Rho-a and Rac-1 GTPases Play Major and Differential Roles in SDF1α- Induced Cell Adhesion and Chemotaxis in Multiple Myeloma.

Abstract Multiple Myeloma (MM) is characterized by disseminated involvement of the bone marrow (BM), implying that its progression involves a continuous circulation of cells into the peripheral blood and homing to the BM. Moreover, the interaction of MM cells with the BM microenvironment plays a crucial role in MM pathogenesis and drug resistance. Previous studies have shown that SDF-1a regulates migration and homing of MM cells in and out of the bone marrow. Rho-A and Rac-1 GTPases are known regulators of cell adhesion and migration in hematopoeitic stem cells. Their role in MM, however, has not been previously elucidated. In this study, we examined the role of Rho-A and Rac-1 GTPases in the regulation of adhesion and chemotactic responses of MM cells to SDF-1a. The effects of the Rac- 1 inhibitor NSC23766, ROCK (down stream target of RhoA) inhibitor Y-27632, and their combination on SDF-1a induced chemotaxis of MMcell lines (MM1S, OPM-2, RPMI 8226) and patient samples were tested by the transwell chemotaxis assay and confirmed by live confocal microscopy. While the ROCK inhibitor abolished the chemotactic effect of SDF-1a in all cell lines and patient samples, the Rac-1 inhibitor did not have a significant effect, and the combination of the two showed similar effect to that of the ROCK inhibitor alone. Testing the effect of the inhibitors on surface expression of CXCR4 by flow cytometry revealed that neither of the inhibitors altered the surface expression of CXCR-4. We next studied the effect of the two inhibitors on the adherence of MM cell lines and patient samples to BM stromal cells (BMSCs), and found that both of the inhibitors had a similar significant reduction in the adhesion of MM cells to BMSCs from MM patients, with no further effect of their combination. We then tested the expression of different adhesion molecules on the MM cells and found that VLA-4 was highly expressed, and LFA-1 minimally expressed, on MM cells. In accord with this, the adhesion of the MM cells to Fibronectin and VCAM was far higher than their adhesion to ICAM. None of the inhibitors altered the surface expression of VLA-4 or LFA. However both inhibitors reduced the adhesion of MM cells to VCAM and fibronectin, but not to ICAM. These results are in agreement with our in vivo homing studies which utilized in vivo flow cytometry and in vivo confocal microscopy to show that both of the inhibitors delayed the homing of MM cells to the BM in a similar manner, with no additive effect for their combination. Furthermore, we investigated the role or Rho-A and Rac-1 in the downstream signaling of SDF-1a/CXCR-4 pathway by immunoblotting as well as Rho and Rac kinase assays. We found that Pertussis Toxin abolished the stimulatory effect of SDF-1a on Rho-A and Rac-1 GTPase, indicating that they are both downstream of CXCR-4. Inhibitors of PI3K (LY294002), AKT (triciribine) and ROCK reduced the activity of Rac-1, indicating that Rac-1 is downstream of PI3K, AKT, Rho-A, and ROCK. However, the inhibitors of PI3K and AKT increased the activity of Rho-A, and inhibition of the Rho-A pathway by the ROCK inhibitor increased p-AKT. These results indicate that Rho-A has a parallel pathway to that of PI3K downstream of CXCR4. Inhibition of Rac-1 did not alter the activation of Rho-A. Inhibition of either ROCK or Rac-1 similarly decreased the SDF-1a induced activation of focal adhesion kinase (FAK) and cofilin, with no additive affect of the combination. While the inhibition of ROCK abolished the SDF1a-induced activation of myosin light chain (MLC), actin polymerization and fillopodia (detected by confocal microscopy), the inhibition of Rac-1 did not alter the activation of MLC, reduced actin polymerization, and had a minimal effect on fillopodia. In conclusion, our results show that Rac-1 GTPase and its downstream targets FAK, cofilin and actin polymerization are major regulators of SDF-1a induced adhesion of MM cells through VLA-4, while fillopodia and chemotaxis are controlled by Rho-A GTPase through its effect on MLC and actin polymerization. These results suggest Rho-A and Rac-1 are potential therapeutic targets for the disruption of MM cells interaction with the BM microenvironment. Figure Figure

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Evaluation Of Bcma As a Therapeutic Target In Multiple Myeloma Using An Antibody-Drug Conjugate

Blood ◽

10.1182/blood.v122.21.4447.4447 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4447-4447 ◽

Cited By ~ 5

Author(s):

Kwee L Yong ◽

Fiona M Germaschewski ◽

Manuel Rodriguez-Justo ◽

Danton Bounds ◽

Lydia Lee ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Confocal Microscopy ◽

