ALBERT

Description: Multiple myeloma (MM) is a hematological malignancy characterized by a plasma cell accumulation in the bone marrow (BM), for which novel treatment options are urgently needed. Epigenetic modulating agents such as histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) are under intense investigation for cancer therapy. As shown in numerous functional in vitro studies, HDACi and DNMTi affect various biological processes important for tumor control including tumor cell survival, proliferation, differentiation and DNA repair. Given the broad mechanisms of action of these agents, it remains important to continue pre-clinical evaluation to identify in vivo relevant mechanisms of action. This may lead to the identification of novel biologically relevant targets and predictive biomarkers allowing clinical trial optimization. Recently, we developed gene expression based risk scores after treating human MM cell lines with TSA (HA-score) or decitabine (DM-score). These scores were predictive for MM patient survival using two independent cohorts (Heidelberg-Montpellier (HM) and University of Arkansas for Medical Sciences-Total Therapy 2) and identified potential biomarkers predictive for drug sensitivity. However, the transcriptional response of MM cells in vivo may be influenced by the close contact with the BM-environment. Therefore, we here aimed to characterize the transcriptional response of MM cells after in vivo treatment with the HDACi JNJ-26481585 (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 isolated from the BM and subjected to microarray analysis. Using Significance Analysis of Microarray, we identified 574 and 180 probesets deregulated by respectively quisinostat and decitabine (of which 111 are in common). To assess the prognostic value of the deregulated genes, we performed MaxStat analysis in the HM cohort. JNJ-585 deregulated 31 genes associated with good prognosis and 31 associated with bad prognosis. Decitabine altered expression of 20 genes linked with poor prognosis while 5 genes were linked with good prognosis. 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 MGUS and healthy bone marrow plasma cells. In addition, the scores were useful to separate patients of the 2 cohorts into a low risk and high risk group. Patients from the proliferation subgroup had a higher score compared to all other subgroups. In concordance, the scores were highest in patients with a high gene-expression based proliferation index. Using gene ontology (GO) tools (DAVID) and pathway tools (Reactome, STRING and Pathway-guide), we next explored the association of in vivo deregulated genes with biological processes and pathways. GO analysis showed that quisinostat-deregulated genes were mainly involved in immune modulation. Pathway analysis revealed associations with lymphocyte activation and proliferation, immune effector mechanisms and T-helper-1 development through processes like cytokine interactions, chemokine signaling and T-cell receptor- and NK-cell-signaling. In concordance, the signature represented elevated presence and signaling of interferon, tumor necrosis factor, interleukin-1 (IL-1), IL-2 and IL-12. The second most prominent alterations were genes linked with transcriptional misregulation in cancer. Pathways predicted to be affected by these alterations are linked with differentiation, resistance and cell survival. For decitabine, the gene list was substantially smaller and for more than half shared by JNJ-585. The pathway analysis also identified genes linked to immune system, gene expression regulation and metabolism pathways. In conclusion, in vivo treatment with epigenetic modulating agents identified a prognostic gene signature. In addition, HDACi (and to a lesser extent DNMTi) deregulated immunomodulatory genes and genes involved in transcriptional regulation. This indicates that immune regulation is an important in vivo anti-tumor property of HDACi and supports the rational to combine HDACi with immunomodulatory therapies such lenalidomide or cellular/peptide vaccination strategies. Disclosures Hose: Novartis: Research Funding. Seckinger:Novartis: Research Funding.

Description: Hypoxia is associated with increased metastatic potential and poor prognosis in solid tumors. In this study, we demonstrated in the murine 5T33MM model that multiple myeloma (MM) cells localize in an extensively hypoxic niche compared with the naive bone marrow. Next, we investigated whether hypoxia could be used as a treatment target for MM by evaluating the effects of a new hypoxia-activated prodrug TH-302 in vitro and in vivo. In severely hypoxic conditions, TH-302 induces G0/G1 cell-cycle arrest by down-regulating cyclinD1/2/3, CDK4/6, p21cip-1, p27kip-1, and pRb expression, and triggers apoptosis in MM cells by up-regulating the cleaved proapoptotic caspase-3, -8, and -9 and poly ADP-ribose polymerase while having no significant effects under normoxic conditions. In vivo treatment of 5T33MM mice induces apoptosis of the MM cells within the bone marrow microenvironment and decreases paraprotein secretion. Our data support that hypoxia-activated treatment with TH-302 provides a potential new treatment option for MM.

