Ganglioside GD3 Research Articles

Sialic Acid-Dependent Inhibition of T Cells by Exosomal Ganglioside GD3 in Ovarian Tumor Microenvironments.

The tumor microenvironment is rendered immunosuppressive by a variety of cellular and acellular factors that represent potential cancer therapeutic targets. Although exosomes isolated from ovarian tumor ascites fluids have been previously reported to induce a rapid and reversible T cell arrest, the factors present on or within exosomes that contribute to immunosuppression have not been fully defined. In this study, we establish that GD3, a ganglioside expressed on the surface of exosomes isolated from human ovarian tumor ascites fluids, is causally linked to the functional arrest of T cells activated through their TCR. This arrest is inhibited by Ab blockade of exosomal GD3 or by the removal of GD3+ exosomes. Empty liposomes expressing GD3 on the surface also inhibit the activation of T cells, establishing that GD3 contributes to the functional arrest of T cells independent of factors present in exosomes. Finally, we demonstrate that the GD3-mediated arrest of the TCR activation is dependent upon sialic acid groups, because their enzymatic removal from exosomes or liposomes results in a loss of inhibitory capacity. Collectively, these data define GD3 as a potential immunotherapeutic target.

ST8SIA1 Regulates Tumor Growth and Metastasis in TNBC by Activating the FAK-AKT-mTOR Signaling Pathway.

Breast cancer stem-like cells (BCSC) are implicated in cancer recurrence and metastasis of triple-negative breast cancer (TNBC). We have recently discovered that ganglioside GD2 expression defines BCSCs and that ST8SIA1 regulates GD2 expression and BCSC function. In this report, we show that ST8SIA1 is highly expressed in primary TNBC; its expression is positively correlated with the expression of several BCSC-associated genes such as BCL11A, FOXC1, CXCR4, PDGFRβ, SOX2, and mutations in p53. CRISPR knockout of ST8SIA1 completely inhibited BCSC functions, including in vitro tumorigenesis and mammosphere formation. Mechanistic studies discovered activation of the FAK-AKT-mTOR signaling pathway in GD2+ BCSCs, and its tight regulation by ST8SIA1. Finally, knockout of ST8SIA1 completely blocked in vivo tumor growth and metastasis by TNBC cells. In summary, these data demonstrate the mechanism by which ST8SIA1 regulates tumor growth and metastasis in TNBC and identifies it as a novel therapeutic target.

Exceptionally Potent Cytotherapy Using T Cells Armed with Novel Tetravalent Recombinant Bispecific Antibodies Specific for GD2 and HER2

▪Introduction: T-cell based therapies have emerged as one of the major breakthroughs in anticancer treatment: Immune checkpoint inhibitors, chimeric antigen receptor gene-modified T-cells (CAR-T-cells), and T-cell engaging bispecific antibodies (BsAb) are leading the advances. In the era of personalized medicine, T-cells offer alternative strategies to overcome resistance to chemotherapy or small molecules. Yet, hurdles for such therapy can be crippling, such as inability of T cells to infiltrate “cold tumors”, cytokine release syndrome following T cell-based therapies, neurologic toxicity, and on-target off-tumor effects. To address these hurdles, polyclonal T-cells armed with GD2xCD3 or HER2xCD3 BsAb for cytotherapy hold promise. Ganglioside GD2 and HER2 are tumor associated surface antigens expressed in a broad spectrum of aggressive malignancies, while being restricted in normal tissues. Phase I trials of T-cells armed with a chemical conjugate of hu3F8 x mouse OKT3 (NCT02173093) or trastuzumab x mouse OKT3 (NCT00027807) demonstrated the safety of 160 x 106/kg/injection x 8 doses (or 1.28 x 109/cycle) with suggestion of clinical benefit. Here, we report the safety and efficacy of adoptive T-cell therapy armed with the recombinant forms of these BsAb for the treatment of GD2(+) and/or HER2(+) tumors in preclinical models.Methods: Recombinant anti-GD2 BsAb and anti-HER2 BsAb were made using the IgG(L)-scFv platform (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from normal volunteer donors were isolated, activated and expanded by CD3/CD28 beads in the presence of 100 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T cells (ATCs) were harvested and armed for 20 minutes at room temperature with -GD2-BsAb or HER2-BsAb. After washing, armed ATCs were tested for cell surface density of BsAb and antibody dependent T cell mediated cytotoxicity (ADTC) in vitro. In vivo anti-tumor potencies of armed T cells were tested against GD2(+) or HER2(+) cell lines or patient derived xenografts (PDXs) in BALB-Rag2-/-IL-2R-γϲ-KO (DKO) mice.Results: GD2-BsAb of the IgG(L)-scFv form showed superior potency over other bispecific platforms in vitro and in vivo. GD2-BsAb or HER2-BsAb armed ATCs showed potent antigen-specific cytotoxicity against GD2 or HER2 positive tumors such as neuroblastoma, melanoma and osteosarcoma in vitro over a range of antibody dose (5 to 500 ng/106 cells). Optimal arming per T cell required 25,000 to 45,000 idiotype(+) molecules. There was no evidence of activation induced cell death when confronted by antigen or tumor target. In vivo GD2-BsAb or HER2-BsAb armed ATCs could ablate neuroblastoma, malignant melanoma, and osteosarcoma tumors over a range of cell doses (10x106, 20x106 and 40x106 per injection, one to three times a week for 2 to 4 weeks) with a range of BsAb doses (5 ng to 500 ng/million of T-cells) without significant toxicities in DKO mice. By immunohistochemistry, the frequency of tumor infiltrating CD3(+) T-cells strongly correlated with tumor response.Conclusions:Using the IgG(L)-scFv format, GD2-BsAb or HER2-BsAb armed ATCs could provide a potent and economical cytotherapy platform against GD2(+) or HER2(+) tumors without the complexity of gene modification (as in chimeric antigen receptor modified T cells). At such low arming doses, where BsAb is T cell bound, where ADTC is not induced, and T cell expansion is not required for anti-tumor effect, clinical toxicity is expected to be low. DisclosuresCheung:Ymabs: Patents & Royalties.

