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Aims: Medulloblastoma (MB) is one of the most common malignant central nervous system tumors of childhood. Despite intensive treatments that often leads to severe neurological sequelae, the risk for resistant relapses remains significant. In this study we have evaluated the effects of the omega 3-long chain polyunsaturated fatty acids (omega 3-LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on MB cell lines and in a MB xenograft model.Main methods: Effects of omega 3-LCPUFA treatment of MB cells were assessed using the following: WST-1 assay, cell death probes, clonogenic assay, ELISA and western blot. MB cells were implanted into nude mice and the mice were randomized to DHA, or a combination of DHA and EPA treatment, or to control group. Treatment effects in tumor tissues were evaluated with: LC-MS/MS, RNA-sequencing and immunohistochemistry, and tumors, erythrocytes and brain tissues were analyzed with gas chromatography.Key findings: omega 3-LCPUFA decreased prostaglandin E2 (PGE(2)) secretion from MB cells, and impaired MB cell viability and colony forming ability and increased apoptosis in a dose-dependent manner. DHA reduced tumor growth in vivo, and both PGE(2) and prostacyclin were significantly decreased in tumor tissue from treated mice compared to control animals. All omega 3-LCPUFA and dihomo-gamma-linolenic acid increased in tumors from treated mice. RNA-sequencing revealed 10 downregulated genes in common among omega 3-LCPUFA treated tumors. CRYAB was the most significantly altered gene and the downregulation was confirmed by immunohistochemistry.Significance: Our findings suggest that addition of DHA and EPA to the standard MB treatment regimen might be a novel approach to target inflammation in the tumor microenvironment.

Medulloblastoma (MB) is one of the most common malignant central nervous system tumors of childhood. Despite intensive treatments that often leads to severe neurological sequelae, the risk for resistant relapses remains significant. In this study we have evaluated the effects of the ω3-long chain polyunsaturated fatty acids (ω3-LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on MB cell lines and in a MB xenograft model.Effects of ω3-LCPUFA treatment of MB cells were assessed using the following: WST-1 assay, cell death probes, clonogenic assay, ELISA and western blot. MB cells were implanted into nude mice and the mice were randomized to DHA, or a combination of DHA and EPA treatment, or to control group. Treatment effects in tumor tissues were evaluated with: LC-MS/MS, RNA-sequencing and immunohistochemistry, and tumors, erythrocytes and brain tissues were analyzed with gas chromatography.ω3-LCPUFA decreased prostaglandin E2 (PGEOur findings suggest that addition of DHA and EPA to the standard MB treatment regimen might be a novel approach to target inflammation in the tumor microenvironment.

Omega-3 fatty acids have been suggested as a complement in cancer treatment, but doses are not established. We performed a dose-finding study in 33 children in remission from cancer. Participants were allocated to a body surface area (BSA) adjusted dose (mg/m2) of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (40:60), ranging 233–3448 mg/m2 daily for 90 days. Fatty acid concentration in plasma phospholipids and red blood cells were determined by GC. Supplementation was well tolerated and correlated strongly with blood ω3-fatty acid concentrations and EPA showed the highest increase. Using the ω3-index disregards docosapentaenoic acid (DPA), which increased 30–43% in our study motivating an EDD-index (∑EPA,DPA,DHA). The ratio between arachidonic acid and EPA or DHA showed negative exponential trends. Dose per BSA enabled an individualized omega-3 supplementation decreasing the variation referred to interindividual differences. Based on our results, we suggest a dose of 1500 mg/m2 BSA for further studies.