Surface Expression ◽

Tumour Cells ◽

Time Dependent ◽

Antibody Drug Conjugate ◽

Drug Conjugate ◽

Antibody Drug

B-cell maturation antigen (BCMA) is upregulated at the terminal stages of plasma cell (PC) differentiation, and is expressed on normal and malignant PC. Apart from low levels of mRNA detected on dendritic cells, expression appears absent on other tissues, indicating the potential as a target for novel antibody therapeutics for multiple myeloma (MM). We generated a humanised anti-BCMA antibody, modified for Fc-enhanced function, and conjugated to mcMMAF (anti-BCMA antibody drug conjugate, ADC). Flow cytometric studies on human myeloma cell lines (HMCL) showed rapid internalisation of anti-BCMA antibody by flow cytometry and confocal microscopy. The internalised antibody was transported to the lysosome and was nearly undetectable by confocal microscopy after 6 hours, indicating the suitability of BCMA as a target for an antibody drug conjugate (ADC). BCMA expression reached original surface levels by 6 hours post antibody treatment, thus maintaining the cell as a target for effector mediated killing. Evaluation of BCMA expression on HMCL revealed variable surface expression (1/11 high, 5/11 moderate, 5/11 low). Tumour cell killing by the ADC was expression level, dose and time dependent. The highest expressing HMCL, H929, showed significant killing (60% at 100ng/mL) at 24 hours, and up to 90% after 2 days. Cells expressing moderate levels of BCMA required incubation for up to 4 days to show maximal levels of cell death, suggesting the importance of continued internalisation of the antibody/antigen complexes over this period. ARH77 cells were transduced to express varying levels of BCMA, and killing at 3 days (200ng/ml) was directly proportional to level of surface expression (NT 0% killing, Low BCMA 75% killing, High BCMA 90%). We studied surface antigen levels in a cohort of patients to ascertain the need for patient selection. Like the HMCL, patient CD138+ plasma cells (PC) displayed a range of expression. Of 67 patients tested, CD138+ PC from 12 expressed high levels, 52 expressed intermediate, and 3 had low/negative surface BCMA as determined by MFI ratio of specific antibody to isotype control. Non-CD138+ cells from the bone marrow (BM) were negative for BCMA. Immuno-histochemistry (IHC) on paraffin-embedded BM sections, using a murine antibody and dual staining with anti-Blimp1 to identify tumour cells, revealed both membrane and diffuse, or punctate, cytoplasmic staining. Expression levels varied, from high uniform, to heterogeneous and patchy, to uniform low level. In no patient were the tumour cells entirely negative for BCMA. There was broad correlation between FACS analysis and IHC, thus patients were divided into high, moderate and low expressing groups. Examination of patient and disease characteristics revealed no correlation between BCMA expression and disease stage, response to last treatment, time from diagnosis, isotype, CD56 expression, or cyclin D-type, but there was a trend towards higher BCMA levels in tumours with adverse genetics (90% of patients with adverse genetics had high/moderate levels cf 64% of patients with standard CGN (p=0.06, Fisher’s exact test, 2-tailed). CD138+ cells in LPL (n=3) were positive for BCMA, but CD20+ lymphocytes were negative. Serum BCMA levels in MM patients (175.6±242.6ng/mL; mean±SD, n=34) were higher than in normal subjects (9.28±1.9ng/mL; n=38) but no correlation with bone marrow plasmacytosis or surface BCMA was noted. Levels appeared similar between new diagnosis (147.6±190.8ng/mL; mean±SD, n=8) and relapsed disease (184.3±259.1ng/mL; n=26). We tested ADC activity on primary tumour cells in whole BM cultures, enumerating viable CD138+ cells by flow cytometry. As with the HMCL, ADC mediated cytotoxicity was expression level, dose and time dependent, with a slower time course than with HMCL, perhaps reflecting the slower turn-over of these cells. In samples expressing moderate levels of BCMA, ADC-mediated cytotoxicity increased from 23.1±2.9% (mean±SEM, n=6) at 1-2 days to 48.3±5.1% at 4 days, and 61.2±6.2% by 6-7 days. Optimal doses of ADC ranged from 500ng-1ug/ml. In summary, these preclinical data in MM support the potential utility of an anti-BCMA ADC across the whole MM population, perhaps with particular efficacy in patients with adverse genetics, for whom an unmet need remains. Disclosures: Yong: GSK: Research Funding. Germaschewski:GSK: Employment. Mayes:GlaxoSmithKline: Employment. Sully:GlaxoSmithKline: Employment. Seestaller-Wehr:GlaxoSmithKline: Employment. Fieles:GlaxoSmithKline: Employment. Tunstead:GlaxoSmithKline: Employment. McCahon:GlaxoSmithKline: Employment. Craigen:GlaxoSmithKline: Employment.

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CXCR7 Regulates SDF-1 Induced Adhesion and Homing in Multiple Myeloma.

Blood ◽

10.1182/blood.v112.11.1674.1674 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1674-1674 ◽

Cited By ~ 2

Author(s):

Nicholas Burwick ◽

Anne-Sophie Moreau ◽

Xiaoying Jia ◽

Xavier Leleu ◽

Judith Runnels ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Cell Adhesion ◽