Description: Insulin-like growth factor-1 (IGF-1) is an important factor in proliferation, cell survival and migration of multiple myeloma (MM) cells. Bcl-2 like 11 (Bim) is a pro-apoptotic protein belonging to the BH3-only group of Bcl-2 family members. Three major forms of Bim are known: BimEL, BimL and BimS. In leukemia, beta1 integrin mediated adhesion has been shown to reduce Bim levels by enhanced proteasome activation. We used the 5T33MM syngeneic, immunocompetent murine model for MM. Both the exclusively in vivo growing 5T33MMvv cells and the stroma independent in vitro growing 5T33MMvt cells were used. The present study showed that Bim levels were reduced in MM cells after IGF-1 treatment. IGF1-mediated regulation of Bim expression in 5T33MMvv cells was demonstrated to be a result of reduced gene expression on the one hand and increased proteasomal-mediated degradation of Bim protein levels on the other hand. Reduced expression was related to activation of the Akt pathway and inactivation of the transcription factor FoxO3a. Increased degradation of Bim was related to activation of the mitogen-activated protein kinase pathway, as IGF-1 treatment resulted in phosphorylation of ERK1/2. Reducing Bim levels by transducing 5T33MMvt cells with a lentiviral vector with a short hairpin RNA cassette inhibited serum starved and the clinical relevant deacetylase inhibitor panobinostat (LBH589, Novartis) induced cell death. Together our data indicate that disrupting IGF-1-mediated regulation of Bim degradation may increase the efficacy of drugs. In the past few years, methylation of DNA and acetylation of histones have been proven to be important regulators of gene expression. Moreover, acetylation of non-histone proteins has emerged as a dynamic posttranslational modification regulating numerous cellular processes. Co-treatment of 5T33MMvv and 5T33MMvt cells for 24h with the demethylation agent 5-Aza-2′deoxycytidine and the deacetylase inhibitor panobinostat resulted in a strong induction of Bim expression at protein level. In the future, we will investigate the contribution of IGF-1 to the epigenetic silencing of Bim by i) comparing the Bim promoter methylation pattern of both human IGF-1 responsive cell lines and murine 5T33MMvv cells treated or not with IGF-1 using bisulfite PCR sequencing and ii) using the epigenetic modulating agents 5-Aza-2′deoxycytidine and panobinostat.