Differential roles of gangliosides in malignant properties of melanomas

Ganglioside GD3 is widely expressed in human malignant melanomas, and has been reported to be involved in the increased cell proliferation and invasion. In this study, we established GM3-, GM2-, GM1-, GD3-, or GD2-expressing melanoma cell lines by transfecting cDNAs of glyscosyltransferases, and effects of individual gangliosides on the cell phenotypes and signals were examined. The phenotypes of established ganglioside-expressing cells were quite different, i.e. cell growth increased as following order; GD2+, GD3+ > GM1+, GM2+, GM3+ cells. Cell invasion activity increased as GD3+ ≧ GM2+ > GM1+, GM3+, GD2+ cells. Intensity of cell adhesion to collagen I (CL-I) and spreading increased as GD2+ >> GD3+, GM1+ > GM2+, GM3+ cells. In particular, cell adhesion of GD2+ cells was markedly strong. As for cell migration velocity, GD2+ cells were slower than all other cells. The immunocytostaining revealed close localization of gangliosides and F-actin in lamellipodia. Immunoblotting of phosphorylated p130Cas and paxillin by serum treatment reveled that these phosphorylations were more increased in GD3+ cells than in GD2+ or GM3+ cells, while phosphorylation of Akt underwent similarly increased phosphorylation between GD3+ and GD2+ cells compared with GM3+ cells. While GD2 and GD3 enhanced cell growth, GD3 might also contribute in cell invasion. On the other hand, GD2 might contribute in the solid fixation of melanoma cells at metastasized sites. These results suggested that individual gangliosides exert distinct roles in the different aspects of melanomas by differentially regulating cytoskeletons and signaling molecules.

Carbohydrate Targets for CAR T Cells in Solid Childhood Cancers.

Application of the CAR targeting strategy in solid tumors is challenged by the need for adequate target antigens. As a consequence of their tissue origin, embryonal cancers can aberrantly express membrane-anchored gangliosides. These are carbohydrate molecules consisting of a glycosphingolipid linked to sialic acids residues. The best-known example is the abundant expression of ganglioside GD2 on the cell surface of neuroblastomas which derive from GD2-positive neuroectoderm. Gangliosides are involved in various cellular functions, including signal transduction, cell proliferation, differentiation, adhesion and cell death. In addition, transformation of human cells to cancer cells can be associated with distinct glycosylation profiles which provide advantages for tumor growth and dissemination and can serve as immune targets. Both gangliosides and aberrant glycosylation of proteins escape the direct molecular and proteomic screening strategies currently applied to identify further immune targets in cancers. Due to their highly restricted expression and their functional roles in the malignant behavior, they are attractive targets for immune engineering strategies. GD2-redirected CAR T cells have shown activity in clinical phase I/II trials in neuroblastoma and next-generation studies are ongoing. Further carbohydrate targets for CAR T cells in preclinical development are O-acetyl-GD2, NeuGc-GM3 (N-glycolyl GM3), GD3, SSEA-4, and oncofetal glycosylation variants. This review summarizes knowledge on the role and function of some membrane-expressed non-protein antigens, including gangliosides and abnormal protein glycosylation patterns, and discusses their potential to serve as a CAR targets in pediatric solid cancers.

Abstract B38: Tetravalent bispecific antibodies specific for HER2 and disialoganglioside GD2 to engage polyclonal T cells for osteosarcoma therapy

