TRAIL Receptor Research Articles

The Transcription Factor PU.1 Controls Alternative Splicing of Cflip By Binding to SRSF2/10 Splice Factors in AML Therapy Responses

cFLIP (CFLAR) is an inactive homolog of caspase 8 that functions downstream of death-receptor mediated apoptosis. CFLAR gene is alternatively spliced and exists in three isoforms: a long isoform (cFLIPL), which partially inhibits CASP8 activity, and two short forms (cFLIPR and cFLIPS) that entirely block caspase 8 activity. Moreover, cFLIPL is crucial for macrophage differentiation. Therefore, the ratio between cFLIPL and cFLIPS/R may regulate myeloid differentiation. The transcription factor PU.1 (SPI1) is necessary for myeloid differentiation as well as myeloid cell survival. PU.1 is expressed at low levels in hematopoietic progenitors and increasing levels of PU.1 promote macrophage and granulocyte differentiation. Additionally, aberrant low PU.1 expression contributes to an immature myeloid phenotype, e.g. seen in acute myeloid leukemia (AML). Interestingly, two studies indicated that high PU.1 protein levels were associated with alternative splicing in a murine erythroleukemia model by either direct binding to splice factors or RNA. Based on these observations we hypothesized that PU.1 controls alternative splicing of cFLIP during myeloid differentiation. In line with above hypothesis, differentiating primary CD34+ hematopoietic progenitors into granulocytes and monocytes or AML cells into granulocytes shifted cFLIP splicing in favor of cFLIPL paralleled by increased PU.1 mRNA and protein levels. Next, we assessed the direct impact of PU.1 protein on splicing during granulocytic differentiation by knocking down PU.1 in NB4 acute promyelocytic leukemia (APL) and MOLM13 AML cells. Silencing PU.1 in NB4 and MOLM13 cells decreased cell death and differentiation during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation. Moreover, knocking down PU.1 reduced cFLIPL/S protein and mRNA ratios during ATRA treatment. Interestingly, increasing the cFLIPL/S ratio by knocking down specifically cFLIPS or overexpressing cFLIPL reduced MOLM13 cell viability during ATRA treatment. Since the canonical function of CFLAR isoforms is the inhibition of extrinsic apoptosis, we next tested if increased cFLIPS levels found in AML cells depleted for PU.1 would show reduced sensitivity to TRAIL treatment. Indeed, TRAIL-induced extrinsic apoptosis via TRAIL receptor was significantly reduced in PU.1 depleted MOLM-13 AML cells. To investigate if PU.1 is directly involved in CFLAR splicing, we analyzed the CFLAR pre-mRNA sequence encompassing the alternative splicing site for PU.1 and splice factor binding motifs. We identified one putative PU.1 and SRSF2 as well as SRSF10 splice factor binding sites in the CFLAR pre-mRNA sequence. Using RNA immunoprecipitation, PU.1 pull-down and proximity ligation assays, we showed that all three proteins bind the CFLAR mRNA and physically interact with each other. Lastly, we asked if PU.1 additionally influences cFLIP alternative splicing via regulating splice factor transcription. Knocking down PU.1 in MOLM13 cells resulted in significantly reduced SRSF2 and SRSF10 mRNA and protein levels. We confirmed PU.1 binding to their promoter using ChIP assays. Accordingly, these splice factors showed altered mRNA expression when comparing mature granulocytes to primary AML cells. Since silencing SRSF2 and SRSF10 switched the CFLARL/S isoform ratio towards the long isoform, we suggest that PU.1-mediated induction of these splice factores represents a negative feedback loop. In conclusion, we provide evidence that PU.1 directly regulates alternative splicing of CFLAR during ATRA-mediated neutrophil differentiation and TRAIL-induced cell death in human AML cells. We propose a model where PU.1 binds to CFLAR pre-mRNA and blocks SRSF2 and SRSF10-mediated splicing ultimatively leading to exon 7 skipping and predominant expression of cFLIPL.

Abstract B076: βIII-Tubulin is a brake on extrinsic cell-death in pancreatic cancer

