Abstract
Introduction
A major challenge in the development of effective myeloma (MM) therapy is addressing tumor heterogeneity, with the presence of sub-clones that exhibit resistance to standard therapy. An ongoing area of investigation focuses on identification of myeloma initiating cells that demonstrate greater capacity for self-renewal and serve as a potential reservoir for disease recurrence. It has been postulated that MM cells arise from a primitive B cell precursor population distinct from the more differentiated malignant plasma cell population. The critical feature of these myeloma-propagating cells is thought to be the ability to efficiently recapitulate MM in immunocompromised mice. MUC1 is an oncoprotein aberrantly expressed in malignant cells, including multiple myeloma, that interacts with multiple transcription factors, such as NF-κB and the β-catenin/TCF4 complex, that regulate cell survival and proliferation critical for malignant transformation. We have previously demonstrated that MUC1 is expressed by AML leukemic blasts, as compared to normal hematopoietic stem cells, and blockade of MUC1 signaling prevents establishment of leukemia in immunocompromised animals. In the present project, we identify a unique population of CD34+/MUC1+/CD138+/CD20+ cells in primary MM bone marrow samples that exhibit features of myeloma initiating cells, as manifested by high levels of enzymatic ALDH activity, the ability to efflux Hoechst dye represented as “side population” (SP), and the ability to establish disease in immunocompromised mice. Of note, MM engraftment of unselected primary myeloma cells in a xenograft model has a low success rate, and typically requires the introduction of an artificial stromal support network.
Methods and results
Bone marrow aspirates were obtained from newly diagnosed MM patients using an established protocol approved by the IRB. Expression of MUC1, myeloid and lymphoid markers was assessed using multicolor flow cytometric analysis. While MUC1 shows only a minimal expression (<5%, n=8) in normal CD34+ hematopoietic progenitors, we have demonstrated that on average 54% of CD34+ cells isolated from bone marrow samples of MM patients expressed MUC1 (n=7, p<0.05), in addition to other MM and lymphoid markers. MM derived CD34+MUC1+ cells segregated with SP by the ability to efflux Hoechst dye and expressed high levels of ALDH as assessed by the Aldefluor assay (11% of CD34+MUC+ cells had high ALDH activity as opposed to less than 1% in bulk MM marrow cells, n=3). CD34+MUC+ cells co-expressed CD138+, CD20+, and were CD38 dim (n=7), consistent with the phenotypic markers that have been previously described in association with myeloma propagating cells.
In order to study the capacity of CD34+MUC1+ cells to recapitulate MM in a murine model, a bone marrow sample was obtained from a patient with newly diagnosed MM with a cytogenetic abnormality characterized by the rearrangement of the CCND1/IGH loci. Primary bone marrow cells were fluorescently labeled and CD34-MUC+ (consistent with mature CD138+CD38hi plasma cells) and CD34+MUC+ populations of cells were isolated using FACS sorting. Cells from each population were injected into an irradiated NOD/SCID mouse (0.5x106cells/mouse). After 13 weeks, no human engraftment was detected in the 4/4 mice injected with CD34-MUC+ population of mature plasma cells. In contrast, 2/2 mice injected with CD34+MUC+ cells demonstrated human engraftment. Engrafted cells were isolated by FACS sorting and transferred onto glass slides for cytogenetic analysis by FISH. Notably, the engrafted cells harbored rearrangement at CCND1/IGH loci consistent with the originating MM clone. In another experiment, 2/2 NOD/SCID mice inoculated with CD34+MUC1+ primary MM cells demonstrated MM engraftment after 12 weeks, characterized by the presence of CD138+CD45- human plasma cells in the murine bone marrow.
Conclusions
We have identified a subpopulation of primitive myeloma cells that coexpress CD34 and the MUC1 oncoprotein. CD34+MUC1+ cells express CD20 and CD138, and express high levels of ALDH. CD34+MUC1+ cells demonstrate the capacity to engraft human MM cells in immunocompromised mice, even without an artificial stromal framework. Inhibition of MUC1 signaling thus may offer new avenues to target critical myeloma subclones.
Disclosures:
No relevant conflicts of interest to declare.
Induction of unscheduled DNA synthesis in human myeloma cells