// Conny Tummler 1 , Igor Snapkov 1 , Malin Wickstrom 2 , Ugo Moens 1 , Linda Ljungblad 2 , Lotta Helena Maria Elfman 2 , Jan-Olof Winberg 3 , Per Kogner 2 , John Inge Johnsen 2 and Baldur Sveinbjornsson 1, 2 1 Molecular Inflammation Research Group, Department of Medical Biology, Faculty of Health Science, University of Tromso, Tromso, Norway 2 Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, Stockholm, Sweden 3 Tumor Biology Research Group, Department of Medical Biology, Faculty of Health Science, University of Tromso, Tromso, Norway Correspondence to: Conny Tummler, email: conny.tummler@uit.no Keywords: pediatric cancer, neuroblastoma, inflammation, GPCR, chemerin Received: December 10, 2016 Accepted: July 06, 2017 Published: July 27, 2017 ABSTRACT Pro-inflammatory cells, cytokines, and chemokines are essential in promoting a tumor supporting microenvironment. Chemerin is a chemotactic protein and a natural ligand for the receptors CMKLR1, GPR1, and CCRL2. The chemerin/CMKLR1 axis is involved in immunity and inflammation, and it has also been implicated in obesity and cancer. In neuroblastoma, a childhood tumor of the peripheral nervous system we identified correlations between high CMKLR1 and GPR1 expression and reduced overall survival probability. CMKLR1, GPR1, and chemerin RNA and protein were detected in neuroblastoma cell lines and neuroblastoma primary tumor tissue. Chemerin induced calcium mobilization, increased MMP-2 synthesis as well as MAP-kinase- and Akt-mediated signaling in neuroblastoma cells. Stimulation of neuroblastoma cells with serum, TNFα or IL-1β increased chemerin secretion. The small molecule CMKLR1 antagonist α-NETA reduced the clonogenicity and viability of neuroblastoma cell lines indicating the chemerin/CMKLR1 axis as a promoting factor in neuroblastoma tumorigenesis. Furthermore, nude mice carrying neuroblastoma SK-N-AS cells as xenografts showed impaired tumor growth when treated daily with α-NETA from day 1 after tumor cell injection. This study demonstrates the potential of the chemerin/CMKLR1 axis as a prognostic factor and possible therapeutic target in neuroblastoma.

Herold, Nikolas et al., The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP), which causes DNA damage through perturbation of DNA synthesis. Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment. Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient-derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies., This work was supported by grants from the Swedish Children’s Cancer Foundation (TJ2016-0040 (to N.H.); 2015-0005 (to J.-I.H.); PR2015-0009 (to D.G.); and PR2013-0002 and PR2014-0048 (both to T.H.)), the Swedish Cancer Society (CAN 2016/837 to J.W.; CAN 2014/814 to S.L.; CAN 2015/768 to D.G.; CAN 2013/396 to J.-I.H.; and CAN 2012/770 and CAN 2015/255 to T.H.), the Swedish Research Council (2014-1839 to M.U.; 2015-02498 to S.L.; 2012-2037 to D.G.; and 2012-5935 and 2013-3791 to T.H.), Radiumhemmet’s Research Foundations (154242 to G.R. and 144063 to D.G.), the Knut and Alice Wallenberg Foundation (KAW2014.0273 to T.H.), the Swedish Pain Relief Foundation (SSF/01-05 to T.H.), the Torsten and Ragnar Söderberg Foundation (to T.H.), the David and Astrid Hagelén Foundation (C24702193 to B.D.G.P.) and the Stockholm County Council (ALF project) (20150353 to S.L. and 20150016 to J.-I.H.). This work was supported by the German Research Foundation (DFG) (SCHA1950/1-1 to T.S.) and partially through the Federal Ministry of Education and Research of Germany (BMBF)–supported Immunoquant project (0316170 C to T.S.) and HIVERA: EURECA project (01KI1307B to T.S.). S.G.R. is supported by an EMBO Long-Term Fellowship (ALTF-605-2014). Chemical Biology Consortium Sweden is funded by the Swedish Research Council, Science for Life Laboratories and Karolinska Institutet (829-2009-6241 to H.A. and T.L.).