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) urgently needs more effective therapies, with a 5-year survival rate <11%. We previously demonstrated βIII-tubulin knockdown in PDAC cells reduced tumor growth and metastasis in vivo, increased chemosensitivity, and induced apoptosis, making it a potent therapeutic target [McCarroll et al, Oncotarget, 6, 2235 (2015)]. However, mechanisms driving these effects are unknown. Apoptosis consists of intrinsic (caspase-9) and extrinsic (caspase-8) pathways. To better understand mechanism and inform therapeutic combinations, our study investigated apoptotic pathways controlled by βIII-tubulin. Aims: 1) Evaluate apoptotic pathways controlled by βIII-tubulin in PDAC cells a) in vitro and b) in vivo; 2) Assess the effect of silencing βIII-tubulin combined with tumor-specific extrinsic apoptosis inducer (TRAIL) in a patient-derived 3D PDAC model. Methods: 1a) PDAC cells were transfected with control-siRNA or βIII-tubulin-siRNA ± caspase-8 or -9 inhibitors and apoptosis measured. The effect of βIII-tubulin knockdown on PDAC cell sensitivity to extrinsic apoptosis inducers (TRAIL, TNFα, FasL) was tested (n≥3). TRAIL induces extrinsic cell death by binding to the TRAIL-receptor DR5, inducing clustering and intracellular activation of caspase 8. DR5 localization following βIII-tubulin knockdown was assessed by confocal microscopy (n=4). Live cell imaging was used to assess trafficking of GFP-tagged DR5 at the membrane of PDAC cells (n≥17 cells/treatment). 1b) Mice with orthotopic PDAC tumors (n=10/group) were treated (5-weeks post-implantation) with nanoparticle+βIII-tubulin-siRNA or control-siRNA for 3.5-weeks. Tumor volume and cleaved caspase-8 were measured. 2) Explants [validated patient-derived 3D tumor model with complete intact stroma, as described in: Kokkinos et al, Sci Rep, 11,1941 (2021); Sharbeen et al, Cancer Res, 81,3461 (2021)] from surgically resected PDAC tumors (5 patients) were cultured for 12 days and treated with nanoparticle+βIII-tubulin-siRNA or control-siRNA every 3 days and TRAIL once weekly. Tumor explants were fixed and assessed for tumor and viability markers via immunohistochemistry. Results: 1a) βIII-tubulin knockdown-induced apoptosis in PDAC cells was blocked by inhibiting caspase-8, but not caspase 9. Knockdown of βIII-tubulin sensitized PDAC cells to extrinsic apoptosis inducers. βIII-tubulin knockdown increased the movement of TRAIL-receptor DR5 through the cell membrane and triggered its clustering in PDAC cells. 1b) βIII-tubulin knockdown in orthotopic PDAC mouse tumors decreased tumor growth and activated extrinsic apoptosis. 2) In patient-derived tumor explants, βIII-tubulin-siRNA plus TRAIL reduced tumor cell frequency. Conclusions: 1) Silencing βIII-tubulin in PDAC cells sensitizes them to extrinsic apoptosis inducers and facilitates increased membrane clustering of the TRAIL receptor DR5. 2) βIII-tubulin knockdown represents an innovative therapeutic strategy to unleash a suicide signal in PDAC cells and render them sensitive to a tumor-specific therapeutic. Citation Format: John Kokkinos, George Sharbeen, Janet Youkhana, Cyrille Boyer, David Goldstein, Koroush S. Haghighi, Joshua A. McCarroll, Phoebe A. Phillips. βIII-Tubulin is a brake on extrinsic cell-death in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B076.

Limiting glutamine utilization activates a GCN2/TRAIL-R2/Caspase-8 apoptotic pathway in glutamine-addicted tumor cells

Oncogenic transformation leads to changes in glutamine metabolism that make transformed cells highly dependent on glutamine for anabolic growth and survival. Herein, we investigated the cell death mechanism activated in glutamine-addicted tumor cells in response to the limitation of glutamine metabolism. We show that glutamine starvation triggers a FADD and caspase-8-dependent and mitochondria-operated apoptotic program in tumor cells that involves the pro-apoptotic TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2), but is independent of its cognate ligand TRAIL. In glutamine-depleted tumor cells, activation of the amino acid-sensing general control nonderepressible-2 kinase (GCN2) is responsible for TRAIL-R2 upregulation, caspase-8 activation, and apoptotic cell death. Interestingly, GCN2-dependent ISR signaling induced by methionine starvation also leads to TRAIL-R2 upregulation and apoptosis. Moreover, pharmacological inhibition of transaminases activates a GCN2 and TRAIL-R2-dependent apoptotic mechanism that is inhibited by non-essential amino acids (NEAA). In addition, metabolic stress upon glutamine deprivation also results in GCN2-independent FLICE-inhibitory protein (FLIP) downregulation facilitating caspase-8 activation and apoptosis. Importantly, downregulation of the long FLIP splice form (FLIPL) and apoptosis upon glutamine deprivation are inhibited in the presence of a membrane-permeable α-ketoglutarate. Collectively, our data support a model in which limiting glutamine utilization in glutamine-addicted tumor cells triggers a previously unknown cell death mechanism regulated by GCN2 that involves the TRAIL-R2-mediated activation of the extrinsic apoptotic pathway.

Induction of apoptosis in lung carcinoma cells (A549) by hydromethanolic extract of Acorus calamus L

Acorus calamus L. of the Acoraceae family referred to as 'Sweet flag' is a well-known plant used to treat fever, epilepsy, liver, and renal problems in the traditional Indian and Chinese medicine systems. In the present study, we prepared the hydromethanolic extract by using the cold maceration method and performed its phytochemical screening. Extract of A. calamus L. showed the presence of many secondary metabolites including the phenolics [i.e., 275.51 ± 0.001 (mg/ml) Gallic acid equivalence] as well as showed good antioxidant activities reflected through DPPH and ABTS assay with IC50 16.32 ± 0.31 μg/ml, 27.62 ± 1.53 μg/ml respectively. It also possesses good total Nitric oxide, and Nitrite reducing activities. Furthermore, we have studied the cytotoxic behavior of AC hydromethanolic extract against human lung carcinoma cells (A549). It demonstrated potential cytotoxicity toward the lung carcinoma cell line (A549) with IC50 of 92.83 ± 1.98 μg/ml. Various staining techniques (DAPI, AO/EB, Giemsa) and RT-PCR techniques were used to identify the mechanism of cytotoxicity. We found that the A. calamus L. hydromethanolic extract caused characteristic apoptotic morphological changes, ROS generation, induction of apoptosis through activation of the TRAIL receptors, caspase 9 and 3 and the inhibition of colony formation in A549 cells.