Cell Lines ◽

Cxcr4 Expression ◽

Rt Pcr ◽

Growth And Survival ◽

Tumor Types

Abstract BACKGROUND: Multiple myeloma (MM) is a plasma cell malignancy that depends on interactions with the bone marrow (BM) microenvironment for growth and survival. In turn, adhesion of MM cells to the BM stroma provides a mechanism of resistance from standard chemotherapeutic agents. Recently, our lab has shown that by disrupting this adhesion using a selective CXCR4 inhibitor named AMD3100, MM cells are more sensitive to the proteasome inhibitor Bortezomib (Ghobrial lab, unpublished data). CXCR4 has been a particularly attractive target because its ligand SDF-1 is known to induce p42/44 MAPK, AKT, and the down-stream anti-apoptotic protein bad in MM cells, leading to increased MM growth and survival. Until recently, CXCR4 was thought to be a canonical receptor for the SDF-1 ligand. However, a second chemokine receptor for SDF-1 was subsequently discovered and named CXCR7. CXCR7 is a novel chemokine receptor that is important in cell adhesion, growth and survival in several tumor types. However, the role of CXCR7 in multiple myeloma (MM) has yet to be explored. Furthermore, the ability of SDF-1 ligand to regulate MM function via CXCR7 has not been studied. METHODS: The MM cell lines (U266, MM1.S, RPMI, OPM2, OPM1) were used. After informed consent was obtained, primary bone marrow samples from MM patients were collected. CD138 positive mononuclear cells were isolated by microbead selection. The expression of CXCR7 on MM cell lines and patient samples was confirmed using flow cytometry and RT-PCR analysis. For functional in vitro and ex-vivo assays, the CXCR7 selective antagonist 733 was used (ChemoCentryx Inc., Mountain View, CA). RESULTS: Here we show that CXCR7 was expressed on all tested MM cell lines and primary patient samples as demonstrated by flow cytometry and RT-PCR. Furthermore, CXCR7 was found to regulate SDF-1 induced MM cell adhesion, as demonstrated by in vitro assays using a small molecule compound specific for CXCR7 (733). The CXCR7 antagonist showed significant inhibition of adhesion of MM cell lines and patient samples to fibronectin, endothelial cells and stromal cells, with 50% reduction of adhesion at 5nM of the CXCR7 inhibitor, and with similar activity compared to 20uM of AMD3100 (CXCR4 inhibitor). However, unlike CXCR4, CXCR7 did not effect trans-well migration to SDF-1 chemokine. Interestingly, both receptors were found to be important for trans-endothelial migration of MM cells. Moreover, pre-treatment with 733 reduced homing of MM cells to the BM niche in vivo. Previous studies have failed to show signaling in response to CXCR7 in many tumor types. Here, we demonstrate that treatment with 733 inhibited SDF-1 induced pERK and pAKT, ribosomal pS6Kinase, pGSK3, pSTAT3, pFAK and pPAK signaling pathways, confirming a role for CXCR7 in facilitating SDF-1 signaling. This effect was further confirmed using immunofluorescence. To investigate whether CXCR7 and CXCR4 interact directly, we examined the effect of 733 and AMD3100 on CXCR4 expression and found that AMD3100 significantly inhibited CXCR4 expression, while 733 had no effect on CXCR4 expression, even in the presence of SDF-1. The CXCR7 inhibitor had no effect on the survival of MM cells using MTT and flow cytometry analysis, while high doses of 733 (1uM) had modest inhibition of proliferation. Interestingly, 733 prevented the growth advantage induced by 30nM SDF-1 at 24 hrs. CONCLUSION: Together, these results demonstrate the importance of CXCR7 in regulating MM adhesion and homing, and highlight the differential effects of CXCR4 and CXCR7 in regulating SDF-1 signaling in MM, thus providing a rationale for targeting the SDF-1/CXCR7 axis in MM.

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Role of Selectins in the Pathogenesis of Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.951.951 ◽

2009 ◽

Vol 114 (22) ◽

pp. 951-951 ◽

Cited By ~ 1

Author(s):

Abdel Kareem Azab ◽

Phong Quang ◽

Feda Azab ◽

Costas M Pitsillides ◽

John T Patton ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Endothelial Cells ◽

Cell Adhesion ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Advisory Committees

