Despite advances in therapeutic strategies for multiple myeloma (MM), long-term outcomes remain poor, largely due to inevitable relapse and acquired drug resistance. Reciprocal interactions between malignant MM plasma cells (PCs) and the bone marrow microenvironment (BMME) drive disease progression, immune evasion, and therapeutic resistance, positioning the BM niche as a focus for targeted therapeutics. Myeloperoxidase (MPO) has recently emerged as a key regulator of MM progression via modification of the BMME. Here, we evaluate the efficacy of AZD5904, an orally bioavailable, irreversible MPO inhibitor, in preclinical models of MM. Initiation of MPO inhibition with AZD5904 during the early stages of MM tumour development significantly reduced tumour burden in the KaLwRij/5TGM1 and Vk*Myc murine models, however had no effect when initiated in established disease. Furthermore, AZD5904 modulated immune responses by decreasing PD1+ T cells in vivo and restoring CD8+ T cell cytotoxicity in vitro. While combining AZD5904 with the frontline agent bortezomib did not provide additional benefit in limiting disease progression, adjuvant AZD5904 following bortezomib treatment markedly delayed 5TGM1 tumour relapse. These findings suggest that while MPO inhibition may not enhance efficacy of bortezomib induction therapy, it holds promise as a maintenance strategy to improve long-term outcomes in MM. Collectively, our data support further investigation of AZD5904 as a novel maintenance therapy targeting the BM microenvironment, with potential to enhance and sustain the effectiveness of existing, standard of care regimens.

Multiple myeloma (MM) is an incurable hematologic malignancy characterized by the uncontrolled proliferation of bone marrow resident plasma cells (PCs). Two members of the TAM (TYRO3, AXL, and MER) receptor family have previously been implicated in distinct aspects of neoplastic PC biology. AXL expression in MM PCs has been associated with the induction of a dormant, noncycling state within the bone marrow, whereas expression of MER has been implicated in PC proliferation and survival. Here, the generation of single TAM receptor-expressing 5TGM1 murine MM cell lines enabled the individual functional assessment of the effects of Axl and Mer receptor expression on MM development. Axl expression did not affect proliferation, cell cycling, or stromal cell-induced dormancy in vitro. Development of 5TGM1 tumors in C57BL/KaLwRij mice was also unaltered by Axl expression. By contrast, Mer expression conferred an increase in cell proliferation to 5TGM1 cells in vitro and increased 5TGM1 tumor burden in C57BL/KaLwRij mice. The protumorigenic properties of Mer were only observed following intravenous cell delivery into mice with an intact adaptive immune system. Thus, Axl is neither necessary nor sufficient for the induction of MM cancer cell dormancy, whereas MER remains a promising target for therapeutic intervention in patients with MM.

Paediatric patients with relapsed B cell acute lymphoblastic leukaemia (B-ALL) have poor prognosis, as relapse-causing clones are often refractory to common chemotherapeutics. While the molecular mechanisms leading to chemoresistance are varied, significant evidence suggests interactions between B-ALL blasts and cells within the bone marrow microenvironment modulate chemotherapy sensitivity. Importantly, bone marrow mesenchymal stem cells (BM-MSCs) and BM adipocytes are known to support B-ALL cells through multiple distinct molecular mechanisms. This review discusses the contribution of integrin-mediated B-ALL/BM-MSC signalling and asparagine supplementation in B-ALL chemoresistance. In addition, the role of adipocytes in sequestering anthracyclines and generating a BM niche favourable for B-ALL survival is explored. Furthermore, this review discusses the role of BM-MSCs and adipocytes in promoting a quiescent and chemoresistant B-ALL phenotype. Novel treatments which target these mechanisms are discussed herein, and are needed to improve dismal outcomes in patients with relapsed/refractory disease.