Abstract PO032: Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-TRAIL-R Mediates Cholangiocarcinoma Tumor Immune Evasion by Enhancing Myeloid-Derived Suppressive Cell Population

Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of TNF superfamily, is predominantly expressed on immune cells. Engagement of TRAIL with its cognate receptor, TRAIL-R (TR), leads to cancer cells apoptosis. Although the anti-tumor role of TRAIL-TR has been extensively studied, TRAIL agonists have had very limited anti-cancer activity in human clinical trials. TRAIL signaling can facilitate the myeloid response. However, this potential immunosuppressive function of TRAIL has not been examined extensively in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, has a dense immunosuppressive microenvironment. Accordingly, CCA provides a model to examine the potential immune regulatory function of TRAIL in cancer biology. Methods: Using syngeneic, orthotopic murine models of CCA, (PMID: 29464042), murine CCA cells (SB cells) that express both Trail and Trail receptor (Tr) were implanted into livers of WT Tr− / − and LyzcreTrf/f mice, the latter have a specific deletion of TRAIL-R on myeloid cells. Hence, in this model the host immune cells express Trail but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr− / − mice had a significant reduction in tumor burden compared to WT mice. Myeloid-derived suppressor cells (MDSCs) were significantly decreased in Tr− / − tumors compared to WT tumors. Implantation of SB cells into LyzcreTrf/f mice resulted in a marked reduction in tumor burden and attenuation of MDSC infiltration compared to Lyzcre mice. In vitro functional studies employing MDSCs from mice deficient in Tr (MDSC-Tr −/−) were carried out. Compared to WT MDSCs, coculture of MDSC-Tr− /- with SB cells resulted in a significant reduction in the proliferation and immunosuppressive function of MDSCs. Moreover, following cocultured with SB cells, immunofluorescence analysis demonstrated that MDSC-Tr− / − had a reduction in the nuclear translocation of the NFkB subunit P65 compared to WT MDSCs. These data suggest that TRAIL-TR augments MDSC proliferation via NFkB. In conclusion, we have demonstrated that Tr− / − mice have a significant reduction in CCA tumor burden and MDSC infiltration. These results indicate that TRAIL-TR facilitates tumor immune escape and progression by fostering MDSC immunosuppressive function and infiltration, in an NFkB-dependent manner. Direct targeting of TRAIL on CCA cells is a potential anti-tumor strategy. Citation Format: Emilien Loeuillard, Jingchun Yang, Dong Haidong, Gregory Gores, Sumera Ilyas. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-TRAIL-R Mediates Cholangiocarcinoma Tumor Immune Evasion by Enhancing Myeloid-Derived Suppressive Cell Population [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO032.

Beyond BCL-2 Inhibition in Acute Myloid Leukemia: Other Approaches to Leverage the Apoptotic Pathway.

BCL-2 inhibition has transformed the therapeutic landscape of acute myeloid leukemia (AML) but is not curative for the majority of patients. Consequently, there has been growing interest in targeting other facets of the apoptotic machinery to improve outcomes. These approaches include targeting the intrinsic and extrinsic apoptotic pathway, inducing apoptosis via p53 activation, and others. Targeting the intrinsic apoptotic pathway includes MCL-1 antagonism and BCL-xL inhibition. MCL-1 can be targeted via direct inhibitors as well as via indirect mechanisms to downregulate MCL-1 including inhibition of cyclin dependent kinases and Nedd8 activating enzyme. The extrinsic apoptotic pathway could be harnessed via inhibition of inhibitor of apoptosis proteins, agonism of the TNF-related apoptosis-inducing ligand receptors and inhibiting FLICE-like inhibitor protein. Approaches inducing p53-mediated apoptosis are being evaluated using inhibitors of MDM2, dual inhibitor of MDM2/X in TP53 wild-type AML and p53 reactivators in TP53-mutant myeloid disorders. Several such agents are in early clinical development and rationale combinations of these agents may help improving outcomes for patients with AML.

Lewis glycosphingolipids as critical determinants of TRAIL sensitivity in cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death and contributes to tumor rejection by cytotoxic lymphocytes in cancer immunosurveillance and immunotherapy. TRAIL and TRAIL receptor agonists have garnered wide popularity as promising agents for cancer therapy. We previously demonstrated that the loss of fucosylation in cancer cells impairs TRAIL sensitivity; however, the precise structures of the fucosylated glycans that regulate TRAIL sensitivity and their carrier molecules remain elusive. Herein, we observed that Lewis glycans among various fucosylated glycans positively regulate TRAIL-induced cell death. Specifically, Lewis glycans on lacto/neolacto glycosphingolipids, but not glycoproteins including TRAIL receptors, enhanced TRAIL-induced formation of the cytosolic caspase 8 complex, without affecting the formation of the membranous receptor complex. Furthermore, type I Lewis glycan expression in colon cancer cell lines and patient-derived cancer organoids was positively correlated with TRAIL sensitivity. These findings provide novel insights into the regulatory mechanism of TRAIL-induced cell death and facilitate the identification of novel predictive biomarkers for TRAIL-related cancer therapies in future.

Role of TRAIL-R in Primary and Secondary Genital and Respiratory Chlamydia muridarum Infections in Mice.