Introduction: Prostaglandin E2 (PGE2) is a key lipid mediator of inflammation and carcinogenesis. Apart from promoting direct growth of cancer cells, PGE2 has been shown to be crucial in maintaining the immunosuppressive tumor microenvironment. Drugs targeting cyclooxygenase (COX)-1 and -2, enzymes upstream in the production of PGE2 and other prostanoids, have successfully reduced tumor growth in many cancer models but these drugs are all associated with adverse long-term effects. The aim of this study is to demonstrate an anti-tumor effect in medulloblastoma by selective inhibition of microsomal prostaglandin E1 (mPGES-1), the enzyme downstream of COX-1/2 specifically required for the synthesis of PGE2. Experimental procedures: Human medulloblastoma cell lines (DAOY and D283) were treated with a selective small-molecule inhibitor against mPGES-1 or COX-2 in the presence or absence of pro-inflammatory interleukin (IL)-1â. Prostaglandins in supernatants were extracted and quantified with LC-MS/MS. Drug efficacy was determined and cell toxicity was measured with cell viability assay. Mice with human medulloblastoma xenograft will function as proof-of-concept for the anti-tumor activities associated with inhibition of PGE2 production. Key inflammatory enzymes were quantified with Western blot. Results: Medulloblastoma cells express high levels of COX-2, mPGES-1, and produce PGE2. The increased production of PGE2 by IL-1â was completely blocked when cells were co-treated with inhibitor for mPGES-1 or COX-2. Both inhibitors showed cell toxicity. We did not observe any shunting towards other prostanoids. Conclusion: We investigate the effect of selective inhibition of mPGES-1 or COX-2 on medulloblastoma growth. We aim to further characterize the mPGES-1 inhibitor and compare to selective COX-2 inhibitor in preclinical medulloblastoma models. Specific inhibition of PGE2 production, rather than general inhibition of prostanoid production, would potentially prove useful as a complement to current medulloblastoma treatments. Citation Format: Filip Bergqvist, Linda Ljungblad, Malin Wickström, Karin Larsson, Anna Kock, Marina Korotkova, John Inge Johnsen, Per Kogner, Per-Johan Jakobsson. Specific inhibition of prostaglandin E2 production in medulloblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 761.

Background: The embryonic brain tumor medulloblastoma is a highly malignant tumor of the cerebellum and the most common CNS malignancy of childhood. By combining neurosurgery, chemotherapy and radiation overall survival is about 70% but the intensive treatment regimen often leads to severe neurological sequelae and the risk of resistant relapses is significant. Hence, there is a need to improve treatment to reduce and alleviate late side effects. Inflammation plays an important role in the tumor microenvironment by suppressing immune response, promoting angiogenesis, facilitating tumor invasion, and metastasis. Prostaglandin E2 (PGE2) is a pro-inflammatory lipid mediator derived from the omega-6 fatty acid arachidonic acid (AA) through conversion by the key enzymes COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1). Previous work from our group showed that medulloblastomas express high levels of COX-2 and mPGES-1 as well as all four PGE2 receptors (EP1-4) and most importantly that PGE2 promotes tumor proliferation. The ω-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) show anti-inflammatory characteristics in general and exert anti-tumoral properties in several cancer types. Here we investigate the potential role of DHA and EPA in treatment of medulloblastoma and their effect on prostaglandins in vitro and in vivo. Methods: In six medulloblastoma cell lines the cytotoxic activity of DHA and EPA was analyzed using cell viability assays. PGE2 levels were assessed in three medulloblastoma cell supernatants after 24 hrs of DHA treatment by ELISA. The prostaglandins were evaluated in human medulloblastoma xenografts in nude mice treated with DHA or a combination of DHA and EPA for 30 days from time of tumor take. Control mice were fed with normal chow. The levels of prostaglandins in the tumor tissue were analyzed with LC-MS/MS. Furthermore, the incorporation of fatty acids in tumors, erythrocytes and brain tissue was analyzed with GC-MS/MS. Results: Both DHA and EPA exerted cell toxicity in a dose dependent manner in vitro. DHA reduced the PGE2 production in all three cell lines investigated. In vivo, the treatment with DHA alone and the combination of DHA and EPA significantly increased the omega-3 index in tumors, erythrocytes and normal brain. The PGE2, and prostacyclin was significantly reduced in both treatment groups while thromboxane levels remained unchanged. The in vivo treatment was non-toxic. Conclusions: Lowering the pro-inflammatory PGE2 levels could mitigate the ongoing inflammation in the tumor microenvironment. We show that the omega-3 fatty acids DHA and EPA exert toxic effects on medulloblastoma cells in vitro and reduce PGE2 both in vitro and in vivo. We propose that DHA and EPA are interesting candidates to improve current medulloblastoma therapy. Citation Format: Linda Ljungblad, Filip Bergqvist, Teodora Andonova, Per-Johan Jakobsson, John Inge Johnsen, Per Kogner, Malin Wickström. Omega-3 fatty acids DHA and EPA decrease oncogenic PGE2 in medulloblastoma in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2454.

The aim of this study was to assess the use of human papillomavirus (HPV) E6/E7 mRNA testing in the follow-up of women treated for cervical intraepithelial neoplasia (CIN) by conization and to compare the prognostic value of HPV E6/E7 mRNA to HPV L1 DNA and cytology. One hundred and forty-three women underwent cytological/histological testing, HPV DNA genotyping by Linear Array, and HPV E6/E7 mRNA testing by APTIMA HPV assay during follow-up after surgical treatment for histologically verified CIN. High-grade residual/recurrent disease (CIN2+/HSIL+) was identified in 7 (4.9%) women, and low-grade disease (CIN1/LSIL) in 25 (17.5%). At the inclusion visit 33 (23%) women were HPV DNA-positive; 13 (9.0%) were HPV E6/E7 mRNA-positive. HPV E6/E7 mRNA did not identify three women with high-grade disease. Presence of high-risk HPV DNA at the inclusion visit predicted 100% (95% CI 64.6-100) of high-grade residual/recurrent disease, with a specificity of 80.9% (95% CI 73.5-86.6); cytology had a sensitivity of 85.7%, and a specificity of 87.5%. HPV E6/E7 mRNA testing was a poor predictor of treatment failure, with a sensitivity of 57.1% (95% CI 25.0-84.2), but high specificity (93.4%; 95% CI 87.9-96.5). Detection of high-risk HPV DNA after treatment by conization identified 100% of women with residual/recurrent high-grade disease, whereas HPV E6/E7 mRNA testing was a poor predictor of treatment failure. This study suggests that a negative HPV mRNA result cannot exclude the risk of malignant progression, and that HPV E6/E7 mRNA testing by APTIMA HPV assay is not useful in the follow-up of women treated for CIN.

Background: The omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exert antitumoral properties shown in several cancer types. DHA is found in abundance in the brain and is of importance in normal brain development and maintenance of function including cognitive functions. Medulloblastoma is the most common malignant CNS tumor of childhood. Intensive multimodal treatment is necessary including surgery, radiotherapy and chemotherapy. However, treatment often results in significant neurological sequelae and the risk of resistant relapses is still significant. The neuroprotective and antitumoral properties of omega-3 could therefore be of great benefit. Our aim was to investigate the effect of DHA and EPA on medulloblastoma in vitro and in vivo. Methods: In a panel of medulloblastoma cell lines the cytotoxic activity of DHA and EPA was studied using cell viability assays. The molecular mechanisms were characterized using cell- and molecular biology techniques. Mice with human medulloblastoma xenografts were treated with; DHA alone or with DHA and EPA for 30 days from time of tumor take while tumor growth was monitored. Control mice were fed with normal chow. The incorporation of fatty acids was analyzed in tumors, erythrocytes and brain tissue by LS-MS/MS. Results: DHA and EPA induced medulloblastoma cell toxicity with IC50 values ranging from 1.9 to 68 μM in six medulloblastoma cell lines. DHA reduced the prostaglandin E2 production, indicating a possible mechanism of action. In vivo, omega-3 supplementation resulted in significant reduction of tumor growth when established tumors were treated. DHA levels were increased in both tumor tissue and erythrocyte membrane compared to control. The in vivo treatment was non-toxic. Conclusions: DHA and EPA exert toxic effect on medulloblastoma cells in vitro and reduce tumor growth in vivo. These omega-3 fatty acids could therefore be good candidates for improving current therapy and at the same time protect healthy neurons from therapy-induced toxicity promoting cognitive function in survivors. Thus, omega-3 fatty acid supplementation has the potential of reducing tumor growth as well as reducing sequelae. Citation Format: Linda M. Ljungblad, John-Inge Johnsen, Malin Wickström, Per Kogner, Helena Gleissman. A novel approach to treat medulloblastoma: The omega-3 fatty acids DHA and EPA reduce medulloblastoma tumor growth in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3275. doi:10.1158/1538-7445.AM2015-3275

