Homing Of Multiple Myeloma Cells Research Articles

HAPLN1 matrikine: a bone marrow homing factor linked to poor outcomes in patients with MM

AbstractThe bone marrow (BM) microenvironment is critical for dissemination, growth, and survival of multiple myeloma (MM) cells. Homing of myeloma cells to the BM niche is a crucial step in MM dissemination, but the mechanisms involved are incompletely understood. In particular, any role of matrikines, neofunctional peptides derived from extracellular matrix proteins, remains unknown. Here, we report that a matrikine derived from hyaluronan and proteoglycan link protein 1 (HAPLN1) induces MM cell adhesion to the BM stromal components, such as fibronectin, endothelial cells, and stromal cells and, furthermore, induces their chemotactic and chemokinetic migration. In a mouse xenograft model, we show that MM cells preferentially home to HAPLN1 matrikine–conditioned BM. The transcription factor STAT1 is activated by HAPLN1 matrikine and is necessary to induce MM cell adhesion, migration, migration-related genes, and BM homing. STAT1 activation is mediated by interferon beta (IFN-β), which is induced by NF-κB after stimulation by HAPLN1 matrikine. Finally, we also provide evidence that higher levels of HAPLN1 in BM samples correlate with poorer progression-free survival of patients with newly diagnosed MM. These data reveal that a matrikine present in the BM microenvironment acts as a chemoattractant, plays an important role in BM homing of MM cells via NF-κB–IFN-β–STAT1 signaling, and may help identify patients with poor outcomes. This study also provides a mechanistic rationale for targeting HAPLN1 matrikine in MM therapy.

Delivery of Therapeutic RNA to the Bone Marrow in Multiple Myeloma Using CD38-Targeted Lipid Nanoparticles.

Multiple myeloma (MM) is a cancer of differentiated plasma cells that occurs in the bone marrow (BM). Despite the recent advancements in drug development, most patients with MM eventually relapse and the disease remains incurable. RNA therapy delivered via lipid nanoparticles (LNPs) has the potential to be a promising cancer treatment, however, its clinical implementation is limited due to inefficient delivery to non-hepatic tissues. Here, targeted (t)LNPs designed for delivery of RNA payload to MM cells are presented. The tLNPs consist of a novel ionizable lipid and are coated with an anti-CD38 antibody (αCD38-tLNPs). To explore their therapeutic potential, it is demonstrated that LNPs encapsulating small interference RNA (siRNA) against cytoskeleton-associated protein 5 (CKAP5) lead to a ≈90% decrease in cell viability of MM cells in vitro. Next, a new xenograft MM mouse model is employed, which clinically resembles the human disease and demonstrates efficient homing of MM cells to the BM. Specific delivery of αCD38-tLNPs to BM-residing and disseminated MM cells and the improvement in therapeutic outcome of MM-bearing mice treated with αCD38-tLNPs-siRNA-CKAP5 are shown. These results underscore the potential of RNA therapeutics for treatment of MM and the importance of developing effective targeted delivery systems and reliable preclinical models.

Sialofucosylation Enables Platelet Binding to Myeloma Cells via P-Selectin and Suppresses NK Cell-Mediated Cytotoxicity

Multiple myeloma (MM) is a plasma cell disorder that develops in the bone marrow (BM) and is characterized by uncontrolled proliferation and the ability to disseminate to different sites of the skeleton. Sialofucosylated structures, particularly Sialyl Lewis a/x (SLea/x), facilitate the homing of MM cells into the BM, leading to resistance to bortezomib in vivo. Platelets have been shown to play an important role in tumor metastasis. Platelets can bind to the surface of cancer cells, forming a "cloak" that protects them from the shear stress of the bloodstream and natural killer (NK) cell-mediated cytotoxicity. In this study, we showed that the presence of SLea/x induced a strong binding of MM cells to P-selectin, leading to specific and direct interactions with platelets, which could be inhibited by a P-selectin-blocking antibody. Importantly, platelets surrounded SLea/x-enriched MM cells, protecting them from NK cell-mediated cytotoxicity. The interactions between the platelets and MM cells were also detected in BM samples obtained from MM patients. Platelet binding to SLea/x-enriched MM cells was increased in patients with symptomatic disease and at relapse. These data suggest an important role of SLea/x and platelets in MM disease progression and resistance to therapy.

Abstract 2962: Investigation of the relationship between obesity, weight cycling, and tumor progression in a murine myeloma model

Abstract Background: The purpose of this study was to investigate physiologic effects of body weight regulation patterns and myeloma progression in vivo. Obesity and obesogenic behaviors are positively associated with both monoclonal gammopathy of unknown significance and multiple myeloma (MM). As the only known modifiable risk factor, this association has emerged as an optimal target for MM prevention, but little is known about the mechanistic relationship of weight patterns with MM progression. In two large prospective cohorts, we have reported an association of episodes of weight cycling (WC), e.g., intentional weight loss followed by unintentional weight gain, with increased MM risk compared to weight-stable individuals (Marinac et al. JNCI Cancer Spectrum, 2019). Experimental Procedures: We used two murine MM models (C57BL/6J Vk*Myc and SCID-Beige MM.1Sluc+ xenograft); male mice were fed a control diet (CD; 10% kcal from fat), high fat diet (HFD; 60% kcal from fat), or a diet cycling regimen (DC; 4 weeks on CD then 4 weeks on HFD) for 16 weeks and thereafter injected them intravenously with tumor cells (1x106 Vk*Myc or 2x106 MM.1S). Body composition was assessed via PIXIMUS-DEXA (SCID-Beige) or NMR (C57BL/6J). Tumor burden was assessed via CD138+ FACS (Vk*Myc) or in vivo bioluminescent imaging (IVIS; MM.1S). Results: The C57BL/6J mice (n=20) on HFD consistently gained weight and sustained the gains throughout the study duration; mice on the DC regimen exhibited dramatic weight cycling responses to diet switching. We observed significant differences in body fat (%) between HFD and CD mice, as assessed by NMR (p<= 0.0001), while body fat fluctuated with diet in the DC mice. At the time of tumor cell injection, HFD mice had higher body weights versus CD (p<0.000001) and DC (p<0.000001) mice; however, weights of DC mice were comparable to CD despite weight fluctuation throughout the study. Three weeks after MM inoculation, we detected elevated tumor burden in DC mice vs. CD mice (n-10, p=0.0061) and a trend toward elevated tumor burden in HFD mice (p=0.145). C57BL/6J mice on the DC regimen also had greater Vk*Myc tumor burden (p=0.0413) within the bone marrow (BM), specifically. In the SCID-Beige MM.1Sluc+ model (n=10), we observed similar trends for weight gain and cycling, and greater early tumor burden in both the HFD (p<0.0001) and DC (p=0.0214) groups compared to CD. Tumor burden was also elevated (p=0.0004) in the heaviest mice (vs. the lightest), regardless of diet, later in the SCID-Beige study. Conclusions: Combined, these results suggest that HFD and WC may contribute to MM risk via changes in the BM microenvironment that affect MM cell homing, engraftment and early expansion. This study lays the groundwork for the future characterization of the relationship between weight patterns, the BM, and MM pathogenesis, which may affect MM prevention and treatment strategies. Citation Format: Heather Fairfield, Catherine Marinac, Mariah Farrell, Brenda Birmann, Michaela Ruth Reagan. Investigation of the relationship between obesity, weight cycling, and tumor progression in a murine myeloma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2962.