Abstract Abstract 951 INTRODUCTION: Multiple Myeloma (MM) is characterized by widespread disease at diagnosis with the presence of multiple lytic lesions and disseminated involvement of the bone marrow (BM), implying that the progression of MM involves a continuous re-circulation of the MM cells in the peripheral blood and re-entrance into the BM. Selectins are adhesion molecules expressed by activated endothelium of venules and leukocytes, and are involved in the primary interaction of lymphocytes with the endothelium of blood vessels. The binding of selectins serves as a biologic brake, making leukocyte quickly decelerate by rolling on endothelial cells, as the first step of extravasation. In this study, we have investigated the role of selectins and their ligands in the regulation of homing of MM Cells to the BM and the therapeutic implications of this role. METHODS AND RESULTS: We have used flow cytometry to characterize the expression of E, L and P-selectins and their ligands on MM cell lines, patient samples and on plasma cells from normal subjects. We found that all MM cell lines and patient samples showed high expression of L and P, but little of no E-selectin. While normal plasma cells showed low expression of all selectins and ligands.(give numbers) A pan-selectin inhibitor GMI-1070 (GlycoMimetics Inc., Gaithersburg, MD) inhibited the interaction of recombinant selectins with the selectin-ligands on the MM cells in a dose response manner. We have tested the role of the selectins and their ligands on the adhesion of MM cells to endothelial cells and found that MM cells adhered preferentially to endothelial cells expressing P-selectin compared to control endothelial cells and endothelial cells expressing E-selectin (p<0.05). Moreover, we found that blockade of P-selectin on endothelial cells reduced their interaction with MM cells (p<0.01), while blockade of E and L-selectin did not show any effect. Treating endothelial cells with GMI-1070 mimicked the effect of blocking P-selectin. Moreover, we found that treating endothelial cells with the chemokine stroma cell-derived factor-1-alpha (SDF1) increased their expression of P but not E or L-selectin detected by flow cytometry. Neither the blockade of each of the selectins and their ligands nor the GMI-1070 inhibited the trans-well chemotaxis of MM cells towards SDF1-alpha. However, blockade of P-selectin (p<0.001) on endothelial cells by GMI-1070 inhibited the trans-endothelial chemotaxis of MM cells towards SDF1-alpha. Both adhesion to endothelial cells and activation with recombinant P-selectin induced phosphorylation of cell adhesion related molecules including FAK, SRC, Cadherins, Cofilin, AKT and GSK3. GMI-1070 decreased the activation of cell adhesion molecules induced by both recombinant P-selectin and endothelial cells. Using in vivo flow cytometry we found that both anti P-selectin antibody and GMI-1070 prevented the extravasation of MM cells out of blood vessels into the bone marrow in mice. Moreover, we found that, in a co-culture system, endothelial cells protected MM cells from bortezomib induced apoptosis, an effect which was reversed by using GMI-1070, showing synergistic effect with bortezomib. CONCLUSION: In summary, we showed that P-selectin ligand is highly expressed in MM cells compared to normal plasma cells, and that it plays a major role in homing of MM cells to the BM, an effect which was inhibited by the pan-selectin inhibitor GMI-1070. This provides a basis for testing the effect of selectin inhibition on tumor initiation and tumor response to therapeutic agents such as bortezomib. Moreover, it provides a basis for future clinical trials for prevention of MM metastasis and increasing efficacy of existing therapies by using selectin inhibitors for the treatment of myeloma. Disclosures: Patton: GlycoMimetics, Inc: Employment. Smith:GlycoMimetics, Inc: Employment. Sarkar:GlycoMimetics, Inc: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Magnani:GlycoMimetics, Inc.: Employment. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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A Novel Mouse Model of Multiple Myeloma Representative of Human Disease and Its Use in Preclinical Therapeutic Assessment

Blood ◽

10.1182/blood.v118.21.2907.2907 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2907-2907

Author(s):

Rosemary A Fryer ◽

Timothy J Graham ◽

Emma M Smith ◽

Brian A Walker ◽

Gareth J Morgan ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Cell Lines ◽

Myeloma Cell ◽

Human Condition ◽

Myeloma Cells ◽

Treatment Groups ◽

Hyperintense Signal

Abstract Abstract 2907 In order to aid the pre-clinical development of novel therapeutics for multiple myeloma, an in vivo model which recapitulates the human condition in particular tumor growth patterns and response to treatment is required. An important feature of such a model is the interaction of the myeloma cells with the bone marrow microenvironment as this is known to modulate tumor activity and protect against drug-induced apoptosis. We have developed a model with myeloma restricted to the bone marrow, which proceeds rapidly from initial inoculation to disease progression, and possesses a range of chemo-sensitive markers with which to monitor anti-tumor response. Female NOD/SCID γcnull mice were injected inta-osseously with luciferase-tagged myeloma cell lines. Disease progression was monitored weekly by bioluminescent imaging (BLI) and measurement of paraprotein levels (ELISA). These methods were compared to histological assessment of tumor infiltration and MRI which provided a quantitative measurement of progression. On T2-weighted images tumor was identified as a hyperintense signal enclosed within cortical bone. Tumor burden was quantified from regions of interest drawn on the periphery of the hyperintense signal. Luciferase-tagged cells engrafted by 3 weeks at the injection site and progressed to the femurs, spine and pelvis from week 4. BLI showed a significant increase in radiance from 5.6×105 to 43.0×105p/s/cm2/sr between weeks 5 and 7 (p<0.05). Quantification of tumor volume by MRI showed a significant increase from 6.4mm3 to 27.6mm3 between weeks 4 and 8 (p<0.05) and μCT demonstrated lytic disease. Serum levels of Igλ increased from 860ng/ml to 4325ng/ml during this period (p<0.05), which mirrored the changes seen with BLI and MRI. Flow cytometry and histology confirmed the confinement of CD138 positive myeloma cells within the bone. These results indicate successful engraftment of human myeloma cell lines with induction of myeloma in a pattern similar to the human condition. We have adapted this model to study primary patient material. 10 mice were implanted with samples from 3 cases of plasma cell leukemia with complex cytogenetics. 5 of these developed myeloma confined to the bone marrow, 2 with additional plasmacytoma localized at the injection site, over a period of 1–5months. We have characterized the original patient cells with gene expression, SNP based gene mapping and have characterized the nature of the engrafted cells using similar technology. We have also shown the model is suitable for preclinical assessment of anti-myeloma agents using bortezomib and a novel aminopeptidase inhibitor, tosedostat (CHR-2797). Non-treated mice displayed a significant increase in radiance from 16.13×105 to 69.00×105p/s/cm2/sr (p<0.01). In comparison, in the bortezomib and tosedostat treated groups no significant increase in radiance was seen (bortezomib: 5.22×105 to 1.12×105 p/s/cm2/sr; tosedostat: 9.92×105 to 13.78×105p/s/cm2/sr). Paraprotein levels mimicked these changes in BLI. At the end of treatment Igλ levels in control, bortezomib and tosedostat treated mice were 2473.7, 132.5 and 923.0ng/ml, respectively. Igλ levels in both treatment groups were significantly different from control (p<0.001). Average tumor volumes derived from MRI were significantly different in bortezomib (14.7mm3) and tosedostat treated (23.4mm3) groups compared to non-treatment (33.0mm3). The volumes for the bortezomib treated group showed no significant difference from control mice. In addition, there was a decrease in CD138 expression by flow cytometry in bone aspirates from treatment groups compared to control which was mirrored in histological samples. In conclusion using both myeloma cell lines and primary patient cells, we have developed a model which recapitulates human myeloma with secretion of paraprotein, disease confined to the bone marrow, lytic bone lesions and spinal compression. In addition, this model is suitable for assessing the efficacy of novel therapeutics in vivo, using a number of non-invasive tumor markers such as BLI and MRI. Disclosures: Morgan: J&J: Honoraria, Speakers Bureau. Davies:J&J: Honoraria, Speakers Bureau.