Description: Multiple myeloma (MM) is ultimately fatal in part because no effective cell cycle-based therapy has been available to control tumor cell proliferation. Loss of cell cycle control in MM cells stems from coordinated deregulation of Cdk4-cyclin D1 or Cdk6 (Cdk4)-cyclin D2. PD 0332991 is the first orally bioactive small molecule that potently and specifically inhibits Cdk4 and Cdk6. It represents a promising cell cycle-based therapy for myeloma owing to its ability to potently inhibit Cdk4/6 and induce G1 cell cycle arrest in primary human myeloma cells in BM stromal cell co-cultures and to control tumor progression in a xenograft model. However, the efficacy of PD 0332991 in the presence of an intact immune system is unknown. To optimize therapeutic targeting of Cdk4/6 with PD 0332991, we investigated the effectiveness of PD 0332991 in inhibiting Cdk4/6 and controlling myeloma tumor progression in the immunocompetent, bone migrating 5T33MM model. By quantitative real-time PCR analysis, we found that these myeloma cells express a normal plasma cell transcription program such as upregulated Blimp-1 and loss of Bcl-6 expression. However, they proliferate aggressively due to Cdk4 overexpression and impaired p27Kip1 expression, thereby mimicking relapse disease in human myeloma. PD 0332991 potently inhibits Cdk4/6 phosphorylation of Rb in primary 5T33MM cells and induces G1 cell cycle arrest, both in vivo and ex vivo. Accordingly, treatment with PD 0332991 significantly prolongs the survival of tumor-induced 5T33MM mice; a mean of 35 days in the PD 0332991-treated group (N=9) versus 25 days in the vehicle-treated group (N=9, p〈 0.003). These findings demonstrate for the first time that PD 0332991 targets Cdk4/6 and controls tumor expansion in the presence of an intact immune system. To further optimize Cdk4/6 targeting in MM, we combined PD 0332991 with bortezomib, a cytotoxic drug widely used in MM treatment. Pretreatment of 5T33MMvt cells with PD 0332991 for 48 hours markedly augmented bortezomib killing, to the same level of twice the dose of bortezomib when it was used alone. These studies of PD 0332991 in the immunocompetent 5T33MM model demonstrate that, as a consequence of Cdk4/6 inhibition and induction of G1 arrest, PD 0332991 sensitizes MM cells to killing by a cytotoxic agent. Targeting Cdk4/6 in combination therapy, therefore, represents a novel and promising strategy for myeloma treatment.

Description: Destructive bone lesions due to osteolytic bone disease are a major cause of morbidity and mortality in multiple myeloma patients, occurring in more than 80% of cases. Underlying osteolytic bone disease is an uncoupling of the bone remodeling process, with an increased activity of osteoclasts and a decreased activity of osteoblasts. Current strategies to treat osteolytic bone disease focus on anti-resorptive agents, which do not rebuild bone loss. Src kinase has been implicated in both osteoclast and osteoblast function. In this study, we assessed the effect of Src inhibition with AZD0530 (saracatinib, Astra Zeneca) on the development of multiple myeloma and its associated osteolytic bone disease. We first determined Src family kinase expression in the multiple myeloma microenvironment and found that patient-derived myeloma cells express Src at low levels but disease stage does not correlate with Src expression levels. In accordance with the literature, Src mRNA expression was found to increase during osteoclast differentiation and decrease during osteoblast differentiation in publicly available microarray datasets. Next, we validated an inhibitory role of AZD0530 on osteoclast differentiation and function. At a pharmacological relevant concentration of 1 micromolar, AZD0530 inhibited the differentiation of RAW264.7 osteoclasts (Oc.N/FOV: 15.5+-1.6 treated vs. 53+-1.5 non-treated). AZD0530 treatment appeared to hamper efficient progenitor cell fusion and osteoclast polarization, reflected by a decrease of CTSK and DC-STAMP mRNA levels and a defective actin ring formation in treated cultures, which culminated in a complete inhibition of bone resorption. When assessing the effect of AZD0530 on osteoblast function we found that AZD0530 inhibits osteoblast differentiation, with a decreased expression of OSX and OCN, and alters osteoblast morphology. In vivo, AZD0530 did not alter myeloma cell bone marrow infiltration in both