Proliferation and Molecular Risk Score of Low Risk Myeloma Cells Are Increased in High Risk Microenvironment Via Augmented Bioavailability of Growth Factors
Authors: Mehdi Syed J. , Johnson Sarah K , Khan Sharmin , Ling Wen , Shelton Randal , Epstein Joshua , van Rhee Frits , Zangari Maurizio , Schinke Carolina D. , Thanendrarajan Sharmilan , Walker Brian A , Ashby Cody , Davies Faith E. , Morgan Gareth , Yaccoby Shmuel 
Published: October 2019

Abstract
Introduction:
Multiple myeloma (MM) cells from patients with smoldering MM (SMM) and low-risk (LR) MM harbor genetic alterations typically seen in patients with high-risk (HR) disease. To test whether the bone marrow (BM) microenvironment plays a role in controlling growth of LR MM cells, we established an experimental model that mimics a HR microenvironment by co-culturing normal mesenchymal stem cells (MSCs) with HR MM cells. We previously have shown that MSC conditioned media (CM) promotes growth of MM cells more effectively than cell-cell contact, as adhesion to MSCs often promotes survival at the expense of proliferation. Therefore, we utilized CM and
hypothesized that MSC CM is enriched with bioactive growth factors that facilitate proliferation of LR MM cells.
The aim of the study was to test the effect of CM from unprimed and primed MSCs on the survival, growth, and molecular properties of LR MM cells, and identify molecular pathways that mediate these effects.
Methods:
Primed MSCs were prepared by co-culturing normal MSCs with BM-dependent MM lines for 5 days. MSCs were trypsinized, replated for 40 min followed by serial washing to remove MM cells.
Molecularly classified CD138-selected LR MM cells from 8 newly diagnosed patients were treated with 50% primed CM or unprimed CM, or growth media (CONT) for 5 days. Growth and survival of primary MM cells was assessed by MTT assay and detection of annexin V/PI and KI67 by flow cytometry.
Microarrays were performed on primed and unprimed MSCs (n=7) and on primary LR MM cells treated with primed and unprimed MSCs CM (n=3). Pathways were analyzed using Ingenuity. Ultra low depth WGS was performed to assess copy number variation. Protein arrays were performed to test levels of secreted factors in CM (n=7).
Results:
Growth of primary LR MM cells (n=8) was increased by primed CM 5.1±0.05 (p<0.0001) and 7.0±0.6 (p<0.0001) folds compared to unprimed CM or CONT, respectively. In contrast, unprimed MSC CM increased growth in these cells by 1.3 fold. Flow cytometry analyses revealed no differences in proportion of annexin V/PI+ cells. However, the proportion of KI67+ cells was increased from 0.95±0.1% in unprimed CM group to 4.6±1.5% in primed CM group (p<0.04).
Primed MSC CM caused MM cell GEP70 score to increase resulting in change from LR to HR in 2 experiments and from an ultra LR score to an intermediate score in another. Pathway analyses on genes differentially expressed between primed CM- and unprimed CM-treated MM cells identified oxidative phosphorylation with mitochondrial dysfunction, cell cycle, mitosis and p53 as the most significantly altered pathways. Top transcription regulators included FOXO3, TP53, E2F4, MYC and E2F1, whereas mir-16-5p and let-7 were the top microRNAs. Top significantly upregulated genes (>2 fold) by primed MSC CM included proliferation-related factors (MKI67, TOP2A, CCNB1, BIRC5 and RRM2), whereas underexpressed genes (< 2 fold) involved regulators of cell dormancy including BCL2 (survival), RICTOR (mTOR), HEY1 (NOTCH), JUN (AP-1) and CXCR4 (adhesion). Four genes we reported to powerfully predict progression of SMM to MM (Khan et al., Haematologica 2015) were highly upregulated in MM cells by primed MSC CM.
WGS revealed similar copy number variation in MM cells treated with unprimed and primed CM, suggesting other mechanisms produced the observed gene expression changes.
IGF1 is a central MM growth factor and IGF binding proteins (IGFBPs) control its bioavailability. We recently reported that mesenchymal cells are the main source of IGFBPs in BM, with IGFBP2 being the most downregulated gene in MM bone (Schinke et al., CCR 2018). Expression and secretion of IGFBPs (particularly IGFBP2) by MSCs were significantly reduced by priming these cells with MM cells, whereas IGF1 levels remained unchanged. IGFBP2 markedly blocked IGF1-induced MM cell growth (p<0.0003). Addition of IGF1R inhibitor significantly blocked primed CM-induced MM cell growth (p<0.02).
Conclusions:
MSCs primed by HR MM cells mimic a HR microenvironment, reflected by reduced level of factors that restrain bioavailability of MM growth factors such as IGF1, resulting in shutdown of master regulators of cell dormancy, which then enable a MM cells to proliferate. Such a scenario is particularly applicable in SMM and LR disease where MM cells exhibit a low proliferative index and their expansion is accelerated in distinct HR BM microenvironmental niches such as focal lesions.

Disclosures
Epstein: University of Arkansas for Medical Sciences: Employment. Davies:Abbvie: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; MMRF: Honoraria; Janssen: Consultancy, Honoraria; TRM Oncology: Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding.