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For most people, being in good health represents the most important factor to wellbeing. While environmental elements such as physical, chemical, biological, social and psychosocial factors in the environment are key for a person’s wellbeing, also the genome of the individual and its interaction with the environment play an important role. In this paper, we will focus on attitudes towards genomics in the field of personalised medicine in oncology. We will document opinions encountered by patients and citizens on sharing health-related information for various purposes (e.g., research, cost-effectiveness, patient support) aiming to maximise the benefits for cancer patients. We will discuss ethical and legal considerations to be taken into account at the Belgian level to provide a secure, transparent framework for the use of genomics in the healthcare system.

Despite current therapy improvements for mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL), most MCL patients and ± 40% of the DLBCL patients experience relapses. Both MCL and DLBCL are aggressive B cell lymphomas, characterized with a higher proliferation rate. The anaphase promoting complex/cyclosome (APC/C) is an important checkpoint in mitosis, regulating the onset of the anaphase and cell cycle. This E3 ubiquitin ligase becomes activated upon interaction with the co-factor CDC20. CDC20 is frequently overexpressed in different human cancers (prostate cancer, breast cancer, multiple myeloma ...). This overexpression is associated with an aggressive course of the disease and a shorter overall survival. So far little is known about CDC20 in B cell lymphomas. Here, we investigated the inhibition of APC/C-CDC20 MCL and DLBCL. Using publicly available gene expression profiling data, we found that the majority of the DLBCL patients (>77%) exhibits a high CDC20 mRNA expression and that this overexpression is correlated with a worse prognosis, indicating that CDC20 is also involved in . Next, we evaluated the anti-lymphoma activity of the APC/C-CDC20 inhibitor proTAME (pT) using human MCL (Jeko-1, Mino and Rec-1) and DLBCL (OCI-Ly1, OCI-Ly7, SU-DHL-6, OCI-Ly3 and OCI-Ly10) cell lines. The pT treatment significantly increased the percentage of cells in metaphase. This metaphase arrest was accompanied by a strong reduction in MCL and DLBCL viability and a significantly increase in apoptosis. The induced apoptosis was found mediated by phosphorylation of the anti-apoptotic proteins Bcl-2/Bcl-XL, degradation/cleavage of the anti-apoptotic protein Mcl-1 and activation of the pro-apoptotic protein Bim. In addition, pT treatment induced phosphorylation of H2AX, suggesting that pT could also induce DNA damage. Finally, APC/C-CDC20 inhibition significantly enhanced the sensitivity of MCL and DLBCL cells the BH3 mimetic ABT-737 and ABT-199, as evidenced by a strong increase in caspase 3-dependent apoptosis compared to either agent alone. In conclusion, we identified APC/C-CDC20as a promising new target in the treatment of the aggressive B cell lymphomas MCL and DLBCL. In the future, the potential of specific CDC20 targeting, either alone or in combinations with currently available anti-cancer therapies, will continue to be investigated.

In Multiple Myeloma (MM), several reports demonstrate the presence and activity of myeloid-derived suppressor cells (MDSC) and their contribution to immunosuppression. S100A9 belongs to a low-molecular-weight calcium-binding protein family with its main function in cancer the induction of both MDSC accumulation and angiogenesis. S100A9 knockdown reduces MDSC accumulation as well as MM cell growth in the bone marrow (BM). S100A9 is therefore proposed as an attractive drug target and compounds inhibiting S100A9 interactions are developed. The goal of this project was to investigate the effect of targeting S100A9 in the immunocompetent 5T33MM mouse model with small molecule compounds that inhibit S100A9 (Active Biotech, Sweden). Western blot analysis demonstrated a higher presence of S100A9 in granulocytic Ly6Ghigh cells compared to monocytic Ly6Glow cells from 5T33MM-diseased BM and no expression in 5T33MM cells. Treatment of 5T33MM inoculated mice with the S100A9 inhibitors ABR-215757 (paquinimod) and ABR-238901 (daily gavage, 30 mg/kg) slightly diminished tumor cell percentages in BM with ABR-238901 being the most potent one (27% relative reduction). Interestingly, ABR-238901 in combination with bortezomib (0.6 mg/kg subcutaneously, 2 times/week) resulted in a higher reduction in tumor load compared to single treatments. We investigated the effect on MDSC accumulation and immunosuppressive activity after ABR-238901 treatment. Flow cytometry analysis demonstrated no differences in MDSC and CD8+ IFNy+ cell numbers in treated and control mice. In addition, blocking S100A9 did not inhibit the suppressive activity of MDSC. Interestingly however, a significant reduction in IL6 and IL10 was observed by PCR in MDSC from ABR-238901 treated mice compared to vehicle. Since S100A9 is involved in angiogenesis, microvessel density was analyzed in the BM by CD31 stainings. 5T33MM mice treated with ABR-238901 had a significant decreased angiogenesis compared to 5T33MM mice treated with vehicle. In conclusion, blocking S100A9 interactions in MM BM does not affect MDSC accumulation and activity, however it influences the expression of IL6 and IL10 in MDSC. Furthermore, S100A9 blockade decreases angiogenesis and tumor load especially in combination with bortezomib.

