Multiple Myeloma Cell Dissemination Research Articles

Characterisation of the Tumour Proteome in Primary Extramedullary Multiple Myeloma Identifies Key Proteins Associated with Transendothelial Migration

Introduction: Extramedullary multiple myeloma (EMM) refers to the spread of clonal plasma cells to tissues extending outside of the bone marrow microenvironment. EMM is present at the time of diagnosis in 6-10% of patients, however, this increases to 13-26% in patients with disease progression and relapse. Cancer cells are suspected to spread to new tissues and organs via the circulatory system as a result of molecular changes that allow malignant cells to escape the bone marrow (BM). For example, downregulation of CXCR4 (C-X-C Motif Chemokine Receptor 4), an important factor in cellular homing to the BM, has frequently been reported to be linked to the EMM phenotype. In addition, the majority of patients presenting with EMM have highly complex cytogenetic abnormalities and high-risk cytogenetic markers such as t(14;16). As EMM is an indicator of a more aggressive disease, more intensive treatment, including combination chemotherapy, is often recommended. Many of the underlying molecular mechanisms accompanying EMM are yet to be characterised. Our mass-spectrometry (MS)-based proteomic study provides insight into the unique molecular mechanisms associated with EMM, identifying key proteins linked to the progression of medullary multiple myeloma (MM) to EMM.Methods: Label-free liquid chromatography mass spectrometric analysis of age and gender matched medullary MM (n=8) and EMM (n=9) bone-marrow derived mononuclear cells (MNCs) was carried out using a Thermo Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Proteome Discoverer 2.2 using Sequest HT (Thermo Fisher Scientific) and a percolator were employed for the identification of peptides and proteins. For protein identification, the following search parameters were used: (i) 0.02 Da for MS/MS mass tolerance, (ii) 10 ppm for peptide mass tolerance, (iii) variable modification settings for methionine oxidation, (iv) fixed modification settings in relation to carbamido-methylation and (v) tolerance for up to two missed cleavages. Peptide probability was set to high confidence. Datasets were imported into Progenesis QI (version 2.0) software for further analysis. Data was filtered based on an ANOVA p-value of ≤0.05, fold change >1.5 between experimental groups, and proteins with ≥1 unique peptides contributing to the identification. Proteins with less than 70% valid values were removed from the analysis. G:profiler and STRING were utilized for functional enrichment and the characterisation of protein interaction patterns.Results: Our quantitative MS-based proteomic analysis identified a total of 492 proteins with significantly altered abundances between EMM and MM bone marrow MNC. Of these significant proteins, 275 were found to be increased in EMM compared to medullary MM and 217 were found to be decreased in EMM compared to medullary MM. Hierarchical clustering was performed to highlight the proteomic profile associated with extramedullary disease (Figure 1A). KEGG pathway analysis and gene ontology (GO) analysis of proteins found to be increased in EMM indicated an increase in proteins associated with cell adhesion, invasion, and migration pathways (Figure 1B). Interestingly, several proteins involved in leukocyte transendothelial migration were significantly increased in EMM indicating their potential involvement in the dissemination of MM cells from the bone marrow microenvironment to distal tissues (Figure 1C). Among the proteins found to be involved in this biological pathway was junctional adhesion molecule-A (F11R), a protein previously reported to play a role in EMD pathophysiology [1]. Other proteins involved in MM invasion and migration including Rho-associated protein kinase 2 (ROCK2), Ras-related C3 botulinum toxin substrate 1 (Rac1) and platelet endothelial cell adhesion molecule (PECAM-1) were significantly increased in EMM.Conclusion: Using high-resolution mass spectrometry to characterise the tumour proteome of MM patients with extramedullary disease, we have identified a significant increase in the abundance of proteins associated with leukocyte transendothelial invasion in primary EMM samples. Our study provides further insight into the molecular mechanisms within EMM and thus holds potential to enhance current efforts to provide a more personalised therapeutic approach for EMM patients.

FGF Trapping Inhibits Multiple Myeloma Growth through c-Myc Degradation-Induced Mitochondrial Oxidative Stress.