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Modulation of CD81 By Epigenetic Drug Combination Sensitizes Acute Lymphoblastic Leukemia Via Decreased BTK Signaling

Blood ◽

10.1182/blood-2019-129198 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2628-2628

Author(s):

Anthony Quagliano ◽

Anilkumar Gopalakrishnapillai ◽

E. Anders Kolb ◽

Sonali Barwe

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Adhesion ◽

Drug Combination ◽

Fold Increase ◽

Surface Expression ◽

Cellular Adhesion ◽

P53 Expression ◽

Epigenetic Drug ◽

Pre Treatment

Background We previously discovered that the epigenetic drug combination - azacitidine (aza, DNMTi) and panobinostat (pano, HDACi) can sensitize ALL cells to chemotherapy (Quagliano et al., Leuk Res, 44:101, 2017). This sensitization occurred through a decrease in cellular adhesion and a modulation of the cell surface expression of the tetraspanin protein CD81 (Quagliano et al., Blood, 132:3957, 2018). This study aims to elucidate the molecular mechanism by which CD81 surface modulation by pre-treatment with aza/pano leads to chemosensitization. Methods CD81 knockout Nalm6 (CD81KO) cells were generated by CRISPR/Cas9 mutagenesis. IHC was performed using anti-human mitochondria antibody on femurs harvested from NSG-SGM3 mice at 72 h post transplantation with WT or CD81KO cells. For adhesion assay, ALL cells stained with VPD450 were co-cultured with Saos-2 cells for 24 h. Unbound cells were washed, and bound cells were collected for flow cytometry analysis. CD19 surface expression was determined after 48 h treatment with aza/pano (500 nM/1.5 nM) using flow cytometry. For western blot analysis, aza/pano treated cells were transferred on to Saos-2 monolayers and treated with Ara-C (30 nM) for 16 h. Results CD81KO cells not only had decreased homing and engraftment visualized by reduced ALL cell presence in bone marrow (Fig. 1A), but also had 45% reduction in cellular adhesion to osteoblasts compared to WT Nalm6 cells (Fig. 1B), indicating that CD81 downregulation results in reduced bone marrow interactions. To investigate the signaling effects downstream of aza/pano-mediated CD81 modulation, we first tested if CD19 was affected due to its prominent role in the formation of the B-cell co-receptor and because CD19 requires CD81 for proper membrane trafficking (Cherukuri et al., J Immunol, 172:370, 2004). CD19 surface expression was reduced in aza/pano-treated cells and completely knocked out in CD81KO cells (Fig. 1C). CD19 is known to prolong and amplify the activation of Bruton's tyrosine kinase (BTK), so we also analyzed the phosphorylation of BTK in aza/pano-treated cells and found it to be reduced by 63% (Fig. 1D). This reduction in BTK phosphorylation may be responsible for the decrease in cell adhesion following treatment with aza/pano because prior studies observed reduced adhesion following BTK inhibition (Herman et al., Clin Cancer Res, 21:4642, 2015). Taken together, these data suggest that aza/pano induced reduction in cell adhesion was mediated by downregulation of CD81 and BTK dephosphorylation. We studied how the expression of p53 and its target BCL2 associated X protein (BAX), which are known to be induced by Ara-C, were affected following pre-treatment with aza/pano. Cells with Ara-C treatment alone or the aza/pano pre-treatment both had minor induction of p53 expression compared to control, while aza/pano pre-treated cells that received Ara-C had further increase in p53 protein (1.7-fold increase compared to Ara-C alone, Fig. 1E). Bax mRNA and protein were increased 4.2-fold in aza/pano-treated cells following Ara-C treatment compared to Ara-C alone. Increased Bax expression following Ara-C treatment in aza/pano pre-treated cells was accompanied by a 3.9-fold increase in cleavage of caspase-3 compared to cells treated with Ara-C alone, which in turn causes cleavage of Poly-ADP Ribose Polymerase (PARP) (Fig. 1E). Both p53 and Bax protein levels were higher in CD81KO cells than in WT cells (Fig. 1E), suggesting that aza/pano-mediated reduction of CD81 is involved in inducing the expression of p53. CD81KO cells that were treated with Ara-C also had further increase in p53 and Bax expression compared to Ara-C treated WT cells (Fig. 1E), consistent with an increased sensitivity to chemotherapy. To confirm the role of BTK inhibition in the induction of p53, Nalm6 cells were treated with the BTK inhibitor fenebrutinib for 24 h (10 nM). Following treatment, expression of p53 was induced and Bax expression was 2.6-fold higher in treated cells compared to untreated cells (Fig. 1E). Taken together, these data suggest that modulation of the surface expression of CD81 and the phosphorylation of BTK by aza/pano induces sensitization via upregulation of p53 and overexpression of Bax. Conclusion We identify a novel mechanism by which aza/pano treatment induces chemosensitization by reducing cell adhesion via modulating CD81 surface expression, reducing BTK phosphorylation, and inducing p53. Figure 1 Disclosures No relevant conflicts of interest to declare.

