Abstract

Novel therapeutic agents have substantially improved the clinical outcomes of multiple myeloma (MM), although the majority of MM patients eventually relapse and ultimately die from therapy-refractory disease. Early studies documented the presence of clonogenic populations with MM stem-like properties in MM patients, which presumably arise from post-germinal center B (GCB) cells and are resistant to current therapies. However, both biologic properties and their ability of these cells have remained unclear. In this study, we attempted to isolate these cells from phenotypically defined (CD38+138+19-45-) MM cells (hereafter PhMMs) in bone marrow (BM) and characterized them.

BM samples were obtained from 48 MM patients, including 26 newly diagnosed (NDMM) and 22 with relapsed or refractory disease (RRMM) according to the protocol approved by the Institutional Review Board of the Kindai University Faculty of Medicine. Since the side population (SP) phenotype has been considered to represent the stem cell population in various types of normal and tumor tissues, we first analyzed the expression of HSC/HPCs markers (e.g. CD34, 49f, 90, 133) in the PhMMs SP population. Among the HSC/HPCs markers, the expression levels of CD34 was significant higher in SP cells compared with those in the main population (MP) cells (mean±SD, MFI of SP vs MP in PhMMs: 925.0±84.5 vs 29.2±4.2, n=5, p<0.01). The median count of CD34+ cells in PhMMs was 0.63% (interquartile range, 0.20), and a large number of these CD34+ PhMMs displayed the same Ig light chain with the unseparated myeloma cell population. Furthermore, FISH analyses on these cell populations confirmed the presence of cytogenetic abnormalities, such as t(11;14) and del(17p) in almost examined cells, while these abnormalities were not observed in the CD34+38-45dim HSC/HPCs subsets of the same patient, suggesting that CD34+ PhMMs contain clonotypic cells with genetic abnormalities. Next, we investigated the % of CD34+ PhMMs at various disease status. Notably, the % of CD34+ PhMMs was significantly higher in RRMM than in NDMM samples. Also, paired analysis showed that the % of CD34+ fraction was significantly higher after therapy than before therapy in PhMMs, indicating that CD34+ PhMMs are more resistant to anticancer drugs than CD34- PhMMs.

Next, we performed a colony forming cell assay by platting CD34+ and CD34- PhMMs isolated from 12 patients (2.5×103/dish), respectively. While CD34- PhMMs developed no colony, CD34+ PhMMs vigorously generated many large colonies in all cases, demonstrating that CD34+ PhMMs but not CD34- PhMMs have strong clonogenic and proliferative capacities. When CD34+ and CD34- PhMMs from 4 MM patients were injected intravenously into sublethally irradiated NOD/shi-scid, IL-2RγKO Jic (NOG) mice (6.0×103-1.0×104 cells/mice), CD34+ PhMMs from all 4 patients were fund to be engrafted in the BM by 24 weeks after transplant, while none of the mice transplanted with CD34- PhMMs showed no engraftment by the same time point. Importantly, all engrafted cells from CD34+ PhMMs had the same Ig light chain with patient myeloma cells. Interestingly, both CD34+ and CD34- PhMMs were identified in BM of mice receiving CD34+ PhMMs. These results indicate that myeloma-propagating activity in PhMMs can be distinguished based on the CD34 expression. To gain insights into the cell origin of CD34+ PhMMs, we quantify the expression levels of selected markers associated with the development of B cell (ex, GATA2, PU1, EBF1, OCT2, BCL6, PAX5, IRF4, BLIMP1, and XBP1) by qRT-PCR analysis using purified CD34+ and CD34- PhMM pairs from 6 patients. Gene expression profiling revealed that CD34- PhMMs had a plasma cell signature (down-regulation of Pax5, and up-regulation of IRF4, BLIMP1, and XBP1), whereas CD34+ PhMMs demonstrated a more immature pre-GCB signature(down-regulation of BCL6, IRF4, and XBP1), except for the aberrant suppression of PAX5 and the expression of GATA2 .

In conclusion, this study revealed that phenotypically restricted cell population (PhMMs) contains a minor CD34+ myeloma cell fraction that originate from pre-GCB cells and possess myeloma-propagating activities, as well as resistance to anticancer drugs. Thus, the identification of these clonogenic MM propagating cells provides a basis for better understanding the pathogenesis of MM and for designing novel therapeutic strategies aimed to eradicate total MM cells.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.