Multiple myeloma, the second most common hematologic malignancy, frequently relapses because of chemotherapeutic resistance. Fibroblast growth factors (FGF) act as proangiogenic and mitogenic cytokines in multiple myeloma. Here, we demonstrate that the autocrine FGF/FGFR axis is essential for multiple myeloma cell survival and progression by protecting multiple myeloma cells from oxidative stress-induced apoptosis. In keeping with the hypothesis that the intracellular redox status can be a target for cancer therapy, FGF/FGFR blockade by FGF trapping or tyrosine kinase inhibitor impaired the growth and dissemination of multiple myeloma cells by inducing mitochondrial oxidative stress, DNA damage, and apoptotic cell death that were prevented by the antioxidant vitamin E or mitochondrial catalase overexpression. In addition, mitochondrial oxidative stress occurred as a consequence of proteasomal degradation of the c-Myc oncoprotein that led to glutathione depletion. Accordingly, expression of a proteasome-nondegradable c-Myc protein mutant was sufficient to avoid glutathione depletion and rescue the proapoptotic effects due to FGF blockade. These findings were confirmed on bortezomib-resistant multiple myeloma cells as well as on bone marrow-derived primary multiple myeloma cells from newly diagnosed and relapsed/refractory patients, including plasma cells bearing the t(4;14) translocation obtained from patients with high-risk multiple myeloma. Altogether, these findings dissect the mechanism by which the FGF/FGFR system plays a nonredundant role in multiple myeloma cell survival and disease progression, and indicate that FGF targeting may represent a therapeutic approach for patients with multiple myeloma with poor prognosis and advanced disease stage. SIGNIFICANCE: This study provides new insights into the mechanisms by which FGF antagonists promote multiple myeloma cell death. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/11/2340/F1.large.jpg.

A Highly Robust Model for Predicting Outcome of Multiple Myeloma Patients By Inferring Patient-Specific Transcription Factor Activity

Objective: Multiple myeloma (MM) is the second most common blood cancer in the United States. Clinical and pathological indicators are inadequate for accurate assessing prognosis of MM patients1. The objective of this study was to develop a robust computational model for predicting prognosis of MM patients by inferring transcription factor (TF) activities based on global gene expression measurements. Methods: The workflow is illustrated in Figure 1. Gene expression data from MM patients were retrieved from the Multiple Myeloma Research Foundation CoMMpass study2 (MMRF) and Gene Expression Omnibus (GEO). MM-specific TF-target gene relationships were derived from the ENCODE database3 and co-expression analysis was based on MMRF data. The activity for each TF was inferred using a rank-based enrichment approach based on the target gene expression measurements in individual patients4. A prognostic model was developed using Cox regression analysis based on the inferred TF activities that demonstrated the highest association with patient survival. The TF model performance was further evaluated using data from four independent MM cohorts, in which gene expression levels were measured using different platforms. Results: Based on MMRF data (767 patients), the inferred activity of four TFs, BATF, E2F1, MYBL2, and RAD21, showed the strongest association with patient survival. The derived prognostic model was validated in four independent MM data sets with a total of 1501 patients. Importantly, gene expression levels in the validation data sets were measured using microarray, which is different from the RNAseq used in training data sets, and supports the robustness of the TF activity model across different platforms (Figure 2). In addition, the TF activity model was able to predict clinical outcomes of patients that received bortezomib treatment, and it also revealed an association with metastasis in myeloma cell line xenograft experiments5 (Figure 3). Conclusion: We developed a highly robust prognostic model for predicting MM clinical outcomes. The inferred TF activity further improves our understanding of the underlying regulatory basis of MM progression. Reference 1. Rajkumar SV. Multiple myeloma: 2012 update on diagnosis, risk-stratification, and management. Am J Hematol. 2012;87(1):78-88. 2. l, a longitudinal study in multiple myeloma relating clinical outcomes to genomic and immunophenotypic profiles: Am Soc Hematology; 2013. 3. Consortium EP. The ENCODE (ENCyclopedia of DNA elements) project. Science 2004;306(5696):636-640. 4. Moerman T, Aibar Santos S, Bravo González-Blas C, et al. GRNBoost2 and Arboreto: efficient and scalable inference of gene regulatory networks. Bioinformatics. 2018;35(12):2159-2161. 5. Shen Y, Mishima Y, Shi J, et al. Deciphering Clonal Evolution and Dissemination of Multiple Myeloma Cells In Vivo: Am Soc Hematology; 2018. Disclosures Abonour: Celgene: Consultancy, Research Funding; BMS: Consultancy; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Roodman:Amgen: Membership on an entity's Board of Directors or advisory committees.