the 5TGM.1 (37+-6.3% AZD0530 treated vs. 25.2+-6.7% non-treated) and 5T2MM (26.1+-7.7% vs. 29.1+-6.4%) murine multiple myeloma models. However, bone health was significantly improved in both models following treatment with AZD0530. In the 5TGM.1 model multiple trabecular bone parameters were restored to levels observed in healthy control mice following AZD0530 treatment, including BV/TV (11.7+-0.3% treated vs. 6.4+-0.3% non-treated), Tb.N. (2.5+-6x10^-2/mm vs. 1.7+-9x10^-2/mm) and Tb.Th (46.2+-1micron vs. 37+-0.8micron). These results were confirmed in the 5T2MM model, which displays a more severe osteolytic bone disease. In addition, AZD0530 treatment resulted in an increase in cortical thickness (157.8+-0.8micron treated vs. 151.4+-0.7micron non-treated) and a decrease in the number and size of cortical lesions in 5TGM.1 mice. Finally, our findings were corroborated by histomorphometric analyses. In conclusion, we report a potent inhibitory effect of the Src inhibitor AZD0530 on the development of osteolytic bone disease in multiple myeloma. Our results indicate that AZD0530 exerts this effect via the modulation of both osteoclast and osteoblast function. These findings warrant further study of the feasibility and efficacy of AZD0530 to treat osteolytic bone disease in multiple myeloma patients. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2966 The great challenges in multiple myeloma (MM) treatment are to overcome drug resistance and to prevent relapse. The bone marrow (BM) microenvironment plays a critical role in MM cell growth and survival. The Notch pathway was found to be activated in MM. In this study we aimed at investigating the role of Dll1/Notch interaction in MM clonogenic growth and in vivo engraftment, as well as the role of Notch pathway in BM-induced drug resistance. Dll1 is a Notch ligand expressed in BM stromal cells. Cocultures were performed using murine 5T33MM cells with Dll1 ligand or MS5.Dll1 (Dll1-overexpressing) stromal cells. Notch downstream target genes (Hes1, Hes5, Hey1, Hey2, and HeyL) were investigated for Notch pathway activation, showing that 5T33MM cells expressed Notch target genes and that Hes5 and HeyL were up-regulated both on mRNA (Real-Time PCR) and protein level (western blot) after Dll1/Notch interaction. This up-regulation could be reverted by blocking Notch signaling with DAPT. Moreover, only adhesive cell-cell direct interaction was necessary for MM Notch activation and no soluble factors in the coculture system were involved. Colony-forming-cell (CFC) assays and in vivo engraftment studies were performed to investigate MM clonogenic ability and MM disease initiation. 5T33MM cells cocultured for several days on MS5.Dll1 cells were able to form more tumor colonies compared to 5T33MM cells cocultured on MS5 control cells. Furthermore, these cocultured 5T33MM cells were injected intravenously into naive mice to investigate disease development. Mice injected with Dll1-activated 5T33MM cells developed faster MM disease than those injected with control cells, and also had a higher paraprotein concentration (1.98 ± 0.44 g/dl, n=3) compared to control mice (0.95 ± 0.02 g/dl, n=3). 5T33MM cells pretreated with DAPT for 6 days, to inhibit constitutive Notch pathway activation, had a decreased ability to form colonies in the CFC assay compared to control cells. In addition, mice were injected with 5T33MM cells pretreated with DAPT or DMSO. After 59 days, the DMSO control group became sick with an average M paraprotein of (1.78±0.14g/dl, n=3), while in the DAPT group, all mice were healthy and no M paraprotein could be detected at that time. The DAPT treated group did develop MM disease, but significantly delayed (average of 161 days after injection) with a lower M paraprotein amount (0.35±0.13g/dl, n=3). All these results suggest that the Notch pathway is involved in MM in vitro clonogenic growth and in vivo engraftment. Further studies revealed that Notch pathway activation with Dll1 could induce drug resistance to Bortezomib treatment. Cell viability assay showed that 72.6% of 5T33MM cells stimulated with Dll1 remained alive after Bortezomib treatment compared to only 24.9% alive in IgG control. This protective effect could be reverted by inhibiting Notch pathway with DAPT. After cocultures of MM cells with MS5.Dll1 stromal cells, we detected an increased percentage of CD138- MM cells and a