CD95 Receptor Research Articles

Cell death-independent activities of the death receptors CD95, TRAILR1, and TRAILR2.

Since their identification more than 20 years ago, the death receptors CD95, TRAILR1, and TRAILR2 have been intensively studied with respect to their cell death-inducing activities. These receptors, however, can also trigger a variety of cell death-independent cellular responses reaching from the activation of proinflammatory gene transcription programs over the stimulation of proliferation and differentiation to induction of cell migration. The cell death-inducing signaling mechanisms of CD95 and the TRAIL death receptors are well understood. In contrast, despite the increasing recognition of the biological and pathophysiological relevance of the cell death-independent activities of CD95, TRAILR1, and TRAILR2, the corresponding signaling mechanisms are less understood and give no fully coherent picture. This review is focused on the cell death-independent activities of CD95 and the TRAIL death receptors and addresses mainly three questions: (a) how are these receptors linked to noncell death pathways at the molecular level, (b) which factors determine the balance of cell death and cell death-independent activities of CD95 and the TRAIL death receptors at the cellular level, and (c) what are the consequences of the cell death-independent functions of these receptors for their role in cancer and inflammatory diseases.

MicroRNA Expression in Sentinel Nodes from Progressing Melanoma Patients Identifies Networks Associated with Dysfunctional Immune Response.

Sentinel node biopsy (SNB) is a main staging biomarker in melanoma and is the first lymph node to drain the tumor, thus representing the immunological site where anti-tumor immune dysfunction is established and where potential prognostic immune markers can be identified. Here we analyzed microRNA (miR) profiles in archival tumor-positive SNBs derived from melanoma patients with different outcomes and performed an integrated analysis of transcriptional data to identify deregulated immune signaling networks. Twenty-six miRs were differentially expressed in melanoma-positive SNB samples between patients with disease progression and non-progressing patients, the majority being previously reported in the regulation of immune responses. A significant variation in miR expression levels was confirmed in an independent set of SNB samples. Integrated information from genome-wide transcriptional profiles and in vitro assessment in immune cells led to the identification of miRs associated with the regulation of the TNF receptor superfamily member 8 (TNFRSF8) gene encoding the CD30 receptor, a marker increased in lymphocytes of melanoma patients with progressive disease. These findings indicate that miRs are involved in the regulation of pathways leading to immune dysfunction in the sentinel node and may provide valuable markers for developing prognostic molecular signatures for the identification of stage III melanoma patients at risk of recurrence.

CORRELATION OF THE SHEDDING OF LYMPHOCYTES RECEPTORS WITH PARAMETERS OF IMMUNOLOGIC REACTIVITY IN RESIDENTS OF THE NORTH

The study retrospectively investigated correlation characteristics of membrane and soluble forms of lymphocyte receptors in 78 healthy people living in the city of Arkhangelsk. The results of a statistical analysis allowed to evaluate the relationship of dynamic indices of shedding lymphocyte receptors CD23 and CD80 to the immunological reactivity indices. It was found that the receptors' shedding was not associated with the processes of proliferation and activation of immune cells, and the receptors abscission occurred by postmitotic mature cells. A high ratio reflecting the relationship of shedding activity of membrane forms of lymphocyte with cytokine IL-1 and IL-10 in the extracellular medium was a reflection of the biological processes regulating the in the form of feedback: increasing of receptor concentration in the extracellular medium by means of cytokine blocked cell receptor expression. Low values of the analyzed indices for CD95 receptor indicated that the cells got free from unnecessary receptor substances in the course of performing its function did not undergo apoptosis. Accumulation of receptors extracellular pool was provided by phagocytosis deficiency. Increase of phagocytosis intensity at a maximum concentration of free receptors was due to the necessity of urgent prevention of unwanted substances accumulation in the blood serum.

A Bak-dependent mitochondrial amplification step contributes to Smac mimetic/glucocorticoid-induced necroptosis.