Specific Targeting of KRAS Using a Novel High-Affinity KRAS Antisense Oligonucleotide in Multiple Myeloma

Specific Targeting of KRAS Using a Novel High-Affinity KRAS Antisense Oligonucleotide in Multiple Myeloma

Deciphering Clonal Evolution and Dissemination of Multiple Myeloma Cells In Vivo

Introduction: Multiple Myeloma (MM) is a genetically complex and evolutionary process with well defined precursor states, which offer a unique opportunity to study the sequential evolution of the disease. A small number of detectable pre-malignant clones are present in early stage and continue to acquire more genomic abnormalities leading to overt disease. The interaction between cancer cells and their environment is reciprocal, multiple components in the tissue environment can influence cancer clonal evolution and cancer cells in turn can also remodel the microenvironment and further disseminate to spatially separated areas of BM. To accurately predict the course of disease with the presence of BM environment, we require methods to estimate clone-specific growth rates and define clones that have the propensity of dissemination.Methods: We developed a novel ‘bone chip’ MM metastatic xenograft model using fluorescent protein tagged ‘rainbow’ system which enables both molecular profiling and functional tracking of clonal dissemination of tumor cells by performing tumor-bearing bone chip implantation subcutaneously to SCID-beige mice (SCID-murine model). Rainbow MM cells with equal proportion of all 15 colors were injected into donor femurs and implanted into recipient mice. After paralysis, the mice were sacrificed and tumor cells were analyzed using flow cytometry and confocal microscopy. Tumor clones in the implanted bone chip (primary sites) and distant host BM (metastatic sites) were purified by sorting and underwent RNA sequencing. By intersecting differentially expressed genes, we identified a set of genes, the expression of which were altered during disease dissemination and designated this set of genes as ‘metastatic signature’. In addition, we also performed genome-wide CRISPR/Cas9-mediated loss-of-function screen in a subcutaneous xenograft mouse model to investigate the essential drivers of tumor growth and metastasis in MM. The cell library infected with human sgRNA library was injected subcutaneously into SCID-Beige mice on both flanks. When metastasis was established, the fractions of each sgRNA of the primary and metastatic tumors were calculated to identify genes that facilitate tumor metastasis.Results: We found that the 15 rainbow subpopulations were present with equal distribution in the primary sites but not at the metastatic sites. Confocal imaging showed the difference in cluster structures between primary and metastatic tumors. Most of the clusters in the metastatic sites consisted of cells of single colors. RNA sequencing analysis of two human MM cell lines derived from SCID-murine model demonstrated a distinct gene expression profile of the metastatic tumors. Gene Set Enrichment Analysis of the metastatic signature in publicly available MM patient datasets (GSE6477 and GSE2658) demonstrated that this signature is significantly correlated with overall survival and with clinical progression from MGUS/smoldering MM to overt myeloma and relapsed disease. Through genome-wide CRISPR screening in vivo, we found that the gene targets of the most enriched sgRNAs in the BM samples were preferentially involved in important cellular processes, such as cell cycle regulation and several oncogenic signaling pathways. Additionally, many sgRNAs that remained the implanted sites until late stage were depleted during dissemination, indicating their targeted genes were important for progression. These depleted sgRNAs mainly targeted genes involved in mTORC1 and DNA repair pathways, many of which are regulated by MYC and cell cycle related targets of E2F transcription factors. By using a network-based inference of protein activity method, we chose 4 genes (HMGA1, KLF6, TRIM28 and PA2G4) and validated in SCID-murine model using CRISPR mediated loss-of-function screen which prioritized HMGA1 as the key regulator in MM dissemination.Conclusions: Here, we demonstrate that in vivo clonal evolution can be characterized using an in vivo model of MM. The data defines specific subclones that have a higher metastatic potential and are likely driver clones for tumor metastasis in MM. We then established a platform for future invivo CRISPR screens to investigate essential genes of response to targeted therapies and/or immunotherapies. Furthermore, a metastatic gene signature was identified and among these, HMGA1 was validated as potential regulator of MM metastasis. DisclosuresRoccaro:AMGEN: Other: Advisory Board; GILEAD: Research Funding. Ghobrial:Takeda: Consultancy; Celgene: Consultancy; BMS: Consultancy; Janssen: Consultancy.