down-regulation of CD138 mRNA expression compared to cocultures with MS5 stromal cells. This could be reverted by preventing cell-cell interaction using transwells or by adding DAPT. Similar results were found with human RPMI-8226 cells. Matsui et al. (Cancer Res 2008; 68: (1) 190–197) and our lab demonstrated that CD138- MM cells were less sensitive to drugs than CD138+ MM cells. Interestingly, CD138- MM cells showed a higher level of Notch activation than CD138+ MM cells, suggesting that Notch activation is involved in the protection of CD138- MM cells from drug induced cell death. In conclusion, our data show that Dll1/Notch pathway activation could promote MM clonogenic growth and accelerate MM development. Inhibiting Notch pathway can significantly suppress MM initiation and delay the engraftment. Moreover, Dll1/Notch interaction could also induce drug resistance to Bortezomib, by altering MM cells to a more resistant CD138- phenotype. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1840 One of the greatest challenges in multiple myeloma (MM) treatment is to overcome drug resistance. More and more evidence showed that not only the MM tumor cells should be targeted but also the bone marrow (BM) micro-environment. Interactions of MM cells with the BM micro-environment have a pivotal role in MM cell proliferation, survival, migration, angiogenesis as well as drug resistance. Many pathways are involved including the conserved Notch signaling pathway. The interaction of Notch receptors and ligands between adjacent cells induces proteolytic cleavage and release of the intracellular domain of the Notch receptor, also called Notch intracellular domains (NICD). NICD will then enter the nucleus and modify the expression of downstream target genes. Notch receptors are expressed by MM cells and Notch ligand Dll1 is present on bone marrow (BM) stromal cells. We investigated whether Notch activation in myeloma cells by the interaction with Dll1 on stromal cells contributes to bortezomib resistance. We analyzed Notch1 and Notch2 surface expression by flow cytometry on MM cells after Dll1 interaction using a stromal cell line modified to overexpress Dll1. Notch1 surface expression was not disturbed on mouse 5T33MMvt and human MMS1 and LP-1 cells while Notch2 expression on MM cells was significantly decreased after Dll1 interaction for 2 days. Next, we investigated NICD1 and NICD2 expression by western blot after Dll1/Notch interaction. NICD1 did not change in murine 5T33MMvt and human LP-1 and MMS-1 cells, while NICD2 is increased after Dll1 interaction. These results suggest that Dll1 can activate Notch signaling likely through the Notch2 receptor. We investigated whether Dll1/Notch activation could contribute to MM bortezomib resistance. MM cells were cocultured on immobilized recombinant Dll1 ligand and treated with 5 nM bortezomib for 48h. Compared to control, MM cells cocultured with Dll1 ligand were less sensitive to bortezomib. Furthermore, blocking the Notch pathway by DAPT (a gamma secretase inhibitor, GSI) could reverse this effect and increased the sensitivity to bortezomib. To delineate the molecular mechanism of Dll1-induced bortezomib resistance, we performed a drug resistance and metabolism gene array and found that CYP1A1 was significantly upregulated by Dll1/Notch interaction. CYP1A1 is a member of the cytochrome P450 family and regulates drug metabolism. We further demonstrated that inhibiting CYP1A1 by either α-Naphthoflavone (inhibitor) or CYP1A1-siRNA increases the sensitivity of MM cells to bortezomib, suggesting that CYP1A1 is involved in bortezomib resistance. As also previously demonstrated, CD138- 5T33MM cells are less sensitive to bortezomib than CD138+ 5T33MM cells. We analyzed CYP1A1 expression and activity and observed a higher CYP1A1 amount in CD138- cells compared to CD138+ MM cells. The higher CYP1A1 expression in CD138- cells might be a possible mechanism for their decreased bortezomib sensitivity compared to CD138+ cells. In addition, an in vivo experiment was performed. Combination treatment of DAPT with bortezomib was able to increase bortezomib sensitivity and prolonged overall survival in the 5T33MM mouse model. In conclusion, our results suggest that Dll1/Notch activation contributes to bortezomib resistance by upregulating CYP1A1, a molecule involved in drug metabolism. Our data provide a potential strategy to overcome bortezomib resistance by combination with a Notch pathway inhibitor. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2794 Poster Board II-770 Myeloid-derived suppressor cells (MDSCs) are a heterogeneous mix of myeloid cells in different maturation stages generated in the bone marrow. The role of MDSCs in cancer is to suppress T-cell responses, thereby likely regulating tumor progression. In mice, MDSCs are identified by the expression of the surface markers CD11b and Gr-1. Recently, Ly6G+ granulocytic (PMN-MDSC) and Ly6G− monocytic (MO-MDSC) subsets could be distinguished (Movahedi et al. Blood 2008, 111:4233-44). In multiple myeloma patients, the immune function is impaired and this is caused by an immunologically hostile microenvironment and cellular defects, such as decreased numbers of immune cells, and DC or T-cell dysfunction. However, the role of MDSCs in immune suppression in multiple myeloma is not yet described. In this study, we investigated the immunosuppressive activity and mechanism of MDSC subsets in the syngeneic and immunocompetent 5TMM mouse model (5T2 and 5T33 models). In first instance, CD11b+Ly6G− and CD11b+Ly6G+ lineage-committed myeloid MDSC subsets were detected in 5TMM-diseased bone marrow by flow cytometry. These subsets were purified via MACS from the bone marrow of naïve and 5TMM tumor-bearing mice, and analyzed for T-cell suppressive activity. Hereto, CD8+ TCR-transgenic OT-1 splenocytes were stimulated with ovalbumin protein in the presence of purified MDSC subsets, after which T-cell proliferation was measured via 3H-thymidine incorporation. Both MDSC subsets from 5TMM bone marrow were able to suppress antigen-specific T-cell responses at a higher level compared to purified MDSC subsets from normal bone marrow. On average, Ly6G− MDSCs were more suppressive than Ly6G+ MDSCs. The 5T2MM model has a tumor take of approximately 12 weeks. Three weeks after intravenous inoculation of the tumor cells, the suppressive effect of the myeloid subsets was already observed (while the plasmacytosis in the BM was very low and no detectable serum M spike was observed), indicating that T-cell suppression is an early event in MM development. To unravel the suppressive mechanism of the MDSC subsets, inhibitors were used in ovalbumin-stimulated cocultures. Ly6G− MDSC-mediated suppression was partially reversed by the iNOS inhibitor L-NMMA and the COX-2 inhibitor sc-791, both of which lower the NO concentration in culture. In contrast, superoxide dismutase and especially catalase enhance NO concentrations, resulting in enhanced T-cell suppression. None of these inhibitors had any impact on the Ly6G+ MDSC-mediated suppression. In conclusion, these data reveal the presence of MDSCs as a novel immune suppressive strategy employed by multiple myeloma cells in the bone marrow, already occurring early in the disease process. Disclosures: No relevant conflicts of interest to declare.

Description: The discovery of novel agents such as the proteasome inhibitor bortezomib has significantly increased the survival of multiple myeloma (MM) patients. However MM remains an incurable disease mainly due to relapse, associated with significant resistance to therapy including bortezomib. Therefore further investigation to elucidate the disease and the mechanisms leading to drug resistance is necessary. The success of bortezomib highlights the importance of the ubiquitin-proteasomal system (UPS) in MM. The UPS regulates protein turnover and plays a key role is several cellular processes such as apoptosis, cell cycle progression, cell proliferation and DNA replication. The Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase protein complex involved in controlling cell cycle progression. The regulation of APC/C is dependent on 2 co-activators: Cdc20 and Cdh1. The APCCdc20 complex is controlling the metaphase to anaphase transition in mitosis, while APCCdh1 controls mitotic exit and early G1 phase. During metaphase, the activity of APCCdc20 is inhibited by the spindle assembly checkpoint. When all kinetochores are properly attached, the spindle assembly checkpoint is silenced and APCCdc20 becomes activated. When APCCdc20is active, cell cycle proteins are targeted for degradation by the proteasome such as securin and cyclin A and B leading to mitotic exit. Recent studies described that spindle assembly checkpoint is defective in MM cells and