Necroptosis is a form of programmed cell death that critically depends on RIP3 and MLKL. However, the contribution of mitochondria to necroptosis is still poorly understood. In the present study, we discovered that mitochondrial perturbations play a critical role in Smac mimetic/Dexamethasone (Dexa)-induced necroptosis independently of death receptor ligands. We demonstrate that the Smac mimetic BV6 and Dexa cooperate to trigger necroptotic cell death in acute lymphoblastic leukemia (ALL) cells that are deficient in caspase activation due to absent caspase-8 expression or pharmacological inhibition by the caspase inhibitor zVAD.fmk, since genetic silencing or pharmacological inhibition of RIP3 or MLKL significantly rescue BV6/Dexa-induced necroptosis. In addition, RIP3 or MLKL knockout mouse embryonic fibroblasts (MEFs) are protected from BV6/Dexa/zVAD.fmk-induced cell death. In contrast, antagonistic antibodies against the death receptor ligands TNFα, TRAIL or CD95 ligand fail to rescue BV6/Dexa-triggered cell death. Kinetic studies revealed that prior to cell death BV6/Dexa treatment causes hyperpolarization of the mitochondrial membrane potential (MMP) followed by loss of MMP, reactive oxygen species (ROS) production, Bak activation and disruption of mitochondrial respiration. Importantly, knockdown of Bak significantly reduces BV6/Dexa-induced loss of MMP and delays cell death, but not ROS production, whereas ROS scavengers attenuate Bak activation, indicating that ROS production occurs upstream of BV6/Dexa-mediated Bak activation. Consistently, BV6/Dexa treatment causes oxidative thiol modifications of Bak protein. Intriguingly, knockdown or knockout of RIP3 or MLKL protect ALL cells or MEFs from BV6/Dexa-induced ROS production, Bak activation, drop of MMP and disruption of mitochondrial respiration, demonstrating that these mitochondrial events depend on RIP3 and MLKL. Thus, mitochondria might serve as an amplification step in BV6/Dexa-induced necroptosis. These findings provide new insights into the role of mitochondrial dysfunctions during necroptosis and have important implications for the development of novel treatment approaches to overcome apoptosis resistance in ALL.

ANTI-APOPTOTIC EFFECT OF CD95 RECEPTOR IN NA VE CD8+ T-LYMPHOCYTES IN CHILDREN WITH ACUTE INFECTIOUS MONONUCLEOSIS

Acute infectious mononucleosis is a widespread viral disease, which most often manifests in childhood. The development of acute infectious mononucleosis is accompanied by the change of the CD4+/CD8+ T-lymphocytes ratio and the increase of the virus-specific CD8+ cytotoxic T-lymphocytes number. One of the T-lymphocytes number regulation mechanisms is the modulation of their progenitor cells apoptosis. The death receptor CD95 takes part in the regulation of T-lymphocytes apoptosis, including naïve T-cells. We studied the effect of CD95 receptor activation on apoptosis of naïve CD4+ and naïve cytotoxic CD8+ T-lymphocytes in healthy children and children with acute infectious mononucleosis. In this study children with acute infectious mononucleosis at the age of 9 to 16 years were included. For comparison healthy children of the same age with no clinical and laboratory signs of the disease were used. Naïve CD4+ and naïve cytotoxic CD8+ T-lymphocytes were isolated by negative magnetic immunoseparation. The analysis of naïve T-cells apoptosis and the CD95 receptor surface expression density was performed by using the flow cytometry analysis. The analysis of T-cells was performed in three variants: freshly isolated naïve CD4+ T-lymphocytes and naïve cytotoxic CD8+ T-lymphocytes, and also cells after 24 hours of the cultivation with anti-CD95 monoclonal antibodies or without them. In healthy children both CD95– and CD95+ naïve CD4+ T-lymphocytes underwent apoptosis. In children with acute infectious mononucleosis CD95– naïve CD4+ T-lymphocytes lost their susceptibility to apoptosis induction. In healthy children and children with acute infectious mononucleosis CD95– naïve cytotoxic CD8+ T-lymphocytes were resistant to apoptosis in contrast to CD95+ naïve CD4+ T-lymphocytes. In healthy children CD95 receptor did not induce apoptosis of isolated naïve CD4+ T-lymphocytes and naïve cytotoxic CD8+ T-lymphocytes. In children with acute infectious mononucleosis CD95 receptor was involved in inhibition of apoptosis of naïve cytotoxic CD8+ T-lymphocytes and did not effect on the level of apoptosis of naïve CD4+ T-lymphocytes. We suggest that CD95-dependent suppression of naïve cytotoxic CD8+ T-lymphocytes apoptosis is a protective mechanism for the maintenance of a sufficient number of cytotoxic T-lymphocytes in the blood for the realization of effective antiviral immune response.