Copper 64–labeled daratumumab as a PET/CT imaging tracer for multiple myeloma

AbstractAs a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose (18F-FDG) is a function of the metabolic activity of both malignant and nonmalignant cells, the results frequently lack sufficient specificity. Radiolabeled antibodies targeting MM tissue may detect disease irrespective of cell metabolism. Hence, we conjugated the clinically significant CD38-directed human antibody daratumumab (Darzalex [Dara]) to the DOTA chelator and labeled it with the positron-emitting radionuclide copper 64 (64Cu; 64Cu-DOTA-Dara). Here, we show that 64Cu-DOTA-Dara can efficiently bind CD38 on the surface of MM cells and was mainly detected in the bones associated with tumor in a MM murine model. We also show that PET/CT based on 64Cu-DOTA-Dara displays a higher resolution and specificity to detect MM cell dissemination than does 18F-FDG PET/CT and was even more sensitive than were bioluminescence signals. We therefore have supporting evidence for using 64Cu-DOTA-Dara as a novel imaging agent for MM.

Heparanase promotes myeloma progression by inducing mesenchymal features and motility of myeloma cells.

Bone dissemination and bone disease occur in approximately 80% of patients with multiple myeloma (MM) and are a major cause of patient mortality. We previously demonstrated that MM cell-derived heparanase (HPSE) is a major driver of MM dissemination to and progression in new bone sites. However the mechanism(s) by which HPSE promotes MM progression remains unclear. In the present study, we investigated the involvement of mesenchymal features in HPSE-promoted MM progression in bone. Using a combination of molecular, biochemical, cellular, and in vivo approaches, we demonstrated that (1) HPSE enhanced the expression of mesenchymal markers in both MM and vascular endothelial cells; (2) HPSE expression in patient myeloma cells positively correlated with the expression of the mesenchymal markers vimentin and fibronectin. Additional mechanistic studies revealed that the enhanced mesenchymal-like phenotype induced by HPSE in MM cells is due, at least in part, to the stimulation of the ERK signaling pathway. Finally, knockdown of vimentin in HPSE expressing MM cells resulted in significantly attenuated MM cell dissemination and tumor growth in vivo. Collectively, these data demonstrate that the mesenchymal features induced by HPSE in MM cells contribute to enhanced tumor cell motility and bone-dissemination.

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.

CXCR4 Regulates Extra-Medullary Myeloma through Epithelial-Mesenchymal-Transition-like Transcriptional Activation

Extra-medullary disease (EMD) in multiple myeloma (MM) is associated with poor prognosis and resistance to chemotherapy. However, molecular alterations that lead to EMD have not been well defined. We developed bone marrow (BM)- and EMD-prone MM syngeneic cell lines; identified that epithelial-to-mesenchymal transition (EMT) transcriptional patterns were significantly enriched in both clones compared to parental cells, together with higher levels of CXCR4 protein; and demonstrated that CXCR4 enhanced the acquisition of an EMT-like phenotype in MM cells with a phenotypic conversion for invasion, leading to higher bone metastasis and EMD dissemination in vivo. In contrast, CXCR4 silencing led to inhibited tumor growth and reduced survival. Ulocuplumab, a monoclonal anti-CXCR4 antibody, inhibited MM cell dissemination, supported by suppression of the CXCR4-driven EMT-like phenotype. These results suggest that targeting CXCR4 may act as a regulator of EMD through EMT-like transcriptional modulation, thus representing a potential therapeutic strategy to prevent MM disease progression.