Multiple myeloma (MM) is characterized by an accumulation of monoclonal plasma cells in the bone marrow (BM) and progression of the disease is the result of a complex crosstalk between the different cell types present in this microenvironment. Although fatty deposits can occupy up to 70% of the BM cavity with aging, little attention has been given to the role of adipocytes in MM development. Moreover, immune cells can be influenced by adipokines, i.e. cytokines and hormones produced by white adipose tissue. The aim of this study is to investigate the role of the adipokine leptin and its receptor in supporting MM disease development, not only by a direct effect on MM cell proliferation but also by modulation of the protective tumor immunity. Our results show that leptin serum levels increase progressively during the course of the disease in the 5T33MM mouse model, an immunocompetent model for MM. Moreover, leptin was able to increase 5T33MM cell proliferation in vitro. We also detected a progressive upregulation of the leptin receptor on Natural Killer T cells (NKT) and different subsets of T cells during MM development. Functional analyses in vitro showed that leptin had inhibitory effects on the NKT cell function, immune cells which are important for anti-tumor immunity. Subsequently, a nanobody targeting the leptin receptor was tested in combination with an NKT stimulating agent, α-GalCer (α galactosylceramide), in order to examine the impact on MM disease in vivo. This combination prevented the occurrence of NKT cell anergy associated with the use of α-GalCer as a single agent and led to a complete inhibition of tumor development as measured by the M spike in the serum. Taken together, our preliminary data support the hypothesis that leptin could play an important function in MM pathology, not only on the MM cells themselves but also on the immune system. Pharmacologic manipulation of adipokines such as leptin in combination with immunotherapy could be an attractive novel strategy to target MM.

Macrophages are an abundant component of the myeloma bone marrow microenvironment and a prognostic factor for multiple myeloma (MM) patients. They express more M2 markers like CD206 (mannose scavenger receptor) and CD163 (hemoglobin scavenger receptor) which correlates with an “alternatively activated state” and immunosuppressive and pro-angiogenic function. They are also involved in drug resistance to several anti-myeloma agents like melphalan (alkylating agent) and bortezomib (proteasome inhibitor). The goal of our project is to further unravel survival mechanisms in MM cells induced by tumor-associated macrophages (TAMs) and to target TAMs. An increase in survival of murine MM cells (5T33MMvv and 5T33MMvt) is observed after 24h of coculture with macrophages independently of their polarization state. The biggest effects were observed with TAMs obtained from bone marrow derived macrophages of C57BL/KaLwRij mice and polarized with 5T33MMvt conditioned medium. This survival benefit for myeloma cells is maintained when bortezomib and melphalan are added for 24h and is confirmed by a reduced cleavage of caspase-3. This drug resistance is cell contact mediated and independent of interleukin-6 and -10. Western blot analysis revealed an increased phosphorylation of STAT3 in 5T33MMvv and 5T33MMvt cells and also an increased phosphorylation of Akt in 5T33MMvv cells in coculture with TAMs while no changes in (p)ERk or MCL-1 were observed. We further investigated the involvement of pSTAT3 in the induced survival. AZD1480 (an ATP-competitive JAK2 inhibitor) targets both MM cells and TAMs. Interestingly, 5T33MMvv/vt cells were more sensitive to AZD1480 induced apoptosis when cocultured with TAMs. The inhibition of phosphorylation STAT3 in 5T33MMvv/vt cells could be a possible explanation for this increased sensitivity. In conclusion, TAMs induce the survival of MM cells with the activation of STAT3 as a possible underlying mechanism. STAT3 activation is an interesting target not only for targeting MM cells but also TAMs.