CD95-mediated apoptosis in Burkitt's lymphoma B-cells is associated with Pim-1 down-regulation

B-cells of the high-grade non-Hodgkin lymphoma Burkitt's lymphoma (BL) overexpress survival oncoproteins, including the proviral integration site for Moloney murine leukaemia virus kinase (Pim)-1, and become apoptosis resistant. Activated death receptor CD95 after ligation with anti-CD95 monoclonal antibody (mAb) resulted in the regression of BL via induction of apoptosis, suggesting a decrease of survival protein expression. Here, CD95-mediated apoptotic pathways in BL B-cell lines (Raji and Daudi) following treatment with anti-CD95 mAb was investigated with the cause-and-effects on pim-1 gene expression, in comparison with leukemic cell line (K562) used as CD95-negative cells. Immunohistochemical staining for CD95 and Pim-1 was performed, and the effects of anti-CD95 mAb on apoptotic signalling using western blotting, on caspase activity and cell survival of BL B-cell and leukemic cell lines were determined. We showed that Raji cells expressed more CD95 receptors than Daudi cells. Half of each population underwent apoptosis accompanied by decreased cell viability after anti-CD95 mAb treatment. Distinct extrinsic and intrinsic CD95-mediated apoptotic pathways in Raji and Daudi cells were revealed by high caspase activity and mitochondrial outer membrane permeabilization, respectively. We observed decreased Pim-1 transcript and protein expression levels with increased heat-shock protein (Hsp)70 and decreased Hsp90 expression in anti-CD95 mAb-treated cells. Throughout the study, K562 cells did not undergo apoptosis upon anti-CD95 mAb treatment. Pim-1 knockdown following to stable transfection with plasmid vectors induced apoptosis and decreased viability of BL and K562 cells. Therefore, CD95-mediated apoptosis induces Pim-1 down-regulation in BL B-cells, but Pim-1 down-regulation cannot fully eradicate BL and leukaemia.

IL-4 downregulates expression of the target receptor CD30 in neoplastic canine mast cells.

CD30 is a novel therapeutic target in human mast cell (MC) neoplasms. In this 'comparative oncology' study, we examined CD30 expression and regulation in neoplastic canine MC using a panel of immunomodulatory cytokines [interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-13 and stem cell factor (SCF)] and the canine mastocytoma cell lines NI-1 and C2. Of all cytokines tested IL-4 was found to downregulate expression of CD30 in NI-1 and C2 cells. We also found that the CD30-targeting antibody-conjugate brentuximab vedotin induces growth inhibition and apoptosis in both MC lines. Next, we asked whether IL-4-induced downregulation of CD30 interferes with brentuximab vedotin-effects. Indeed, pre-incubation of NI-1 cells with IL-4 decreased responsiveness towards brentuximab vedotin. To overcome IL-4-mediated resistance, we applied drug combinations and found that brentuximab vedotin synergizes with the Kit-targeting drugs masitinib and PKC412 in inhibiting growth of NI-1 and C2 cells. In summary, CD30 is a new marker and IL-4-regulated target in neoplastic canine MC.

THE EXPRESSION LEVEL OF THE MOLECULES OF THE RECEPTOR COMPLEX CD95 AND CD8 HUMAN BLOOD LYMPHOCYTES UNDER CARBON MONOXIDE AND ULVTRAVIOLET LIGHT INFLUENCE

By means of enzymoiimmunoassay and flow cytofluorometry the effect of carbon monoxide and ultraviolet (UV)-light on the expression level of the receptor complex molecules (CD95 and CD8 markers) on the membranes of human blood lymphocytes has been studied. It was stated that the carbon monoxide causes decrease of CD95 receptors on the surface immune cells during (60 ^ 90 min). However, strengthening of this effect was registered in 24 hours. It is shown that UV-light in 453 and 755 J/m2 doses provides the pro-apoptotic effect as related to the expression level of CD95 receptors of analyzed cells. After daily thermostating of modified lymphocytes, different receptiveness of CD8 molecules to carbon monoxide was revealed. Thus, in patients with initially higher levels of CD8 markers - decrease is registered, and in individuals with initially low - increase in this marker levels. The obtained data of blood lymphocytes receptor profile changes in the presence of low molecular ligand (CO) must be taken into account in the Ultraviolet Blood Irradiation-therapy in patients with different etiologies.