ABSTRACTThe tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor (TRAIL-R) suppresses inflammation and could therefore affect the course of Chlamydia infections and their long-term sequelae. Wild-type (WT) and TRAIL-R−/− C57BL/6 mice were inoculated vaginally with Chlamydia muridarum; the course of the infection was followed with vaginal cultures and the presence of hydrosalpinx determined. To evaluate the role of TRAIL-R following a secondary infection, the mice were vaginally reinfected. WT and TRAIL-R−/− male mice were also infected and reinfected in the respiratory tract, and the course of the diseases and the infections were followed. Following the primary and secondary vaginal infection, no significant differences in vaginal shedding or hydrosalpinx formation were observed between the WT and TRAIL-R−/− mice. The WT and TRAIL-R−/− mice mounted antibody responses in serum and vaginal washes that were not significantly different. After the primary and secondary intranasal infections of the male mice, changes in body weight were determined, and no significant differences were observed between the WT and TRAIL-R−/− mice. Ten days after the primary and the secondary infections, the weight of the lungs and number of C. muridarum inclusion forming units (IFU) were determined. The lungs of the WT mice weighed less compared with the TRAIL-R−/− mice following a primary infection but not after a secondary infection. No differences in the number of C. muridarum IFU in the lungs were observed between the two groups of mice. In conclusion, despite playing a role in inflammation cell-signaling pathways in vitro, TRAIL-R does not appear to play a major role in the susceptibility, clinical outcomes, or long-term sequelae of C. muridarum infections in vivo.IMPORTANCE TNF-related apoptosis-inducing ligand receptor (TRAIL-R) is involved in suppressing inflammatory responses. Bacterial pathogens such as Chlamydia spp. elicit inflammatory responses in humans following genital, ocular, and respiratory infections. The inflammatory responses are important to control the spread of Chlamydia. However, in certain instances, these inflammatory responses can produce long-term sequelae, including fibrosis. Fibrosis, or scarring, in the genital tract, eye, and respiratory system results in functional deficiencies, including infertility, blindness, and chronic obstructive lung disease, respectively. The goal of this study was to determine if mice deficient in TRAIL-R infected in the genital and respiratory tracts with Chlamydia spp. suffer more or less severe infections, infertility, or lung diseases than wild-type mice. Our results show no differences between the immune responses, infection severity, and long-term sequelae between TRAIL-R knockout and wild-type animals following a genital or a respiratory infection with Chlamydia.

Proscillaridin A Sensitizes Human Colon Cancer Cells to TRAIL-Induced Cell Death.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces cancer cell death by binding to TRAIL receptors. Because of its selective cytotoxicity toward cancer cells, TRAIL therapeutics, such as recombinant TRAIL and agonistic antibodies targeting TRAIL receptors, have garnered attention as promising cancer treatment agents. However, many cancer cells acquire resistance to TRAIL-induced cell death. To overcome this issue, we searched for agents to sensitize cancer cells to TRAIL-induced cell death by screening a small-molecule chemical library consisting of diverse compounds. We identified a cardiac glycoside, proscillaridin A, as the most effective TRAIL sensitizer in colon cancer cells. Proscillaridin A synergistically enhanced TRAIL-induced cell death in TRAIL-sensitive and -resistant colon cancer cells. Additionally, proscillaridin A enhanced cell death in cells treated with TRAIL and TRAIL sensitizer, the second mitochondria-derived activator of caspase mimetic. Proscillaridin A upregulated TRAIL receptor expression, while downregulating the levels of the anti-cell death molecules, cellular FADD-like IL-1β converting enzyme-like inhibitor protein and Mcl1, in a cell type-dependent manner. Furthermore, proscillaridin A enhanced TRAIL-induced cell death partly via O-glycosylation. Taken together, our findings suggest that proscillaridin A is a promising agent that enhances the anti-cancer efficacy of TRAIL therapeutics.

Selective modulation of cell surface proteins during vaccinia infection: A resource for identifying viral immune evasion strategies.

The interaction between immune cells and virus-infected targets involves multiple plasma membrane (PM) proteins. A systematic study of PM protein modulation by vaccinia virus (VACV), the paradigm of host regulation, has the potential to reveal not only novel viral immune evasion mechanisms, but also novel factors critical in host immunity. Here, >1000 PM proteins were quantified throughout VACV infection, revealing selective downregulation of known T and NK cell ligands including HLA-C, downregulation of cytokine receptors including IFNAR2, IL-6ST and IL-10RB, and rapid inhibition of expression of certain protocadherins and ephrins, candidate activating immune ligands. Downregulation of most PM proteins occurred via a proteasome-independent mechanism. Upregulated proteins included a decoy receptor for TRAIL. Twenty VACV-encoded PM proteins were identified, of which five were not recognised previously as such. Collectively, this dataset constitutes a valuable resource for future studies on antiviral immunity, host-pathogen interaction, poxvirus biology, vector-based vaccine design and oncolytic therapy.

Abstract 1315: Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma

Abstract Background and Aims: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable attention as a potential anti-cancer strategy, TRAIL agonists have had very limited anti-cancer activity in humans. TRAIL signaling can attenuate the T lymphocyte response. However, this potential immunosuppressive function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, has a dense immunosuppressive microenvironment. Accordingly, CCA provides a model to examine the potential immune regulatory function of TRAIL in cancer biology. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both Trail and Trail receptor (Tr) were implanted into livers of WT and Tr-/- mice. Hence, in this model the host immune cells express Trail but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr-/- mice had a significant reduction in tumor burden compared to WT mice. Moreover, tumor bearing Tr-/- mice had a significant increase in cytotoxic T lymphocytes (CTLs) and enhanced CTL effector function. Myeloid-derived suppressor cells (MDSCs) were significantly decreased in Tr-/- tumors compared to WT tumors. Furthermore, implantation of SB cells devoid of Trail (SB-Trail-/-) into WT mice resulted in a significant reduction in tumor burden and MDSC infiltration. In vitro functional studies employing SB cells deficient in Tr (SB-Tr-/-) and MDSCs were carried out. There was a significant reduction in proliferation and immunosuppressive function of MDSCs when cocultured with SB-Tr-/- compared to SB cells. These results indicate that TRAIL-TR fosters MDSC growth and immunosuppressive function. In conclusion, we have demonstrated that Tr-/- mice have a significant reduction in CCA tumor burden and MDSC infiltration. Consequently, Tr-/- tumors have enhanced CTL infiltration and function. These data suggest that the TRAIL-TR system mediates tumors immune evasion via MDSCs, and direct targeting of TRAIL on CCA cells is a potential anti-tumor strategy. Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera I. Ilyas. Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1315.