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Altered Iron Metabolism Is a New Targetable Hallmark for Multiple Myeloma

Blood ◽

10.1182/blood-2019-123473 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3059-3059

Author(s):

Can Li ◽

Xuelian Tan ◽

Qierra Brockman ◽

Yogesh Jethava ◽

Marta Chesi ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Western Blot ◽

Plasma Cells ◽

Macrophage Polarization ◽

Mouse Bone Marrow ◽

Iron Dextran

Conventional therapies to multiple myeloma (MM) are not aimed at specific molecular targets leading ultimately to treatment resistance. Recent reports have shown that iron is instrumental in cancer development and progression and that high intracellular iron levels are associated with poor prognosis. We have demonstrated that MM cells exhibit dysregulated iron homeostasis and that limitation of cytosolic iron inhibits MM cell growth both in vitro and in vivo. The potential therapeutic role of iron should be further investigated to better understand how targeting high-iron MM cells could prevent or delay MM development and recurrence. Our study will provide crucial insights into the iron biology of MM pathogenesis and may lead to novel MM therapy. In this study, two mouse models, young Vk*MYC and old KaLwRij mice, were injected with iron dextran (1.25 mg/kg, IP, once a week). Tumor burden was monitored by serial Serum Protein Electrophoresis (SPEP) tests, flow cytometry, and immunohistochemistry. In vitro co-culturing of ARP1 MM cells with macrophages was employed to determine iron transfer. To determine iron's roles in MM evolution, we injected iron dextran into Vk*MYC mice at 8-week age. Vk*MYC mice develop MGUS around 40-50 weeks with plasma cell (PC) bone marrow infiltration and kidney damage etc. Iron-dextran was used because it is primarily taken up by macrophages. After 14-16 weeks of iron injection, M spike was detected in the injected Vk*MYC mice. The percentage of bone marrow plasma cells (CD138+) were significantly increased to 9% in the Vk*MYC mice injected with iron compared to control mice injected with vehicle by flow cytometry and immunohistochemistry. The acceleration of disease progression via iron injection was also tested in KaLwRij mice, which also spontaneously develops MGUS in old age. M protein was detected in 12 of 15 mice (80%) injected with iron dextran for 10 weeks and 1 of 5 KaLwRij (20%) control mice at 18-months of age. CD138+ B220- plasma cells were determined by flow cytometry. A significant increase of CD138+B220- plasma cells in iron treated mice (4% versus 2%) was observed compared to vehicle control mice. Deparaffined sections of bone marrow from the above mice were stained with Prussian blue and confirmed positive staining of macrophages from iron administrated mice. These results indicate that iron accelerates MGUS development in vivo. We next evaluated whether MM cells accumulate iron from the microenvironment. ARP1 MM cells were co-cultured with primary macrophages derived from mouse bone marrow to mimic disease environment in vitro. Under these conditions, MM cells induced macrophage polarization from M0 to M1 and M2. Furthermore, increased macrophage polarization was confirmed in vivo from the KaLwRij mice injected with 5TGM1 MM cells. To confirm that MM cells uptake iron from macrophages, increased intracellular ferritin levels were observed by western blot in ARP1 MM cells following co-culture with iron-loaded macrophages. We observed that this increase in intracellular ferritin was mediated via the transferrin receptor. This iron mobilization was prevented by iron chelation. Additionally, we confirmed that ferritin levels were higher in CD138+ primary human MM cells compared to CD138- non-MM cells by western blot. Our data indicate that MM cells promote macrophage polarization resulting in the transferring of iron into MM cells. The blockade of iron trafficking between MM cells and macrophages might hold a promise for the prevention and therapy in MM. Disclosures Bergsagel: Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Zhan:BIPHARM LLC: Consultancy, Other: % Allocation of Profit.

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Targeting S100A9 Interactions in the Multiple Myeloma Bone Marrow Environment Reduces Angiogenesis and Tumor Growth

Blood ◽

10.1182/blood.v128.22.3248.3248 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3248-3248

Author(s):

Kim De Veirman ◽

Els Van Valckenborgh ◽

Nathan De Beule ◽

Hendrik De Raeve ◽

Ken Maes ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Microvessel Density ◽