Abstract Introduction: Osteosarcoma is the most common pediatric high-grade bone tumor. Although multimodal therapeutic approaches have significantly improved patient survival to more than 60%, the prognosis for patients with metastatic or relapsed disease remains dismal—an urgent unmet need. Its genomic complexity and higher mutational burden compared to other pediatric cancers should provide neoantigens as potential targets for T cell-based immunotherapy; however, these tumors are in general “cold,” with insufficient or inactive tumor-infiltrating lymphocytes. Ganglioside GD2 is a tumor-associated surface antigen expressed in a broad spectrum of pediatric malignancies, including neuroblastoma, brain tumors, Ewing’s sarcoma, rhabdomyosarcoma, and osteosarcoma, while being restricted in normal tissues. Another promising target, HER2 or ErbB2, is overexpressed in many aggressive malignancies, including pediatric medulloblastoma, nephroblastoma, osteosarcoma, and desmoplastic small round cell tumor (DSRT). Fully humanized tetravalent bispecific antibodies (BsAb) specific for human GD2 and CD3 (hu3F8-BsAb) or HER2 and CD3 (HER2-BsAb), built on an IgG(L)-scFv platform, have been developed (Oncoimmunology 2017; Can Immunol Res 2015). They induce rapid and quantitative T-cell homing to tumors, mediating potent T-cell dependent cytotoxicity (TDCC) against GD2 or HER2 expressing tumor cells, effecting cures of xenografts in SCID mice. In this study, we evaluated the in vitro and in vivo antitumor properties of these BsAbs in the treatment of osteosarcoma. Methods: Hu3F8-BsAb and HER2-BsAb were tested both in vitro and in vivo against a panel of osteosarcoma cell lines (RG143B, U2OS, CRL1427, HOS, and SaOS2). Using these cell lines, FACS analysis for surface antigen (GD2 and HER2) expression (MFI), sensitivity to TDCC (EC50), and antitumor activity in vivo were evaluated. Two in vivo tumor models with different effector routes were used to simulate different clinical situations: 1) subcutaneous (sc) tumor cells/sc effector peripheral blood mononuclear cells (PBMCs), and 2) sc tumor cells/intravenous (iv) PBMCs. In vivo tumor responses were measured by Peira TM900 imaging device or by bioluminescence. Results: The majority of osteosarcoma cell lines express GD2 and HER2 on their surface, and their in vitro sensitivity to TDCC (EC50) was inversely correlated with MFI of their respective antigen. Both hu3F8-BsAb and HER2-BsAb mediated potent TDCC against osteosarcoma cell lines. In vivo, both hu3F8-BsAb and HER2-BsAb exerted a significant antitumor effect compared to control BsAb (P=0.001). Conclusions: Hu3F8-BsAb and HER2-BsAb induced strong TDCC and had significant antitumor effect against osteosarcoma cell lines both in vitro and in vivo. Considering the limited therapeutic options currently available in advanced osteosarcomas, these results support their further clinical development as potential T cell-based immunotherapeutics. Citation Format: Jeong A. Park, Hong Xu, Irene Cheung, Nai-Kong V. Cheung. Tetravalent bispecific antibodies specific for HER2 and disialoganglioside GD2 to engage polyclonal T cells for osteosarcoma therapy [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B38.

Red blood cell-derived extracellular vesicles communicate with megakaryocyte–erythrocyte progenitor cells in bone marrow

Melanomas often accumulate gangliosides, sialic acid-containing glycosphingolipids found in the outer leaflet of plasma membranes, as disialoganglioside GD3 and its derivatives. Here, we have transfected the GD3 synthase gene (ST8Sia I) in a normal melanocyte cell line in order to evaluate changes in the biological behavior of non-transformed cells. GD3-synthase expressing cells converted GM3 into GD3 and accumulated both GD3 and its acetylated form, 9-O-acetyl-GD3. Melanocytes were rendered more migratory on laminin-1 surfaces. Cell migration studies using the different transfectants, either treated or not with the glucosylceramide synthase inhibitor d-1-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (PPPP), allowed us to show that while GM3 is a negative regulator of melanocyte migration, GD3 increases it. We showed that gangliosides were shed to the matrix by migrating cells and that GD3 synthase transfected cells shed extracellular vesicles (EVs) enriched in GD3. EVs enriched in GD3 stimulated cell migration of GD3 negative cells, as observed in time lapse microscopy studies. Otherwise, EVs shed by GM3+veGD3−ve cells impaired migration and diminished cell velocity in cells overexpressing GD3. The balance of antimigratory GM3 and promigratory GD3 gangliosides in melanocytes could be altered not only by the overexpression of enzymes such as ST8Sia I, but also by the horizontal transfer of ganglioside enriched extracellular vesicles. This study highlights that extracellular vesicles transfer biological information also through their membrane components, which include a variety of glycosphingolipids remodeled in disease states such as cancer.

Abstract A07: Changes in immune profiles of osteosarcoma dogs receiving a GD3-based vaccine concurrently with carboplatin chemotherapy and surgery