Abstract 2783: Increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression in normal colonic mucosa with imipridone ONC201/TIC10 treatment: A potential biomarker for chemoprevention studies

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States and currently the most effective method at reducing risk of death is through screening. Although these strategies have reduced the incidence of CRC, they are invasive, costly, and have low rates of compliance. Chemoprevention aims to prevent or delay the onset of CRC through eradication or prevention of precursor colonic adenomas, ideally with affordable and nontoxic drugs. The imipridone ONC201, which induces apoptosis, in part, through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), is currently undergoing preclinical evaluation as a potential chemopreventive agent. In preclinical models, we found that treatment with ONC201 is capable of increasing TRAIL expression in normal cells and can improve tumor suppression through immune surveillance by the innate immune system. To further assess potential biomarkers of response to ONC201, we evaluated expression of TRAIL and death receptor 5 (DR5), as well as markers related to cellular stemness such as LGR5 in colonic mucosa obtained from mice treated with ONC201 and vehicle controls. In this study, 16-week-old female C57BL/6 mice were administered vehicle or ONC201 50mg/kg (n=11) body weight by oral gavage twice a week for a total of five doses. Both immunohistochemistry and western blotting were used to evaluate differential expression of various potential biomarkers in both fresh frozen and FFPE colonic tissue. Using western blotting we found a significant increase in TRAIL expression in normal colonic mucosa obtained from mice treated with ONC201 as compared to vehicle controls. This finding was further validated using immunohistochemistry. Other markers, such as DR5 appeared unaffected with treatment. As efforts are currently underway to develop a CRC chemoprevention trial using ONC201, TRAIL may represent a biomarker of interest. Further analysis of stemness markers, as well as serum analyses are currently in process and may reveal other potential biomarkers for use in chemoprevention trial development. Citation Format: Alexander G. Raufi, Arielle De La Cruz, Andrew George, Sean Hacking, Venkateshwar Madka, Varun Prabhu, Howard Safran, Lanlan Zhou, Dean Brenner, Chinthalapally V. Rao, Wafik S. El-Deiry. Increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression in normal colonic mucosa with imipridone ONC201/TIC10 treatment: A potential biomarker for chemoprevention studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2783.

Abstract 3147: Natural killer cells mediate tumor-inhibitory functions of USP6 in Ewing sarcoma

Abstract INTRODUCTION: Understanding how Natural Killer (NK) cell activity is regulated by cancer cells is critical for developing NK-cell based immunotherapies. Our studies show that the deubiquitinating enzyme ubiquitin-specific protease-6 (USP6) can induce an immune-stimulatory hot tumor microenvironment in Ewing sarcoma (ES), the 2nd most common pediatric bone cancer. High USP6 expression in ES is associated with significantly improved overall and progression-free survival. We found that USP6 expression in ES cells inhibits xenograft growth in nude mice, but not NSG mice, implicating a role for the innate immune system, particularly cytotoxic NK cells. Notably, USP6 expression enhanced intra-tumoral abundance, activation, and proliferative capacity of NK cells. In vitro cytotoxicity assays confirm that USP6 can directly augment the ability of ES cells to activate NK cells. Based on these observations, we hypothesize that NK cells are essential for USP6-mediated tumor suppression and seek to determine the mechanism by which their cytolytic function is activated by USP6. METHOD: We used ES cell lines A673 and RD-ES expressing USP6 in a doxycycline (dox)-inducible manner, the immortalized human NK cell line NK-92, and primary human and mouse NK cells. Athymic nude or RAG2-/- mice were xenografted subcutaneously with ES cell lines and fed dox-infused water to induce USP6. Immune cell abundance and activation were monitored both in tumors and peripheral blood by flow cytometry. For in vivo depletion of NK cells, 300ug of anti-NK1.1 (PK136) ab was injected i.p. into RAG2-/- mice one day prior to ES xenografting, then once weekly thereafter. RESULTS: USP6 induces in ES cells the surface expression of multiple NK activating ligands, as well as receptors for the apoptotic factor TRAIL (DR5) and Type I/II interferons (IFNAR1 and IFNGR1), both in vitro and in vivo. This leads to enhanced NK activation and renders ES cells hypersensitive to NK-mediated cytotoxicity. IFNy released by activated NK cells feeds back on the USP6-expressing ES cells, inducing synergistic expression of multiple IFNy-inducible genes, including chemokines CXCL9/10 (chemoattractants for NK cells) and ICAM-1 (essential for immunological synapse formation between NK cells and their targets). A potent feed forward loop for NK activation/ES cell killing is thus generated. Concordantly, in RAG2-/- mice, USP6 induces increased intratumoral infiltration and activation of NK cells, and also, strikingly, systemic activation of NK cells. Depletion of NK cells using anti-NK1.1(PK136) completely abrogates USP6-mediated inhibition of ES xenograft growth. CONCLUSION: NK cells are essential for mediating the tumor inhibitory functions of USP6 in ES. We posit that USP6’s ability to activate NK cells both intratumorally and systemically underlies its association with improved ES patient prognosis, which we seek to exploit for therapeutic benefit in future studies. Citation Format: Kanika Jain, Ian C. Henrich, Laura Nicole Quick, Robert A. Young, Margaret M. Chou. Natural killer cells mediate tumor-inhibitory functions of USP6 in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3147.

