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Programmed cell death 1 ligand 1 (PD-L1) is a key driver of tumor-mediated immune suppression, and targeting it with antibodies can induce therapeutic responses. Given the costs and associated toxicity of PD-L1 blockade, alternative therapeutic strategies are needed. Using reverse-phase protein arrays to assess drugs in use or likely to enter trials, we performed a candidate drug screen for inhibitors of PD-L1 expression and identified verteporfin as a possible small-molecule inhibitor. Verteporfin suppressed basal and IFN-induced PD-L1 expression in vitro and in vivo through Golgi-related autophagy and disruption of the STAT1–IRF1–TRIM28 signaling cascade, but did not affect the proinflammatory CIITA-MHC II cascade. Within the tumor microenvironment, verteporfin inhibited PD-L1 expression, which associated with enhanced T-lymphocyte infiltration. Inhibition of chromatin-associated enzyme PARP1 induced PD-L1 expression in high endothelial venules (HEV) in tumors and, when combined with verteporfin, enhanced therapeutic efficacy. Thus, verteporfin effectively targets PD-L1 through transcriptional and posttranslational mechanisms, representing an alternative therapeutic strategy for targeting PD-L1.

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Figure S1. Aspirin and Aspirin-PC does not block the release of TGF from platelets following co-culture with colon cancer cells. Figure S2. Aspirin and Aspirin-PC block platelet induced upregulation of the EMT marker SNAIL in MC-26 cells. Figure S3. Aspirin and Aspirin-PC decrease collagen-induced activation of platelets obtained from the AOM/DSS cancer model. Figure S4. Time course of aspirin (ASA) test drugs on body weight in AOM/DSS mouse colon cancer model. Figure S5. Aspirin and Aspirin-PC do not alter changes in body weight induced by AOM/DSS. Figure S6. Effect of aspirin (ASA) test drugs on fecal hemoglobin in AOM/DSS mouse cancer model. Figure S7. Effect of aspirin (ASA) test drugs on hematocrit in AOM/DSS mouse cancer model.

Figure S-1. Chemical structures of aspirin and the predominant phospholipid in soybean phosphatidylcholine (PC).Figure S-2. Evaluation of the effects of aspirin-PC and aspirin on tube-formation. Figure S-3. In vitro evidence that the growth inhibitory activity of aspirin-PC and aspirin of ovarian cancer cell lines.

Figure legends for Supplementary Figures 1-7.

Figure S5 shows heat maps from brain immune cells.

Table S1 shows KINOMEscan profiling of WP1066.

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Purpose:Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma.Experimental Design:C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood–brain barrier–penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment.Results:The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control P < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions.Conclusions:This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.

Supplementary Figure 8 from Deubiquitinase Inhibition by Small-Molecule WP1130 Triggers Aggresome Formation and Tumor Cell Apoptosis

Supplementary Figure 5 from Deubiquitinase Inhibition by Small-Molecule WP1130 Triggers Aggresome Formation and Tumor Cell Apoptosis

Supplementary Figure 2 from Deubiquitinase Inhibition by Small-Molecule WP1130 Triggers Aggresome Formation and Tumor Cell Apoptosis

Supplementary Figure 1 from Deubiquitinase Inhibition by Small-Molecule WP1130 Triggers Aggresome Formation and Tumor Cell Apoptosis

Supplementary Figure 3 from Deubiquitinase Inhibition by Small-Molecule WP1130 Triggers Aggresome Formation and Tumor Cell Apoptosis

Supplementary Figure 7 from Deubiquitinase Inhibition by Small-Molecule WP1130 Triggers Aggresome Formation and Tumor Cell Apoptosis

Recent evidence suggests that several deubiquitinases (DUB) are overexpressed or activated in tumor cells and many contribute to the transformed phenotype. Agents with DUB inhibitory activity may therefore have therapeutic value. In this study, we describe the mechanism of action of WP1130, a small molecule derived from a compound with Janus-activated kinase 2 (JAK2) kinase inhibitory activity. WP1130 induces rapid accumulation of polyubiquitinated (K48/K63-linked) proteins into juxtanuclear aggresomes, without affecting 20S proteasome activity. WP1130 acts as a partly selective DUB inhibitor, directly inhibiting DUB activity of USP9x, USP5, USP14, and UCH37, which are known to regulate survival protein stability and 26S proteasome function. WP1130-mediated inhibition of tumor-activated DUBs results in downregulation of antiapoptotic and upregulation of proapoptotic proteins, such as MCL-1 and p53. Our results show that chemical modification of a previously described JAK2 inhibitor results in the unexpected discovery of a novel DUB inhibitor with a unique antitumor mechanism. Cancer Res; 70(22); 9265–76. ©2010 AACR.