Ligand stimulation of CD95 induces activation of Plk3 followed by phosphorylation of caspase-8

Upon interaction of the CD95 receptor with its ligand, sequential association of the adaptor molecule FADD (MORT1), pro-forms of caspases-8/10, and the caspase-8/10 regulator c-FLIP leads to the formation of a death-inducing signaling complex. Here, we identify polo-like kinase (Plk) 3 as a new interaction partner of the death receptor CD95. The enzymatic activity of Plk3 increases following interaction of the CD95 receptor with its ligand. Knockout (KO) or knockdown of caspase-8, CD95 or FADD prevents activation of Plk3 upon CD95 stimulation, suggesting a requirement of a functional DISC for Plk3 activation. Furthermore, we identify caspase-8 as a new substrate for Plk3. Phosphorylation occurs on T273 and results in stimulation of caspase-8 proapoptotic function. Stimulation of CD95 in cells expressing a non-phosphorylatable caspase-8-T273A mutant in a rescue experiment or in Plk3-KO cells generated by CRISPR/Cas9 reduces the processing of caspase-8 prominently. Low T273 phosphorylation correlates significantly with low Plk3 expression in a cohort of 95 anal tumor patients. Our data suggest a novel mechanism of kinase activation within the Plk family and propose a new model for the stimulation of the extrinsic death pathway in tumors with high Plk3 expression.

MOAP-1 Mediates Fas-Induced Apoptosis in Liver by Facilitating tBid Recruitment to Mitochondria.

Fas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM), is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the OMM. Here, we show that MOAP-1-deficient mice are resistant to Fas-induced hepatocellular apoptosis and lethality. In the absence of MOAP-1, mitochondrial accumulation of tBid is markedly impaired. MOAP-1 binds to MTCH2, and this interaction appears necessary for MTCH2 to engage tBid. These findings reveal a role for MOAP-1 in Fas signaling in the liver by promoting MTCH2-mediated tBid recruitment to mitochondria.

NF-κB deregulation in Hodgkin lymphoma.

Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (HL) show constitutive activity of both the canonical and non-canonical NF-κB signaling pathways. The central pathogenetic role of this activity is indicated from studies with HL cell lines, which undergo apoptosis upon NF-κB inhibition. Multiple factors contribute to the strong NF-κB activity of HRS cells. This includes interaction with other cells in the lymphoma microenvironment through CD30, CD40, BCMA and other receptors, but also recurrent somatic genetic lesions in various factors of the NF-κB pathway, including destructive mutations in negative regulators of NF-κB signaling (e.g. TNFAIP3, NFKBIA), and copy number gains of genes encoding positive regulators (e.g. REL, MAP3K14). In Epstein-Barr virus-positive cases of classical HL, the virus-encoded latent membrane protein 1 causes NF-κB activation by mimicking an active CD40 receptor. NF-κB activity is also seen in the tumor cells of the rare nodular lymphocyte predominant form of HL, but the causes for this activity are largely unclear.

Manipulations of the TNF-Receptor Affinity and Oligomerization Characterized by Fluorescence Lifetime Measurements

Tumor necrosis factor (TNF) receptor 1 (TNFR1) and Death Receptor 5 (DR5) are members of the TNF receptor superfamily that are activated by binding to their ligands, TNFα and TNF-related apoptosis-inducing ligand (TRAIL) respectively. The TNFα-TNFR1 signaling pathway culminates with NF-κB activation and inflammatory response. The TRAIL-DR5 signaling pathway culminates in apoptosis. Recently, we have shown that TRAIL-induced apoptosis is initiated through the formation of large and highly structured networks held together by receptor dimers. In our current work, we use fluorescence lifetime measurements to study TNFR1. As an initial control to validate our approach, we monitored receptor-receptor binding affinity as a function of temperature and detergent concentration. Here, we have extended these initial studies to include investigation of the role of cholesterol and cytochalasin B. We have recently shown that cholesterol depletion in cells does not inhibit the formation of DR5 networks but, interestingly, does fail to initiate apoptosis. Others have shown that disruption of actin cytoskeleton by cytochalasin B in death receptor CD95, another member of the TNF receptor superfamily, induces and enhances apoptosis. Our lifetime measurements are used to investigate any potential connection between these cell-level observations and the biophysics of TNF-Receptor oligomerization.