Calcium Binding ◽

Conflicts Of Interest ◽

Relative Reduction ◽

Immunosuppressive Activity ◽

Tumor Load

Abstract S100A9 belongs to a family of low-molecular-weight calcium-binding proteins and is involved in many biological processes including inflammation, cell migration and angiogenesis. It has been reported that S100A9 knockdown reduced myeloid-derived suppressor cell (MDSC) accumulation and Multiple Myeloma (MM) cell growth in MM models. MDSC are a heterogeneous population of immature myeloid cells and contribute to MM disease by immunosuppression, induction of angiogenesis and secretion of cytokines and growth factors. S100A9 is therefore proposed as an attractive drug target and compounds inhibiting the interaction of S100A9 with its receptors RAGE and TLR4 have been developed. In this study we evaluated the therapeutic relevance of S100A9 inhibition in MM using the preclinical immunocompetent murine 5T33MM model and two different small molecule inhibitors for S100A9 interactions (Active Biotech AB, Sweden). The presence of S100A9 was investigated in different cell populations. S100A9 is expressed in monocytic and granulocytic MDSC in murine and MM patient derived bone marrow (BM) samples, as demonstrated by western blot analysis and flow cytometry. S100A9 levels were higher in granulocytic MDSC compared to monocytic MDSC. Human MM cell lines and patient derived MM cells demonstrated low S100A9 expression, while no expression could be observed in 5T33MM cells. Treatment of 5T33MM mice with 30 mg/kg/day ABR-215757 (paquinimod) (quinoline-3-carboxamide analog) or ABR-238901 (N-(heteroaryl)-sulfonamide derivate) significantly diminished tumor cell percentages in the BM with ABR-238901 being the most potent one (27% relative reduction, p<0.01). Interestingly, ABR-238901 in combination with bortezomib (0.7 mg/kg subcutaneously, 2 times/week) resulted in a higher reduction in tumor load compared to single treatments (55% relative reduction compared to bortezomib alone). We investigated the effect on MDSC accumulation and immunosuppressive activity after ABR-238901 treatment in vivo. Flow cytometry analysis demonstrated no differences in MDSC and CD8+ IFNy+ cell numbers in ABR-238901 treated mice compared to vehicle mice. In addition, blocking S100A9 did not inhibit the immunosuppressive activity of MDSC. Interestingly, a significant reduction in IL6 and IL10 was observed by PCR in MDSC from ABR-238901 treated mice compared to vehicle. Both cytokines are produced by MDSC and are involved in MM cell proliferation and survival. To confirm the effects of S100A9 on cytokine expression, we treated 5T33MM derived MDSC with recombinant S100A9 in vitro and observed an increased expression of IL-6 and IL-10. In addition, transwell migration assays demonstrated that recombinant S100A9 has a chemoattractive effect on 5T33MM cells with a 3.5-fold induction of migration compared to control. Since S100A9 is also involved in angiogenesis, microvessel density was analyzed in the BM by CD31 staining. 5T33MM diseased mice treated with ABR-238901 had a significant decreased angiogenesis compared to 5T33MM mice treated with vehicle (p<0.01). We plotted for each mouse the microvessel density versus the percentage of tumor cells in the BM. Interestingly, the effect of ABR238901 on angiogenesis seems independent of tumor load. In conclusion, according to our study S100A9 is not involved in direct MDSC accumulation and activity; however it influences the expression of IL6 and IL10 by MDSC and act as a chemoattractant for MM cells. Interestingly, blocking S100A9 interactions in vivo affects angiogenesis and reduces tumor load especially in combination with bortezomib. Disclosures No relevant conflicts of interest to declare.

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The Effect of the CXCR4 Inhibitors, AMD3100 and AMD3465, in Multiple Myeloma Cell Homing and Its Interaction with the Bone Marrow Microenvironment.

Blood ◽

10.1182/blood.v114.22.1826.1826 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1826-1826

Author(s):

Judith M. Runnels ◽

Abdel Kareem Azab ◽

Costas Pitsillides ◽

Jonathan Farag ◽

Juwell Wu ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Combined Treatment ◽

Xenograft Model ◽

Antibody Binding ◽

Disease Spread ◽

Cell Trafficking ◽

In Vivo Flow Cytometry

Abstract Abstract 1826 Poster Board I-852 Patients with multiple myeloma (MM) typically present with the disease spread diffusely throughout the bone marrow (BM). This fact points to the role MM cell trafficking plays in disease progression. Like normal plasma cells, MM cell trafficking is directed by the cytokine, SDF-1 and its receptor, CXCR4. Using the small bicyclam molecule, AMD3100, to block the SDF-1/CXCR4 interaction, and in vivo flow cytometry to measure circulation time of MM cells in a xenograft model, we have previously shown that homing of injected AMD3100-treated MM cells to the bone marrow is perturbed. Soon after injection fewer AMD3100-treated than untreated MM cells are detected in mouse skull BM using in vivo fluorescence confocal microscopy. Furthermore, the combined treatment of established tumors with AMD3100 and bortezomib enhanced survival. Here we present data that the 2nd generation SDF-1/CXCR4 small monomacrocyclic inhibitor, AMD3465 behaves like its predecessor in blocking antibody binding to CXCR4 on MM cells, migration of MM cells toward SDF-1, and in vivo homing, but at concentrations 10-50 fold lower than AMD3100. Incubation with either 1uM AMD3465 or 50uM AMD3100 reduced antibody binding to CXCR4 on MM cells to isotype control levels. Likewise, treatment with AMD3465 reduced MM1S migration to 15% of that of untreated cells in a transwell migration assay. These in vitro effects translated in vivo into longer circulation times for AMD3465-treated MM cells than control cells. In vivo flow cytometry revealed that 40-50% of AMD3465-pre-treated cells remained in the circulation one hr after injection whereas untreated cells depleted to 20% of their original circulating cell count by that time. Future experiments will further describe the effect of AMD 3465 on MM cells in the BM microenvironment to answer whether AMD3465 can be used to mobilize MM cells from the BM environment or enhance survival when used in conjunction with therapeutic drugs. Disclosures Ghobrial: Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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Potent in vitro and in vivo activity of an Fc-engineered humanized anti-HM1.24 antibody against multiple myeloma via augmented effector function