Runx2 Deficiency in Committed Osteoblasts Promotes Myeloma Cell Homing to and Progression in New Bone Sites

Multiple myeloma (MM) is a plasma cell malignancy that arises and preferentially grows in the bone marrow (BM), with frequent progression to new local and distant bone sites. Our previous studies demonstrated that primary MM cells secrete soluble factors that orchestrate changes in distant bone sites prior to the arrival of metastatic tumor cells. One such alteration is the simultaneous suppression of osteoblastogenesis and bone formation via inhibition of the critical Runt-related transcription factor 2 (Runx2) in osteoblast (OB) progenitors and committed OBs. While the mechanisms regulating MM-induced Runx2 suppression in these cells have been described, how this change in distant sites provides feedback to regulate MM cell homing and survival has not been determined.To study the effect of OB-Runx2 suppression at distant bone sites on MM dissemination and progression to these sites, we generated a syngeneic mouse model of MM in which Runx2 is specifically deleted in the committed OBs of C57BL6/KaLwRij mice (OB-Runx2-/- mice). Bone-homing and growth of luciferase-expressing 5TGM1 (5TGM1-Luc) MM cells was tested by tail vein injection in 5-week-old OB-Runx2+/+ (controls) and OB-Runx2-/- mice (n=7 per group). Compared with controls, OB-Runx2-/- mice showed significantly enhanced bone homing of MM cells and increased tumor growth in bone, which was confirmed by weekly luciferin imaging and ELISA for serum levels of IgG2bκ (a soluble marker of 5TGM1 MM cells). These results demonstrate that Runx2 deficiency in committed OBs in new bone sites promotes MM metastasis to and progression in these areas.To investigate the molecular mechanism by which OB-Runx2 deficiency promotes MM bone-homing and tumor progression, we harvested BM from 5-week-old OB-Runx2+/+ controls and OB-Runx2-/- mice with 1 ml PBS (n=3-4 per group) and centrifuged it to collect BM cells and supernatants. Measurement of 48 cytokines related to tumor cell bone-homing and bone remodeling revealed substantially increased levels of pro-metastatic cytokines in the BM supernatants of OB-Runx2-/- mice, including EGF, GM-CSF, M-CSF, ICAM-1, IGF-1, MMP-3, OPN, SDF-1, TGF- β, RANKL, VCAM-1, and IL-10. Flow cytometric analysis of the immune cell profile showed significantly more immune inhibitory cells, including myeloid-derived suppressor cells (MDSCs) and regulatory T and B cells, in the BM of OB-Runx2-/- mice. These data indicate that Runx2 deficiency in committed OBs induces an inflammatory and immunosuppressive pre-condition in the BM microenvironment, which attracts MM cells and allows their invasion into the bone tissue.Next, we evaluated the survival and proliferation status of MM cells and the immune cell response to MM cells in the BM under the condition of OB-Runx2 suppression. We injected 2 x 106 5TGM1-Luc cells into 5-week-old OB-Runx2+/+ control and OB-Runx2-/- mice via tail vein (n=5 per group). After 4 weeks, MM and BM cells isolated from the BM of these mice were analyzed by immunostaining and flow cytometry with antibodies specific for CD138, a marker of MM cells; Ki-67, a marker of cell proliferation; and Bcl-2, an anti-apoptotic protein. Results showed significantly more proliferating and non-apoptotic MM cells in the BM of Runx2 OB-/- mice. Additional flow cytometry analysis of isolated BM cells revealed a striking increase in immune-suppressive cell populations, significantly higher expression of CD8+ T cell inhibitory molecules (Arginase 1, iNOS, IL-10) in MDSCs, and dramatically decreased production of tumor-killing factors (Perforin, Granzyme B, and IFN-γ) in T cells in OB-Runx2-/- mice. Together, these findings indicate that Runx2 deficiency in committed OBs impairs antitumor immunity, leading to tumor proliferation and progression in bone.In conclusion, this study demonstrated that Runx2 inhibition in committed OBs in distant bone sites, induced by primary MM, can feed back to invite MM cell metastasis to and progression in these new bone areas. The increase in pro-metastatic cytokines and immunosuppressive cells induced by OB-Runx2 deficiency in new bone sites play a critical role in this process. These novel discoveries provide insights into the mechanisms of MM dissemination and progression and may inform the prognosis of outcomes in patients with MM. DisclosuresNo relevant conflicts of interest to declare.

A Novel in-Vivo Model to Examine Homing of Multiple Myeloma Cells in Postnatal, Inducible, and Reversible Loss of Mature Osteoblasts