The emerging role of CD30 and p53 as novel targets for therapy in anaplastic large cell lymphoma.

ALK+ anaplastic large cell lymphoma (ALCL). frequently carries the t(2;5).(p23;q35). resulting in expression of NPM-ALK oncogenic kinase, which is capable of activating multiple oncogenic pathways. ALK+ ALCL is also characterized by overexpression of CD30 receptor, a member of the tumor necrosis factor (TNF). receptor superfamily, which has been targeted for therapy using conjugated anti-CD30 antibodies with clinical success. Also, the tumor suppressor p53 is frequently non-mutated in ALK+ ALCL allowing for therapeutic modulation of p53 reactivation in this lymphoma type. Therefore, this review is focused on the role of CD30 receptor and p53 as novel targets for therapy in ALK+ ALCL, and also provides an update on their potential involvement in ALK+ ALCL pathogenesis.

Targeting CD30 Using Brentuximab Vedotin in the Treatment of Hodgkin Lymphoma.

The expression of CD30 receptors is one of the defining characteristics of the malignant Reed-Sternberg cells of Hodgkin lymphoma (HL). CD30 is rarely expressed by normal cells and is rapidly internalized, making it an ideal therapeutic target for monoclonal antibodies and for antibody-drug conjugates. Brentuximab vedotin is the first antibody-drug conjugate to be approved by regulatory agencies for the treatment of patients with relapsed HL, with a single-agent response rate of 75%. In this review article, we discuss the current and ongoing development of brentuximab vedotin in patients with relapsed and newly diagnosed HL.

Sodium benzoate, a food preservative, affects the functional and activation status of splenocytes at non cytotoxic dose

Sodium benzoate (SB) is a widely used food preservative due to its bacteriostatic and fungistatic properties. The acceptable daily intake of SB is 5 mg/kg-bw, however, it has been found to be used in the food commodities at relatively high levels (2119 mg/kg). Earlier studies on SB have shown its immunosuppressive properties, but comprehensive immunotoxicity data is lacking. Our studies have shown that SB was non cytotoxic in splenocytes up to 1000 μg/ml for 72 h, however at 2500 μg/ml it was found to be cytotoxic. Thus, 1000 μg/ml dose of SB was chosen for the subsequent experiments. SB significantly suppresses the proliferation of Con A and LPS stimulated splenocytes at 72 h, while allogenic response of T cells was significantly decreased after 96 h. SB did not affect the relative expression of CD3e or CD4 molecules following 72 h exposure, however, it downregulated the relative expression of CD8 co-receptor. Further, exposure of splenocytes to SB for 72 h led to reduced expression of CD28 and CD95, which play a vital role in T cell activation. SB also suppresses the relative expression of CD19, CD40 and CD95 receptors on B cells after 72 h. In addition to the functional responses, SB lowered the expression of IL4, IL6, IFNγ and IL17 cytokines in Con A stimulated splenocytes; and IL6, IFNγ and TNFα in LPS stimulated splenocytes following 48 h of exposure. Taken together, the present study is suggestive of the immunomodulatory potential of SB.

TGR5 is essential for bile acid-dependent cholangiocyte proliferation in vivo and in vitro

ObjectiveCholestatic liver diseases in humans as well as bile acid (BA)-feeding and common bile duct ligation (CBDL) in rodents trigger hyperplasia of cholangiocytes within the portal fields. Furthermore, elevation of...

Increased extrafollicular expression of the B-cell stimulatory molecule CD70 in HIV-1-infected individuals.

CD70 molecules expressed by activated T cells provide potent B cell stimulatory signals. We hypothesized that an altered CD70 expression might contribute to B cell abnormalities during HIV-1 infection. CD70 expression and the functional and migratory properties of the CD4CD70 T lymphocytes were analyzed in HIV-1-infected patients and in humanized mice. Correlations were tested between CD70 expression and features of B-cell activation, apoptosis sensitivity and functional exhaustion. CD4CD70 T cells were analyzed in cohorts of CD4 T-cell lymphopenic, viremic or nonlymphopenic, nonviremic HIV-1-infected patients and in noninfected individuals. CD70 upregulation was also followed in HIV-1-infected humanized mice. CD38, CD95, LAIR1 and PD-1 expressions were monitored on B-cell subpopulations, Ki67 was assessed to estimate B-cell proliferation and antibody levels were measured in plasma. Blood CD4CD70 T-cell frequencies increased in response to CD4 T-cell depletion or high viremia levels as a possible consequence of increased activation and proliferation in this subset. CD4CD70 T cells produced T-helper 1-type cytokines and expressed chemokine receptors mobilizing toward sites of inflammation but not to lymphoid follicles. High CD70 expression was observed in HIV-1-infected humanized mice at extrafollicular sites (peritoneum, bone-marrow). CD4CD70 T-cell frequencies correlated with the expression of the activation marker CD38 and the death receptor CD95 on various memory B-cell subsets, with B-cell proliferation and with plasma IgG levels. CD4CD70 T cells may contribute to B cell hyperactivation and accelerated memory B-cell turnover during HIV-1 infection.