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

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.

CXCR4 Monoclonal Antibody, BMS-936564 (MDX-1338), Modulates Epithelial to Mesenchymal Transition (EMT) in Multiple Myeloma Cells

AbstractAbstract 4009 Background.The SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. Also, hypoxia leads to EMT activation as well as CXCR4 up-regulation in MM cells. We therefore hypothesized that CXCR4 may represent a crucial regulator of EMT in MM and an important target for preventing MM disease dissemination. Methods.Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Dissemination of MM.1S/GFP+ cells to distant bone marrow niches was evaluated in vivo, by using in vivo confocal microscopy. CXCR4-loss of function studies were performed by transfecting MM cells with either a scrambled probe or CXCR4-siRNA. A novel HuMAb anti-CXCR4 (BMS-936564; Bristol Myers Squibb, NY) was used. Migration towards SDF-1 and BMSCs was evaluated. Cytotoxicity and DNA synthesis were measured by MTT and 3H-thymidine uptake, respectively. Cell signaling, apoptotic- and EMT-related pathways were studied by Western Blot. Synergism was calculated by using the Chou-Talalay method. In vivo, MM tumor growth was evaluated by using xenograft mouse models and a melanoma xenograft mouse model was used to validate the effect of anti-CXCR4 antibody on modulating tumor cell metastasis. Results.We demonstrated down-regulation of Twist, Snail and Slug, together with up-regulation of E-Cadherin in CXCR4-siRNA-transfected cells, compared to scrambled probe-transfected cells. These findings were next validated by using the new selective CXCR4 antibody (BMS-936564); and confirmed that BMS-936564-dependent inhibition of CXCR4 led to inhibition of Twist, Snail, and Slug; with up-regulation of E-Cadherin. These data were further corroborated in vivo, by using in vivo confocal microscopy: mice treated with BMS-936564 presented with less MM cell dissemination to distant bone marrow niches, compared to vehicle-treated mice, supporting the hypothesis that CXCR4 may represent a crucial modulator of tumor cell dissemination. These data were also confirmed in vivo, by using a xenograft melanoma model, where BMS-936564-treated mice presented with a reduced number of metastasis, compared to vehicle-treated mice. These in vivo data were supported by in vitro evidence showing the ability of BMS-936564 to functionally target MM cells in terms of migration, adhesion and survival. BMS-936564 inhibited migration of MM cells towards SDF-1a and primary MM BMSCs, in a dose-dependent manner. In addition, survival and adhesion of primary MM cells to BMSCs were inhibited by BMS-936564 in a dose-dependent manner. BMS-936564 targeted MM cells in the context of BM milieu, by overcoming BMSCs-induced proliferation of tumor cells. Moreover, BMS-936564 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS-936564-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorylation were inhibited by BMS-936564 in a dose-dependent manner. Importantly, BMS936564 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion.These findings indicate that CXCR4 represents a valid therapeutic target due to its ability to modulate EMT, and that BMS-936564 functionally targets MM cell migration, adhesion and survival; thus providing evidence for using the anti-CXCR4 antibody, BMS-936564, as a therapeutic modality for MM. Disclosures:Kuhne:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Novartis: Advisory Board Other; Onyx: Advisory Board, Advisory Board Other; Millennium: Advisory Board, Advisory Board Other; Bristol Myers Squibb: Advisory Board, Advisory Board Other.

