“Antitumor Activity of Novel Anti-MM Agents and Combinations, the Proteasome Inhibitor Bortezomib and Multikinase Inhibitor Sorafenib, Both Applied as Monotherapy and in Combination in NOD/SCID-IL2-Receptor-Gamma-chain−/− (NSG) Mice Using a Intratibial Tumor Dissemination approach”.
Authors: Schüler Julia , Wider Dagmar , Pfeifer Dietmar , Udi Josefina , Fiebig Heinz-Herbert , Waesch Ralph M. , Engelhardt Monika 
Published: October 2019

Abstract
Abstract 4912
Since novel treatment options are needed in multiple myeloma (MM), novel anti-MM agents and combinations are eagerly pursued to further improve the prognosis for MM patients. For potentially novel therapeutic agents, functional in vivo models are highly valuable. We have established a cell line-based, disseminated MM model in NOD/SCID-IL2-receptor-gamma-chain−/− (NSG) mice. In our current analysis, the multikinase inhibitor sorafenib was validated alone and in combination with the well-established anti-MM agent bortezomib in 6 independent experiments. Optimized dose and schedule were determined as follows: 1. sorafenib (100mg/kg/d; d0-11) alone, 2. bortezomib (0.7mg/kg/day (d); d0,4,11) alone, 3. both in combination with the respective doses and schedules compared to 4. a control group. L363 cells were injected intratibialy into NSG mice and respective therapies were started 7 days after L363-injection (d0). Tumor growth was monitored with daily monitoring of MM-symptoms, flow-cytometry (FACS) and fluorescence-based in vivo imaging (FI). Tumor inhibition was calculated as the proportional reduction of mean MM-cell-infiltration at the respective compartment of the test- compared to the control-group (optimal T/C in %). Furthermore, hollow bones of the injected mice were retrieved when mice were sacrificed, cells flushed out and MM cells purified by MACS microbeads. Total RNA was isolated from these cells and gene expression profiles analyzed using the HG-U133 Plus 2.0 array (Affymetrix) and the Expressionist software (Genedata AG, Basel). L363 engrafted reliably (take rate=100%) at the injection site and in distant organs, such as bone marrow (BM; 100%), spleen (38%) and rarely liver (8%); in the latter organs as previously reported. Control mice developed MM symptoms, such as hind limb pareses, weight loss and osteolyses. At the respective doses and schedules, the examined compounds were well tolerated in tumor-bearing mice. No acute toxicity could be observed and maximal body weight loss was 4% with mono- and 11% with combined therapy. Primary tumor development was markedly reduced by sorafenib (optimal T/C of 11% on d11), as well as with bortezomib, albeit to a lesser extend (optimal T/C: 22% on d5). BM metastases were also significantly reduced by sorafenib with an optimal T/C value of 21% on d11. Bortezomib reduced BM infiltration to an optimal T/C value of 46% on d5 as compared to the control. Combined therapy of sorafenib and bortezomib showed most pronounced anti-tumor and anti-metastatic effects, inducing T/C values of 17% (primary tumor) and 7% (BM) on day 11, respectively.
Table 1. Antitumor effect of Sorafenib and Bortezomib in mono- and combined therapy in the L363-xenograft model Compound Side effects Primary tumor Bone marrow Dose Mortality Max. median bwc1 FI2 tumor inhibition FI2 tumor inhibition [mg/kg/d] [n] [%] [%] [%] Sorafenib 100 0 / 5 96 11 21 Bortezomib 0.7 0 / 5 97 22 46 Soraf. / Bortez. 100 / 0.7 0 / 5 89 17 7 1 bwc=body weight changes 2 Tumor inhibition was calculated as the median % of MM cells determined by FI at respective compartments of the test vs. control group multiplied by 100 (optimal test/control (T/C) in %)
L363 engraftment into NSG is a valuable in vivo MM model which exhibits high reproducibility, take- and metastases-rates and closely mimics the clinical situation. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor MM growth. Sorafenib showed promising results in our MM model, in particular in combination with bortezomib. Amongst others, a detailed characterization of the anti-tumor activity of both compounds will be provided by gene expression analysis of L363 cells isolated from untreated vs. treated mice. Further investigations to validate other innovative anti-MM agents as well as their combinations are currently also pursued.

Disclosures
No relevant conflicts of interest to declare.