Abstract Introduction: The disialyl gangliosides GD2/GD3 have been implicated in the enhancement of malignancy in a number of human and animal cancers and as a tumor antigen target for immunotherapy. In a recent abstract presented at an AACR Conference we reported on the coexpression of GD2/GD3 in four canine osteosarcoma (OSA) cell lines (1). In a prospective IACUC approved clinical trial we vaccinated dogs with a GD3-based vaccine with naturally occurring OSA receiving surgery and carboplatin chemotherapy to investigate the expression profiles of immune modulating cells overtime. Methods: Dogs will be entered into the study only if they meet the following inclusion criteria: have a confirmed diagnosis of OSA and no other life-threatening diseases. The study will accrue 40 cases; 20 will receive the vaccine plus standard of care and 20 dogs will receive only the standard of care (amputation and intent to treat with 6 doses of carboplatin). On admission blood will be collected according to the protocol for monitoring of the immune response. The dogs will then be vaccinated according to a predetermined protocol during chemotherapy and staged. The immune response profile of the vaccine group will be compared to dogs receiving standard of care alone and normal dogs. Flow cytometric platforms were developed to monitor changes in immune cells (CD5, CD21, CD4, CD8, CD14, CD11b, MHCII, and Foxp3). In addition, IHC arrays and RNA Scope will be developed for checkpoints of immunity PD1, PDL-1 and expression of intratumoral immune cells. Results: Currently twenty dogs with osteosarcoma have enrolled into the study and have received standard of care and vaccination. Complete flow cytometric immune profiles are available for nine dogs with osteosarcoma. On admission, all dogs with OSA showed elevated cell counts of Treg (FoxP3+/CD4+) cells, Monocytic (m-) and Granulocytic (g-) myeloid derived suppressor cells (MDSCs) when compared to normal dogs (all dogs had normal CBCs). All m-MDCSs and g-MDSCs and Treg cells decreased significantly after the first dose of chemotherapy. Serial sampling over weeks showed sustained inhibition even after chemotherapy was completed (18 weeks). Two OSA cases which relapsed with metastasis to the lungs showed significant increases in Treg cells at the time of restaging. Complete necropsy post-therapy in three dogs showed changes in metastatic profiles; 2/3 showed no metastatic disease to the lungs, but metastases occurred to bone and kidneys. Discussion: MDSCs series of cells and Treg cells are increased in OSA compared to normal dogs and may be a factor in maintaining a welcoming tumor microenvironment and resistance to immunotherapy. MDSCs are reported to be elevated in other cancers, but not in naturally occurring OSA. Furthermore, canine OSA cell lines show increased expression of CCL2 and COX2 (data not shown). Recruitment of MDSCs to the cancer micro-tumor environment are thought to be mediated by the chemokine CCL2 and COX2. Abrogation of these pathways with chemotherapy and possibly immunotherapy may enhance overall survival. Early necropsy data seem to support the attenuation of the metastatic profile in dogs receiving standard of care and GD3-based vaccine. Conclusions: Anticipated results from the study will be used to adjust the vaccine protocol according to the changes in immune profiles. Data is not mature enough to evaluate survival at this time. Acknowledgment: The study is funded by a grant from the American Kennel Club Health Foundation and The UF CVM. Reference: 1. Milner RJ, Chimura N, Bowles KD, Salute M. Abstract A29: Differential expression of the gangliosides GD3 and GD2 in canine and human osteosarcoma cell lines: An immunotherapy target. Cancer Immunol Res 2015 Oct 1;3(10 Supplement):A29. Citation Format: Bikash Sahay, Shana Hutchison, Matt Cascio, Amandine Lejeune, Carlos Souza, Anna Szivek, Keijiro Shiomitsu, Kayla Harding, Stacey Fox-Alvarez, Mia Livaccarri, Lindsay Powers, Rowan James Milner. Changes in immune profiles of osteosarcoma dogs receiving a GD3-based vaccine concurrently with carboplatin chemotherapy and surgery [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A07.

Transcriptional Activation of Human GD3 Synthase (hST8Sia I) Gene in Curcumin-Induced Autophagy in A549 Human Lung Carcinoma Cells.

Curcumin, a natural polyphenolic compound isolated from the plant Curcuma longa, is known to induce autophagy in various cancer cells, including lung cancer. In the present study, we also confirmed by LC3 immunofluorescence and immunoblotting analyses that curcumin triggers autophagy in the human lung adenocarcinoma A549 cell line. In parallel with autophagy induction, the gene expression of human GD3 synthase (hST8Sia I) responsible for ganglioside GD3 synthesis was markedly elevated in response to curcumin in the A549 cells. To investigate the transcriptional activation of hST8Sia I associated with the autophagy formation in curcumin-treated A549 cells, functional characterization of the 5′-flanking region of the hST8Sia I gene was carried out using the luciferase reporter assay system. Deletion analysis demonstrated that the -1146 to -646 region, which includes the putative c-Ets-1, CREB, AP-1, and NF-κB binding sites, functions as the curcumin-responsive promoter of hST8Sia I in A549 cells. The site-directed mutagenesis and chromatin immunoprecipitation assay demonstrated that the NF-κB binding site at -731 to -722 was indispensable for the curcumin-induced hST8Sia I gene expression in A549 cells. Moreover, the transcriptional activation of hST8Sia I by the curcumin A549 cells was strongly inhibited by compound C, an inhibitor of AMP-activated protein kinase (AMPK). These results suggest that curcumin controls hST8Sia I gene expression via AMPK signal pathway in A549 cells.