Abstract 5607: Combining PD-1 inhibition with the TRAIL receptor agonist TLY012 promotes pancreatic ductal adenocarcinoma tumor regression in an immunocompetent mouse model

Abstract Introduction: An immunosuppressed, apoptosis-resistant phenotype characterizes pancreatic ductal adenocarcinoma (PDAC). Both inflammation and tumorigenesis are influenced by the Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) pathway. TLY012 is pegylated recombinant TRAIL protein and is an orphan drug for systemic sclerosis and chronic pancreatitis. The potential synergy between TLY012 and anti-mouse PD-1 antibody was investigated in a syngeneic immunocompetent heterotopic mouse model. Methods: KPC-luc PDAC cells were subcutaneously implanted into C57Bl/6 mice. Treatment combined 10 mg/kg TLY012 three times per week with 10 mg/kg anti-mouse PD-1 antibody twice weekly and were compared to individual treatment and control groups. Cytokine profiling via 32-plex Luminex immunoassay was completed after 7 days and after three months of treatment. Flow cytometry at three months evaluated NK and T cell profiles in the spleen and tumors. Results: PDAC tumor-bearing mice treated with the combination of anti-mouse PD-1 and TLY012 had slower tumor growth and reduced tumor volume at 70 days compared to either drug alone (all p<0.005). B-cell activating factor, which can be a marker of PDAC tumor progression, was decreased in single and dual treatment groups at three months of treatment. This factor showed the earliest decrease in the dual treatment group, with a cytokine level 44% of that of control mice at 7 days. Long-term dual treatment mice also had the highest levels of proinflammatory cytokines interferon gamma (average 5.6 times control level, p=0.046), CCL5 (average 14.1 times control level, p=0.048), and interleukin-3 (IL-3, average 71.1 times control level, p=0.0053). Flow cytometry showed trends toward decreased circulating regulatory T cells, increased NK cells, and a higher proportion of CD8+ T cells within tumors in the dual treatment group. Conclusions: The combination of anti-mouse PD-1 and TLY012 prevents growth of PDAC in an immunocompetent mouse model. The combination also increases tumor-infiltrating CD8+ T cells, decreases circulating Treg cells and alters cytokine expression. CCL5 is a T-cell chemokine, and both interferon gamma and IL-3 are produced by activated T cells and macrophages, and have proinflammatory, antitumor effects. Combining TLY012 and anti-mouse PD-1 creates these changes in immune cell and cytokine levels, suggesting induction of a more proinflammatory immune environment. The proinflammatory state may contribute to decreased tumor growth. Citation Format: Anna D. Louie, Kelsey E. Huntington, Young Lee, Jared Mompoint, Jinxuan Wu, Seulki Lee, Wafik S. El-Deiry. Combining PD-1 inhibition with the TRAIL receptor agonist TLY012 promotes pancreatic ductal adenocarcinoma tumor regression in an immunocompetent mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5607.

Abstract 3577: First-in-human phase I study of intraperitoneally administered interferon-activated autologous monocytes in platinum-resistant ovarian cancer

Abstract Background: Ovarian cancer is the most lethal gynecologic cancer and is intrinsically resistant to immunotherapies. Previous work has demonstrated superior response rates with intraperitoneally (IP) directed therapies, providing the rationale for IP immunotherapy. Methods: A previously published model system in which OVCAR ovarian cancer spheroids are killed by IFN-activated human monocytes was further characterized using confocal microscopy, global gene expression analysis, flow cytometry, and knockout/knockdown of key genes in the TRAIL-DR4/DR5 pathway. These results were translated into a phase I dose escalation trial in which 3-6 patients with platinum-resistant or refractory ovarian cancer were enrolled into 4 cohorts and treated IP q28 days with Peginterferon alfa-2b (25-250 mcg) and Interferon gamma-1b (5-50 mcg), with or without autologous monocytes (75-750 x 106 cells), in order to determine the recommended phase II dose (RP2D). Results: Confocal microscopy of IFN-activated monocytes demonstrated migration into OVCAR spheroids and destruction of the spheroids and global gene expression analysis identified TRAIL as a ligand on monocytes upregulated by IFN treatment. Disruption of TRAIL receptors DR4/DR5 or deletion of CASPASE8 on OVCAR cells largely abrogated killing. IFN-activated human monocytes were able to kill IP-injected OVCAR tumor cells in a mouse xenograft model, and IFN-activated monocytes from ovarian cancer patients were able to kill OVCAR spheroids as effectively as those from healthy donors. 18 patients were subsequently enrolled on a phase I trial (median age, 61 years; median 5 prior therapies). 1 of 3 patients at the second dose level experienced a dose-limiting toxicity (grade 3 anemia) and no subsequent DLTs were observed. The RP2D was defined as 250mcg IFNa/50mcg IFNg/750 x 106 cells. The only treatment-related grade 3 or higher adverse events occurring in more than one patient were lymphocyte decrease (33.3%) and abdominal pain (11.1%). The best response observed was partial response (PR) in 2/9 RECIST-evaluable patients, and 1 additional patient had a CA-125 response with >50% reduction. 4/9 patients had stable disease. Four patients were able to receive 5+ cycles (range 1-10 cycles). Biomarker analyses demonstrated that these long-term responders had a lower level of T-regulatory cells at baseline, but exhibited marked increase in myeloid derived suppressor cells (MDSC) at the time of tumor progression. Conclusion: These findings characterize the mechanism of a novel cellular immunotherapy for ovarian cancer which is well tolerated with evidence of clinical activity in a heavily pretreated patient population. This therapy may serve as the backbone for a combination immunotherapy regimen incorporating additional agents targeting T-regulatory cells and/or MDSCs. Citation Format: Christopher B. Cole, Daniel S. Green, Franklin Ning, Kathryn Zoon, Lindsey B. Rosen, Steven M. Holland, Christina M. Annunziata. First-in-human phase I study of intraperitoneally administered interferon-activated autologous monocytes in platinum-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3577.