Mutual communication between multiple myeloma (MM) cells and mesenchymal stromal cells (MSC) plays a pivotal role in supporting MM progression. In MM, MSC exhibit a different genomic profile and dysregulated cytokine secretion compared to normal MSC, however the mechanisms involved in these changes are not fully understood. Here, we examined the miRNA changes in human MSC after culture with conditioned medium of MM cells and found 19 dysregulated miRNAs, including upregulated miR-146a. Moreover, exosomes derived from MM cells contained miR-146a and could be transferred into MSC. After overexpressing miR-146a in MSC, secretion of several cytokines and chemokines including CXCL1, IL6, IL-8, IP-10, MCP-1, and CCL-5, was elevated, resulting in the enhancement of MM cell viability and migration. DAPT, an inhibitor of the endogenous Notch pathway, was able to abrogate the miR-146a-induced increase of cytokines in MSC, suggesting the involvement of the Notch pathway. Taken together, our results demonstrate a positive feedback loop between MM cells and MSC: MM cells promote the increase of miR146a in MSC which leads to more cytokine secretion, which in turn favors MM cell growth and migration.

Multiple myeloma (MM) is a hematological malignancy characterized by a plasma cell accumulation in the bone marrow (BM). Pre-clinical research showed that epigenetic modulating agents have promising therapeutic and prognostic applications in MM. Early clinical work however has been disappointing. To provide new possibilities for combinatory therapies, reduce side effects and identify biomarkers, better understanding of the therapeutic and prognostic features of HDACi and DNMTi is needed, especially in vivo where MM cells are protected by the BM microenvironment. Therefore, we aimed to characterize the transcriptional response of MM cells after in vivo treatment with the HDACi quisinostat or the DNMTi decitabine using the syngeneic immunocompetent murine 5T33MM model. 5T33MM mice (n=4/group) with established disease were treated with quisinostat or decitabine for 5 days after which tumor cells were subjected to microarray analysis. Quisinostat or decitabine deregulated respectively 61 and 25 genes with a prognostic value in newly diagnosed patients. The prognostic value of these genes was then implemented in a murine (Mu)-DM and Mu-HA score. The score values were significant higher in MM patients and human myeloma cell lines compared to healthy bone marrow plasma cells. In addition, the scores could separate newly diagnosed as well as relapsed patients into a low risk and high risk group. A high score was furthermore identified in the proliferation subgroup, in patients with a high gene-expression based proliferation index and in immature plasma cell stages. Gene ontology analysis showed that the deregulated genes encoded for proteins involved in apoptosis, regulation of cytoskeleton, immune regulation, metabolism, development and regulation of transcription. Pathway analysis revealed associations with lymphocyte activation and proliferation, immune-effector mechanisms, T-helper-1 development and elevated signaling of interferon, tumor necrosis factor, interleukin-1 (IL-1), IL-2 and IL-12. In conclusion, the in vivo transcriptional response to epigenetic modulating agents provided a prognostic gene signature and was significantly associated with immune regulation. This provides the rationale for testing epigenetic modulating agents in combination with immunotherapies.