Blood ◽

10.1182/blood-2011-06-364521 ◽

2012 ◽

Vol 119 (9) ◽

pp. 2074-2082 ◽

Cited By ~ 33

Author(s):

Yu-Tzu Tai ◽

Holly M. Horton ◽

Sun-Young Kong ◽

Erik Pong ◽

Hsing Chen ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cells ◽

Surface Expression ◽

Cell Surface Expression ◽

Immune Effector ◽

Bone Marrow Plasma ◽

Therapeutic Monoclonal Antibodies

Abstract HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B–dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.

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A Novel CD49d Targeting Antisense, ATL1102, Effectively Mobilizes Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood.v126.23.3807.3807 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3807-3807

Author(s):

Yann Duchartre ◽

EunJi Gang ◽

Hye Na Kim ◽

Stephanie Nicole Shishido ◽

Muller Fabbri ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Cell Line ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Surface Expression ◽

Leukemia Cells ◽

Acute Myeloid

Abstract BACKGROUND: Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Acute myeloid leukemia comprises approximately one-fifth of pediatric leukemias and is the seventh most common pediatric malignancy. In children, relapse following primary therapy approaches 40%, and the 5-year event-free survival (EFS) rate is only approximately 50%. Treatment is dominated by generic chemotherapeutic agents. Novel therapeutic strategies are highly warranted. The bone marrow microenvironment has been shown to promote cell adhesion-mediated drug resistance in leukemia cells. Breaking adhesive bonds of AML cells with their protective niche to mobilize them from the bone marrow to the peripheral blood may make drug treatment more efficient. Our studies have suggested the adhesion molecule CD49d as an anchor molecule for ALL and AML cells in the bone marrow. However, as of today, no drug targeting CD49d is approved for use in leukemia. Here, we evaluate a novel human specific CD49d targeting antisense, ATL1102, in clinical development for Multiple Sclerosis, in human AML cells. METHODS: We determined CD49d expression in the human AML cell line HL-60 treated with a CD49d targeting antisense ATL1102 and antisense control by qPCR and flow cytometry. Annexin V/DAPI and BrdU stainings were used for viability determination and cell cycle assay respectively by flow cytometry. A NOD/SCID IL2Rγ-/- (NSG)xenograft model of human HL-60 cell line was used for an in vivo mobilization assay. RESULTS: To assess the on-target effect of ATL1102 on CD49d, HL-60 cells were nucleoporated with either ATL1102 or control antisense.mRNA expression of CD49dwas significantly decreased by ATL1102 treatment cells (85.2%±15.4 expression inhibition using ATL1102 1µM after 24h compared to control, p<0.001) as assessed by RT-PCR. The FACS analysis 72 hours after treatment revealed a significant decrease of surface expression of CD49d in a dose-dependent manner (99%±0.4 (1µM, *), 87.9%±8.7 (3µM) and 57.8%±7.2 ATL1102 (10µM, ***), 55.9±13.5 (30µM, **) vs 99.7%±0.1 for control antisense (30 µM), P<0.001, n=3). No significant effect on apoptosis or cell cycle was observed after ATL1102 treatment. We also evaluated the in vivo effect of ATL-1102 on mobilization of leukemia cells in a pilot experiment. For this purpose, HL-60 cells (5x106/per mouse) were injected via the tail vein in sublethally irradiated NSG mice. Presence of human ALL cells (hCD45) was determined weekly by flow cytometry of white blood cells isolated from peripheral blood (PB). 23 Days post-leukemia injection, mice were treated with either antisense control (CTRL) (n=3), ATL1102 (50mg/kg, n=2). Peripheral blood was drawn before and 24 hours after ATL1102-treatment. ATL1102 induced a strong mobilization of AML cells to the PB of leukemia-recipient mice compared to control antisense treated-mice (69.1% and 87.7% vs 1.1%, 0.2% and 28.1% for ATL1102 (50mg/ml) and CTRL treated-mice respectively. The mobilized cells show a decrease of surface expression of CD49d (16.8%±9.2% vs 32.8%±16.7%), although this was not of statistical significance in this pilot experiment. Experiments to repeat this assay with large numbers of mice are in progress as well as experiments to determine the initial location of the mobilized AML cells and synergy of ATL1102 with chemotherapy are ongoing. CONCLUSION: We demonstrate that ATL1102 can efficiently decrease CD49d expression in AML cell line in vitro and in vivo, and that ATL1102 leads to mobilization of AML cells to the peripheral blood. Disclosures Wayne: NIH: Patents & Royalties; Medimmune: Honoraria, Other: travel support, Research Funding; Kite Pharma: Honoraria, Other: travel support; Pfizer: Honoraria; Spectrum Pharmaceuticals: Honoraria, Other: travel support, Research Funding. Tachas:Antisense Therapeutics Ltd: Employment, Equity Ownership, Patents & Royalties.

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