Abstract 1766: Dinutuximab targets GD2+ breast cancer stem cells and inhibits TNBC tumor growth

Abstract Breast cancer stem-like cells (BCSCs), also known as breast cancer initiating cells, are a fraction of cells in the primary tumor that are highly tumorigenic, chemotherapy resistant, and are able to cause metastases. Therefore, targeting BCSCs is an important strategy that could complement standard chemotherapy for breast cancer. We have identified that ganglioside GD2 as an important maker for BCSCs and that ST8SIA1 (GD3-synthase) is an enzyme that regulates GD2 expression in BCSCs (Battula et al., JCI, 2012). GD2 is over expressed in triple negative breast cancer (TNBC) compared to non-TNBC. Inhibition of GD2 expression by knockdown of ST8SIA1 completely inhibited tumor growth and metastases in xenograft models. Therefore, we hypothesize that targeting GD2+ BCSCs will inhibit TNBC tumor growth. To test our hypothesis, we used the chimeric anti-GD2 monoclonal antibody (Dinutuximab) that has been FDA approved for the treatment of neuroblastoma in children. The main mode of action for dinutuximab is antibody dependent cell-mediated cytotoxicity (ADCC). To test the activity of dinutuximab against GD2+ BCSCs, we treated MDA-MB-231 and SUM159 breast cancer cells with dinutuximab alone at different concentrations (1, 12.5, 25, 50, and 100µg/ml) for 24, 48, and, 72hrs or in combination with IL-21 activated NK cells. As control, cells were treated with rituximab (anti-CD20 antibody) and NK cells. Although only a moderate effect was observed on GD2+ cells when treated with dinutuximab alone, we found a dramatic decrease in the number of live (annexin-vneg and DAPIneg) GD2+ cells when treated with dinutuximab in combination with NK-cells. This combination decreased the percentage of GD2+ cells from 20.1±0.3% to 5.8±0.2% (p<0.001). No significant effect on GD2+ cells was observed in the rituximab + NK cell group, indicating that the dinutuximab + NK-cell combination is most affective in inducing cytolysis of GD2+ cells. Next, to investigate the effect of dinutuximab on in-vivo tumor growth, we implanted firefly luciferase expressing MDA-MB-231 cells into nude mice (these mice were chosen because of the presence of NK cells). When palpable tumors were formed (tumor volume 25-30mm3), the mice were treated with dinutuximab or rituximab at 1.4mg/kg twice a week for 6 weeks. Tumor growth was measured weekly by bioluminescence imaging as well as by calipers. We found a >10 fold decrease in tumor growth in mice treated with dinutuximab compared to control. In contrast, tumor volumes reached up to 250mm3 within 10 weeks of tumor implantation in the rituximab treated group (p<0.001). Conclusion: Our data suggest that dinutuximab targets GD2+ BCSCs with the help of NK cells by ADCC and inhibits tumor growth. We are currently in the process of developing a phase I/II clinical trial using dinutuximab in patients with TNBC. In addition, preclinical testing of dinutuximab in combination with NK cells using TNBC PDX models is underway. Citation Format: Venkata Lokesh Battula, Khoa Nguyen, Anitha Somanchi, Hong Mu, Naoto T. Ueno, Dean Lee, Michael Andreeff. Dinutuximab targets GD2+ breast cancer stem cells and inhibits TNBC tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1766.

Abstract CT145: A Cancer Research UK phase I trial of anti-GD2 chimeric antigen receptor (CAR) transduced T-cells (1RG-CART) in patients with relapsed or refractory neuroblastoma

Abstract Background: Treatment of high risk neuroblastoma remains challenging; current multimodal treatment regimens achieve long term survival in <50% of patients and are associated with significant morbidity. Ganglioside GD2 is abundantly expressed on almost all neuroblastomas whilst expression on normal tissue is highly limited, providing a suitable CAR target. Here, we report the preliminary results of a Phase I clinical study of GD2-CART for refractory/relapsed neuroblastoma (NCT02761915). Trial design: The therapeutic (1RG-CART) is autologous T-cells transduced with a gamma-retroviral vector encoding both an anti-GD2 CAR and the RQR8 suicide gene. The CAR comprises a humanized anti-GD2 single chain variable fragment derived from the K666 antibody and CD28/CD3ζ signalling domains. Both lymphodepletion and CART dose were escalated as follows: dose level (DL) 1 without lymphodepletion, DL2 with 1.2 g/m2 cyclophosphamide and DL3 and beyond 1.2 g/m2 cyclophosphamide and 125 mg/m2 fludarabine followed by administration of a single intravenous dose of 1x107/m2 (DL1-3) or 1x108/m2 (DL4) 1RG-CART. Primary objectives are to assess safety and tolerability. Results: To date, 12 patients have been enrolled. All had relapsed/refractory neuroblastoma with measurable disease in bone (n=11), bone marrow (n=7) and/or soft tissue sites (n=9). Cell products were successfully manufactured for all patients. Median transduction efficiency was 34.5% (range 16-54%). Nine patients have been treated on DL1 (n=4), DL2 (n=1), DL3 (n=1) and DL4 (n=3) respectively. No dose limiting toxicity (DLT) was seen. For patients treated on DL1-3 (1x107/m2), 1RG-CART could not be detected in peripheral blood, and no clinical responses were seen. In contrast, expansion of 1RG-CART cells as detected by flow cytometry and qPCR was seen in the 3 patients treated on DL4 (1x108/m2). In two DL4 patients, 1RG-CART expansion was still limited and transient (marking levels <10,000 copies/μg DNA). These patients had disease progression as measured at Day +28. In one DL4 patient however, 1RG-CART marking levels of >40,000 copies/μg DNA were achieved. This patient developed Grade 2 cytokine release syndrome (Day +5) and biochemical evidence of tumour lysis (Day +21). Disease reassessment on Day +28 showed response in many sites of bone/marrow disease as measured by mIBG scintigraphy, and near complete tumour clearance in bone marrow which at baseline was heavily infiltrated with neuroblastoma. Disease progression occurred on Day +45 at which time 1RG-CART were no longer detectable. In the absence of DLT this prompted us to continue with DL5 (1x109/m2). Conclusions: These preliminary results are the first to demonstrate on-target activity in bone and bone marrow of GD2-CART in this childhood solid tumour. Further 1RG-CART dose escalation is warranted, and under way. Citation Format: Karin Straathof, Barry Flutter, Rebecca Wallace, Simon Thomas, Gordon Cheung, Angela Collura, Talia Gileadi, Jack Barton, Gary Wright, Sarah Inglott, David Edwards, Claire Barton, Karen Dyer, Nigel Westwood, Thalia Loka, Sarita Depani, Karen Howe, Giuseppe Barone, Martin Pule, John Anderson. A Cancer Research UK phase I trial of anti-GD2 chimeric antigen receptor (CAR) transduced T-cells (1RG-CART) in patients with relapsed or refractory neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT145.

