Transcriptome Alterations of Prospectively Isolated Bone Marrow Stromal Cells Provide Potential Cues to MGUS and Multiple Myeloma Pathobiology

Multiple myeloma (MM) is the second most common hematopoietic malignancy characterized by the clonal population of plasma cells that invade and destroy adjacent bone tissue. Monoclonal gammopathy of undetermined significance (MGUS) is the premalignant precursor of MM with 1% of patients progressing to MM every year. Although both MGUS and MM plasma cells depend on interactions with bone marrow stromal cells for their survival and growth, little is known about the exact mechanisms of this crosstalk and if specific genetic events in the stromal compartment are correlated with disease state.

Primary bone marrow mesenchymal stromal cells (BMSC) are essential components of the hematopoietic microenvironment and they give rise to the hematopoietic stroma in vivo. We have previously reported the phenotype of the bona-fide stroma stem cells in human bone marrow (Li et al., Stem Cell Reports, 3(6):965-74, 2014), and surface marker expression profiling studies on primary lin^-CD45^-CD71^-CD235a^-CD31^-CD271⁺ BMSC from patients with MM (n=5), MGUS (n=8) and healthy age-matched controls (n=7) demonstrated clear differences in the expression of signature MSC surface markers, such as CD146, CD105, CD106, CD230, CD51, CD73, and W5C5.

Motivated by these striking findings we then went on to transcriptionally interrogate prospectively isolated primary BMSC from MGUS and MM patients using RNAseq. Primary lin^-CD45^-CD71^-CD235a^-CD31^-CD271⁺ stromal stem cells from patients undergoing diagnostic bone marrow examination (n=43) and age-matched healthy donors (n=7) were isolated from aspirated bone marrow samples by FACS. Fifteen and five of these patients were diagnosed with MGUS and MM, respectively, and RNA was extracted from bulk sorted populations of primary MM-BMSC, MGUS-BMSC and control-BMSC. The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing was used for cDNA synthesis. Sequencing libraries were prepared using Nextera XT DNA Library Prep kit (Illumina) and run on a NextSeq500 system (Illumina) using the NextSeq 500/550 High Output Kit v2 (300 cycles).

Unsupervised clustering of the data showed that MGUS-BMSC samples clustered partly with healthy controls, but partly also with MM-BMSC or as a distinct and completely separate cluster. Clearly, some MGUS-BMSC showed features of healthy donor BMSC and some exhibited already a transcriptome profile that was more similar to MM-BMSC. In the next step, we therefore restricted the analysis to those BMSC samples that clustered separately and thus represented the MGUS-, MM- and normal BMSC specific transcriptional profiles. Comparison of the transcription patterns of these three distinct groups (MGUS-BMSC, n=3; MM-BMSC, n=5; control-BMSC, n=5) showed that a total of 3,758 genes were differentially expressed between the three groups. Further clustering of the data into 16 non-overlapping subsets of differentially expressed gene groups showed intriguing expression patterns. In one cluster, 78 genes were downregulated only in the MGUS-BMSC group, whereas three clusters contained genes that were downregulated specifically in MM-BMSC, and two clusters showed genes that were upregulated in both, MGUS-BMSC and MM-BMSC compared to control-BMSC. GSEA analysis revealed enrichment of genes reported to be downregulated in MM plasma cells, genes involved in cell adhesion and angiogenesis as well as genes involved in osteoclast differentiation in MM-BMSC versus MGUS-BMSC. Furthermore, MM-BMSCs were enriched for genes involved in integrin-mediated signaling pathway, cell-adhesion, and growth factors and chemokines associated with MM support compared to both control-BMSC and MGUS-BMSC.

Taken together, surface marker profiling of prospectively isolated BMSC identified marked differences between these important stromal elements in MGUS, MM, and normal bone marrow. RNAseq of the stroma stem cells identified genes that were specifically upregulated/suppressed in MM-BMSC compared to MGUS-BMSC and control-BMSC. These genes thus represent potential candidates that might be implicated in the disruption of normal stroma function, thus leading to disease development and progression. Accordingly, studies are underway to investigate the effects of selected genes on stroma-myeloma crosstalk (dys)function.