, and IL-11 and chemokines CXCL2 and CXCL5, or genes associated with an “inflammatory” phenotype. Strikingly, these gene changes are highly similar to those changes previously identified in cellular senescence. Brief exposure of apoptosis-resistant renal, lung and prostate cancer cell lines to ABT-737, although not capable of inducing cell death, causes the induction of senescence-associated β-galactosidase and inhibition of cell growth consistent with the induction of cellular senescence. Evidence indicates that the induction of senescence occurs as a result of reactive oxygen species elevation followed by low-level activation of the caspase cascade, insufficient to induce apoptosis, but sufficient to lead to minor DNA damage and increases in p53, p21, IL-6 and 8 proteins. By overexpression of a dominant-negative p53 protein, we show that ABT-737-induced cellular senescence is p53-dependent. Thus, in multiple cancer types in which ABT-737 is incapable of causing cell death, ABT-737 may have additional cellular activities that make its use as an anticancer agent highly attractive. Cancer Res; 71(2); 506–15. ©2010 AACR.

Aberrant activation of the PI3K pathway leading to increases in AKT activity is seen in many prostate cancer patients refractory to androgen blockade therapy. A number of small-molecule inhibitors of PI3K, including BKM120 (Novartis), have entered clinical trials. As with PI3K-AKT activation, highly expressed Pim kinase has been shown to drive the growth of prostate cancer. Recent studies have demonstrated that Pim kinase plays a critical role in promoting resistance of breast cancer to PI3K inhibition. Similarly, when the Pim-1 protein kinase is overexpressed, prostate cancer cell lines become resistant to PI3K inhibitor. In both tissue culture and animal models, the combination of a Pim inhibitor, PIM447, and BKM120 acted synergistically in blocking the survival and growth of prostate cancer cells. We therefore investigated the biochemical mechanism by which Pim-1 mediates cell resistance to the PI3K inhibitor. Previously, we have demonstrated that knockout of Pim kinases led to accumulation of reactive oxygen species (ROS) accompanied by a lower level of expression of ROS scavengers. To explore whether Pim-1 overexpression reverses this phenotype in prostate epithelial cancer, using lentiviral transduction, we expressed Pim-1 in mouse prostate epithelial cancer cells derived from spontaneous prostate adenocarcinomas that arose in a mouse model in which Tp53 and Rb1 are deleted in prostate epithelium. Pim-1 overexpression promoted anchorage-independent growth and formation of mouse prostate epithelial tumors in vivo. Gene expression arrays demonstrated that Pim-1 induced an Nrf2 antioxidant response element gene signature, including increases in Heme oxygenase-1 (Hmox1), NAD(P)H quinone oxidoreductase 1 (Nqo1), and glutathione S-transferase kappa1 (Gstk1) genes. Similarly, overexpression of Pim-1 in human prostate cancer LNCaP cells results in increased expression of Nrf2 and Hmox1 protein. As expected, depletion of Nrf2 with shRNA prevented Pim-1-mediated increases in Hmox1 expression, indicating that Nrf2 activation is required for the Pim-1 induction of ROS scavengers. In line with this result, in Pim-1 overexpressing cell lines knockdown of either Hmox1 or Nrf2 restored sensitivity to BKM120 treatment. A vital role of ROS scavengers in mediating the resistance to BKM120 was further confirmed by the treatment of cells and xenografted tumors with buthionine sulfoximine (inhibitor of glutathione synthesis), which resensitized the Pim-1 overexpressing cells to BKM120 via ROS induction. Our data further demonstrated that Pim-1 overexpression decreases the ubiquitination of Nrf2 protein, thus preventing its degradation, and increasing its half-life. Conversely, inhibition of Pim kinase promoted Nrf2 ubiquitination and proteasomal degradation. Taken together, our results show that the increased expression of Pim kinases in prostate cancer blocks therapeutic efficacy of PI3K inhibitor by inducing Nrf2 and antioxidant scavengers that inhibit the induction of ROS. Citation Format: Jin H. Song, Libia A. Leuvano, Neha Singh, Andrew S. Kraft. NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B097.