Abstract 4631: Expression of ganglioside GD2 in Ewing sarcoma cells is not associated with specific functional characteristics or stem cell features

Abstract Safe and effective targeting of cancer with chimeric antigen receptor (CAR) engineered T cells relies on the presence of adequate tumor-associated surface antigens. We have found that Ewing sarcomas (EwS) express ganglioside GD2 and are recognized by T cells engineered with GD2-specific CARs. In an extended series of 97 EwS patient samples, 52 were GD2pos by immunohistochemistry. Based on reports that GD2 in breast cancer defines a malignant population with stem cell characteristics, we hypothesized that GD2 expression in EwS is associated with high capacity to self-renew and reinitiate tumor growth. Among 15 individual EwS cell lines with variable surface expression of GD2 by flow cytometry, GD2 expression levels were not associated with the capacities to proliferate and expand in vitro, form colonies in semi-solid medium, nor with their chemosensitivity, assessed by cell viabilities in the presence of increasing concentrations of the cytotoxic drug doxorubicin. Subpopulations with GD2hi phenotype selected from GD2pos and from GD2low EwS cell lines by cell sorting maintained their GD2hi and GD2low expression status during subsequent cultures over several weeks. The two subpopulations did not have different in vitro growth, colony-formation capacity, or chemosensitivity. In xenografting experiments, GD2hi and GD2low subpopulations of two EwS cell lines initiated tumors with comparable efficacies. To obtain direct evidence that GD2 surface expression is irrelevant for the biology of EwS cells, we performed genetic knockdown of the GD3S gene which drives biosynthesis of GD2 by CRISPR/Cas9 gene editing. GD3S gene editing resulted in effective elimination of GD2 surface expression in the GD2hi EwS cell lines TC-71, VH-64 and A4573. The knockdown did not affect the capacity of the cells to proliferate, form colonies in soft agar in vitro, nor their chemosensitivity compared to wild-type EwS cells from the individual cell lines. We conclude that GD2 expression in EwS cells, other than reported in breast cancer, is not associated with distinct functional features. Specifically, GD2 does not affect the growth characteristics, clono- and tumorigenicity and chemosensitivity of the tumor cells. Elimination of GD2pos subpopulations from heterogeneous tumors by CAR T cell targeting is therefore unlikely to eradicate the disease and will have to be combined with additional targeting strategies. Citation Format: Sareetha Kailayangiri, Bianca Altvater, Silke Jamitzky, Stefanie Lesch, Jan-Henrik Mikesch, Sonja Schelhaas, Schaefers Michael, Jutta Meltzer, Nicole Farwick, Wolfgang Hartmann, Eva Wardelmann, Petra Fischer, Jendrik Hardes, Claudia Rossig. Expression of ganglioside GD2 in Ewing sarcoma cells is not associated with specific functional characteristics or stem cell features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4631.

Abstract 1929: ST8SIA1 is a novel therapeutic target in TNBC: Regulates FAK-PI3K-AKT-mTOR signaling to promote tumor growth and metastasis