P-123 A phase Ia/Ib, open-label, dose-escalation study of the TRAILR2 agonist BI 905711 in combination with chemotherapy in patients with advanced gastrointestinal cancers

Activation of the tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAILR2) induces apoptosis via the extrinsic pathway. Targeting TRAILR2 is therefore an attractive therapeutic strategy in oncology, but some early TRAILR2 agonists showed limited clinical success due to low efficacy or severe hepatotoxicity. The tetravalent bispecific antibody BI 905711 represents a novel, liver-sparing, TRAILR2 agonist, which cross-links TRAILR2 with cadherin 17 (CDH17) to induce CDH17-dependent TRAILR2 oligomerisation. CDH17, a membrane protein highly expressed in gastrointestinal (GI) cancers, is not expressed in normal hepatocytes. Thus, using CDH17 as a liver-sparing anchor may avoid hepatotoxicity while increasing selectivity to cancer cells. In preclinical models of GI cancer, including colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC), BI 905711 showed activity both as a single agent and in combination with chemotherapeutic agents (Garcia-Martinez et al., Mol Cancer Ther 2021;20:96–108). Preclinical studies also showed synergism of BI 905711 and SoC chemotherapy, particularly irinotecan. This phase Ia/Ib open-label, multicentre study (NCT05087992) aims to determine the maximum tolerated dose (MTD) and recommended dose for expansion (RDE), pharmacokinetics (PK), and preliminary efficacy of BI 905711 in combination with chemotherapy (FOLFIRI; irinotecan, leucovorin, and fluorouracil) with or without bevacizumab in patients with advanced, refractory GI cancers. Up to 100 patients with histologically or cytologically confirmed, advanced, unresectable or metastatic GI cancers will be enrolled. In phase Ia, approximately 20 patients with CRC will receive intravenous BI 905711 at escalating doses (starting dose 0.6 mg/kg) on day 3 of 14-day cycles, plus FOLFIRI and bevacizumab (5 mg/kg IV infusion over 30 mins) on day 1. BI 905711 dose escalation will be guided by a Bayesian logistic regression model with overdose control based on dose-limiting toxicities (DLTs) during the MTD evaluation period (first two 14-day cycles). BI 905711 will be administered until disease progression, unacceptable toxicities or another reason requiring treatment discontinuation. In phase Ib, patients with CRC or PDAC will be enrolled to one of two dose expansion cohorts. In the CRC cohort, patients who have progressed after prior oxaliplatin-based therapy will be randomised in a 2:1 ratio to receive FOLFIRI and bevazicumab with or without BI 905711. In the PDAC cohort, patients with CDH17-positive PDAC who have progressed after prior platin/gemcitabine-based first-line therapy will receive FOLFIRI plus BI 905711. In both cohorts, BI 905711 will be administered at the RDE determined in phase Ia. A safety run-in will be included in the PDAC cohort to confirm the RDE defined in phase Ia prior to enrolling further patients. Primary endpoints are the MTD based on the occurrence of DLTs (phase Ia), and confirmed objective response (OR) according to RECIST 1.1 (phase Ib). Secondary endpoints include PK assessments (phase Ia), progression-free survival, tumour shrinkage, duration of OR and disease control (phase Ib). Further endpoints include assessment of immunogenic response and pharmacodynamic biomarker modulation (including caspase activation and changes in circulating tumour DNA levels). Patient recruitment is active and the study is ongoing. NCT05087992. Medical writing assistance, supported financially by Boehringer Ingelheim Pharmaceuticals Inc., was provided by Jane Saunders of Ashfield MedComms, an Ashfield Health Company, during the preparation of this abstract. The authors received no direct payment. Boehringer Ingelheim.