Human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is widely overexpressed in human cancers including prostate cancer. Along with its regulation of the telomere, hTERT was also shown to regulate various signal transduction mechanisms involving DNA damage response, cell cycle checkpoint, and apoptosis. The majority of human solid tumors display telomerase activity, which was found to be well correlated with hTERT expression, and hTERT inhibition led to reduced telomerase activity and telomere length. However, approaches that directly inhibit the action of telomerase have not produced a therapeutically useful response because of long lag times for subsequent cell division to produce telomere shortening. Alternatively, recent studies demonstrated that this delay problem can be solved through downregulation of hTERT transcription. It was shown that telomeric DNA is capable of folding into four-stranded guanine quadruplex (G4) structures, which then lock the hTERT promoter activity that leads to robust telomere shortening. Our new approach has focused on the discovery of small molecules that could stabilize G-quadruplexes by interacting with the telomeric G-rich overhang stem loop, producing a lead candidate compound that enhances the kinetic folding rate of the G4 silencer element. Our in vitro studies demonstrate that this singular mechanism of action permitted reduced telomerase production and telomere shortening. Significantly, hTERT downregulation with the identified molecule caused prostate cancer cell death, which was characterized by activation of the caspase cascade and increased Annexin V- positive cells. In contrast, this compound did not harm normal prostate epithelial cells. Similarly, mouse prostate cancer cells that do not contain a G-quadruplex were not killed, demonstrating that the unique mechanism of action is mediated through the hTERT promoter G-quadruplex element. Our in vivo study demonstrated that administration of this significantly delayed tumor growth on xenografted mice. The rapid onset of cell death by G4 stands in contrast to the delayed action of classic telomerase inhibition. Therefore, our current study provides important data to support developing inhibitors of hTERT as a cancer drug target. Citation Format: Jin H. Song, Libia A. Luevano, Hyunjin Kang, Vijay Gokhale, Laurence H. Hurley, Andrew S. Kraft. Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4841.

The serine/threonine Pim kinases are up-regulated in specific hematologic neoplasms, and play an important role in key signal transduction pathways, including those regulated by MYC, MYCN, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. We demonstrate that SMI-4a, a novel benzylidene-thiazolidine-2, 4-dione small molecule inhibitor of the Pim kinases, kills a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre–T-LBL/T-ALL) being highly sensitive. Incubation of pre–T-LBL cells with SMI-4a induced G1 phase cell-cycle arrest secondary to a dose-dependent induction of p27Kip1, apoptosis through the mitochondrial pathway, and inhibition of the mammalian target of rapamycin C1 (mTORC1) pathway based on decreases in phospho-p70 S6K and phospho-4E-BP1, 2 substrates of this enzyme. In addition, treatment of these cells with SMI-4a was found to induce phosphorylation of extracellular signal-related kinase1/2 (ERK1/2), and the combination of SMI-4a and a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor was highly synergistic in killing pre–T-LBL cells. In immunodeficient mice carrying subcutaneous pre–T-LBL tumors, treatment twice daily with SMI-4a caused a significant delay in the tumor growth without any change in the weight, blood counts, or chemistries. Our data suggest that inhibition of the Pim protein kinases may be developed as a therapeutic strategy for the treatment of pre–T-LBL.

Because Bcl-2 family members inhibit the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce apoptosis, we investigated whether ABT-737, a small molecule Bcl-2 inhibitor, enhances TRAIL killing. We demonstrate that a combination of ABT-737 and TRAIL induced significant cell death in multiple cancer types, including renal, prostate, and lung cancers, although each agent individually had little activity in these tumor cells. All of these cell lines expressed the Mcl-1 protein that is known to block the activity of ABT-737 and TRAIL but did not block the synergy between these agents. However, Bax-deficient cell lines, including DU145 and HCT116 cells and those cell lines expressing low levels of TRAIL receptor, were resistant to apoptosis induced by these agents. To understand how ABT-737 functions to markedly increase TRAIL sensitivity, the levels of specific death-inducing signaling complex components were evaluated. Treatment with ABT-737 did not change the levels of c-FLIP, FADD, and caspase-8 but up-regulated the levels of the TRAIL receptor DR5. DR5 up-regulation induced by ABT-737 treatment occurred through a transcriptional mechanism, and mutagenesis studies demonstrated that the NF-κB site found in the DR5 promoter was essential for the ability of ABT-737 to increase the levels of this mRNA. Using luciferase reporter plasmids, ABT-737 was shown to stimulate NF-κB activity. Together, these results demonstrate that the ability of ABT-737 and TRAIL to induce apoptosis is mediated through activation of both the extrinsic and intrinsic pathways. Combinations of ABT-737 and TRAIL can be exploited therapeutically where antiapoptotic Bcl-2 family members drive tumor cell resistance to current anticancer therapies.