Supplementary Figure 6 from Deubiquitinase Inhibition by Small-Molecule WP1130 Triggers Aggresome Formation and Tumor Cell Apoptosis

Supplementary Figure 4 from Deubiquitinase Inhibition by Small-Molecule WP1130 Triggers Aggresome Formation and Tumor Cell Apoptosis

Background: Chimeric antigen receptor (CAR) T cells have recently been demonstrated to extract and express cognate tumor antigens through trogocytosis. This process may contribute to tumor antigen escape, T cell exhaustion, and fratricide, which plays a central role in CAR dysfunction. We sought to evaluate the importance of this effect in epidermal growth factor receptor variant III (EGFRvIII) specific CAR T cells targeting glioma. Methods: EGFRvIII-specific CAR T cells were generated from various donors and analyzed for cytotoxicity, trogocytosis, and in vivo therapeutic activity against intracranial glioma. Tumor autophagy resulting from CAR T cell activity was evaluated in combination with an autophagy inducer (verteporfin) or inhibitor (bafilomycin A1). Results: CAR T cell products derived from different donors induced markedly divergent levels of trogocytosis of tumor antigen as well as PD-L1 upon engaging target tumor cells correlating with variability in efficacy in mice. Pharmacological facilitation of CAR induced-autophagy with verteporfin inhibits trogocytic expression of tumor antigen on CARs and increases CAR persistence and efficacy in mice. Conclusion: These data propose CAR-induced autophagy as a mechanism counteracting CAR-induced trogocytosis and provide a new strategy to innovate high-performance CARs through pharmacological facilitation of T cell-induced tumor death.

Purpose: The blood–brain barrier (BBB) inhibits adequate dosing/penetration of therapeutic agents to malignancies in the brain. Low-intensity pulsed ultrasound (LIPU) is a safe therapeutic method of temporary BBB disruption (BBBD) to enhance chemotherapeutic delivery to the tumor and surrounding brain parenchyma for treatment of glioblastoma. Experimental Design: We investigated if LIPU could enhance therapeutic efficacy of anti–PD-1 in C57BL/6 mice bearing intracranial GL261 gliomas, epidermal growth factor receptor variant III (EGFRvIII) chimeric antigen receptor (CAR) T cells in NSG mice with EGFRvIII-U87 gliomas, and a genetically engineered antigen-presenting cell (APC)-based therapy producing the T-cell attracting chemokine CXCL10 in the GL261-bearing mice. Results: Mice treated with anti–PD-1 and LIPU-induced BBBD had a median survival duration of 58 days compared with 39 days for mice treated with anti–PD-1, and long-term survivors all remained alive after contralateral hemisphere rechallenge. CAR T-cell administration with LIPU-induced BBBD resulted in significant increases in CAR T-cell delivery to the CNS after 24 (P < 0.005) and 72 (P < 0.001) hours and increased median survival by greater than 129%, in comparison with CAR T cells alone. Local deposition of CXCL10-secreting APCs in the glioma microenvironment with LIPU enhanced T-cell glioma infiltration during the therapeutic window (P = 0.004) and markedly enhanced survival (P < 0.05). Conclusions: LIPU increases immune therapeutic delivery to the tumor microenvironment with an associated increase in survival and is an emerging technique for enhancing novel therapies in the brain.

Purpose: Activation of STING (stimulator of interferon genes) can trigger a robust, innate antitumor immune response in immunologically “cold” tumors such as glioblastoma. Patients and Methods: A small-molecule STING agonist, IACS-8779, was stereotactically administered using intraoperative navigation intratumorally in dogs with spontaneously arising glioblastoma. The phase I trial used an escalating dose design, ascending through four dose levels (5–20 μg). Treatment was repeated every 4–6 weeks for a minimum of two cycles. Radiographic response to treatment was determined by response assessment in neuro-oncology (RANO) criteria applied to isovoxel postcontrast T1-weighted MR images obtained on a single 3T magnet. Results: Six dogs were enrolled and completed ≥1 cycle of treatment. One dog was determined to have an abscess and was removed from further analysis. One procedure-related fatality was observed. Radiographic responses were dose dependent after the first cycle. The first subject had progressive disease, whereas there was 25% volumetric reduction in one subject and greater than 50% in the remaining surviving subjects. The median progression-free survival time was 14 weeks (range: 0–22 weeks), and the median overall survival time was 32 weeks (range: 11–39 weeks). Conclusions: Intratumoral STING agonist (IACS-8779) administration was well tolerated in dogs with glioblastoma to a dose of 15 μg. Higher doses of IACS-8779 were associated with radiographic responses.