Abstract We identified ganglioside GD2 as a breast cancer stem cell (BCSC) marker and that GD3 synthase (ST8SIA1) regulates GD2 expression and BCSC function. We have reported that GD2 is up-regulated in TNBC and inhibition of its expression by knockdown of ST8SIA1 inhibits tumor growth and metastasis. Here we hypothesize that ST8SIA1 is overexpressed in TNBC and regulates cell signaling downstream of GD2 to promote tumor growth and metastasis. RNA sequencing data analysis of TCGA dataset with over 1100 primary and metastatic breast tumors revealed that ST8SIA1 was overexpressed in around 10% of all breast cancer patients. Basal type tumors expressed the highest levels of ST8SIA1 compared to luminal-A, luminal-B, and HER2-enriched (p<0.01) tumors. In addition, TNBC, (n=115) had 4.63-fold higher expression of ST8SIA1 than did hormone receptor-positive tumors, including ER+, PR+, and HER2+ tumors (n=852, p<0.001). Further analysis of ST8SIA1 expression in different TNBC subtypes revealed that the mesenchymal-subtype of TNBC had the highest expression (p<0.001) among the 7 TNBC subtypes. A survival analysis by univariant analysis indicated that patients with ST8SIA1high tumors had significantly lower overall and disease-free survival rates than did patients with ST8SIA1low tumors (p=0.0148 and p=0.0109, respectively with 60months follow-up). To investigate the mechanism of the ST8ISA1-mediated regulation of tumor growth and metastasis, we performed a proteomic analysis of GD2+ and GD2- SUM159 and MDA-MB-231 cells using antibody micro-arrays (Kinexus). After systematic data analysis and functional validation by western blotting, we found that FAK, PI3K, AKT, ERK, mTOR, and 4EBP1 were highly phosphorylated in GD2+ compared to in GD2- SUM159 cells. As it is known that GD2 acts as a co-receptor on the cell surface and activates downstream signaling, we hypothesized that ST8SIA1 regulates PI3K-AKT-mTOR signaling in BCSCs through GD2. To test our hypothesis, we knocked out ST8SIA1 in SUM159 cells using CRISPR-Cas9 approach. As expected knockout (KO) of ST8SIA1 reduced percentage of GD2+ cells from 20±5% to <1% in SUM159 cells. In addition, ST8SIA1-KO cells did not induce tumor growth or cause metastases in-vivo. Interestingly, we found that lack of ST8SIA1 led to inhibition of FAK, AKT, ERK, 4EBP1, and mTOR phosphorylation, suggesting that this signaling axis is regulated by ST8SIA1 in TNBC cells. Conclusion: ST8SIA1 is up-regulated in basal-like and mesenchymal subsets of TNBC tumors and is associated with poor prognosis in TNBC patients. Furthermore, we found that ST8SIA1 tightly regulates PI3K-AKT-mTOR signaling pathway downstream of GD2. Targeting ST8SIA1 could be a novel therapeutic strategy in TNBC therapy. Citation Format: Khoa Nguyen, Bin Yuan, Yuanqing Yan, Kim-Anh Do, Naoto T. Ueno, Michael Andreeff, Venkata Lokesh Battula. ST8SIA1 is a novel therapeutic target in TNBC: Regulates FAK-PI3K-AKT-mTOR signaling to promote tumor growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1929.

Donation of neural stem cells? Post mortal viability of spinal cord neuronal cells.

Transplantation of cells into central nervous system (CNS) shows a potential for treatment of post-traumatic and neurodegenerative diseases. Cadaver-derived neural cells can help reducing deficit of allogeneic material ready for transplantation. In this study we analyze post-mortal survival of spinal cord neural cells. Maximal time when alive neuronal cells can be recovered form spinal cord of the animals was determined as 56hr for human-size animal and 18hr for rat. Cells with surface expression of ganglioside GD2 and antigen CD24 constituted up to one percent of all recovered alive cells in earlier samples with time dependent decline in percentage. GD2-positive cells from rat spinal cord demonstrated spontaneous and induced electrical activity, which reduces with time post mortem.

GD3 ganglioside-enriched extracellular vesicles stimulate melanocyte migration

Melanomas often accumulate gangliosides, sialic acid-containing glycosphingolipids found in the outer leaflet of plasma membranes, as disialoganglioside GD3 and its derivatives. Here, we have transfected the GD3 synthase gene (ST8Sia I) in a normal melanocyte cell line in order to evaluate changes in the biological behavior of non-transformed cells. GD3-synthase expressing cells converted GM3 into GD3 and accumulated both GD3 and its acetylated form, 9-O-acetyl-GD3. Melanocytes were rendered more migratory on laminin-1 surfaces. Cell migration studies using the different transfectants, either treated or not with the glucosylceramide synthase inhibitor d-1-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (PPPP), allowed us to show that while GM3 is a negative regulator of melanocyte migration, GD3 increases it. We showed that gangliosides were shed to the matrix by migrating cells and that GD3 synthase transfected cells shed extracellular vesicles (EVs) enriched in GD3. EVs enriched in GD3 stimulated cell migration of GD3 negative cells, as observed in time lapse microscopy studies. Otherwise, EVs shed by GM3+veGD3−ve cells impaired migration and diminished cell velocity in cells overexpressing GD3. The balance of antimigratory GM3 and promigratory GD3 gangliosides in melanocytes could be altered not only by the overexpression of enzymes such as ST8Sia I, but also by the horizontal transfer of ganglioside enriched extracellular vesicles. This study highlights that extracellular vesicles transfer biological information also through their membrane components, which include a variety of glycosphingolipids remodeled in disease states such as cancer.

A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest.

