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Background Multiple myeloma (MM) disease progression is influenced by signals from the bone marrow (BM) microenvironment. Recently, we showed that the MM BM is characterized by inflammatory mesenchymal stromal cells (iMSCs) that transcribe MM survival factors and are predicted to recruit proliferating myeloma cells via CCL2-CCR2 interactions (de Jong et al. Nat Immunol. 2021). iMSCs also transcribed high levels of chemokines that can bind to CXCR1 and 2. Myeloid cells are known to express CXCR1/2, and have been implicated in both pro- and anti-tumor responses in various malignancies. Therefore, we hypothesized that iMSCs attract and influence myeloid populations in the MM BM. Methods Using flow cytometry, we verified expression of CXCR1/2 on myeloid cell populations in the BM of 5 newly diagnosed MM (NDMM) patients. Results CD15+ neutrophils were the most dominant population expressing these receptors, as 22.4% (± 9.8%) of cells expressed CXCR2 alone, and 72.6% (± 8.0%) expressed both CXCR1 and CXCR2. CD14+ monocytes only expressed CXCR2 (86.9% ± 15.8%). Importantly, less than 1% of myeloma cells expressed these receptors (n = 17 NDMM). As these findings suggested neutrophils as a potential target of iMSC-mediated chemotaxis, we set out to identify MM-associated alterations in this population by performing single cell RNA sequencing of the full neutrophilic lineage (n = 5 NDMM and 2 controls). Interestingly, CXCR1 and CXCR2 transcription was increased in mature neutrophils of MM patients compared to controls. Additionally, mature neutrophils of MM patients had an activated transcriptome as defined by increased transcription of C3AR1, SLPI, and IL6R, the plasma cell supportive factor TNFSF13B (encoding BAFF), and the inflammatory cytokines IL1B and IL18. Transcription of IL1B and IL18 can be regulated by pattern-recognition receptors (PRRs) binding damage-associated molecular patterns (DAMPs) resulting from e.g. matrix breakdown. Transcription of PRRs as TLR1, 2 and 4 was increased in mature neutrophils of MM patients compared to controls. Secretion of IL-1 β and IL-18 relies on the cleavage of pro-forms of these cytokines by the inflammasome, a multiprotein complex that is assembled in response to alarmins. Transcription of inflammasome components PYCARD, NLRP3 and CASP1 was increased in mature neutrophils of patients with MM. Additionally, protein levels of both IL-18 and IL-1 β are increased in BM plasma from MM patients, implicating activated neutrophils as a potential source of these cytokines. Conclusion In MM, mature neutrophils are activated and might interact with iMSCs via CXCR1/2. Moreover, neutrophils are inflammasome-primed and are likely to be a source of increased IL-1 β levels in the MM BM. As IL-1 β can activate normal MSCs to become iMSC, neutrophils and iMSCs may form a feed-forward loop in which activated neutrophils contribute to a pro-MM environment by maintaining iMSC and by directly providing BAFF to tumor cells.

Background Multiple Myeloma (MM) disease progression and therapy response are influenced by cues of the microenvironment including tumor control by a cytotoxic immune response. Natural killer (NK) cells are notable mediators of the cytotoxic immune response to MM, and important effector cells in recent immune-mediated therapies. NK cells are drivers of antibody-dependent-cellular cytotoxicity in therapies based on anti-CD38 monoclonal antibodies such as Daratumumab. Classically, NK cells are divided based upon CD56 expression into a cytokine-producing CD56bright subset, releasing cytokines such as IFN-γ, TNF-a and GM-CSF, and a cytotoxic CD56dim subset. However, accumulating evidence suggests much larger heterogeneity in the NK cell compartment and modulation of these NK cell subsets could impact response to NK cell-driven immunotherapies. Here, we used single-cell RNA sequencing to investigate the heterogeneity and MM-driven alterations of the NK cell compartment in the bone marrow of newly diagnosed MM patients undergoing first-line Daratumumab-containing therapy. Methods We performed single-cell RNA sequencing of the CD38+ and CD38- fractions of viably frozen bone marrow aspirates from 19 newly diagnosed MM patients and 5 non-cancer control patients. NK cells were identified in silico by transcription of KLRF1, KLRD1, GNLY and NKG7 resulting in a single-cell transcriptomic dataset of 30,373 NK cells from MM patients and 8,865 NK cells from control patients. Results After integration of the datasets, bone marrow NK cells formed eight distinct transcriptomic clusters. Conventional CD56bright and CD56dim NK-cells were identified by increased transcription of GZMK or GZMB, respectively. The GZMK+CD56bright NK cells contained both a cluster of naive and a cluster of activated NK cells. The GZMB+CD56dim NK cells consisted of 5 subclusters. To identify MM-induced alterations in NK cell subsets we compared GZMK+CD56bright vs GZMB+CD56dim cluster composition and distribution between control and MM patients. Control bone marrow was dominated by GZMB-transcribing cytotoxic CD56dim NK cells, represented by a low cytokine-producing GZMK+CD56bright vs cytotoxic GZMB+CD56dim ratio. In contrast, MM bone marrow was characterized by heterogeneity in the ratio of cytokine-producing GZMK+CD56bright vs cytotoxic GZMB+CD56dim NK cells. A subset of patients presented with a complete reversal of this ratio. This altered composition was due to a loss of cytotoxic GZMB+CD56dim NK cells, and more specifically a loss of NK cells with a transcriptome suggesting recent activation. Conclusion Here we present a transcriptomic overview of NK cells in MM bone marrow at the single-cell level. A subset of MM patients has a loss of activated cytotoxic GZMB+CD56dim NK cells, suggestive of reduced cytotoxic anti-tumor responses. Current analyses are focused on clinical implications of NK cell alterations in response to Daratumumab-based therapies.