We implicated the X-chromosome THOC2 gene, which encodes the largest subunit of the highly-conserved TREX ( Tr anscription- Ex port) complex, in a clinically complex neurodevelopmental disorder with intellectual disability as the core phenotype. To study the molecular pathology of this essential eukaryotic gene, we generated a mouse model based on a hypomorphic Thoc2 exon 37–38 deletion variant of a patient with ID, speech delay, hypotonia, and microcephaly. The Thoc2 exon 37–38 deletion male ( Thoc2 Δ/Y ) mice recapitulate the core phenotypes of THOC2 syndrome including smaller size and weight, and significant deficits in spatial learning, working memory and sensorimotor functions. The Thoc2 Δ/Y mouse brain development is significantly impacted by compromised THOC2/TREX function resulting in R-loop accumulation, DNA damage and consequent cell death. Overall, we suggest that perturbed R-loop homeostasis, in stem cells and/or differentiated cells in mice and the patient, and DNA damage-associated functional alterations are at the root of THOC2 syndrome.

the mechanisms driving the high-risk disease progression associated with 1q21+ have not been identified.This study aims to identify new druggable targets in the 1q21 region.Methods: We purified PCs from BM samples of 18 newly diagnosed MM patients.In addition, a cohort of 11 smoldering (SMM) was included in the study.Fluorescent in situ hybridization analysis was performed in purified CD138+ PCs in all patients to access copy number alterations in the 1q21 region.The expression profile of all 29 samples was generated using GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA).The smar package was used to identify differentially expressed genes between 1q21+ and control samples.Results: Among the most significant upregulated genes in our analysis, we identified the expression of PYGOPUS 2 (PYGO2), a downstream component of the Wnt signaling pathway, to be significantly upregulated in CD138+ MM cells with 1q21+ as compared to those without 1q21+ (p= 0.0008).Additionally, we found a significant positive correlation between gene expression and the 1q21 copy number (p= < 0.0001, r= 0.6738), correlated with 1q21 copy number but not with disease stage (SMM vs. MM).Moreover, we assessed PYGO2 mRNA and protein expression levels in a panel of MM human cell lines (HMCLs) carrying 1q21+.HMCL OCI-MY5 was used as a negative control.Our results showed that PYGO2 was higher in HMCLs with 1q21+.Furthermore, we evaluated the mRNA and protein expression levels of PYGO2 in HMCLs resistant to carfilzomib, showing that the expression of PYGO2 was consistently upregulated.Next, to investigate the functional role of PYGO2 in patients with 1q21, we generated a knockdown of PYGO2 in HMCL JJN3 using short hairpin RNA (shRNA).We found that PYGO2 transcripts were significantly downregulated in shPYGO2 when compared to scramble control.Notably, cells lacking PYGO2 have an increased rate of cell death when compared with the scramble cell line.Conclusions: In conclusion, our results show that the expression of PYGO2 is significantly upregulated in MM patients carrying 1q21+, and that PYGO2 inhibition by shRNA leads to an increase in cell death in HMCLs with 1q21+.Overall, our data indicate that targeting PYGO2 could represent a novel strategy to treat MM patients with 1q21+.