The initiation of Multiple myeloma (MM) coincides with a decrease in osteoblasts and an increase in osteoclasts and adipocytes. However, the contribution of osteoblasts to the initiation and progression of MM remains mostly unknown. In-vitro studies have shown that co-cultures with MC3T3 murine osteoblast cell-line induced quiescence in 5TGM1 murine myeloma cells. In SCID or C57BL/KaLwRijHsd mice, the fluorescently labeled MM cell lines, injected through tail-vein, co-localized with endosteal osteoblasts and were found to be quiescent. We previously demonstrated that increased osteoblast numbers using Activin A inhibition in SCID mice with human-origin MM.1S cells resulted in inhibition of MM growth (Vallet et al., PNAS 2010). Here we aimed to develop a genetic in-vivo model for a better understanding of the interactions between osteoblasts and MM cells. Osteolineage cells develop through the stages of pre-osteoblasts, committed osteoblasts, mature osteoblasts, and osteocytes; each stage governed by a distinct molecular signature and function. To delineate these differences, individual osteoblast populations were FACS-sorted from long bones of Osterix-GFP+ (Osx+) mice (pre-osteoblasts), Collagen 2.3-GFP+ (Col2.3+) mice (committed osteoblasts), and Osteocalcin-YFP+ (OCN+) mice (mature osteoblasts). Co-cultured of these osteoblast populations with 5TGM1 MM cells showed that the OCN+ mature osteoblasts suppressed MM proliferation the most. Therefore, we focused our studies on the role of mature osteoblasts in MM homing and engraftment. We next generated mice in which mature osteoblasts were postnatally deleted in an inducible and reversible manner to better understand the molecular mechanisms of engraftment, proliferation, and migration of 5TGM1 injected intra-tibialy. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre). This led to the expression of DTR in mature osteoblasts (OC-Cre/iDTR) only. The control mice were littermates lacking the OC-Cre allele (iDTR). The OC-Cre/iDTR mice were indistinguishable from the controls until treated with diphtheria toxin (DT). We chose to induce mature osteoblast-deficiency at 8-weeks to allow skeletal maturation which is assumed to be completed by 6-7weeks of age. To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treated with 50 µg/Kg DT once a week, beginning at 8-weeks of age. Micro-CT analysis showed a significant increase in cortical porosity within 1-week after DT injection. 8-weeks of DT treatment significantly reduced trabecular bone fraction (BV/TV), trabecular numbers (Tb.N), and bone mineral density (BMD) with a significant increase in trabecular spacing (Tb.Sp). Immunohistochemistry for osteocalcin showed rapid loss of mature and endosteal osteoblasts (N.Ob/T.Ar). This was accompanied with a marked decreased in serum sclerostin and serum osteocalcin levels suggesting reduced osteocytes and mature osteoblasts, respectively. Importantly, serum CTX levels or numbers of osteoclasts (N.Oc/T.Ar) were unchanged. To study MM engraftment and progression, 3x106 5TGM1 luciferase eGFP positive (5TGM1-Luc-GFP) MM cells were injected into the tibia of OC-Cre/iDTR and control iDTR mice followed by weekly injection of DT for 8-weeks. Flow cytometry analysis showed a 4-fold increase in the 5TGM1-GFP cells in the BM from OC-Cre/iDTR mice compared to controls. Bioluminescence imaging (BLI) for 5TGM1-Luc-GFP cells at 4-weeks showed 600-fold increase in the OC-Cre/iDTR mice compared to controls. Interestingly, by 8-weeks, the BLI imaging showed 5TGM1-Luc-GFP cells in other long-bones of OC-Cre/iDTR mice but not the controls. These data show that MM cells engraft and proliferate rapidly in the absence of mature osteoblasts in-vivo. Moreover, in the absence of mature osteoblasts, MM cells have a propensity to migrate to other long bones. These data further suggest that expanding the mature osteoblast niche may provide novel therapeutic avenues and reduce disease burden and create an environment for long term tumor control. Importantly, this model will allow us to follow and analyze the sequential engraftment, dormancy, reactivation, proliferation, and migration of myeloma cells, and evaluate the effects of osteoanabolic and anti-myeloma therapies. Disclosures Fulzele: FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Raje:BMS: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Astrazeneca: Consultancy; Bluebird, Bio: Consultancy, Research Funding; Takeda: Consultancy; Immuneel: Membership on an entity's Board of Directors or advisory committees; Caribou: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy.

Immunologic Status Evaluated By the Absolute Lymphocyte/Monocyte Ratio Provides a Powerful Prognostic Tool for Newly Diagnosed Multiple Myeloma

The bone marrow (BM) microenvironment contains numerous cell types, including immunosuppressive tumor-associated macrophages (TAM), that promote the growth and survival of multiple myeloma (MM) cells. TAMs are recruited to the site of MM within the BM and help to create a permissive microenvironment that promotes the homing of MM cells. Prior studies have shown that the relative amount of BM-associated TAMs correlates with patient poor outcome in MM (Suyaný et al. 2013). Emerging evidence suggests that the absolute monocyte count (AMC) in peripheral blood may reflect the level of TAM recruitment at the tumor site and its prognostic significance has been shown in other lymphoid malignancies. Absolute lymphocyte count (ALC) as a surrogate marker for host immune system has prognostic significance. Early lymphocyte recovery after induction therapy or autologous stem cell transplant correlates with better survival for MM patients. The aim of the present study was to investigate the prognostic significance of the ratio of ALC, as a surrogate of host immune status, to AMC, which reflects the MM-immune microenvironment, in predicting clinical outcome among newly diagnosed MM patients.Methods: The study included 372 MM patients (pts), diagnosed 2004-2014 at the University Hospitals in Cleveland, OH and University of Cincinnati, Cleveland OH who had CBC differentials at diagnosis. The primary end-point of the study was progression free survival (PFS) calculated from the time of diagnosis. The Cox proportional hazards model was used to evaluate ALC/AMC at diagnosis as a prognostic factor for PFS as well as to assess and adjust of other know prognostic factors.Results: Median pt age was 67.3 years (range: 30-92), 53% were male and 47% female. Distributions of detailed baseline characteristics are presented in Table-1. The median follow up was 33.5 months (range: 1.16 - 122.9). Pts who were lost to follow up were censored from the survival analysis. At the time of this analysis 92 pts (24%) had died, 75 (20%) of them was due MM. The median AMC, ALC and ALC/AMC ratio at diagnosis was 412 cells/µL, 1,461 cells/µL and 3.9, respectively. ALC neither AMC was associate with different PFS or overall survival (OS). ALC/AMC as a continuous variable was identified as a predictor of PFS [Hazard ratio (HR) = 0.62, (95% CI: 0.52 - 0.75), p-value: 0.001]. To define a cut-off point, the choice of ALC/AMC ≥ 3.6 yielded the greatest differential in survival, based on the chai-squarevalue (χ2 = 94·4, P < 0·01) analyzed at different cut-off points between the 25% and 75% quartiles (2.9 - 4.3), from the log-rank test. Pts with an ALC/AMC ≥ 3.6 (n=236) experienced a superior median PFS compared to patients with an ALC/AMC < 3.6 (n=136) [Median PFS, 24 vs. 43 months respectively, P-value < 0.001] (Figure-1). Multivariate analysis including ALC, age, ISS stage and treatment type (autologous stem cell transplant vs. no transplant) showed that ALC/AMC as well as treatment type remained an independent prognostic factor of PFS after the step-wise fashion analysis. Out of 372 patients 192 patients had cytogenetics analysis at diagnosis. Univariate analysis demonstrated that low ALC, high AMC and a low ALC/AMC ratio were correlated with presence of del (17p) and t(4,14) and light chain myeloma. Multivariate analysis indicated that the ALC and AMC were not associated with any specific cytogenetic group. However, the ALC/AMC ratio independently predict 17p and t(4,14) abnormalities (p-value = .041). Low ALC, high AMC, and low ALC/AMC ratio were associated with poor prognostic factors such as high β2-microglobulin.Conclusions: In our cohort, myeloma patients with a higher ALC/AMC at diagnosis enjoyed a longer PFS (either as a continuous or dichotomized variable). Also, multivariate analysis demonstrated that patient with tumors that harbored del (17p) or t(4, 14) had low ALC/AMC ratio suggesting possible immunoparesis. ALC at diagnosis was not associated with OS, in contrast to prior studies, possibly due to the shorter follow-up duration used here and inadequate power to predict OS. Taken together, our data potentially suggest the ALC/AMC ratio as a readily available surrogate marker to assess the relative strength of host immune system to myeloma-induced immunoparesis. The ratio can serve as a biomarker to stratify patients for future immunological therapies. Also, our findings indicate that the host immune status should be carefully monitored. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Effects of Bone Marrow Mesenchymal Stem Cells on the Biological Characteristics of Migrating and Homing in Multiple Myeloma Cells