Neuroblastoma (NB) is an embryonic childhood tumor of the sympathetic nervous system. Despite intensive treatment patients in the high-risk group of NB shows a poor prognosis. We have shown the importance of inflammation in NB development and progression and that the pro-inflammatory lipid mediator prostaglandin E2 (PGE2) acts as a paracrine survival factor in NB. We have also shown that mPGES-1, essential for PGE2 synthesis, promotes growth and survival of several malignancies. In this study we focus on the abundance of prostaglandins and their specific enzymes in primary NB of different biological and clinical subsets in the aim of finding a more specific and targeted NB treatment. Levels of prostaglandins were assayed in 40 primary human NB tumors by mass spectrometry (MS) showing elevated levels of PGE2 in high risk 11q-deleted NB compared to low-risk NB whereas elevated levels of prostaglandin D2 (PGD2) could be coupled to NB with favorable clinical outcome. The expression of COX-1, COX-2, mPGES-1, L-PGDS, H-PGDS and 15-PGDH were analyzed in tumors by immunohistochemistry (IHC). Expression of mPGES-1 and L-PGDS was found in all analyzed tumors. Double stainings of mPGES-1 and the specific NB cell marker GD2 were performed on tumors with different biology using immunofluorescence. In low-risk tumors mPGES-1 is not expressed by the tumor cells whereas co-localization were found in 11q deleted high-risk tumors. To consider the level of inflammation and PGE2 signaling in these tumors we analyzed cell markers of both the innate and adaptive immune system together with mPGES-1. In all tumors mPGES-1 expressing cells are found in clusters closely together with T-cells, B-cells, dendritic cells and M1/M2 macrophages. Together this suggests an involvement of inflammatory prostaglandins in neuroblastoma development and tumor progression opening up new strategies for targeted therapies. Citation Format: Anna Kock, Karin Larsson, Linda Ljungblad, Helena Idborg, Marina Korotkova, Lotta Elfman, John-Inge Johnsen, Per-Johan Jakobsson, Per Kogner. Characterization of prostaglandin signaling in primary neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3986. doi:10.1158/1538-7445.AM2014-3986

Background/Aim: Docosahexaenoic acid (DHA) and eicosapentaenic acid (EPA) are two essential omega-3 polyunsaturated fatty acid that have been shown to exert antitumoral effects in several cancer types, both in vitro and in vivo. DHA has also been shown to protect neural cells from apoptosis and is of importance in the maintenance of normal brain development and function. Medulloblastoma (MB) is the most common malignant brain tumor in children with a poor 5-year survival of only 50-60%. It is a highly invasive embryonal neuroepithelial tumor that arises in the cerebellum and has a tendency to disseminate throughout the CNS early in its course. Multimodal treatment is always necessary in the form of surgery, radiotherapy and chemotherapy. However, treatment for this disease often results in significant endocrinological and intellectual sequelae. The neuroprotective and antitumoral properties of omega-3 fatty acids could therefore be of great benefit in this patient group. Here we aim to investigate the effect of DHA and EPA in medulloblastoma. Methods: Cytotoxic activity of DHA and EPA was studied in cell viability assay in a panel of medulloblastoma cell lines. The molecular mechanisms were characterized using cell- and molecular biology techniques. Results: By adding different concentrations of DHA and EPA we could demonstrate IC50s ranging between 1.9 and 46 µM for DHA and 3.8 and 68 µM for EPA in the six examined medulloblastoma cell lines. All cell lines showed greater sensitivity for DHA than EPA. Preliminary data showed that treatment with DHA inhibited the prostaglandin E2 production in medulloblastoma cells, indicating a possible mechanism of action. Conclusions: Medulloblastoma cells are highly sensitive to omega-3 induced toxicity. Previous studies have showed that DHA can protect neurons from apoptosis. Therefore, omega-3 fatty acids are good candidates for improving medulloblastoma therapy by acting as both “sword and shield” by killing off cancer cells while protecting healthy neurons from therapy induced toxicity, thus possibly both inhibiting tumor growth and reducing the sequelae associated with other modes of brain tumor treatment. Citation Format: Linda M. Ljungblad, Malin Wickström, John-Inge Johnsen, Per Kogner, Helena Gleissman. The omega-3 fatty acids DHA and EPA inhibit medulloblastoma growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3988. doi:10.1158/1538-7445.AM2014-3988