Functional 1D metal oxides have attracted much attention because of their unique applications in electronic, optoelectronic, and spintronic devices. For semiconducting oxide nanowires (NWs) (e.g., ZnO, In2O3, and SnO2 NWs), field-effect transistors and light-emitting diodes have been demonstrated. Metallic oxide nanoscale materials, such as nanoscale RuO2, can be good candidates as interconnects in electronic applications. RuO2 nanomaterials have been produced by chemical vapor deposition (CVD) and through chemical reaction. Recently, RuO2 NWs have been synthesized using pure Ru as metal target under different flux ratios of O2/Ar in a reactive sputtering system. [5]

Purpose: Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is currently under clinical development as a cancer therapeutic agent. Many human malignant glioma cells, however, are resistant to TRAIL treatment. We, therefore, investigated the genomic alterations in TRAIL-resistant malignant glioma cells.Experimental Design: Seven glioma cell lines and two primary cultures were first analyzed for their sensitivity to TRAIL and chemotherapy and then examined for the genomic alterations in key TRAIL apoptotic genes by comparative genomic hybridization (CGH), G-banding/spectral karyotyping, and fluorescence in situ hybridization (FISH).Results: CGH detected loss of the chromosomal regions that contain the following genes: 8p12-p23 (DR4 and DR5), 2q33-34 (caspase-8), 11q13.3 (FADD), 22q11.2 (Bid), and 12q24.1-q24.3 (Smac/DIABLO) in TRAIL-resistant cell lines. Spectral karyotyping showed numerical and structural aberrations involving the chromosomal regions harboring these genes. A combination of G-banding/spectral karyotyping and FISH further defined the loss or gain of gene copy of these genes and further showed the simultaneous loss of one copy of DR4/DR5, caspase-8, Bid, and Smac in two near-triploid cell lines that were resistant to the combination treatment with TRAIL and chemotherapy. Loss of the caspase-8 locus was also detected in a primary culture in correlation with the culture resistance to the combined TRAIL and chemotherapy treatment.Conclusions: The study identifies chromosomal alterations in TRAIL apoptotic genes in the glioma cells that are resistant to the treatment with TRAIL and chemotherapy. These genetic alterations could be used to predict the responsiveness of malignant gliomas to TRAIL-based therapies in clinical treatment of the tumors.

Human astrocytes express Fas yet are resistant to Fas-induced apoptosis. Here, we report that calcium/calmodulin-dependent protein kinase II (CaMKII) is constitutively activated in human astrocytes and protects the cells from apoptotic stimulation by Fas agonist. Once stimulated, Fas recruits Fas-associated death domain and caspase-8 for the assembly of the death-inducing signaling complex (DISC); however, caspase-8 cleavage is inhibited in the DISC. Inhibition of CaMKII kinase activity inhibits the expression of phosphoprotein enriched astrocytes-15 kDa/phosphoprotein enriched in diabetes (PEA-15/PED) and cellular Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (c-FLIP), thus releasing their inhibition of caspase-8 cleavage. Inhibition of PEA-15/PED or c-FLIP by small interfering RNA sensitizes human astrocytes to Fas-induced apoptosis. In contrast, inhibition of CaMKII, PEA-15, or c-FLIP does not affect the sensitivity of human astrocytes to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL death receptors (DR4, DR5) are weakly expressed at mRNA, protein, and cell surface levels and thus fail to mediate the assembly of the DISC in human astrocytes. Overexpression of DR5 restores TRAIL signaling pathways and sensitizes the human astrocytes to TRAIL-induced apoptosis if CaMKII kinase activity or expression of PEA-15 and c-FLIP is inhibited; the results suggest that CaMKII-mediated pathways prevent TRAIL-induced apoptosis in human astrocytes under conditions in which TRAIL death receptors are upregulated. This study has therefore identified the molecular mechanisms that protect normal human astrocytes from apoptosis induced by Fas ligand and TRAIL.

Tumor necrosis factor (TNF) family ligand TNF-α and Fas ligand (FasL) can trigger apoptosis in solid tumors, but their clinical usage has been limited by hepatotoxicity. TNF-related apoptosis-inducing ligand (TRAIL) is a newly identified member of the TNF family, and its clinical application currently is under a similar debate. Here, we report a recombinant soluble form of human TRAIL (114 to 281 amino acids) that induces apoptosis in tumor cells but not human hepatocytes. We first isolated human hepatocytes from patients and showed that the human hepatocytes expressed Fas but no TRAIL death receptor DR4 and little DR5 on the cell surface. Antibody cross-linked FasL, but not TRAIL, triggered apoptosis of the human hepatocytes through cleavage of caspases. We then examined TRAIL hepatotoxicity in severe combined immunodeficient/Alb-uPA chimeric mice harboring human hepatocytes. Intravenous injection of FasL, but not TRAIL, caused apoptotic death of human hepatocytes within the chimeric liver, thus killing the mice. Finally, we showed that repeated intraperitoneal injections of TRAIL inhibited intraperitoneal and subcutaneous tumor growth without inducing apoptosis in human hepatocytes in these chimeric mice. The results indicate that the recombinant soluble human TRAIL has a profound apoptotic effect on tumor cells but is nontoxic to human hepatocytes in vitro and in vivo.