To construct a co-culture system for bone marrow mesenchymal stem cells (BMMSC) and multiple myeloma (MM) cells, and to investigate the effects of co-cultured BMMSC on the migrating and homing of multiple myeloma cells. The BMMSC from the transgenic mice with green fluorescent protein (GFP) fetal bone were cultured by adherent screening. A co-culture system of BMMSC and MM cell line XG-7 cells was constracted, the proliferation and apoptosis of cells were determined by trypan blue exclusion and Annexin V/PI, respectively, MDC staining was employed to detect the autophagy. The moving direction distribution of molecule in BMMSC and XG-7 cells labeled with PE-CD138 in co-culture process were observed dinamically by confocal microscopy. After co-culture with GFP-BMMSC, the resistance of XG-7 cells to apoptosis and autophagy were enhanced; at the same time, their proliferation increased. Apoptosis rates of XG-7 cells directly and indirectly co-cultured with BMMSC were (6.23 ± 0.12)% and (6.97 ± 0.03)% respectively, which were lower than that of XG-7 cells cultured alone (17.90 ± 1.46)% (P < 0. 01). There was low level of autophagy in XG-7 cells co-cultured with BMMSC. XG-7 cells are highly polarized and contained a specialized membrane domain with specific protein and lipid components to contact with BMMSC under confocal microscope. After methyl-β-cyclodextrin treatment, the molecules were normally enriched in the specialized domain. BMMSC can protect XG-7 cells from apoptosis and autophagy, and obviously promote the proliferation of XG-7 cells, and can influence the migrating and homing of multiple myeloma cells.

Sprouty2 regulates proliferation and survival of multiple myeloma byinhibiting activation of the ERK1/2 pathway invitro and invivo.

Multiple myeloma (MM) is an incurable disease, and its pathogenesis remains unclear. MicroRNA (miR)-21 was detected at a high level in MM and plays a key role in the pathogenesis of MM. However, Sprouty2 (spry2), a downstream target of miR-21, has low expression, and its mechanism in MM is unknown. We investigated whether spry2 could exert an antimyeloma effect and further studied the potential pathogenesis and progression of MM. To address the functional consequences of spry2, we assessed the expression levels of spry2 in several myeloma cell lines and detected low expression levels in MMcells. Overexpression of spry2 suppressed growth and colony formation ability and decreased the phosphorylation of extracellular signal-regulated kinases 1 and 2. Spry2 also decreased secretion of vascular endothelial growth factor and partially enhanced the sensitivity of MMcells to an inhibitor of mitogen-activated protein kinases 1 and 2. Additionally, spry2 inhibited the tumorigenesis and angiogenesis of MMcells invivo. In summary, we report for the first time that spry2 can inhibit MMcell growth and survival with a concomitant reduction in phosphorylation of extracellular signal-regulated kinases 1 and 2 invitro and invivo.

The KDM3A-KLF2-IRF4 axis maintains myeloma cell survival.

KDM3A is implicated in tumorigenesis; however, its biological role in multiple myeloma (MM) has not been elucidated. Here we identify KDM3A–KLF2–IRF4 axis dependence in MM. Knockdown of KDM3A is toxic to MM cells in vitro and in vivo. KDM3A maintains expression of KLF2 and IRF4 through H3K9 demethylation, and knockdown of KLF2 triggers apoptosis. Moreover, KLF2 directly activates IRF4 and IRF4 reciprocally upregulates KLF2, forming a positive autoregulatory circuit. The interaction of MM cells with bone marrow milieu mediates survival of MM cells. Importantly, silencing of KDM3A, KLF2 or IRF4 both decreases MM cell adhesion to bone marrow stromal cells and reduces MM cell homing to the bone marrow, in association with decreased ITGB7 expression in MAF-translocated MM cell lines. Our results indicate that the KDM3A–KLF2–IRF4 pathway plays an essential role in MM cell survival and homing to the bone marrow, and therefore represents a therapeutic target.

A New Model for Studying the Dissemination of Myeloma Cells throughout the Bone Marrow Using Embryonic Zebrafish

Multiple myeloma develops from a pre-malignant clonal proliferation of plasma cells. The dissemination of myeloma cells throughout the bone marrow (BM) is an important early step in myeloma pathogenesis. Studies of myeloma cell homing in mouse models are not quantitative and cannot be used for functional genomics or drug screening. To overcome these limitations, we have developed a novel in vivo model to assess multiple myeloma (MM) cell homing, that takes advantage of the optical clarity of zebrafish (Danio rerio) embryos.We performed intra-cardiac (i.c.) injection of GFP+MM.1S cells into zebrafish embryos 48 hpf, and assessed the ability of the injected cells to enter the peripheral circulation and then traced their homing to the trunk region of zebrafish embryos, an area referred as the caudal hematopoietic tissue (CHT), by using intra-vital confocal microscopy. We next injected primary CD138+ cells derived from MM patient BM that had been stained with either DiO or DiD and demonstrated that they also homed to CHT. As a control, we injected DiO-labeled CD41-GFPlow zebrafish hematopoietic stem cells (HSCs) that are known to home to the CHT hematopoietic niche; and observed that zebrafish-derived CD41-HSCs homed to the same area as MM cells.We next analyzed changes in the transcriptome of those MM cells that homed to the CHT-niche. We dissected the zebrafish embryos to separate the CHT from other tissue containing non-adherent MM cells and performed whole human exome enrichment prior to sequencing of total RNA. We had an alignment rate of 10-15%, with a high intragenic rate an exonic rate (> 95%) and a low mismatch rate (~0.5%). RNAseq revealed that the MM cells that homed to the CHT were enriched in transcripts important for cytokine/chemokine mediated signaling, the IL-6 signalling pathway, cell-cell adhesion and angiogenesis (FDR<0.25; P<0.05). Overall, these findings indicate that the changes observed in MM cells that have homed to the CHT mirror those that are seen in MM cells that are resident in the human BM. In order to investigate the functional relevance of the zebrafish model, we established CXCR4-, VLA-4- and FAK-silenced MM cells and compared their ability to home to CHT to that of control scrambled shRNA-transfected cells. DiO-labeled-CXCR4-silenced and DiO-labeled-scrambled-probe control MM cells were mixed in equal numbers and subsequently injected into recipient zebrafish. We found a significant reduction in the number of CXCR4-silenced MM cells homing to the CHT, compared to the control cells (P<0.00). We then examined VLA-4- and FAK-knock-down MM cells and observed that the homing of MM cells to CHT was impaired when either VLA-4 or FAK were silenced (P<0.001; Fig. 3C-D). Having demonstrated the role of CXCF4, FAK and VLA4 in MM cell homing to the CHT niche, we next performed qRT-PCR for those transcripts and confirmed that MM cells harvested from the CHT areas expressed higher levels of CXCR4, FAK and VLA4, compared to MM cells harvested from non-CHT areas. (P<0.05). To ascertain whether homing to zebrafish embryo CHT is occurs in other hematologic malignancies that are known to home to the human and murine BM, we used a cultured cell lined derived from a patient with Waldenstrom's Macroglobulinemia (WM). We injected either CXCR4-overexpressing or CXCR4-silenced WM cells and found that increased CXCR4 expression in WM cells led to enhanced CHT-homing of WM cells (P<0.001), while the homing of CXCR4-silenced WM cells to the CHT was reduced compared to scrambled control (P<0.001).These findings demonstrate that zebrafish can be used to study the homing of human myeloma cells to a hematopoietic niche. The rapidity of homing to CHT, which occurs within seconds of cell injection, suggests that a fraction of the CD138+ harvested from patient bone marrow already express those RNA transcripts and proteins needed for stable adhesion and residence in CHT. This hypothesis is confirmed by the RNA seq and qRT-PCR studies which directly demonstrate increased expression of relevant transcripts in adherent cells. This zebrafish model may provide new insights into the pathogenesis of MM and may be useful as a means to screen for agents which can disrupt homing and dissemination of MM cells. DisclosuresNo relevant conflicts of interest to declare.