Simple Summary Glioblastoma is one of the most treatment-refractory human malignancies, and despite techniques that have allowed scientists and clinicians to better understand the molecular underpinnings of resistance, little progress has been made in improving the survival of patients with glioblastoma. We posit that dysregulated Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling represents one major hub of tumorigenesis and resistance to medical therapies and that clinical study of its targeted inhibition is warranted, as well as highlighting the lessons learned from historical investigation going forward. Abstract Glioblastoma remains one of the deadliest and treatment-refractory human malignancies in large part due to its diffusely infiltrative nature, molecular heterogeneity, and capacity for immune escape. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway contributes substantively to a wide variety of protumorigenic functions, including proliferation, anti-apoptosis, angiogenesis, stem cell maintenance, and immune suppression. We review the current state of knowledge regarding the biological role of JAK/STAT signaling in glioblastoma, therapeutic strategies, and future directions for the field.

Simple Summary MicroRNAs can silence a broad set of target genes that may benefit heterogeneous tumors like glioblastoma. We have previously shown that osteopontin has an oncogenic role and may have immune modulatory effects on macrophages. In the current study, we used miRNAs to target osteopontin in tumor cells and modulate immune cells to elicit an antitumor effect. Intravenous delivery of miR-181a to immune competent mice bearing intracranial glioblastoma demonstrated a 22% increase in median survival duration relative to that of control mice. The overexpression of miR-181a in tumor cells led to decreased OPN production and proliferation and increased apoptosis in vitro, and increased survival duration of the mice when compared to its controls. miR-181a controls osteopontin expression in tumor cells by regulating their proliferation and apoptosis. Abstract MiRNAs can silence a wide range of genes, which may be an advantage for targeting heterogenous tumors like glioblastoma. Osteopontin (OPN) plays both an oncogenic role in a variety of cancers and can immune modulate macrophages. We conducted a genome wide profiling and bioinformatic analysis to identify miR-181a/b/c/d as potential miRNAs that target OPN. Luciferase assays confirmed the binding potential of miRNAs to OPN. Expression levels of miR-181a/b/c/d and OPN were evaluated by using quantitative real-time PCR and enzyme-linked immunosorbent assay in mouse and human glioblastomas and macrophages that showed these miRNAs were downregulated in Glioblastoma associated CD11b+ cells compared to their matched blood CD14b+ cells. miRNA mimicking and overexpression using lentiviruses showed that MiR-181a overexpression in glioblastoma cells led to decreased OPN production and proliferation and increased apoptosis in vitro. MiR-181a treatment of immune competent mice bearing intracranial glioblastoma demonstrated a 22% increase in median survival duration relative to that of control mice.

Background Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and sulindac are effective for colorectal cancer prevention in humans and some animal models, but concerns over gastro-intestinal (GI) ulceration and bleeding limit their potential for chemopreventive use in broader populations. Recently, the combination of aspirin with a phospholipid, packaged as PL-ASA, was shown to reduce GI toxicity in a small clinical trial. However, these studies were done for relatively short periods of time. Since prolonged, regular use is needed for chemopreventive benefit, it is important to know whether GI safety is maintained over longer use periods and whether cancer prevention efficacy is preserved when an NSAID is combined with a phospholipid. Methods As a first step to answering these questions, we treated seven to eight-week-old, male and female C57B/6 Apcmin/+ mice with the NSAID sulindac, with and without phosphatidylcholine (PC) for 3-weeks. At the end of the treatment period, we evaluated polyp burden, gastric toxicity, urinary prostaglandins (as a marker of sulindac target engagement), and blood chemistries. Results Both sulindac and sulindac-PC treatments resulted in significantly reduced polyp burden, and decreased urinary prostaglandins, but sulindac-PC treatment also resulted in the reduction of gastric lesions compared to sulindac alone. Conclusions Together these data provide pre-clinical support for combining NSAIDs with a phospholipid, such as phosphatidylcholine to reduce GI toxicity while maintaining chemopreventive efficacy.

Purpose: Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma. Experimental Design: C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood–brain barrier–penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment. Results: The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control P < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions. Conclusions: This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.