Despite major improvements in therapeutic strategies for patients with multiple myeloma (MM), effective treatment still remains a persistent challenge, as patients ultimately relapse and succumb to the disease. In the last decade, studies have highlighted the reciprocal interaction between MM plasma cells (PC) and the bone marrow (BM) microenvironment in regulating immune evasion, disease progression and persistence. As MM PC rely on BM stromal cells and their secreted factors for their survival and growth, the therapeutic targeting of the BM microenvironment may prove to be a novel and successful strategy for myeloma care in the future. Myeloid-derived suppressor cells (MDSC), a heterogenous population of myeloid cells are described to promote MM progression through immunosuppression and induction of angiogenesis. Myeloperoxidase (MPO), a key inflammatory enzyme important in host defence, is reported to be the most highly upregulated gene in MDSCs in murine cancer models. Recently, the accumulation of MPO within the tumour microenvironment has attracted much attention with a number of studies describing a role for MPO in regulating cancer development due to its potent pro-oxidative and proinflammatory properties. Our most recent findings have revealed new functional roles for myeloid-derived MPO in the BM microenvironment of MM. Specifically, we demonstrate that myeloid cell populations are increased within the BM of 5TGM1 tumour-bearing mice and that MM PC may directly influence Mpo gene expression in BM-derived myeloid cells. Mechanistically, we report that MPO has the capacity to induce the expression of key MM growth factors, and exerts potent immune suppression by inhibiting anti-tumour T-cell responses. Remarkably, in the syngeneic KaLwRij/5TGM1 mouse model of MM, targeted inhibition of MPO with the suicide substrate 4-Aminobenzoic acid hydrazide (4-ABAH) demonstrated a significant reduction in overall MM tumour burden. Here, we investigate for the first time the efficacy of an orally bioavailable irreversible small molecule inhibitor of MPO (MPOi) in the preclinical Vk*MYC murine model of myeloma. Twelve-week-old C57BL/6J mice were intravenously inoculated with Vk*MYC (Vk14451-GFP) cells and tumour progression was monitored by serum paraprotein electrophoresis (SPEP), whilst endpoint GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. To characterise myeloid cell populations and associated Mpo expression in the Vk*MYC tumour landscape, we utilised flow cytometry to quantitate CD11b+ cells and used magnetic activated cell separation to isolate these populations and characterise the expression of Mpo by qPCR. Our studies confirm that CD11b+ myeloid cells are significantly increased in the BM of Vk*MYC tumour-bearing mice (p<0.01), accompanied by an upregulation of MPO mRNA expression (p<0.001). To assess the efficacy of MPOi in MM, mice were treated with MPOi or vehicle alone twice daily by oral gavage, with treatment initiated at the time of Vk*MYC inoculation. Notably, mice receiving MPOi presented with significantly reduced endpoint tumour burden (9 weeks post tumour cell inoculation). We observed a 15.2% and 31.4% reduction in MM tumour as determined by hind limb GFP% (p<0.05) and SPEP (p<0.01) respectively, compared to vehicle control. Additionally, mRNA analysis of complete BM revealed an upregulation of the critical cytokine IFN gamma (p<0.05) and the downregulation of the potent proangiogenic factor VEGF (p<0.05) in mice treated with MPOi, suggesting MPO inhibition in MM may be an advantageous means to regulate the BM microenvironment and impede disease progression. However, when MPOi treatment was initiated at first signs of detectable disease, (as identified by the presence of a monoclonal spike by SPEP; 5 weeks post tumour cell inoculation), no difference in endpoint tumour burden was observed. This suggests that targeted inhibition of MPO using MPOi may be more effective in at early stages of MM development. In conclusion, the findings presented in this study indicate that inhibiting MPO activity with MPOi, as a single agent therapy, attenuates MM tumour growth in the Vk*Myc mouse model. With limited therapies used in the clinic that target the stromal microenvironment, these findings suggest MPOi could be investigated as a potential novel treatment option that may be included in combination with current frontline therapeutic agents.

Expression of myeloperoxidase (MPO), a key inflammatory enzyme restricted to myeloid cells, is negatively associated with the development of solid tumours. Activated myeloid cell populations are increased in multiple myeloma (MM); however, the functional consequences of myeloid-derived MPO within the myeloma microenvironment are unknown. Here, the role of MPO in MM pathogenesis was investigated, and the capacity for pharmacological inhibition of MPO to impede MM progression was evaluated. In the 5TGM1-KaLwRij mouse model of myeloma, the early stages of tumour development were associated with an increase in CD11b+ myeloid cell populations and an increase in Mpo expression within the bone marrow (BM). Interestingly, MM tumour cell homing was increased towards sites of elevated myeloid cell numbers and MPO activity within the BM. Mechanistically, MPO induced the expression of key MM growth factors, resulting in tumour cell proliferation and suppressed cytotoxic T-cell activity. Notably, tumour growth studies in mice treated with a small-molecule irreversible inhibitor of MPO (4-ABAH) demonstrated a significant reduction in overall MM tumour burden. Taken together, our data demonstrate that MPO contributes to MM tumour growth, and that MPO-specific inhibitors may provide a new therapeutic strategy to limit MM disease progression.

Elucidation of the molecular signatures that define hematopoietic tissue resident macrophage specialisation has been challenging. There are no validated markers that differentiate the specialised macrophage subsets in bone marrow (BM) that support erythropoiesis, bone homeostasis and hematopoietic stem cell (HSC) niches. We took an unbiased ex vivo approach to characterise macrophage subsets in mouse BM, spleen and lymph node using a flow cytometry marker panel which allowed analysis of all mature leucocytes, red blood cells and hematopoietic stem and progenitor cells (HSPC) in combination with in situ verified macrophage markers. Despite readily detectable F4/80 staining we were unable to identify any population in hematopoietic tissues that definitively represented intact macrophages. Imaging flow cytometry and confocal microscopy showed macrophage marker staining was derived from membrane-bound subcellular remnants associated with unrelated cell types. Remnant-restricted macrophage membrane markers, cytoplasmic reporters and mRNA were detected in non-macrophage cell populations including HSPC. Of note, HSC-associated detection of a Csf1r-reporter as well as anti-F4/80 and VCAM-1 staining were entirely attributable to membrane-bound subcellular remnants. Distinct marker expression on macrophage subsets within spleen verified that the profile of remnant binding reflected in vivo cell-cell interactions. Macrophage remnant attachment was reduced in Siglec1 deficient mice with frequency of F4/80+ BM events reduced by over 50% in HSPC and neutrophils yet unchanged in lymphocytes. Analysis of published RNA-seq data confirmed that macrophage fragmentation is a general phenomenon in disaggregated hematopoietic tissues. Overall, we have shown that abundant tissue macrophages are absent/under-represented in hematopoietic tissue cell suspensions. Detection of macrophage remnant-restricted cytoplasmic and membrane contents on other cells has confounded interpretation of ex vivo analyses and results in misattribution of macrophage-expressed genes to non-macrophage cells.