Lysosomal accumulation of undegraded materials is a common feature of lysosomal storage diseases, neurodegenerative disorders, and the aging process. To better understand the role of lysosomal storage in the onset of cell damage, we used human fibroblasts loaded with sucrose as a model of lysosomal accumulation. Sucrose-loaded fibroblasts displayed increased lysosomal biogenesis followed by arrested cell proliferation. Notably, we found that reduced lysosomal catabolism and autophagy impairment led to an increase in sphingolipids ( i.e., sphingomyelin, glucosylceramide, ceramide, and the gangliosides GM3 and GD3), at both intracellular and plasma membrane (PM) levels. In addition, we observed an increase in the lysosomal membrane protein Lamp-1 on the PM of sucrose-loaded fibroblasts and a greater release of the soluble lysosomal protein cathepsin D in their extracellular medium compared with controls. These results indicate increased fusion between lysosomes and the PM, as also suggested by the increased activity of lysosomal glycosphingolipid hydrolases on the PM of sucrose-loaded fibroblasts. The inhibition of β-glucocerebrosidase and nonlysosomal glucosylceramidase, both involved in ceramide production resulting from glycosphingolipid catabolism on the PM, partially restored cell proliferation. Our findings indicate the existence of a new molecular mechanism underlying cell damage triggered by lysosomal impairment.-Samarani, M., Loberto, N., Soldà, G., Straniero, L., Asselta, R., Duga, S., Lunghi, G., Zucca, F. A., Mauri, L., Ciampa, M. G., Schiumarini, D., Bassi, R., Giussani, P., Chiricozzi, E., Prinetti, A., Aureli, M., Sonnino, S. A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest.

Helicobacter pylori SabA binding gangliosides of human stomach

ABSTRACT Adhesion of Helicobacter pylori to the gastric mucosa is a prerequisite for the pathogenesis of H. pylori related diseases. In this study, we investigated the ganglioside composition of human stomach as the target for attachment mediated by H. pylori SabA (sialic acid binding adhesin). Acid glycosphingolipids were isolated from human stomach and separated into subfractions, which were characterized by mass spectrometry and by binding of antibodies, bacteria, and Solanum tuberosum lectin. H. pylori SabA binding gangliosides were characterized as Neu5Acα3-neolactohexaosylceramide and Neu5Acα3-neolactooctaosylceramide, while the other acid human stomach glycosphingolipids characterized (sulfatide and the gangliosides GM3, GD3, GM1, Neu5Acα3-neolactotetraosylceramide, GD1a and GD1b) were not recognized by the bacteria. Defining H. pylori binding glycosphingolipids of the human gastric mucosa will be useful to specifically target this microbe-host interaction for therapeutic intervention.

FRI-065 - Cholesterol-mediated expression of ganglioside GD3 acetylation contributes to liver fibrogenesis

FRI-065 - Cholesterol-mediated expression of ganglioside GD3 acetylation contributes to liver fibrogenesis

Prevalence of IgG Autoantibodies against GD3 Ganglioside in Acute Zika Virus Infection

Zika virus (ZIKV) disease has become a global health emergency with devastating effects on public health. Recent evidences implicate the virus as an emergent neuropathological agent promoting serious pathologies of the human nervous system, that include destructive and malformation consequences such as development of ocular and fetal brain lesions, microcephaly in neonates, and Guillain–Barré syndrome (GBS) in adults. These neurological disorders of both central and peripheral nervous systems are thought to be associated to the neurotropic properties of the virus that has ability to infect neural stem cells as well as peripheral neurons, a hallmark of its pathogenicity. The presence of autoantibodies against gangliosides plays a pivotal role in the etiogenesis of GBS and a variety of neurological disorders. Gangliosides are a class of galactose-containing cerebrosides mainly expressed in nervous system tissues playing a critical role in the physiology of neural cells and neurogenesis. Herein, our findings indicate that patients at acute phase of ZIKV infection without any neurological signs show increased levels of IgG autoantibody against GD3 gangliosides, a class of glycolipid found to be highly expressed in neural stem cell acting in the maintenance of their self-renewal cellular capacity. It is possible that a pathological threshold of these antibodies is only acquired in secondary or subsequent infections. In the light of these evidences, we propose that the target of GD3 by autoimmune responses may possibly has an effect in the neuropathy and neurogenesis disorder seen during ZIKV infection.

Mahanine drives pancreatic adenocarcinoma cells into endoplasmic reticular stress-mediated apoptosis through modulating sialylation process and Ca2+-signaling

Endoplasmic reticulum (ER) stress results from protein unfolding/misfolding during cellular maturation, which requires a coordinated action of several chaperones and enzymes and Ca2+ signalling. ER-stress possibly has a positive effect on survival of pancreatic cancer cell. Therefore, detailed insights into this complex signaling network are urgently needed. Here, we systematically analyzed the impact of ER stress-mediated unfolded protein response (UPR) and Ca2+-signaling cross-talk for the survival of pancreatic adenocarcinoma (PDAC) cells. We observed enhanced ER activity and initiation of UPR signaling induced by a carbazole alkaloid (mahanine). This event triggers a time-dependent increase of intracellular Ca2+ leakage from ER and subsequently Ca2+ signaling induced by enhanced reactive oxygen species (ROS) produced by this pro-oxidant agent. In addition, we observed an altered glycosylation, in particular with regard to reduced linkage-specific sialic acids possibly due to decreased sialyltransferase activity. Changes in sialylation entailed enhanced expression of the ganglioside GD3 in the treated cells. GD3, an inducer of apoptosis, inhibited pancreatic xenograft tumor. Taken together, our study describes a molecular scenario how PDAC cells are driven into apoptosis by mahanine by UPR-driven ER stress-associated and ROS-mediated calcium signaling and possibly defective sialylation.