Introduction: In contrast to brain metastases, gliomas have few antigen-presenting cells (APCs) in the tumor microenvironment, which may limit T cell effector responses. We hypothesized that: (1) an APC could be modified to express a T cell attracting chemokine that could enhance localized immune activation, and (2) that ultrasound-based blood-brain barrier (BBB) opening would enhance delivery of these APCs at the site of tumor antigens. Methods: Murine F4/80 and CD11c expressing APCs were isolated from the bone marrow of C57BL/6 mice and modified via lentiviral transduction to encode the cDNA of immune cell chemokines CXCL9 or CXCL10 and methionine-deficient green fluorescent protein (co-expression) for cell tagging. C57BL/6 mice were implanted with GL261 cells and treated with 1 x 106 CXCL9 or CXCL10 over-expressing APCs intracranially (i.c.), or intravenously (i.v.) with or without ultrasound-based BBB opening. To determine how the CXCL10 expressing APCs were altering the tumor microenvironment, immune cells were isolated from the glioma implanted hemisphere of all treatment groups and analyzed by multicolor flow cytometry. Results: Transduced APCs produced 3,392 pg/ml and 2,987 pg/ml of CXCL9 or CXCL10, respectively. Mice tolerated the treatments well without any adverse events, behavioral changes, or neurological toxicity. Survival analysis of mice treated with CXCL9 expressing APCs using i.c., or i.v. with and without BBB opening ultrasound showed no significant survival benefit compared with PBS-treated control mice. Mice treated i.c. with CXCL10-expressing APCs had significantly improved survival relative to the PBS control group (p Conclusions: The delivery of CXCL10-secreting APCs to the glioma microenvironment with ultrasound-based BBB opening was superior to delivery with direct i.c. injection. BBB disruption may have triggered more diffuse dispersal of the APCs throughout the tumor microenvironment or positioned these cells in closer proximity to the T cells emigrating from the vascular space into the localized glioma microenvironment. Citation Format: Aria Sabbagh, Kevin Beccaria, Xiaoyang Ling, Anantha Marisetty, Martina Ott, Ling-Yuan Kong, Dexing Fang, Jun Wei, Carole Desseaux, Guillaume Bouchoux, Michael Canney, Alexandre Carpentier, Amy B. Heimberger. Deposition of genetically engineered T cell attracting antigen presenting cells in the glioma microenvironment with low intensity pulsed ultrasound-based blood-brain barrier opening triggers therapeutic responses in preclinical glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1489.

Introduction: Effective anti-tumor immunity requires the presence of abundant effector T cells in the tumor microenvironment with both cancer cell specificity and cytotoxic potency. Chimeric antigen receptor (CAR) T cells engineered ex vivo to fulfill such quintessence are effective in both experimental and clinical settings with curative potential. However, the efficacy of CAR T cells can be limited by trogocytosis whereby CAR T cells extract cognate antigens from target cells thereby eliciting antigen escape and then express the target antigen on the CAR triggering fratricide that reduces the CAR persistence and efficacy. Methods: Epidermal growth factor variant III (EGFRvIII) specific CAR T cells were generated from various donors and analyzed for EGFRvIII cytotoxicity, trogocytosis, and in vivo therapeutic activity which was modulated with verteporfin, an inducer of autophagy and bafilomycin A1, an inhibitor of autophagy. A three-way co-culture system modeling the glioblastoma microenvironment of the CAR T cell interaction with targets (U87-EGFRvIII) and bystanders (U87-EGFR) was used to evaluate phagocytic and autophagy functions of the CAR T cells. Results: Glioblastoma-specific EGFRvIII CARs induce trogocytosis of the cognate tumor antigen upon engaging target tumor cells within 2 hours. Trogocytic activity varies in a donor-dependent manner and correlates with impaired CAR performance in murine models of orthotopic gliomas expressing EGFRvIII. Trogocytosis is blocked with verteporfin which restores cytolytic capacity of the CAR. In the three-way co-culture system, therapeutically effective in vivo CARs demonstrate superior phagocytic clearance of U87-EGFRvIII released mitochondria that is blocked by autophagy inhibition. The induction of immune checkpoints such as PD-L1 during CAR-target interaction is enhanced when autophagy is blocked. Furthermore, autophagy blockade prevents phagocytic clearance of cytolytic target release of mitochondria. We further demonstrate that pharmacological facilitation of autophagy with verteporfin inhibits trogocytic expression of target tumor antigen on CARs and thereby markedly increases CAR persistence in the glioma microenvironment in mice. Conclusions: Our data establish autophagy as an antagonizing mechanism counteracting CAR-induced trogocytosis and immune checkpoint activation, providing evidence for a new strategy to innovate high-performance CARs. Citation Format: Jiyong Liang, Dexing Fang, Amy B. Heimberger. Chimeric antigen receptor (CAR) T cell donor dependent dysfunction modulation with activation of autophagy and inhibition of trogocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1492.