Activation of invariant natural killer T cells reduces angiogenesis in the 5T33 multiple myeloma model Nur H1,2, Rao L3, Vacca A3, , Elewaut D4, Van Valckenborgh E1, De Bruyne E1, Vanderkerken K1 and Menu E1 1 Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel, Brussels, Belgium. 2 Department of Biology, Hebron University, Hebron, Palestine. 3 Department of Biomedical Sciences and Human Oncology, University of Bari "Aldo Moro" Medical School, Bari, Italy. 4 Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University, Ghent, Belgium. Angiogenesis refers to the generation of new vasculature from pre-existing blood vessels. In cancer, it is required for tumor growth. We used the immunocompetent 5T33MM model to investigate the anti-angiogenic effect of iNKTs. iNKTs respond to glycolipids such as ?-Galactosylceramide (?-GalCer). We first examined whether ?-GalCer-activated iNKTs have an effect on tumor angiogenesis by treating 5T33MM mice with ?-GalCer. A significant decrease of the microvessel density (MVD) was observed in the treated group (23.7%) compared to untreated group (33.3%). This was independent of the reduction in tumor load indicating that the secretory products of iNKTs such as IFN-? can reduce angiogenesis. To proceed further, conditioned media (CM) were prepared by coculturing DC with iNKT in presence (CM+) or absence (CM-) of ?-GalCer. Chorioallantoic membrane (CAM) and matrigel plug (MP) assays were performed by injecting CM into fertilized chicken eggs and NOD-SCID mice, respectively. Results showed a reduction in both blood vessel count in CAM (from 26 in CM- to 13 in CM+) and CD31+ cell count in MP (from 112 in CM- to 25 in CM+). To see whether this inhibition is mediated by IFN-?, endothelial cells (ECs) were cultured in vitro in CM, IFN-? and CM+ ?-IFN-? antibody. Cell Titer-Glo assays showed a significant reduction in the viability of ECs in CM+ and IFN-?. This reduction was partially blocked when CM+ was blocked by ?-IFN-?. To determine if the cells' low viability is related to anti-proliferative and/or apoptotic mechanisms, proliferation and TUNEL assays were performed. We found a reduction in ECs proliferation in CM+. This defect was confirmed using matrigel capillarogenesis assay which was then used for TUNEL stainings. A high number of apoptotic cells was observed in CM+ (18%) compared to CM- (7%) and control (8%). Therefore, we can conclude that anti-angiogenesis can be achieved by both pathways, and IFN-? is involved as an angiostatic factor. Taken together, these preclinical data indicate the possibility of harnessing the anti-angiogenic activity of iNKTs in MM.

Epigenetic modulating agents, including DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi), are of interest for their anti-tumor properties. Decitabine (DAC) is a FDA approved DNMTi for treatment of myelodysplastic syndrome, while JNJ-26481585 (JNJ-585) is a HDACi with potent anti-myeloma activity. Several studies revealed that DNMTi and HDACi exert their anti-tumor response by reversing epigenetic changes resulting in alterations in the transcriptome. In addition, DNMTi and HDACi exert direct cytotoxic effects. Limited studies are available on DAC alone and in combination with HDACi in multiple myeloma. Therefore we assessed the potential synergistic effect of DAC and JNJ-585 in MM. For in vitro experiments, we focused on human (OPM-2, RPMI 8226) and murine (5T33MM) cell lines. Using Cell-Titer-Glo, DAC and JNJ-585 showed synergistic anti-myeloma effects. This was confirmed by an AnnexinV-7AAD apoptosis assay. In general, apoptosis was associated with H2AX phosphorylation, caspase- and PARP-cleavage, which was more pronounced in the combination. In the OPM-2 cells, we also observed Mcl-1 cleavage and Bim upregulation. DAC and JNJ-585 also affected cell cycle progression. In the 5T33MMvt cells, DAC induced a G2/M-phase arrest while JNJ-585 induced a G0/G1-phase arrest. Combining both agents cancelled out the observed arrests and resulted in subG1 increase. In OPM-2 cells, DAC did not induce an arrest while JNJ-585 induced a G0/G1-phase arrest, which was maintained in the combination. In support of the cell cycle arrest, p21 and p27 were upregulated in both cell lines. Lastly, in the 5T33MM murine model, combination treatment resulted in significant lower serum M-spike, bone marrow tumor load and increased survival probability compared to single agent treatment. In conclusion, we demonstrated a synergistic anti-myeloma effect of DAC and JNJ-585. This was associated with a DNA damage response, cell cycle arrest, caspase activation and disruption of Bcl-2 family balance. DAC and JNJ-585 treatment in vivo resulted in slower disease progression suggesting clinical relevance.

