The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.

Multiple myeloma is a fatal plasma cell malignancy that is reliant on the bone marrow microenvironment. The bone marrow is comprised of numerous cells of mesenchymal and hemopoietic origin. Of these, macrophages have been implicated to play a role in myeloma disease progression, angiogenesis, and drug resistance; however, the role of macrophages in myeloma disease establishment remains unknown. In this study, the antimyeloma efficacy of clodronate-liposome treatment, which globally and transiently depletes macrophages, was evaluated in the well-established C57BL/KaLwRijHsd murine model of myeloma. Our studies show, for the first time, that clodronate-liposome pretreatment abrogates myeloma tumor development in vivo . Clodronate-liposome administration resulted in depletion of CD169 + bone marrow–resident macrophages. Flow cytometric analysis revealed that clodronate-liposome pretreatment impaired myeloma plasma cell homing and retention within the bone marrow 24 hours postmyeloma plasma cell inoculation. This was attributed in part to decreased levels of macrophage-derived insulin-like growth factor 1. Moreover, a single dose of clodronate-liposome led to a significant reduction in myeloma tumor burden in KaLwRij mice with established disease. Collectively, these findings support a role for CD169-expressing bone marrow–resident macrophages in myeloma disease establishment and progression and demonstrate the potential of targeting macrophages as a therapy for myeloma patients.

Highlights d Genuine macrophages are absent from hematopoietic tissue single-cell preparations d Fragmented remnants of macrophages containing cytoplasm adhere to other cells d Macrophage remnants convolute hematopoietic tissue flow cytometry analysis d Remnant-derived macrophage-RNA is misattributed to other cells in RNA-seq data

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In most instances, multiple myeloma (MM) plasma cells (PCs) are reliant on factors made by cells of the bone marrow (BM) stroma for their survival and growth. To date, the nature and cellular composition of the BM tumor microenvironment and the critical factors which drive tumor progression remain imprecisely defined. Our studies show that Gremlin1 (Grem1), a highly conserved protein, which is abundantly secreted by a subset of BM mesenchymal stromal cells, plays a critical role in MM disease development. Analysis of human and mouse BM stromal samples by quantitative PCR showed that GREM1/Grem1 expression was significantly higher in the MM tumor-bearing cohorts compared to healthy controls (p < 0.05, Mann–Whitney test). Additionally, BM-stromal cells cultured with 5TGM1 MM PC line expressed significantly higher levels of Grem1, compared to stromal cells alone (p < 0.01, t-test), suggesting that MM PCs promote increased Grem1 expression in stromal cells. Furthermore, the proliferation of 5TGM1 MM PCs was found to be significantly increased when co-cultured with Grem1-overexpressing stromal cells (p < 0.01, t-test). To examine the role of Grem1 in MM disease in vivo, we utilized the 5TGM1/KaLwRij mouse model of MM. Our studies showed that, compared to immunoglobulin G (IgG) control antibody-treated mice, mice treated with an anti-Grem1 neutralizing antibody had a decrease in MM tumor burden of up to 81.2% (p < 0.05, two-way ANOVA). The studies presented here demonstrate, for the first time, a novel positive feedback loop between MM PCs and BM stroma, and that inhibiting this vicious cycle with a neutralizing antibody can dramatically reduce tumor burden in a preclinical mouse model of MM.

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Multiple myeloma, a plasma cell malignancy, is a genetically heterogeneous disease and the genetic factors that contribute to its development and progression remain to be fully elucidated. The tumour suppressor gene GLIPR1 has previously been shown to be deleted in approximately 10% of myeloma patients, to inhibit the development of plasma cell tumours in ageing mice and to have reduced expression levels in the plasma cells of patients with light-chain amyloidosis, a myeloma-related malignancy. Therefore, we hypothesised that GLIPR1 may have tumour suppressor activity in multiple myeloma. In this study, we demonstrate that plasma cell expression of GLIPR1 is reduced in the majority of myeloma patients and Glipr1 expression is lost in the 5TGM1 murine myeloma cell line. However, overexpression of GLIPR1 in a human myeloma cell line did not affect cell proliferation in vitro. Similarly, re-expression of Glipr1 in 5TGM1 cells did not significantly reduce their in vitro proliferation or in vivo growth in C57BL/KaLwRij mice. In addition, using CRISPR-Cas9 genome editing, we generated C57BL/Glipr1-/mice and showed that loss of Glipr1 in vivo did not affect normal haematopoiesis or the development of monoclonal plasma cell expansions in these mice up to one year of age. Taken together, our results suggest that GLIPR1 is unlikely to be a potent tumour suppressor in multiple myeloma. However, it remains possible that the down-regulation of GLIPR1 may cooperate with other genetic lesions to promote the development of myeloma.

Mouse hematopoietic tissues contain abundant and heterogeneous populations of tissue-resident macrophages attributed trophic functions in control of immunity, hematopoiesis and bone homeostasis. A systematic strategy to characterise macrophage subsets in mouse bone marrow (BM), spleen and lymph node, unexpectedly revealed macrophage surface marker staining typically emanated from membrane-bound subcellular remnants associated with unrelated cell types. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters and mRNA were all detected in non-macrophage cell populations including isolated stem and progenitor cells. The profile of macrophage remnant association reflects adhesive interactions between macrophages and other cell types in vivo. Applying this knowledge, reduced macrophage remnant attachment to BM granulocytes in Siglec1 deficient mice was associated with compromised emergency granulocytosis, revealing a function for Siglec1-dependent granulocyte-macrophage interactions. Analysis of published RNA-seq data for purified macrophage and non-macrophage populations indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single cell analysis of disaggregated tissues.