Aims: To study the changes in protein content and protein patterns in platelet activated epithelial-mesenchymal transition of cultured colon cancer cells. Place and Duration of Study: UT McGovern Medical School, Houston, Texas, USA. Between May, 2015 and May, 2017. Methodology: Mouse platelets were added to cultured colon cancer cells and fluorescence deconvolution microscopy was performed with image acquisitions being stacked, volume rendered and modeled to yield protein localizations, quantity and patterns. Results: Studies identified and localized proteins involved in cell transitions. Co-culturing resulted in an increased cell number, increased cell size and increased protein content. Fibronectin content was increased to a greater extent than laminin; G-actin content was also increased indicating up-regulation of synthesis, and the fibroblastic-type cells were much longer with the fibronectin containing fibrillar extensions suggesting increased adhesion was occurring. Greater fibronectin than laminin synthesis indicates cells undergoing a change to matrix protein form rather than a basement membrane, polarized epithelial cell type, observations which suggest platelet activation endows the cells with adhesion and invasion abilities. Conclusions: The study showed that platelet activation of cancer cells drives endothelial cells into a matrix-compatible mesenchymal form and that the epithelial to mesenchymal transition occurs when surface cells lose polarity and cell-cell adhesion properties. These cells become migratory and invasive for rapid and facilitated cancer progression, due to enhanced adhesion and synthesis of specific proteins and cell extensions.

To determine the efficacy of a novel and safer (for gastrointestinal tract) aspirin (aspirin-PC) in preclinical models of ovarian cancer, in vitro dose–response studies were performed to compare the growth-inhibitory effect of aspirin-PC versus aspirin on three human (A2780, SKOV3ip1, and HeyA8) and a mouse (ID8) ovarian cancer cell line over an 8-day culture period. In the in vivo studies, the aspirin test drugs were studied alone and in the presence of a VEGF-A inhibitor (bevacizumab or B20), due to an emerging role for platelets in tumor growth following antiangiogenic therapy, and we examined their underlying mechanisms. Aspirin-PC was more potent (vs. aspirin) in blocking the growth of both human and mouse ovarian cancer cells in monolayer culture. Using in vivo model systems of ovarian cancer, we found that aspirin-PC significantly reduced ovarian cancer growth by 50% to 90% (depending on the ovarian cell line). The efficacy was further enhanced in combination with Bevacizumab or B20. The growth-inhibitory effect on ovarian tumor mass and number of tumor nodules was evident, but less pronounced for aspirin and the VEGF inhibitors alone. There was no detectable gastrointestinal toxicity. Both aspirin and aspirin-PC also inhibited cell proliferation, angiogenesis, and increased apoptosis of ovarian cancer cells. In conclusion, PC-associated aspirin markedly inhibits the growth of ovarian cancer cells, which exceeds that of the parent drug, in both cell culture and in mouse model systems. We also found that both aspirin-PC and aspirin have robust antineoplastic action in the presence of VEGF-blocking drugs. Mol Cancer Ther; 15(12); 2894–904. ©2016 AACR.

Purpose: To evaluate the chemoprevention efficacy and relative safety of sulindac combined with a phospholipid compared to sulindac alone. Methods: Apcmin/+ mice on the C57/B6 background were randomized to receive oral gavage of sulindac, sulindac combined with the phospholipid (phosphatidyl choline, sulindac-PC), saline or no treatment, daily for twenty-one days. At the end of the treatment period, polyp burden and gastric integrity were assessed in a blinded Following formalin fixation, the stomach from each animal was embedded in paraffin and sectioned for microscopic evaluation. In addition to mucosal ulceration, three measures of gastric inflammation were taken. To test biological activity of sulindac and sulindac-PC, nuclear β-catenin immunohistochemistry was performed on small intestinal polyps. Results: Both sulindac and sulindac-PC treated animals had significantly decreased polyp burden compared to controls (Table 1). Additionally, sulindac-PC treated mice had significantly (p=0.02) lower gastric inflammation scores (1.00 ± 0.63) compared to sulindac alone (2.14 ± 0.90). Two out of seven sulindac treated mice were found to have gastric ulcers, compared to zero out of six sulindac-PC treated mice, though this difference was not statistically significant. Both sulindac and sulindac-PC treated mice had significantly lower nuclear β-catenin staining compared to control treated mice (Table 1). Conclusions: Our findings suggest the addition of a phospholipid as an effective strategy to reduce gastric toxicity of non-steroidal anti-inflammatory drugs, such as sulindac, while maintaining chemopreventive efficacy. Citation Format: Jennifer Sarah Davis, Preeti Kanikarla, Patrick L. Yu, Mihai Gagea, Dexing Fang, Manu Sebastian, Peiying Yang, Ernest Hawk, Roderick Dashwood, Lenard M. Lichtenberger, David Menter, Scott Kopetz. Sulindac plus phospholipid is effective for polyp reduction and safer than sulindac alone [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1.