that patient samples after chemotherapy and at relapse displayed an increased chromosomal instability signature including Cdc20. The aim of our study is to elucidate the importance and therapeutic potential of APC/C and its co-activators Cdc20 and Cdh1 in MM. Analysis of gene expression in the data of Zhan et al. (Blood 108, 2020-8, 2006) revealed that the co-activator Cdc20 was higher expressed in certain MM sub-groups (PR, MS, CD1, MF) compared to healthy bone marrow plasma cells. Moreover, high Cdc20 expression is correlated with poor prognosis. Cdh1 on the other hand was significantly lower expressed in all MM sub-groups compared to healthy bone marrow plasma cells. Interestingly, lower Cdh1 expression is correlated with poor prognosis. Next, we analyzed whether blocking APC/C would affect MM cells. For this study the pro-drug of TAME (tosyl-L-arginine methyl ester) that has been described as an inhibitor of the APC/C, was used. When the human myeloma cell lines LP-1 and RPMI-8226 were treated with proTAME, an accumulation of the APCCdc20 substrate cyclin B1 was seen already after 6 hours. However the levels of Skp2, an APCCdh1 substrate, were not affected by proTAME treatment. This suggests that proTAME inhibits the APCCdc20 complex but not the APCCdh1complex. We morphologically assessed the effect on number of metaphases on May-Grünwald Giemsa stained cytospins. ProTAME clearly induced an accumulation of LP-1 and RPMI-8226 cells in metaphase. Since a metaphase arrest can lead to cell death, we investigated the effect of proTAME on the viability and apoptosis. A significant dose-dependent decrease in viability and increase in apoptosis was observed after treatment with proTAME of human myeloma cell lines and primary MM cells purified from human and 5T33MM diseased mice. In contrast, other cells from the bone marrow microenvironment were not affected upon proTAME treatment. The induction of apoptosis was accompanied with caspase 3, 8, 9 and PARP cleavage. Western Blot analysis also showed phosphorylation of H2AX suggesting DNA damage upon proTAME treatment. Previous studies showed that MM is a heterogeneous disease consisting of a bulk CD138+ population and a minor CD138- population which is less sensitivity to drugs such as bortezomib. Interestingly, treatment of CD138+/- 5T33MM cells with proTAME demonstrated an equal targeting of both populations. From these results we can conclude that overexpression of Cdc20 by MM cells is correlated with a bad prognosis. Inhibition of APCCdc20 results in a metaphase arrest in MM cells which is associated with reduced viability and induction of apoptosis. Moreover, APC/C inhibition equally targets CD138+ and the more resistant CD138- 5T33MM cells. This study suggests that APC/C and its co-activator Cdc20 could be a new and promising target in MM. Disclosures No relevant conflicts of interest to declare.

Description: Myeloid derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that accumulate in different cancer types, including Multiple Myeloma (MM). Besides immune regulation, MDSC promote tumor angiogenesis and tumor growth by secretion of cytokines and growth factors. The presence and activation of MDSC in MM patients has been well-documented, however direct actions of MDSC on cancer cells have been poorly defined. In this study, we investigated MDSC distribution in the immunocompetent 5T33MM murine model and the effects of in vivo MDSC targeting by 5-Fluorouracil (5FU) in the presence of bortezomib. In addition, we determined direct effects of MDSC on MM cells in vitroin the presence of currently used anti-MM drugs (Bortezomib and Melphalan) and analyzed downstream pathways. In a first part, we determined the effects of the MM microenvironment on the total MDSC population using the 5T33MM model. The number of MDSC (defined as CD11b+ cells) was only increased the first week after MM cell inoculation, while circulating myeloid cells increased at end-stage of disease. In addition, we evaluated the effect of 5FU, an MDSC depleting chemotherapy agent, on tumor progression. 5T33MM mice (n=7/group) were treated with 50mg/kg 5FU on day 4, with bortezomib (0.7mg/kg, 2 times a week), with the combination of both or with vehicle and tumor load was assessed at day 17. We observed a significant reduction in serum M-spike and idiotype positive cells in the BM and spleen (30% reduction compared to vehicle, p