Multiple myeloma (MM) is an incurable haematological malignancy, caused by the uncontrolled proliferation of plasma cells within the bone marrow (BM). Obesity is a known risk factor for MM, however, few studies have investigated the potential of dietary intervention to prevent MM progression. Calorie restriction (CR) is associated with many health benefits including reduced cancer incidence and progression. To investigate if CR could reduce MM progression, dietary regimes [30% CR, normal chow diet (NCD), or high fat diet (HFD)] were initiated in C57BL/6J mice. Diet-induced changes were assessed, followed by inoculation of mice with Vk*MYC MM cells (Vk14451-GFP) at 16 weeks of age. Tumour progression was monitored by serum paraprotein, and at endpoint, BM and splenic tumour burden was analysed by flow cytometry. 30% CR promoted weight loss, improved glucose tolerance, increased BM adiposity and elevated serum adiponectin compared to NCD-fed mice. Despite these metabolic changes, CR had no significant effect on serum paraprotein levels. Furthermore, endpoint analysis found that dietary changes were insufficient to affect BM tumour burden, however, HFD resulted in an average two-fold increase in splenic tumour burden. Overall, these findings suggest diet-induced BM changes may not be key drivers of MM progression in the Vk14451-GFP transplant model of myeloma.

Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations. The macrophage remnant binding profile reflects interactions between macrophages and other cell types in vivo. Depletion of CD169(+) macrophages in vivo eliminates F4/80(+) remnant attachment. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters, and mRNA are all detected in non-macrophage cells including isolated stem and progenitor cells. Analysis of RNA sequencing (RNA-seq) data, including publicly available datasets, indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single-cell analysis of disaggregated hematopoietic tissues. Hematopoietic tissue macrophage fragmentation undermines the accuracy of macrophage ex vivo molecular profiling and creates opportunity for misattribution of macrophage-expressed genes to non-macrophage cells.

Disease relapse is the greatest cause of treatment failure in paediatric B-cell acute lymphoblastic leukaemia (B-ALL). Current risk stratifications fail to capture all patients at risk of relapse. Herein, we used a machine-learning approach to identify B-ALL blast-secreted factors that are associated with poor survival outcomes. Using this approach, we identified a two-gene expression signature (CKLF and IL1B) that allowed identification of high-risk patients at diagnosis. This two-gene expression signature enhances the predictive value of current at diagnosis or end-of-induction risk stratification suggesting the model can be applied continuously to help guide implementation of risk-adapted therapies.

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.

The protein SAMSN1 was recently identified as a putative tumor suppressor in multiple myeloma, with re-expression of Samsn1 in the 5TGM1/KaLwRij murine model of myeloma leading to a near complete abrogation of intramedullary tumor growth. Here, we sought to clarify the mechanism underlying this finding. Intratibial administration of 5TGM1 myeloma cells into KaLwRij mice revealed that Samsn1 had no effect on primary tumor growth, but that its expression significantly inhibited the metastasis of these primary tumors. Notably, neither in vitro nor in vivo migration was affected by Samsn1 expression. Both knocking-out SAMSN1 in the RPMI-8226 and JJN3 human myeloma cell lines, and retrovirally expressing SAMSN1 in the LP-1 and OPM2 human myeloma cell lines had no effect on either cell proliferation or migration in vitro. Altering SAMSN1 expression in these human myeloma cells did not affect the capacity of the cells to establish either primary or metastatic intramedullary tumors when administered intratibially into immune deficient NSG mice. Unexpectedly, the tumor suppressive and anti-metastatic activity of Samsn1 in 5TGM1 cells were not evidenced following cell administration either intratibially or intravenously to NSG mice. Crucially, the growth of Samsn1-expressing 5TGM1 cells was limited in C57BL/6/Samsn1-/- mice but not in C57BL/6 Samsn1+/+ mice. We conclude that the reported potent in vivo tumor suppressor activity of Samsn1 can be attributed, in large part, to graft-rejection from Samsn1-/- recipient mice. This has broad implications for the design and interpretation of experiments that utilize cancer cells and knockout mice that are mismatched for expression of specific proteins.