The KDM3A-KLF2-IRF4 Axis Maintains Myeloma Cell Survival

Histone methylations are tightly regulated by a balance between methyltransferases and demethylases that mediate the addition and removal of these modifications. Importantly, dysregulation of histone methylation is implicated in pathogenesis of cancers, including multiple myeloma (MM). For example, the t(4;14) (p16;q32) is present in 15 - 20% of MM patients and results in overexpression of WHSC1, a histone H3 lysine 36 (H3K36) methyltransferase. On the other hand, approximately 10% of MM patients without the t(4;14) have inactivating mutations in KDM6A, a H3K27 demethylase.KDM3A is a Jumonji C-domain-containing histone demethylase which catalyzes removal of H3K9 mono- and dimethylation (H3K9me1 and H3K9me2). KDM3A is implicated in pathogenesis of different types of cancers. Here we investigated the biological impact of KDM3A in MM.KDM3A expression was significantly elevated in MM patient samples compared to normal plasma cells in publicly available dataset (GSE5900, GSE6691). To evaluate the functional role of KDM3A, shRNAs targeting KDM3A were transduced into MM cell lines: knockdown of KDM3A significantly inhibited MM cell growth (RPMI8226, MM.1S, U266, H929) in vitro and in xenograft model (MM.1S). Apo2.7 staining showed that apoptotic cells were significantly increased after knockdown of KDM3A.We next examined gene expression profiles after knockdown of KDM3A in RPMI8226 cells. With a cutoff of > 1.5-fold downregulation, a total of 305 probe sets were downregulated in KDM3A-knockdown cells relative to control cells. Among putative KDM3A targets, a gene of particular interest is KLF2 which plays a key role in maintenance of B cell and plasma cell phenotype, and function. Another intriguing gene is IRF4, given its known crucial role in MM cell survival. We confirmed that expression of KLF2 and IRF4 was downregulated after knockdown of KDM3A by quantitative realtime PCR and immunoblots in RPMI82226, MM.1S, and U266 cells. KDM3A binding to KLF2 and IRF4 core promoters was demonstrated by chromatin immunoprecipitation (ChIP) assay in RPMI8226 cells. Moreover, knockdown of KDM3A increased H3K9me1 and me2 levels at both promoter regions, indicating that KDM3A directly regulates KLF2 and IRF4 expression by removing H3K9 methylation marks at their promoters in MM cells.shRNAs targeting KLF2 were next transduced into MM cell lines: silencing of KLF2 significantly reduced cell growth of MM cell lines, associated with decreased IRF4. Promoter reporter assays using human IRF4 promoter showed that KLF2 significantly increased luciferase expression in a dose-dependent manner. Moreover, ChIP assay showed that KLF2 bound to IRF4 promoter in RPMI8226 cells. Since transcription factors could form an autoregulatory feedback loop, we hypothesized that IRF4 might regulate KLF2 expression. As expected, knockdown of IRF4 downregulated KLF2 expression at both the mRNA and protein levels in 3 MM cell lines. In addition, ChIP assays demonstrated that IRF4 bound to KLF2 second intron that contains tandem IRF4 motifs in RPMI8226 cells. Collectively, these results suggest that KLF2 activates IRF4 expression and vice versa, forming an autoregulatory loop in MM cells.KLF2 has been reported to control homing of plasma cells to the bone marrow; we therefore hypothesized that KDM3A-KLF2-IRF4 axis might regulate adhesion and homing of MM cells to the bone marrow. Importantly, knockdown of KDM3A, KLF2, or IRF4 decreased adhesion of 3 MM cell lines to bone marrow stromal cells. Furthermore, bone marrow homing of MM.1S cells was significantly reduced after knockdown of KDM3A, KLF2, or IRF4 in a murine xenograft MM model, indicating that KDM3A-KLF2-IRF4 axis regulates, at least in part, MM cell adhesion and homing to the bone marrow.In conclusion, our study demonstrated that KDM3A is a crucial epigenetic regulator of MM cell survival, and that inhibition of KDM3A represents a novel therapeutic strategy in MM. [Display omitted] DisclosuresRaje:Amgen: Consultancy; Takeda: Consultancy; Novartis: Consultancy; Celgene Corporation: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding; Onyx: Consultancy; AstraZeneca: Research Funding; Millenium: Consultancy. Richardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding. Anderson:Oncopep: Equity Ownership; Gilead: Consultancy; BMS: Consultancy; Millennium: Consultancy; Celgene: Consultancy; Acetylon: Equity Ownership.

The Dual PIM/PI3-K Inhibitor Ibl-202 Overcomes Microenvironmental Mediated Resistance in Multiple Myeloma and Prevents PIM1 Induced CXCR4 Upregulation