Assessment of Apoptosis Level of Naive CD8+ T-lymphocytes in Children with Acute Infectious Mononucleosis in CD95 and DR3 Receptors Activation

Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russian FederationThe aim of the investigation was to estimate the relation of CD95 and DR3 receptors activation with apoptosis level of naive cytotoxic Т-lymphocytes (nCТL) in children with acute infectious mononucleosis (AIM).Materials and Methods. The test materials were peripheral blood samples of healthy children and children with AIM. nCTL were isolated by negative immunоmagnetic separation. Specific activation of CD95 and DR3 receptors was performed using monoclonal antibodies. An apoptosis level and expression of receptors were studied by flow cytometry.Results. The percentage of cell apoptosis decreased in children with AIM in freshly isolated nCTL, as well as in CD95 receptor activation compared to healthy children. nCTL apoptosis in healthy children regardless of culture conditions was accompanied by the reduced quantity of CD95

The tragic fate of group 3 innate lymphoid cells during HIV-1 infection.

HIV-1 infection usually leads to systemic chronic inflammation that is associated with gut microbial translocation. The recently defined group 3 innate lymphoid cells (ILC3s) are critical for maintenance of intestinal barrier function; however, it is not clear whether and how HIV-1 infection influences the function of these cells. In this issue of the JCI, Zhang and colleagues present compelling evidence that the survival and function of ILC3s are dramatically impaired by HIV-1 infection. The authors provide evidence that HIV-1 infection induces persistent activation of plasmacytoid dendritic cells (pDCs) and production of type I IFNs, which together increase expression of death receptor CD95 on ILC3s and thereby promote subsequent ILC3 apoptosis. Together, these results identify a mechanism that explains the impaired intestinal barrier function that results from chronic HIV-1 infection and shed light on the role of pDCs in HIV-1 immunopathogenesis and therapy.

Abstract LB-094: Regorafenib and sildenafil interact to kill tumor cells

Abstract The present studies were to determine whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with phosphodiesterase 5 (PDE5) inhibitors such as sildenafil (Viagra) to kill tumor cells. PDE5 and PDGFR (alpha and beta) were over-expressed in liver tumors compared to normal liver tissue. In multiple cell types in vitro sorafenib/regorafenib and PDE5 inhibitors interacted in a greater than additive fashion to cause tumor cell death. Knock down of PDE5 or of PDGFR (alpha and beta) recapitulated the effects of the individual drugs. The drug combination increased ROS/RNS levels that were causal in cell killing. RNS stimulated the activation of the death receptor CD95 as well as the formation of autophagosomes and autolysosomes. Inhibition of CD95 / FADD / caspase 8 signaling suppressed drug combination toxicity. Knock down of ULK-1, Beclin1 or ATG5 suppressed drug combination lethality. The drug combination inactivated ERK, AKT, p70 S6K and mTOR and activated JNK. The drug combination also reduced mTOR protein expression. Activation of ERK or AKT was modestly protective whereas re-expression of an activated mTOR protein or inhibition of JNK signaling almost abolished drug combination toxicity. Sildenafil and sorafenib / regorafenib interacted in vivo to suppress xenograft tumor growth using liver and colon cancer cells. PDE5 inhibitors and sorafenib / regorafenib are FDA approved agents, and our data is now being translated into the clinic for further determination as to whether this drug combination is a useful anti-tumor therapy for solid tumor patients. Citation Format: Mehrad Tavallai, Laurence Booth, Jane L. Roberts, Andrew Poklepovic, Paul Dent. Regorafenib and sildenafil interact to kill tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-094. doi:10.1158/1538-7445.AM2015-LB-094