Follo, Marie

Abstract Introduction: Novel substances such as the next generation IMiD pomalidomide or the recently approved next generation proteasome inhibitor carfilzomib (Cfz) have considerably expanded our treatment options in MM, both of them influencing multiple myeloma (MM) interaction with bone marrow stroma cells (BMSCs), that provides an interesting target for anti MM therapy. More compounds directed at this disease critical crosstalk are currently under investigation, however the development of novel drugs remains inefficient, displayed by a substantial drop out rate of the 376 preclinical single agents tested since 1961 (Rongvaux, Annu Rev Immunol. 2013; Schüler, Expert Opin Biol Ther. 2013; Kortüm, CLML 2014). Our focus in the projected presented here was to develop a novel bone-derived in vitro 3D co-culture model specifically adapted to mimic the BM niche to more closely study the role of bone and BM bystander cells and to perform more reliable ex vivo compound screening in MM. Methods: Previous 2D models were compared to a novel 3D co-culture model (agarose matrix interlayer, 100 microwells/cm², 1.5mm in depth, permeable for oxygen+cytokines, but not for BMSCs utilizing U266, RPMI-8226, OPM-2 and primary BM patient (pt) cells, with and without HS-5 vs. M210B4 stroma support (Fig. A + Fig. B.a. for pt characteristics). Analyses covered Trypan Blue, Annexin/PI, MTT, FACS, cell cycle analyses and H2B-mCherry/cytochrome c-GFP assays (Udi, Br J Hematol. 2013). In a next step, primary bone-derived stroma cells were acquired from bones of C57BL/6 J mice. Bones were flushed, digested and FACS sorted in order to acquire single BM and bone bystander cell subtypes (MSPCs [mesenchymal stem and progenitor cells], endothelial cells, osteoblasts, PAS [PDFGRalphaSca1] and CaRs [CXCL12-abundant reticular cells]) which were then compared to HS-5 and M210B4 with regard to growth support, cytokine secretion and protection from anti-MM substances. Results: MMCLs and pt specimens were cultured at different concentrations (10 vs. 100 cells per microwell) with and without M210B4 demonstrating a growth advantage with vs. without M210B4 (Fig. B.b). Liquid overlay technique allowed cluster formation of pt specimens leading to more reliable propagation of pt material for up to 20d of culture. Apoptotic changes were assessed by confocal microscopy of RPMI8226 co-expressing fluorescently labelled histone 2B-mCherry (red) and cytochrome c-GFP (green) as indicators of late and early apoptosis. Comparing BMSCLs with regard to their MM growth support capacities, human HS-5 proved even more beneficent than M210B4 stroma (Fig. B.b). Phenotypic analyses of pt specimens showed decreased CXCR4 expression with vs. without BMSCs suggesting a dynamic regulation of homing molecules. The model was then used as an ex-vivo platform allowing both cytotoxicity and cell cycle analyses for the combination of bortezomib (Btz) vs. Cfz with ARRY-520 (kinesin spindle protein inhibitor). Btz (10nM) and Cfz (20nM) proved significantly cytotoxic compared to the control (U266 and pt specimens, respectively) after 48h of single agent treatment Fig.B.c). Compared to Btz (10nM, B10) and Cfz (20nM, C20) as single agents, the additional combination with ARRY-520 showed stronger additive cytotoxicity for Btz (A5+B10, median: 37.5% vs. 13.1%) than for Cfz (A5+C20, 38.6% vs. 34.1%). To note, the model could also be utilized for more profound analyses as depicted for G2/M cell cycle studies in Fig.B.d. 5nM ARRY-520 (A5) led to significant accumulation of OPM-2 cells in G2/M arrest after 48h of treatment confirming prior analyses (Hernández-Garcia Blood Suppl 4710,2014; Fig. B.d). Co-culture studies with different subsets of BM and bone-derived bystander cells are currently ongoing and will be presented at the meeting (Fig. B.e). Conclusions: More complex, 3D bone-derived high-throughput in vitro models are urgently needed to better predict the potency of preclinically tested agents and to better estimate the likelihood of their later clinical adoption into phase I-II trials. With this work, we provide an innovative model which reflects the BM microenvironment as a crucial predictor for in-vivo sensitivity as shown for ARRY-5200. This ex-vivo approach helps to better incorporate MM growth support by bone and BM-derived bystander cells and thus depicts a valid tool to better characterize the role of the BM niche in myeloma. Figure 1. Figure 1. Disclosures Engelhardt: Deutsch Krebshilfe: Other: grant. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Abstract Introduction: In the past decade, substantial progress has been made in the understanding of multiple myeloma (MM) cell biology and its interaction with the bone marrow microenvironment (BMM). Binding of MM cells to BM stroma cells (BMSCs) alters the expression of SDF-1α and its receptor CXCR4, leading to the secretion of anti-apoptotic cytokines, promoting tumor growth, drug resistance and migration. MM cancer stem cells migrate to endosteal BM niches, where they escape therapies in a quiescent state causing relapse in the course of the disease. The development of novel agents that aim to target the MM and BMM interaction includes drugs as promising as 2nd and 3rdgeneration IMIDs or proteasome inhibitors. Despite these profound advances, the failure rate of preclinically proven cytotoxic single substances is sizeable, as preclinical models often lack the biological, genetic, etiological and immunological properties of the disease (Schüler, Expert. Opin. Biol. Ther. 2013; Kortüm. CLML 2014; Rongvaux. Annu Rev Immunol 2013). Methods & Results: We have previously demonstrated that BM interaction and homing to niches, mediated by the adhesion molecules CXCR4, CD49d and CD44, protect MM cell lines (MMCL) and primary plasma cells (PC) from the cytotoxic effect of anti-MM agents, such as bortezomib (Bor), vorinostat (Vor) and pomalidomide (Pom). Our in vitro and in vivo observed cytotoxic effects from Bor, Vor and Pom confirmed their potent cytotoxicity, whereas cocultivation with M2-10B4 substantially reduced apoptosis and induced tumor protective effects. Additional treatment with the CXCR4 inhibitor AMD3100 blocked CXCR4 in coculture, but left CD49d, CD44 and CD11a widely unchanged. Toxic or therapeutic effects from AMD3100 monotherapy were excluded for the doses used. Comparison of the CXCR4 antibody (ab)-clones 12G5, 44717 and 4G10 revealed that AMD3100 treatment of U266 cells reduced CXCR4 expression with use of 12G5 and 44717, whereas binding of both FITC- and PE-coupled 4G10 was not influenced, making the latter the most reliable for CXCR4 analysis. Use of image cytometry (IC) allowed accurate visualization of co-localisation of CXCR4 expression both on the cell surface and within the cytoplasm of MM cells. IC correlated with flow cytometry-determined CXCR4 expression and allowed the detailed assessment of treatment studies with and without anti-MM agents and AMD3100. Of note, AMD3100 resensitized MM cells to Bor, Vor and Pom (Waldschmidt. Blood 2012:2450), whereas carfilzomib (Cfz) reduced CXCR4 expression in MMCL and could not be antagonized by stroma coculture. Cfz sensitivity was not increased by adding AMD3100 (Simon. Blood 2013:3851). These preclinical studies need additional adaptation to the clinical setting in order to surpass prior drug failure rates, and there is a need to develop more broadly available and better predictive preclinical systems. Therefore, we are currently assessing a 3D co-culture MM model composed of agarose matrix interlayers, based on a novel liquid overlay technique. This model has been specifically adapted to MM cell and BM component interactions as described (Udi. BJH 2013; Zlei. Exp Hematol 2007; Schüler. EOBT 2013). MM cells are cultivated in conical microwells of a non-adherent agarose matrix after BMSCs were plated on the bottom of each plate, allowing the diffusion of soluble cytokines but no direct contact between BMSC and MMCL. Therein, we are presently testing novel anti-MM substances in comparison to our standard-coculture system. Conclusion: Targeting microenvironmental mediators, like SDF-1α and CXCR4, is a promising approach to expand the choice of antimyeloma agents and amplify the effects of established antimyeloma drugs, as previously shown by us and others for the combination of AMD3100 and Bor or Pom. However, as our knowledge on MM and its BMM has dramatically increased a great effort has been made in the preclinical testing of promising new anti-MM agents, and more complex high-throughput in vitro models are urgently needed to better predict the potency of these substances in order to reduce dropouts in clinical trials. We hereby provide a novel approach which better reflects the spatial growth of human MM samples in BMSC coculture, and more closely mimics the growth and proliferation of human MM clones in vivo. Disclosures No relevant conflicts of interest to declare.