Background:The interaction of multiple myeloma (MM) cells with bone marrow (BM) cells along with factors in the BM milieu such as chemokines and cytokines play a crucial role in both progression of MM and drug resistance. Activation of the PI3-K/Akt survival pathway is a characteristic of both human MM cell lines and patient samples. This activation can be linked to BM microenvironmental signalling and use of proteasome inhibitors in treatment, suggesting this as a crucial point of therapeutic intervention to abrogate growth and survival signals in MM. However, the efficacy of such therapeutics has been modest and is likely to be compromised by the stimulation of compensatory signalling pathways, such as the PIM kinases, which like the PI3-K/Akt pathway are also induced by BM microenvironmental influences and share similar downstream targets. These proto-oncogenic kinases are constitutively active and play an important role in proliferation and survival in MM. The influence of these kinases on homing and migration has been observed in other malignancies, this has yet to be reported in MM. Here we report the effects of a dual inhibitor of PIM/PI3-K, IBL-202, and provide novel insights into effects on cell survival, signaling and migration.Methods:We investigated the effect of IBL-202 against a panel of MM cell lines (MM.IS, NCI-H929s, KMS11 and RPMI-8226) and primary MM patient samples. The in vitro efficacy of IBL-202 was compared to that of single pan-PIM inhibitors pPIMi and AZD1208 and also the pan-PI3-K inhibitor GDC-0941. Apoptosis was measured with AnnexinV staining and cell cycle analysed with Edu/DAPI staining.To mimic BM microenvironmental conditions MM cells were cultured under hypoxic conditions (1% O2) and in co-culture with the human stromal cell line HS5.Surface expression of CXCR4 was assessed in MM cell lines by flow cytometry. PIM kinases, pCXCR4 and downstream targets of PIM/PI3-K were examined by western blot. Transwell migration assays were carried out in the presence of 50ng SDF-1α for 4h @ 37o C.Results:Simultaneous inhibition of PIM and PI3-K using IBL-202 in vitro was significantly more potent at inducing apoptosis than GDC-0941, pPIMi or AZD1208 in all MM cell lines tested. IC50 values were under 1μM for IBL-202 at 48h whilst in comparison the pan PIM inhibitors pPIMi and AZD1208 scored IC50 values between 5 and 10μM. The IC50 for GDC-0941 was on average 5μM (Figure 1).At the molecular level there was a notable decrease in phosphorylation of known PIM/PI3-K targets Akt (Ser473), Bad (Ser112) and the translational targets S6 (Ser235/236) and 4EBP1 (Thr37/46). The levels of total proteins were unchanged.Treatment with increasing doses of IBL-202 led to a marked reduction in cells in S phase of the cell cycle. These changes were paralled by down regulation of the cell cycle promoting proteins cyclin D1 and c-myc.IBL-202 was also effective in inducing apoptosis in primary MM patient samples (n=4) after just 24h as assessed by Annexin-V staining (Figure 2).To explore the role of the BM microenvironment we co-cultured MM cell lines with HS5s. This led to strong induction of PIM2 in MM cells. While MM cells in this setting were protected from Bortezomib-induced cell death, the apoptotic effect of IBL-202 was enhanced. In a further effort to mimic the tumour microenvironment we cultured MM cell lines in hypoxia. This may be of particular relevance as Pim-1 has been reported to be a pivotal regulator involved in hypoxia-induced chemoresistance. MM cells were further sensitised to IBL-202 in hypoxia. In addition, hypoxia increased the surface expression of CXCR4, a chemokine receptor critical for homing of MM cells to the bone marrow, with a concomitant increase in PIM1. Treatment of MM cell lines with IBL-202 reduced the level of PIM1 and CXCR4 Ser339 phosphorylation, along with down regulation of CXCR4 surface expression resulting in reduced migration of MM cells along an SDF-1 gradient.Conclusion:Together these data provide direct evidence of the potency of IBL-202 in MM in conditions that mimic the BM microenvironment. Moreover, they indicate a potential role for PIM kinases in facilitating dissemination and invasiveness of MM by CXCR4 and provide an added rationale for targeting PIM kinases in MM. [Display omitted] [Display omitted] DisclosuresO'Neill:Inflection Biosciences: Employment.

Multiple myeloma: Mama, I'm coming home.

Zhu et al. find that the homing of multiple myeloma cells to the bone marrow is promoted by cyclophilin A–CD147 signalling between bone marrow endothelial cells and myeloma cells.

The Cyclophilin A-CD147 complex promotes the proliferation and homing of multiple myeloma cells.

B-cell malignancies frequently colonizes the bone marrow (BM). The mechanisms responsible for this preferential homing are not entirely known. Using multiple myeloma (MM) as a model of a terminally differentiated B-cell malignancy that selectively colonizes the BM, we demonstrated that BM endothelial cells (BMECs), secrete cyclophilin A (eCyPA), which promotes migration, proliferation, and BM colonization of MM cells via binding to its receptor, CD147, on MM cells. The clinical and translational implications of this work are highlighted by the observation of significantly higher eCyPA levels in BM serum than in peripheral blood (PB) in MM persons, and that eCyPA-CD147 blockade supresses BM-homing and tumor growth in a mouse xenograft model of MM. eCyPA also promoted migration of CLL and LPL cells, two other B-cell malignancies that colonize the BM and express CD147. These findings offer a compelling rationale for exploring the eCyPA-CD147 axis as therapeutic target for these malignancies.

Abstract PR14: In vivo targeting of stromal-derived factor-1 as a strategy to prevent myeloma cell dissemination to distant bone marrow niches

Abstract Background. Multiple myeloma (MM) patients present with multiple lytic lesions at diagnosis, indicating the presence of continuous dissemination of MM cells from the primary site of tumor development to multiple distant bone marrow (BM) niches. We hypothesized that stromal-derived factor-1 (SDF1) may represent a target for preventing transition from MGUS (micrometastatic-stage) to active-MM (macrometastatic-stage). We therefore evaluated SDF1 expression in the BM of patients with MGUS, MM, compared to healthy individuals; and tested NOX-A12, a high affinity L-oligonucleotide binder to SDF1, looking at its ability to modulate MM cell tumor growth and homing to the BM in vivo and in vitro. Methods. SDF1 levels were evaluated by immunohistochemistry on BM specimens of patients with MGUS, active-MM, or healthy individuals; and confirmed by ELISA, using conditioned-medium of BM-mesenchymal stromal cells (BM-MSCs) from MGUS, active-MM and healthy individuals. BM metastatic lesions from primary epithelial tumors were also evaluated. Co-localization of SDF1 with MM cell (MM.1S-GFP+)-enriched BM niches was evaluated using in vivo confocal microscopy. Effect of NOX-A12 on modulating MM cell dissemination was tested in vivo, by using in vivo MM metastasis model. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) was assessed by using in vivo confocal microscopy and bioluminescence, in mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12+bortezomib. Effects of drug combination on dissemination of MM cells to distant BM niches was evaluated ex vivo by immunofluorescence on explanted femurs. DNA synthesis and adhesion of MM cells in the context of NOX-A12 treated primary MM BM-MSCs in presence or absence of bortezomib were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software. Results. Patients with active-MM present with higher BM SDF1 expression vs MGUS patients and healthy individuals. Similarly, BM presenting with metastasis from epithelial primary tumors had higher SDF1 levels compared to healthy subjects, thus suggesting the importance of SDF1 in favoring tumor cell metastasis to BM niches. SDF1 co-localized at BM level with MM tumor cells in vivo. In vitro, NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BMSCs supported by inhibition of BM-MSC-mediated phosphorylation of ERK1/2 and cofilin. Importantly, NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo by reduction of MM cells in the BM and increased number of MM cells within the PB compared to control mice (P&amp;lt;.05). This was supported by inhibited homing and dissemination of MM cells to the BM of those mice pre-treated with NOX-A12. NOX-A12 enhanced MM cell sensitivity to bortezomib, in vivo: tumor burden was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor growth vs. control (P&amp;lt;.05); importantly, significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 and bortezomib was observed, compared to bortezomib-treated mice (P &amp;lt;.05). Similarly, NOX-A12+bortezomib combination induced significant inhibition of MM cell homing, as shown by in vivo confocal microscopy. In vitro studies confirmed synergism between NOX-A12 and bortezomib in modulating MM cell survival and adhesion to BM-MSCs. Conclusion. SDF-1 represents a valid target for inhibiting MM cell dissemination to distant BM niches, thus providing the evidence for using the SDF1 inhibiting spiegelmer NOX-A12 to target MM cells at the stage of micrometastasis (MGUS), thus preventing development of macrometastatic MM. This abstract is also presented as Poster B80. Citation Format: Aldo M. Roccaro, Antonio Sacco, Michele Moschetta, Patricia Maiso, Yuji Mishima, Yosra Aljawai, Fabio Facchetti, Anna Kruschinski, Giuseppe Rossi, Irene M. Ghobrial. In vivo targeting of stromal-derived factor-1 as a strategy to prevent myeloma cell dissemination to distant bone marrow niches. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR14. doi:10.1158/1538-7445.CHTME14-PR14