Background Circulating tumor cells (CTCs) are present in the blood of all patients with (precursor)stages of Multiple Myeloma (MM). The levels of CTCs vary widely between patients, yet the biology behind this variability is unknown. Importantly, MM patients with a higher percentage of CTCs have an inferior prognosis independent of high-risk cytogenetics, suggesting that CTC numbers are a relevant reflection of tumor cell biology. Here, we set out to identify differences in CTCs and paired bone marrow plasma cells (BM PCs). Additionally, we compared BM PCs from patients with high and low levels of CTCs with the goal of identifying mechanistic drivers of high-risk disease. Methods We isolated PCs from viably frozen mononuclear cells of peripheral blood and bone marrow aspirates of newly diagnosed MM patients. Common high-risk mutations were present in both groups. We performed single cell RNA sequencing of paired CTCs and BM PCs from five patients with a high percentage of CTCs (0.5%-8%). In addition, we generated single cell transcriptomes of BM PCs of eight patients with a high percentage of CTCs (2-22%) and 13 patients with low percentage of CTCs (0.004%-0.08%) Results Single cell transcriptomes were generated from 44,779 CTCs and 35,697 bone marrow PCs. When paired CTCs and BM PCs were integrated, we identified 9 common clusters, no cell specific cluster for either source was detected. Moreover, only 25 genes were significantly differentially expressed between CTCs and BM PCs. The absence of unique clusters in either CTCs or BM PCs, and the transcriptional overlap between these two sources indicate that CTC levels are not driven by the emergence of a transcriptionally different migratory clone, but are likely a reflection of altered BM PC biology. To identify such alterations, we compared bone marrow PCs from patients with high and low percentages of CTCs. Single cell transcriptomes were generated from 74,830 bone marrow PCs. Integration of all patients lead to the identification of 8 distinct PC clusters, one of which was characterized by active proliferation as defined by transcription of STMN1 and MKI67. Interestingly, this proliferative cluster was larger in patients with a high percentage of CTCs. Furthermore, cell cycle analyses based on canonical G2M and S phase markers revealed that actively cycling PCs were more frequent in the BM of patients with a high percentage of CTCs (64% versus 30%, p Conclusions Through single cell transcriptomic analyses we reveal that CTCs and MM cells from BM are transcriptionally similar. Importantly, we identify increased BM PC proliferation as a significant difference between patients with high and low levels of CTCs, implicating an increased entry into the cell cycle as one of the mechanisms driving CTC levels and MM disease pathobiology.

Background Primary plasma cell leukemia (pPCL) is an aggressive subtype of multiple myeloma that is characterized by ≥20% circulating tumor cells (CTCs), whereas CTC levels in newly diagnosed multiple myeloma (NDMM) are Methods Transcriptomic data were generated of CD138-enriched bone marrow (BM) plasma cells from both NDMM patients enrolled in the Cassiopeia (NCT02541383) and HO143 (EudraCT 2016-002600-90) trials and pPCL patients from the EMN12/HO129 (EudraCT 2013-005157-75) trial. NDMM CTC levels were determined with the EuroFlow NGF protocol. HR FISH was defined as the presence of either t(4;14), t(14;16) or del17p.154 NDMM and 29 pPCL patients were divided into a discovery and validation cohort to construct and test a classifier for PCL-like disease. Subsequently, data from 8 additional NDMM cohorts were used to assess the association of PCL-like status with progression-free survival (PFS) and overall survival (OS) in Cox proportional hazards (PH) models including conventional HR markers. Results Baseline CTC levels were determined in 297 NDMM and 51 pPCL patients. CTCs could be detected in 87% of NDMM patients, with a limit of detection Conclusions (1) pPCL cannot only be identified clinically, but also molecularly. (2) PCL-like status is a novel marker for HR disease in NDMM that identifies patients with a tumor transcriptome similar to pPCL and has independent prognostic value in the context of conventional HR markers. (3) PCL-like status could help detect NDMM patients with early stage or borderline pPCL.

behavior, and help identify novel therapeutic targets for the development of molecularly-based therapies to improve outcome in myeloma.