The chemopreventive activity of non-steroidal anti-inflammatory drugs (NSAIDs), particularly aspirin, has been well demonstrated in preclinical and clinical studies. However, the primary side effect from this class of drug is gastrointestinal (GI) bleeding, which has limited the widespread use of NSAIDs for the prevention of cancer. The development of GI-safer NSAIDs, which are associated with phosphatidylcholine (PC) may provide a solution to this therapeutic problem. In the present study, the efficacy of two NSAIDs, aspirin and indomethacin, were compared using murine colon cancer cell line MC-26. Each NSAID was assessed alone and in combination with PC, using in vitro and in vivo systems. The results reveal that the PC-associated NSAIDs had a significantly higher degree of protection against cancer cell growth compared with the unmodified NSAIDs. It was also observed that Aspirin-PC and Indomethacin-PC prevented the metastatic spread of cancer cells in a syngeneic mouse model. These results support the potential use of PC-NSAIDs for the chemoprevention of colorectal cancer.

The chemopreventive activity of non-steroidal anti-inflammatory drugs (NSAIDs), particularly aspirin, has been well demonstrated in preclinical and clinical studies. However, the primary side effect from this class of drug is gastrointestinal (GI) bleeding, which has limited the widespread use of NSAIDs for the prevention of cancer. The development of GI-safer NSAIDs, which are associated with phosphatidylcholine (PC) may provide a solution to this therapeutic problem. In the present study, the efficacy of two NSAIDs, aspirin and indomethacin, were compared using murine colon cancer cell line MC-26. Each NSAID was assessed alone and in combination with PC, using in vitro and in vivo systems. The results reveal that the PC-associated NSAIDs had a significantly higher degree of protection against cancer cell growth compared with the unmodified NSAIDs. It was also observed that Aspirin-PC and Indomethacin-PC prevented the metastatic spread of cancer cells in a syngeneic mouse model. These results support the potential use of PC-NSAIDs for the chemoprevention of colorectal cancer.

Activation of sialic-acid-binding immunoglobulin-like lectin-G (Siglec-G) by noninfectious damage-associated molecular patterns controls innate immune responses. However, whether it also regulates T-cell-mediated adaptive immune responses is not known. Graft-versus-host reaction is a robust adaptive immune response caused by allogeneic hematopoietic cell transplantation that have been activated by antigen-presenting cells (APCs) in the context of damaged host tissues following allogeneic hematopoietic cell transplantation. The role of infectious and noninfectious pattern recognition receptor-mediated activation in the induction and aggravation of graft-versus-host disease (GVHD) is being increasingly appreciated. But the role of pathways that control innate immune responses to noninfectious stimuli in modulating GVHD has heretofore not been recognized. We report that Siglec-G expression on host APCs, specifically on hematopoietic cells, negatively regulates GVHD in multiple clinically relevant murine models. Mechanistic studies with various relevant Siglec-G and CD24 knockout mice and chimeric animals, along with rescue experiments with novel CD24 fusion protein demonstrate that enhancing the interaction between Siglec-G on host APCs with CD24 on donor T cells attenuates GVHD. Taken together, our data demonstrate that Siglec-G-CD24 axis, controls the severity of GVHD and suggest that enhancing this interaction may represent a novel strategy for mitigating GVHD.

The mechanism by which aspirin consumption is linked to significant reductions in the incidence of multiple forms of cancer and metastatic spread to distant tissues, resulting in increased cancer patient survival is not well understood. In this study, using colon cancer as an example, we provide both in vitro (cell culture) and in vivo (chemically induced mouse model of colon cancer) evidence that this profound antineoplastic action may be associated with aspirin's ability to irreversibly inhibit COX-1–mediated platelet activation, thereby blocking platelet–cancer cell interactions, which promote cancer cell number and invasive potential. This process may be driven by platelet-induced epithelial–mesenchymal transition (EMT), as assessed using confocal microscopy, based upon changes in cell morphology, growth characteristics and fibronectin expression, and biochemical/molecular analysis by measuring changes in the expression of the EMT markers; vimentin, β-catenin, and SNAIL. We also provide evidence that a novel, gastrointestinal-safe phosphatidylcholine (PC)-associated aspirin, PL2200 Aspirin, possesses the same or more pronounced actions versus unmodified aspirin with regard to antiplatelet effects (in vitro: reducing platelet activation as determined by measuring the release of thromboxane and VEGF in culture medium; in vivo: inhibiting platelet number/activation and extravasation into tumor tissue) and chemoprevention (in vitro: inhibiting colonic cell growth and invasive activity; in vivo: inhibiting colonic dysplasia, inflammation, and tumor mass). These results suggest that aspirin's chemopreventive effects may be due, in part, to the drug blocking the proneoplastic action of platelets, and the potential use of Aspirin-PC/PL2200 as an effective and safer chemopreventive agent for colorectal cancer and possibly other cancers. Cancer Prev Res; 10(2); 142–52. ©2016 AACR.

Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation remains unclear. We demonstrate here that EGFR activation results in disruption of the complex of beta-catenin and alpha-catenin, thereby abrogating the inhibitory effect of alpha-catenin on beta-catenin transactivation via CK2alpha-dependent phosphorylation of alpha-catenin at S641. ERK2, which is activated by EGFR signaling, directly binds to CK2alpha via the ERK2 docking groove and phosphorylates CK2alpha primarily at T360/S362, subsequently enhancing CK2alpha activity toward alpha-catenin phosphorylation. In addition, levels of alpha-catenin S641 phosphorylation correlate with levels of ERK1/2 activity in human glioblastoma specimens and with grades of glioma malignancy. This EGFR-ERK-CK2-mediated phosphorylation of alpha-catenin promotes beta-catenin transactivation and tumor cell invasion. These findings highlight the importance of the crosstalk between EGFR and Wnt pathways in tumor development.

Suppression of inflammation is critical for effective therapy of many infectious diseases. However, the high rates of mortality caused by sepsis attest to the need to better understand the basis of the inflammatory sequelae of sepsis and to develop new options for its treatment. In mice, inflammatory responses to host danger-associated molecular patterns (DAMPs), but not to microbial pathogen-associated molecular patterns (PAMPs), are repressed by the interaction [corrected] of CD24 and SiglecG (SIGLEC10 in human). Here we use an intestinal perforation model of sepsis to show that microbial sialidases target the sialic acid-based recognition of CD24 by SiglecG/10 to exacerbate inflammation. Sialidase inhibitors protect mice against sepsis by a mechanism involving both CD24 and Siglecg, whereas mutation of either gene exacerbates sepsis. Analysis of sialidase-deficient bacterial mutants confirms the key contribution of disrupting sialic acid-based pattern recognition to microbial virulence and supports the clinical potential of sialidase inhibition for dampening inflammation caused by infection.

Although chronic myelogenous leukemia (CML) is effectively controlled by Bcr-Abl kinase inhibitors, resistance to inhibitors, progressive disease, and incomplete eradication of Bcr-Abl–expressing cells are concerns for the long-term control and suppression of this disease. We describe a novel approach to targeting key proteins in CML cells with a ubiquitin-cycle inhibitor, WP1130. Bcr-Abl is rapidly modified with K63-linked ubiquitin polymers in WP1130-treated CML cells, resulting in its accumulation in aggresomes, where is it unable to conduct signal transduction. Induction of apoptosis because of aggresomal compartmentalization of Bcr-Abl was observed in both imatinib-sensitive and -resistant cells. WP1130, but not Bcr-Abl kinase inhibitors, directly inhibits Usp9x deubiquitinase activity, resulting in the down-regulation of the prosurvival protein Mcl-1 and facilitating apoptosis. These results demonstrate that ubiquitin-cycle inhibition represents a novel and effective approach to blocking Bcr-Abl kinase signaling and reducing Mcl-1 levels to engage CML cell apoptosis. This approach may be a therapeutic option for kinase inhibitor–resistant CML patients.

MK-0457 inhibits aurora, BCR-ABL and other kinases and may be clinically active in imatinib resistant leukemia. To define mediators of MK-0457 responsiveness, kinase inhibitory profiles were examined in multiple cell models of imatinib sensitive and resistant disease. Aurora and BCR-ABL kinase inhibition were consistently measured at 20-100 nM and 2-10 microM MK-0457, respectively, but expression of T315I-BCR-ABL and overexpression of Lyn kinase reduced MK-0457 sensitivity. Aurora kinase inhibition was associated with cell cycle restriction and p53 induction and p53-null cells were far less responsive to MK-0457, requiring BCR-ABL inhibitory concentrations for apoptotic activity. In wild-type p53 expressing CML cells MK-0457 sensitivity was modulation by alterations in p53 levels through HDM-2 inhibition and gene silencing. MK-0457 suppressed aurora kinase activity and induced apoptosis in imatinib resistant clinical specimens expressing T315I and other BCR-ABL mutations without effecting BCR-ABL kinase activity. Together, these results suggest that MK-0457 apoptotic activity in CML cells is primarily associated with aurora kinase inhibition but can be altered by multiple molecular changes associated with disease progression or acquisition of imatinib resistance.