Abstract B62: Stroma-derived exosomes mediate progression in multiple meyloma

Abstract Background. Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) support multiple myeloma (MM) cell growth, but little is known about the putative mechanisms that may regulate the interaction between clonal MM plasma cells and the surrounding BM milieu. We characterized the role BM-MSC-derived exosomes as key regulators of MM pathogenesis. Purpose. 1) To determine the ability of BM-MSCs to release and transfer exosomes to MM cells. 2) To determine the role of BM-MSC-derived exosomes in inducing MM tumor growth and MM cell dissemination Methods. MSCs were collected from BM of healthy subjects and MM patients, showing a multipotent MSC phenotype (CD34-; CD14-; CD45-; CD19-; CD138-; CD73+; CD90+; CD105+; CD106+). Exosomes were collected from conditioned-medium of normal-BM-MSCs and MM-BM-MSCs, or HS-5 cells; and studied using electron microscopy, immunogold-labeling, and western-blot for CD63 and CD81 detection. Transfer of PKH67-fluorescently-labeled-exosomes to MM cells was evaluated by time-lapse confocal microscopy. Transfer of murine-derived miRNA-containing exosomes into human MM cells was evaluated by qRT-PCR (exosomes were collected from BM-MSCs of C57BL/6 miRNA-15a/16-1-/- or C57BL/6 mice). miRNA expression profiling was obtained from normal (n=4) and MM (n=9) BM-MSCs-derived exosomes (TaqMan-human-miRNA-profiling). Normal and MM BM-MSCs-derived exosomes were loaded into tissue-engineered bones (TEB) with MM.1S-GFP+/Luc+ cells: MM cell homing and MM tumor growth have been tested in vivo by using confocal-microscopy and bioluminescence-imaging (BLI), respectively. Loss- and gain-of-function studies were performed using normal-BM-MSCs, MM-BM-MSCs and HS-5 cells transfected with either pre- or anti-miRNA-15a. Results. Normal-BM-MSCs and MM-BM-MSCs released CD63+/CD81+ exosomes, as confirmed by electron microscopy, immunogold labeling, and western blot. BM-MSCs exosomes are transferred into MM cells, as shown by confocal microscopy; and further validated by qRT-PCR in human MM cell lines incubated with murine (C57BL/6 miRNA-15a/16-1-/- and wild-type) BM-MSCs-derived exosomes. The impact of normal-BM-MSC- and MM-BM-MSC-derived exosomes on MM cell behavior in vivo was next evaluated. MM cells co-cultured with MM BM-MSC-derived exosomes induced rapid tumor growth at the site of the TEB scaffold, as well as rapid dissemination to distant BM niches, as compared to MM cells co-cultured with exosomes derived from normal BM-MSCs. We next performed miRNA expression profiling on exosomes isolated from MSCs, and found increased expression of 24 miRNAs and reduced expression of 3 miRNAs in MM-BM-MSCs-derived exosomes versus normal (1.5 fold-change; P&amp;lt;0.05). Specifically, miRNA15a was significantly lower in MM-BM-MSC-derived exosomes, similarly to primary MM cells that present with reduced miRNA-15a expression. We therefore sought to examine whether lack of transfer of the tumor suppressor miRNA15a can lead to significant change in tumor growth and dissemination in MM, and found that by over-expressing miRNA-15a in normal BM-MSCs and HS-5 cells inhibited MM cell proliferation and adhesion to fibronectin. Next MM cells were cultured in presence of BM-MSCs isolated from either C57BL/6 mice or C57BL/6 miRNA15a/16-1-/-: miRNA15a-deficient BM-MSCs significantly induced MM cell proliferation (P&amp;lt;0.05). Moreover, exosomes isolated from HS-5 pre-miRNA15a-transfected cells both inhibited MM cell proliferation and reduced their adhesion properties. Conclusion. These findings demonstrate the existence of exosome-driven interactions between the BM milieu and MM cells, suggesting that exosomes might constitute a novel mechanism for intercellular transfer of miRNAs to MM cells. Citation Format: Aldo M. Roccaro, Antonio Sacco, Patricia Maiso, Michele Moschetta, Salomon Manier, Yuji Mishima, Michaela Reagan, Yosra Aljawai, Irene M. Ghobrial. Stroma-derived exosomes mediate progression in multiple meyloma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B62. doi:10.1158/1538-7445.CHTME14-B62