Dynamic Regulation of the Level of Hypoxia In the Bone Marrow Regulates Cell Dissemination In Multiple Myeloma

AbstractAbstract 4035 INTRODUCTION:The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis, implying that progression of MM occurs through continuous interaction between the BM and MM cells, which controls the ability of MM cells to egress out of the BM and home into new BM niches. We have previously shown that the CXCR4/SDF1 axis as well as Rho GTPases downstream of the receptor was important for chemotaxis, adhesion, homing and egress of MM cells. However, the driving force for MM cells to leave the BM and metastasize to other BM sites is not well understood. Regions of severe oxygen deprivation (hypoxia) arise in tumors due to rapid cell division and are associated with poor patient prognosis, cell motility, associated angiogenesis and metastasis. In this study, we tested the role of hypoxia in the dissemination of MM cells in vivo, as well as regulation of the retention/egress of MM cells in and out of the BM. METHODS:To test the effect of hypoxia on induction of MM egress, MM1s-GFP+/Luc+ cells were injected into 12 SCID mice, and then mice with different stages of tumor development (based on the tumor size detected by bioluminescence) were treated with the hypoxia marker pimonidazole. Blood was drawn and BM was obtained from the femur. Mononuclear cells were then fixed, permeabilized, and stained with antibodies against pimonidazole, followed with an APC- secondary antibody, PE-mouse-anti-human CXCR4, and anti-cadherin antibody followed by an Alexa-Fluor-594 secondary antibody. MM cells in BM and peripheral blood were identified by gating on cells with high GFP signal. To confirm the effects of severe hypoxia found in vivo compared to physiologic mild hypoxia found in the BM, we tested the effect of mild hypoxic conditions (6% O2) and severe hypoxic conditions (0.5% O2) on MM expression of cadherins and CXCR4, as well on functional adhesion of MM cells to stromal cells and chemotaxis. RESULTS:Twelve mice with different stages of MM tumor progression were used. A bi-phasic correlation between tumor progression and the percent of hypoxic cells in BM was found, showing that severe hypoxic conditions in the BM correlated with tumor burden. The correlation between the tumor burden and the number of circulating cells was not linear; however, a direct linear correlation was observed between the number of circulating MM cells and hypoxia in the BM. Moreover, hypoxia in BM correlated directly with the expression of CXCR4 and negatively correlated with the expression of cadherins in MM cells isolated from the BM. To test the effect of the severe hypoxic conditions induced by tumor progression compared to mild hypoxic conditions found physiologically in the BM, we tested the effect of 0.5% O2 (severe hypoxia) and 6% O2 (mild hypoxia) compared to normoxia (21%) on MM cell adhesion to BMSCs, as well as on chemotaxis in response to SDF1, as well as expression of CXCR4 and cadherins. We found that severe hypoxic conditions decreased MM expression of cadherins and adhesion to BMSCs, as well as increased expression of CXCR4 and chemotaxis to SDF1 compared to cells in normoxia. In contrast, mild hypoxic conditions did not alter the expression of CXCR4 and cadherins, adhesion of MM cells to BMSCs, or chemotaxis of MM to SDF1 compared to normoxic cells. CONCLUSION:Hypoxia in the BM directly correlates with the number of circulating MM cells, and with changes in expression of cadherins and CXCR4 in vivo. Severe hypoxic conditions, but not mild hypoxic conditions, induce hypoxic responses in MM cells. Based on these findings, further studies to manipulate hypoxia in order to regulate tumor dissemination as a therapeutic strategy in MM are warranted. Disclosures:Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Part of the multiple myeloma-associated microvessels is functionally connected to the systemic circulation: a study in the murine 5T33MM model.

It is now well established that angiogenesis in multiple myeloma (MM) is associated with poor prognosis. The exact function of the newly formed vessels in MM is, however, a matter of debate. It is believed that, in contrast to solid tumor growth, the bone marrow (BM) is a sufficiently vascularized organ to support expansion of the MM clone with no need for additional blood vessels. From this point of view, it could be that MM-associated angiogenesis is rather an epiphenomenon and that the newly formed microvessels form a chaotic network that does not contribute to the blood flow. We investigated whether these newly formed microvessels in MM are connected to the blood circulation. The intravenously injected ferritin 30 min prior to sacrifice was detected in 100% of the BM vessels of control mice. In MM-bearing mice, the ferritin tracer was found in 31% of the MM-associated vessels, indicating a connection with the peripheral blood circulation in these vessels. We conclude that, comparable to the situation in solid tumors, at least part of the tumor-associated microvessels in MM is functionally connected to the blood circulation and, therefore, can participate in the transport of nutrients and in the dissemination of MM cells.