Eftozanermin alfa (ABBV-621) monotherapy in patients with previously treated solid tumors: findings of a phase 1, first-in-human study

Eftozanermin alfa (eftoza), a second-generation tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAIL-R) agonist, induces apoptosis in tumor cells by activation of death receptors 4/5. This phase 1 dose-escalation/dose-optimization study evaluated the safety, pharmacokinetics, pharmacodynamics, and preliminary activity of eftoza in patients with advanced solid tumors. Patients received eftoza 2.5–15 mg/kg intravenously on day 1 or day 1/day 8 every 21 days in the dose-escalation phase, and 1.25–7.5 mg/kg once-weekly (QW) in the dose-optimization phase. Dose-limiting toxicities (DLTs) were evaluated during the first treatment cycle to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Pharmacodynamic effects were evaluated in circulation and tumor tissue. A total of 105 patients were enrolled in the study (dose-escalation cohort, n = 57; dose-optimization cohort, n = 48 patients [n = 24, colorectal cancer (CRC); n = 24, pancreatic cancer (PaCA)]). In the dose-escalation cohort, seven patients experienced DLTs. MTD and RP2D were not determined. Most common treatment-related adverse events were increased alanine aminotransferase and aspartate aminotransferase levels, nausea, and fatigue. The one treatment-related death occurred due to respiratory failure. In the dose-optimization cohort, three patients (CRC, n = 2; PaCA, n = 1) had a partial response. Target engagement with regard to receptor saturation, and downstream apoptotic pathway activation in circulation and tumor were observed. Eftoza had acceptable safety, evidence of pharmacodynamic effects, and preliminary anticancer activity. The 7.5-mg/kg QW regimen was selected for future studies on the basis of safety findings, pharmacodynamic effects, and biomarker modulations. (Trial registration number: NCT03082209 (registered: March 17, 2017)).

T Cell-Specific Deletion of TRAIL Receptor Reveals Its Critical Role for Regulating Pathologic T Cell Activation and Disease Induction in Experimental Autoimmune Encephalomyelitis.

Recent evidence from several autoimmune animal models has demonstrated that TRAIL suppresses the activation of T cells and inhibits autoimmune inflammation via an apoptosis-independent pathway. However, it remains unclear whether the immunosuppressive effects of TRAIL are dependent on its direct effects on T cells or on other immune cells to regulate T cells for the induction of disease. Therefore, we generated mice with T cell-specific TRAIL receptor (TRAIL-R) conditional knockout to investigate the impact of TRAIL on autoimmune inflammation and disease induction in experimental autoimmune encephalomyelitis (EAE). T cell-specific TRAIL-R knockout mice were found to completely reverse the TRAIL-mediated suppression of inflammation and disease induction, indicating that TRAIL-R on T cells is essential for TRAIL-mediated suppression of inflammation and disease induction in EAE. Moreover, the immune suppression effects were not due to the induction of cell apoptosis, but to the direct inhibition of T cell activation. In addition, RNA sequencing and transcriptome analysis revealed that TRAIL-R signaling significantly downregulated the genes involved in TCR signaling pathways, T cell differentiation, and proinflammatory cytokines. These results indicate that TRAIL-R on T cells is critical for pathologic T cell activation and induction of inflammation in EAE, suggesting that TRAIL-R serves as a novel immune checkpoint receptor in T cell-mediated autoimmune diseases.

Toxoplasma gondii infection induces cell apoptosis via multiple pathways revealed by transcriptome analysis

Toxoplasma gondii is a worldwide parasite that can infect almost all kinds of mammals and cause fatal toxoplasmosis in immunocompromised patients. Apoptosis is one of the principal strategies of host cells to clear pathogens and maintain organismal homeostasis, but the mechanism of cell apoptosis induced by T. gondii remains obscure. To explore the apoptosis influenced by T. gondii, Vero cells infected or uninfected with the parasite were subjected to apoptosis detection and subsequent dual RNA sequencing (RNA-seq). Using high-throughput Illumina sequencing and bioinformatics analysis, we found that pro-apoptosis genes such as DNA damage-inducible transcript 3 (DDIT3), growth arrest and DNA damage-inducible α (GADD45A), caspase-3 (CASP3), and high-temperature requirement protease A2 (HtrA2) were upregulated, and anti-apoptosis genes such as poly(adenosine diphosphate (ADP)-ribose) polymerase family member 3 (PARP3), B-cell lymphoma 2 (Bcl-2), and baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5) were downregulated. Besides, tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1), TRAF2, TNF receptor superfamily member 10b (TNFRSF10b), disabled homolog 2 (DAB2)‍-interacting protein (DAB2IP), and inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) were enriched in the upstream of TNF, TNF-related apoptosis-inducing ligand (TRAIL), and endoplasmic reticulum (ER) stress pathways, and TRAIL-receptor 2 (TRAIL-R2) was regarded as an important membrane receptor influenced by T. gondii that had not been previously considered. In conclusion, the T. gondii RH strain could promote and mediate apoptosis through multiple pathways mentioned above in Vero cells. Our findings improve the understanding of the T. gondii infection process through providing new insights into the related cellular apoptosis mechanisms.

CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity

The cellular processes that govern tumor resistance to immunotherapy remain poorly understood. To gain insight into these processes, here we perform a genome-scale CRISPR activation screen for genes that enable human melanoma cells to evade cytotoxic T cell killing. Overexpression of four top candidate genes (CD274 (PD-L1), MCL1, JUNB, and B3GNT2) conferred resistance in diverse cancer cell types and mouse xenografts. By investigating the resistance mechanisms, we find that MCL1 and JUNB modulate the mitochondrial apoptosis pathway. JUNB encodes a transcription factor that downregulates FasL and TRAIL receptors, upregulates the MCL1 relative BCL2A1, and activates the NF-κB pathway. B3GNT2 encodes a poly-N-acetyllactosamine synthase that targets >10 ligands and receptors to disrupt interactions between tumor and T cells and reduce T cell activation. Inhibition of candidate genes sensitized tumor models to T cell cytotoxicity. Our results demonstrate that systematic gain-of-function screening can elucidate resistance pathways and identify potential targets for cancer immunotherapy.