N-cadherin is a homophilic cell-cell adhesion molecule that plays a critical role in maintaining vascular stability and modulating endothelial barrier permeability. Pre-clinical studies have shown that the N-cadherin antagonist peptide, ADH-1, increases the permeability of tumor-associated vasculature thereby increasing anti-cancer drug delivery to tumors and enhancing tumor response. Small molecule library screens have identified a novel compound, LCRF-0006, that is a mimetic of the classical cadherin His-Ala-Val sequence-containing region of ADH-1. Here, we evaluated the vascular permeability-enhancing and anti-cancer properties of LCRF-0006 using in vitro vascular disruption and cell apoptosis assays, and a well-established pre-clinical model (C57BL/KaLwRij/5TGM1) of the hematological cancer multiple myeloma (MM). We found that LCRF-0006 disrupted endothelial cell junctions in a rapid, transient and reversible manner, and increased vascular permeability in vitro and at sites of MM tumor in vivo. Notably, LCRF-0006 synergistically increased the in vivo anti-MM tumor response to low-dose bortezomib, a frontline anti-MM agent, leading to regression of disease in 100% of mice. Moreover, LCRF-0006 and bortezomib synergistically induced 5TGM1 MM tumor cell apoptosis in vitro. Our findings demonstrate the potential clinical utility of LCRF-0006 to significantly increase bortezomib effectiveness and enhance the depth of tumor response in patients with MM.

Approximately 15% of patients with multiple myeloma (MM) harbour the t(4;14) chromosomal translocation, leading to the overexpression of the histone methyltransferase NSD2. Patients with this translocation display increased tumour dissemination, accelerated disease progression and rapid relapse. Using publicly available gene expression profile data from NSD2high (n = 135) and NSD2low (n = 878) MM patients, we identified 39 epithelial-mesenchymal transition (EMT)-associated genes which are overexpressed in NSD2high MM plasma cells. In addition, our analyses identified Twist-1 as a key transcription factor upregulated in NSD2high MM patients and t(4;14)-positive cell lines. Overexpression and knockdown studies confirmed that Twist-1 is involved in driving the expression of EMT-associated genes in the human MM cell line KMS11 and promoted the migration of myeloma cell lines in vitro. Notably, Twist-1 overexpression in the mouse MM cell line 5TGM1 significantly increased tumour dissemination in an intratibial tumour model. These findings demonstrate that Twist-1, downstream of NSD2, contributes to the induction of an EMT-like signature in t(4;14)-positive MM and enhances the dissemination of MM plasma cells in vivo, which may, in part, explain the aggressive disease features associated with t(4;14)-positive MM.