Novel CXCR4-Targeted Therapy to Inhibit Multiple Myeloma Bone Dissemination

Background. Presence of multiple osteolytic lesions is one of the main clinical features of patients with active multiple myeloma (MM), thus suggesting the ability of clonal plasma cells to disseminate from bone to bone. The bone marrow (BM) homing process of MM cells has been shown to be supported by the activation of the CXCR4/CXCL12 axis. Nevertheless, the role of CXCR4 in mediating MM cell bone metastasis, and the role of CXCR4-targeted therapy in inhibiting MM cell dissemination from bone-to-bone has not been previously reported.Methods. We dissected the in vivo functional relevance of CXCR4 in mediating MM cell dissemination, tumor growth and survival, by using gain- and loss-of function approaches. Tumor growth was monitored by bioluminescence imaging (BLI) and MM cell homing and engraftment within the BM niches was evaluated by performing intravital confocal microscopy. CXCR4-oeverexpressing (CXCR4+) MM cells were tested in vivo using a model of bone-to-bone dissemination. Empty vector or CXCR4+ cells were loaded into murine femurs and subsequently implanted s.q. into recipient mice. The ability of MM cells to disseminate from the implanted bone to the host bones was evaluted by flow cytometry and immunohistochemistry (IHC; CD138; hematoxylin-eosin) on the harvested host bones. Modulation of EMT-related genes (twist, snail, slug, E-cadherin, vimentin) was evaluated ex vivo by qRT-PCR. A novel anti-CXCR4 monoclonal antibody (BMS-936564) was tested both in vivo and in vitro to define the possible role in modulating MM cell dissemination and growth.Results. CXCR4-overexpression led to changes in actin cytoskeleton reorganization of MM cells with protrusion of cell pseudopodia, compared to empty vector-transfected cells, sustained by modulation of EMT-related markers, as shown by up-regulation of Slug, Snail and Twist, together with down-regulation of E-cadherin, in CXCR4-overexpressing (CXCR4+) MM.1S cells, compared to empty vector-transfected cells. CXCR4+ MM cells presented with enhanced invasive properties, compared to control cells. These findings were next recapitulated in vivo using an in vivo model of MM cell dissemination: CXCR4+ MM cells presented with higher ability to metastasize from bone to bone compared to control cells, as confirmed on harvested host femurs by using IHC and flow cytometry. Lower expression of human (h)-E-cadherin, and higher mRNA expression of h-Twist, h-Snail, h-Slug was confirmed within the BM of the host femurs. We next tested the novel monoclonal antibody, anti-CXCR4 (BMS-936564), in vivo. BMS-936564 exerted an anti-MM activity in situ, within the s.q. implanted bones; and also reduced MM cell dissemination from the implanted bone to the host bone. We examined whether BMS-936564 might have modulated EMT-related genes in the MM cells metastasized to the host bones, and found higher mRNA expression of human (h)-E-cadherin, together with reduced mRNA expression of h-Twist, h-Snail, h-Slug, h-vimentin and h-CXCR4 in the BM cells harvested from the host bones of mice treated with BMS-936564. Similar findings were confirmed in a model of breast cancer in vivo. CXCR4 silencing in MM cells led to inhibition of MM cell homing to the bone marrow, as shown by using intra-vital confocal microscopy and inhibition of MM tumor growth in vivo, as shown by BLI. Prolonged survival was documented in those mice that were injected with CXCR4-silenced MM cells compared to control mice injected with scramble probe-infected MM cells.Conclusion. CXCR4 enhanced the acquisition of an EMT-like phenotype in MM cells, leading to higher bone-to-bone dissemination of clonal plasma cells in vivo; while CXCR4-silencing in MM cells led to inhibited tumor growth and improved survival. BMS-936564 led to inhibited MM cell bone-to-bone dissemination, supported by suppression of the CXCR4-driven EMT-like phenotype. These studies suggest that targeting CXCR4 may represent a novel therapeutical strategy to prevent MM cell dissemination. DisclosuresCardarelli:BMS: Employment. Cohen:BMS: Employment. Kuhne:BMS: Employment. Ghobrial:Celgene: Membership on an entity’s Board of Directors or advisory committees; BMS: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Membership on an entity’s Board of Directors or advisory committees; Millennium/Takeda: Membership on an entity’s Board of Directors or advisory committees.

CD166 Identifies Multiple Myeloma Cells That Preferentially Home to the Bone Marrow Microenvironment and Promote Disease Progression and Bone Lytic Disease

Multiple myeloma (MM) is an incurable malignancy characterized by the proliferation of neoplastic plasma cells in the bone marrow (BM) and by multiple lytic lesions throughout the skeleton. We previously reported that CD166 is a functional molecule on normal hematopoietic stem cells that plays a critical role in HSC homing to the BM and engraftment (Blood. 2014; 124(4):519-29.), suggesting that CD166 is involved in trafficking and lodgment of HSC. CD166, which is a member of the immunoglobulin superfamily capable of mediating both homophilic and heterophilic (CD6) interactions, has been shown to enhance metastasis and invasion in several tumors including breast cancer and melanoma. However, whether CD166 is involved in MM and whether it plays a role in MM progression or the bone lytic disease has not been addressed. H929-GFP MM cells were injected intravenously into NSG mice and GFP cells were recovered from the BM 14hr later. While only 29.9%±1.4% of total H929-GFP cells were CD166+, the frequency of CD166+ cells contained in BM-homed H929-GFP cells was significantly higher (80.0%±2.5%, n=9, p<0.01). Knocking-down (KD) CD166 expression on H929-GFP cells with shRNA reduced the homing of MM cells to the BM. The number of BM-homed GFP cells was significantly decreased for CD166KD cells (5658±904, n=6) compared to mock cells (8551±848, n=6; p<0.05). To examine the potential role of CD166 in osteolytic lesions, we used a novel Ex Vivo Organ Culture Assay (EVOCA) in which MM cells are co-cultured over calvariae from 10d-old pups for 7 days creating an in vitro 3D system for the interaction of MM cells with the bone microenvironment. EVOCA data from two MM cells lines, H929 and OPM2 as well as from three primary MM patients’ CD138+ BM cells demonstrated that bone osteolytic lesions were significantly reduced when CD166 was absent on either MM cells (sorted CD166- fraction) or osteoblast lineage cells (calvariae from CD166-/- mice). Osteolytic lesions can be caused by decreased osteoblastogenesis or increased osteoclastogenesis. Co-culturing BM stromal cells (BMSC) from WT or CD166-/- mice with mock or CD166KD H929 cells in osteoblastogenic media revealed that loss of CD166 on either MM cells or BMSC released the inhibition of mRNA expression of RUNX2, a critical transcriptional factor for early osteoblast differentiation, suggesting that interactions through CD166 suppresses osteoblastogenesis. Gene analysis of calvariae co-cultured with mock or CD166KD H929 cells revealed that the ratios of mRNA levels of RANKL to that of OPG, an important inhibitor of osteoclastogenesis, were elevated in cultures containing a CD166+ component suggesting that CD166 enhances osteoclastogenesis. Furthermore, co-cultures of BM monocytes (BMM) from WT or CD166-/- mice with mock or CD166KD H929 cells in the presence of RANKL and MCSF confirmed that the absence of CD166 on either BMM or MM cells results in significantly reduced osteoclast differentiation as evidenced by TRAP staining. Given that Akt signaling is regulated by CD166 and that Akt pathway is important in osteoclast differentiation, we examined whether or not the Akt pathway is involved in CD166-induced osteoclastogenesis in MM. Western blot analysis of BMM from WT or CD166-/- mice starved then stimulated with either mock or CD166 KD H929 cells revealed that phospho-Akt was significantly down-regulated in the absence of CD166 on either BMM or MM cells indicating that CD166 can activate the Akt pathway and promote MM-induced osteoclast differentiation. Finally, we compared in NSG mice the progression of MM initiated with control, mock or CD166KD H929-GFP cells (n=7-13/group, 2 independent experiments). Compared to control and mock groups, NSG mice transplanted with CD166KD cells showed delayed disease progression and prolonged survival as evidenced by serum levels of human IgA (kappa) and Kaplan-Meier survival curves (p<0.05), respectively. Meanwhile, the inhibition of osteocalcin mRNA expression in calvariae of NSG mice was significantly reduced in the presence of CD166KD cells compared to that with control or mock cells, suggesting again that expression of CD166 on MM cells inhibits osteoblastogenesis. These results suggest that CD166 directs MM cell homing and retention in the BM and promotes MM disease progression and propose that CD166 may induce osteoclastogenesis in MM through the Akt pathway. CD166 may serve as a therapeutic target in the treatment of MM. DisclosuresNo relevant conflicts of interest to declare.