Heat shock proteins (Hsps) are broadly classified into distinct subfamilies—Hsp100, Hsp90, Hsp70, Hsp60, Hsp40, small Hsps, and Hsp10—on the basis of their apparent molecular weights, amino acid sequences, and functions. Many members of these subfamilies are present constitutively in cells, whereas others are expressed only after stress (9). Hsps are involved in the normal folding of various polypeptides, assist misfolded proteins to attain or regain their native states, regulate protein stability and degradation, and promote protein translocation (9). Hsp90 is one of the most abundant Hsps in eukaryotic cells, comprising 1% to 2% of cellular proteins under nonstress conditions (25). There are two major cytoplasmic isoforms of Hsp90 in vertebrates, inducible Hsp90α and constitutively expressed Hsp90β (7). Hsp90 is unique because it is not required for the biogenesis of most polypeptides. Instead, many but not all of its cellular substrates or client proteins are conformationally labile signal transducers, including protein kinases and transcription factors that have crucial roles in cell proliferation and differentiation, cell survival and apoptosis, and developmental processes (1, 19, 22, 28). Upon Hsp90 inhibition, client protein substrates are unable to properly fold into their biologically active conformations; therefore, they are destined for degradation via the ubiquitin-proteasomal pathway (3, 13). Because Hsp90 is involved in the modulation of tumor cell apoptosis and its inhibition disrupts multiple pathways essential to the survival, proliferation, and metastasis of transformed cells, Hsp90 has become a promising target for the development of cancer chemotherapeutics (28). Apoptotic stimulation activates at least one of the two major apoptotic pathways, the intrinsic or mitochondrial death pathway and the extrinsic or receptor-mediated cell death pathway. Both of these pathways eventually activate effector caspases to execute cell death (4). The activation of the intrinsic pathway leads to the assembly of functional apoptosomes composed of mitochondrially released cytochrome c, apoptotic protease activating factor 1, and procaspase-9. The apoptosome complex proteolytically processes procaspase-9 to an active form that activates effector caspase-3 and -7 (4, 30). The extrinsic pathway transduces death signals through the binding of death ligands such as tumor necrosis factor alpha (TNF-α), FAS ligand/Apo1L/CD95L, Trail/Apo2L, and Apo3L to their respective cell surface receptors. A homotypic interaction occurs between the death domains of TNF receptor-1 (TNFR-1) and FAS receptors and their respective adaptor molecules, TNFR-1-associated death domain protein (TRADD) and Fas-associated death domain protein (FADD), that leads to the formation of the death-inducing signal complex (DISC), which activates procaspase-8 and, subsequently, caspase-3 and -7. The extrinsic death signals can be linked to the intrinsic pathway through the Bcl-2 family of proteins, which includes both proapoptotic (e.g., Bax, Bad, Bak, and Bid) and antiapoptotic (e.g., Bcl-XL) members (1, 8, 30). Hsp90β is involved in many cellular functions via regulation of its client substrate proteins. However, most studies of Hsp90β regulation are conducted at transcriptional levels (7, 25); its posttranslational modification, especially in response to apoptotic stimulation, is not well understood. In this report, we found that Hsp90β was cleaved by activated caspase-10 under UVB irradiation. Caspase-10 activation, in turn, depended on UVB-induced secretion of FAS ligand and subsequent activation of caspase-8. The downregulation of Hsp90β promoted UVB-induced cell apoptosis.

In primary mammalian cells, expression of oncogenes such as activated Ras induces premature senescence rather than transformation. We show that homozygous deletion of glycogen synthase kinase (GSK) 3β (GSK3β −/− ) bypasses senescence induced by mutant Ras V12 allowing primary mouse embryo fibroblasts (MEFs) as well as immortalized MEFs to exhibit a transformed phenotype in vitro and in vivo . Both catalytic activity and Axin-binding of GSK3β are required to optimally suppress Ras transformation. The expression of Ras V12 in GSK3β −/− , but not in GSK3β +/+ MEFs results in translocation of β-catenin to the nucleus with concomitant up-regulation of cyclin D1. siRNA-mediated knockdown of β-catenin decreases both cyclin D1 expression and anchorage-independent growth of transformed cells indicating a causal role for β-catenin. Thus Ras V12 and the lack of GSK3β act in concert to activate the β-catenin pathway, which may underlie the bypass of senescence and tumorigenic transformation by Ras.