Multiple myeloma (MM) is a fatal malignancy characterised by the clonal proliferation of malignant plasma cells (PC) within the bone marrow (BM). Numerous cells within the BM microenvironment have been shown to influence myeloma pathogenesis. Specifically, phagocytic macrophages have been implicated in MM disease progression, angiogenesis and drug resistance; however, the role of this cell type in the initial establishment of MM disease has not been investigated. In this study, the role of macrophages in MM disease establishment was investigated in the 5TGM1/C57BL.KaLwRijHsd (KaLwRij) murine model of MM. Clodronate-liposomes (clo-lip) were used to globally and transiently deplete macrophages in these mice. We found that within 24 hours of clo-lip treatment, 90% of CD169+ macrophages were depleted from the BM (p<0.01). Notably, a single injection of clo-lip 24 hours prior to tumour cell inoculation resulted in greater than 95% reduction in MM tumour burden at week 4, compared with that observed following control PBS-lip injections (p<0.0001). Moreover, MM PC homing and retention were impaired in clo-lip treated mice compared with PBS-lip, as evidenced by a 2.7-fold reduction in GFP+ 5TGM1 cells within the BM (p<0.001) and a concomitant 5.4-fold increase in circulating tumour cells (p<0.01), 24 hours post-tumour cell inoculation. Taken together, these data suggested the involvement of CD169+ BM-resident macrophages in MM establishment. Next, we investigated whether macrophages play a role in MM PC migration. We determined that BM-macrophage conditioned medium enhances 5TGM1 MM PC migration in a dose-dependent manner in vitro (p<0.01). Moreover, BM-derived macrophages expressed abundant mRNA transcripts encoding the pro-proliferative and migratory factor, insulin-like growth factor 1 (Igf1) (p<0.0001). Consistent with this finding, clo-lip treated mice exhibited significantly reduced Igf1 mRNA levels within the BM compared with PBS-lip treated mice (p<0.001). Western blot analysis revealed that BM-macrophage conditioned medium stimulated IGFR1 phosphorylation in 5TGM1 cells, suggesting that macrophage secreted IGF-1 may stimulate 5TGM1 MM PC homing. Lastly, a single injection of clo-lip in the established disease setting (i.e. 2 weeks after 5TGM1 tumour inoculation) resulted in a 64% reduction in MM tumour burden, compared with PBS-lip controls (p<0.05), thereby demonstrating the potential therapeutic efficacy of targeting macrophages in MM. Collectively, our studies suggest that CD169+ BM-resident macrophages are an important component of the MM niche and are required for disease establishment and progression in vivo. Furthermore, we have demonstrated that macrophage-secreted factors, including IGF-1, play an important role in MM PC migration and homing to the BM. These findings highlight the potential of targeting BM-resident macrophages as a novel therapy for MM.

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.

Multiple myeloma (MM) is a hematological malignancy resulting from the uncontrolled proliferation of antibody-producing plasma cells in the bone marrow. At diagnosis, independent plasma cell tumors are found throughout the skeleton. The recirculation of mutant plasma cells from the initial lesion and their recolonization of distant marrow sites are thought to occur by a process similar to solid tumor metastasis. However, the efficiency of this bone marrow homing process and the proportion of disseminated cells that actively divide and contribute to new tumor growth in MM are both unknown. We used the C57BL/KaLwRij mouse model of myeloma, lentiviral-mediated DNA barcoding of 5TGM1 myeloma cells, and next-generation sequencing to investigate the relative efficiency of plasma cell migration to, and growth within, the bone marrow. This approach revealed three major findings: firstly, establishment of metastasis within the bone marrow was extremely inefficient, with approximately 0.01% of circulating myeloma cells becoming resident long term in the bone marrow of each long bone; secondly, the individual cells of each metastasis exhibited marked differences in their proliferative fates, with the majority of final tumor burden within a bone being attributable to the progeny of between 1 and 8 cells; and, thirdly, the proliferative fate of individual clonal plasma cells differed at each bone marrow site in which the cells “landed.” These findings suggest that individual myeloma plasma cells are subjected to vastly different selection pressures within the bone marrow microenvironment, highlighting the importance of niche-driven factors, which determine the disease course and outcome.

Multiple myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of malignant plasma cells within the bone marrow. We have previously identified pituitary tumour transforming gene 1 (Pttg1) as a gene that is significantly upregulated in the haematopoietic compartment of the myeloma-susceptible C57BL/KaLwRij mouse strain, when compared with the myeloma-resistant C57BL/6 mouse. Over-expression of PTTG1 has previously been associated with malignant progression and an enhanced proliferative capacity in solid tumours.

Multiple myeloma (MM), a hematological malignancy characterized by the clonal growth of malignant plasma cells (PCs) in the bone marrow, is preceded by the benign asymptomatic condition, monoclonal gammopathy of undetermined significance (MGUS). Several genetic abnormalities have been identified as critical for the development of MM; however, a number of these abnormalities are also found in patients with MGUS, indicating that there are other, as yet unidentified, factors that contribute to the onset of MM disease. In this study, we identify a Samsn1 gene deletion in the 5TGM1/C57BL/KaLwRij murine model of myeloma. In addition, SAMSN1 expression is reduced in the malignant CD138+ PCs of patients with MM and this reduced expression correlates to total PC burden. We identify promoter methylation as a potential mechanism through which SAMSN1 expression is modulated in human myeloma cell lines. Notably, re-expression of Samsn1 in the 5TGM1 murine PC line resulted in complete inhibition of MM disease development in vivo and decreased proliferation in stromal cell–PC co-cultures in vitro. This is the first study to identify deletion of a key gene in the C57BL/KaLwRij mice that also displays reduced gene expression in patients with MM and is therefore likely to play an integral role in MM disease development.