The ABT-analogous 737, 263 and 199 are BH3 mimetics showing potent anti-myeloma (MM) activity, but only on defined molecular subgroups of MM patients presenting a Bcl-2high/Mcl-1low profile. IGF-1 is a major survival factor in MM regulating the expression of Bcl-2 proteins and might therefore be a resistance factor to these ABT-analogous. We first show that IGF-1 protected human MM cell lines (HMCLs) against ABT-737. Concurrently, the IGF-1 receptor inhibitor picropodophyllin (PPP) synergistically sensitized HMCL, primary human MM and murine 5T33MM cells to ABT-737 and ABT-199 by further decreasing cell viability and enhancing apoptosis. Knockdown of Bcl-2 by shRNA protected MM cells to ABT-737, while Mcl-1 shRNA sensitized the cells. PPP overcame the Bcl-2 dependency of ABT-737, but failed to completely overcome the protective effect of Mcl-1. In vivo, co-treatment of 5T33MM bearing mice significantly decreased tumor burden and prolonged overall survival both in a prophylactic and therapeutic setting. Interestingly, proteasome inhibitor resistant CD138- 5T33MM cells were more sensitive to ABT-737, whereas PPP alone targeted the CD138+ cells more effectively. After co-treatment, both subpopulations were targeted equally. Together, the combination of an IGF-1R inhibitor and an ABT-analogue displays synergistic anti-myeloma activity providing the rational for further (pre)clinical testing.

Multiple Myeloma (MM) is an incurable malignancy of terminally differentiated plasma cells, which are predominantly localized in the bone marrow. The presence of an immunosuppressive bone marrow microenvironment and dysfunction of immune cells are both described in MM patients; however the mechanisms controlling this immunosuppression are not well defined. Recently, a heterogeneous population of immature myeloid cells, the so called myeloid-derived suppressor cells (MDSC), were identified to be present and active in MM. MDSC are thought to promote cancer progression by their T-cell suppressive capacity, however little is known about the generation and activation of MDSC by MM cells. Also, it is not known whether MDSC are involved in the development of drug resistance. In this study we investigated the effects of myeloma cells on MDSC induction using the 5T33MM mouse model and healthy donor blood samples. In a first instance, myeloid cells (CD11b+) were isolated from the bone marrow of naïve C57BL/KaLwRij mice and cultured in conditioned medium (CM) derived from 5T33MMvt cells to measure survival with CellTiterGlo Assay and AnnexinV staining. Increased viability and reduced apoptosis of CD11b+ cells cultured in MM-CM compared to control medium were observed. In literature GM-CSF is described as a major survival factor for MDSC. The pro-survival effect induced by MM-CM could be abrogated by the use of a GM-CSF blocking antibody. In addition, we analyzed the effect of CM of human myeloma cell lines (HMCL) RPMI8226, LP-1 and OPM2 on the viability of peripheral blood mononuclear cells (PBMC) of healthy individuals. Human myeloma CM increased the viability of PBMC compared to control medium. When we analyzed the phenotype of PBMC using MDSC markers, we did not detect differences in the total CD11b+CD33+ percentage between control condition and HMCL-CM. However, in the CD11b+CD33+ population, the percentages of mononuclear MDSC (identified as CD14+HLA-DRlow) and polymorphonuclear MDSC (identified as CD11b+CD33+CD15+)were increased compared to control conditions. The immunosuppressive capacity of the generated population was analyzed. For mouse as well as for human experiments, we could observe a decreased proliferation of T-cells in CM compared to control medium. To unravel the mechanism of MM-induced MDSC survival, the expression of apoptosis regulating proteins from the Bcl-2 family (bcl-2,bcl-xl,mcl-1) and survival proteins (pSTAT3,STAT3) were investigated by western blot. Mouse CD11b+ cells incubated with 5T33MMvt CM had a clear increase in pSTAT3 compared to control. Moreover the anti-apoptotic proteins mcl-1 and bcl-xl were increased in 5T33MMvt-CM compared to control, while bcl-2 was not affected. We were able to confirm the results of pSTAT3, bcl-2 and mcl-1 using human CD11b+ cells incubated in control or RPMI8226-CM. In addition, we investigated the sensitivity of MM cells to drugs currently used in the clinic (melphalan and bortezomib) in the presence of MDSC. 5T33MMvv cells incubated with MDSC could survive better than 5T33MMvv cells cultured without MDSC. This phenomenon was also observed when drugs were added, indicating a pro-survival effect of MDSC on MM cells. From our experiments we can conclude that soluble factor(s) from myeloma cells are able to reduce T-cell proliferation. Since we could observe a better survival of CD11b+ cells and the appearance of MDSC phenotypes in myeloma CM, this immunosuppression is likely caused by the generation and increased survival of MDSC in myeloma CM with an increased STAT3 phosphorylation, mcl-1 and bcl-xl expression as underlying mechanisms.