In Vivo Targeting of Stromal-Derived Factor-1 As a Strategy to Prevent Myeloma Cell Dissemination to Distant Bone Marrow Niches

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 440-440
Author(s):  
Aldo M. Roccaro ◽  
Antonio Sacco ◽  
Marco Ungari ◽  
Patricia Maiso ◽  
Salomon Manier ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Ex Vivo ◽  
Tumor Burden ◽  
Advisory Board ◽  
In Vivo Confocal Microscopy ◽  
Healthy Individuals ◽  
Cell Homing ◽  
Cell Dissemination

Abstract Abstract 440 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 (SDF-1) may represent a target for preventing transition from MGUS (micrometastatic stage) to active-MM (macrometastatic stage); thus resulting in inhibition of MM progression. We therefore evaluated SDF-1 expression in the BM of patients with MGUS, MM, compared to healthy individuals; and tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1 in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vitro and in vivo . Methods. SDF-1 levels were evaluated by immunohistochemistry on BM specimens obtained from patients with MGUS, active-MM, or healthy individuals; and confirmed by ELISA, using conditioned-medium of BM-mesenchymal stromal cells obtained from MGUS, active-MM and healthy individuals. BM metastatic lesions from primary epithelial tumors were also considered. Co-localization of MM tumor cells (MM.1S-GFP+) with SDF-1 was tested in vivo by in vivo confocal microscopy, using both AlexaFluor633-conjugated-anti-SDF-1 monoclonal antibody and AlexaFluor647-conjugated-NOX-A12 oligonucleotide. Effect of NOX-A12 on modulating MM cell dissemination was tested in vivo, by using in vivo confocal microscopy. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12+bortezomib. Detection of mobilized MM-GFP+ cells ex vivo was performed by flow cytometry. Effects of drug combination on dissemination of MM cells to distant BM niches was evaluated ex vivo by immunofluorescence on femurs obtained from each cohort of mice. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–100nM) treated primary MM BM stromal cells (BMSCs) in presence or absence of bortezomib (2.5–5nM) were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software. NOX-A12-dependent-modulation of signaling was evaluated by western blot on MM cells exposed or not to primary BM-MSCs. Results. Patients with active-MM present with higher BM SDF-1 expression vs. MGUS patients and healthy individuals. Similarly, BM presenting with metastasis from epithelial primary malignancies had higher SDF-1 levels compared to healthy subjects, thus suggesting the importance of SDF-1 in favoring tumor cell metastasis to BM niches. SDF-1 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. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the peripheral blood of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). This was supported by inhibited homing of MM cells to the BM of those mice pre-treated with NOX-A12. We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 and bortezomib was observed (C.I.: .57-.76). These findings were validated in vivo: tumor burden was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly, significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 and bortezomib was observed, compared to bortezomib alone-treated mice (P <.05). Similarly, NOX-A12+bortezomib combination induced significant inhibition of MM cell homing, as shown by in vivo confocal microscopy. Conclusion. SDF-1 represents a valid target for inhibiting MM cell dissemination to distant BM niches, thus providing the evidence for using the SDF-1 inhibiting Spiegelmer NOX-A12 to target MM cells at the stage of micrometastasis (MGUS), thus preventing development of symptomatic macrometastatic MM. Disclosures: Zboralski: NOXXON Pharma AG, Berlin, Germany: Employment. Kruschinski:NOXXON Pharma AG, Berlin, Germany: 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.

L-Stereoisomer RNA Oligonucleotide Anti-SDF-1 (Nox-A12) Disrupts the Interaction of Multiple Myeloma Cells with the Bone Marrow Milieu In Vivo, Leading to Enhanced Sensitivity to Bortezomib

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 887-887
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Phong Quang ◽  
AbdelKareem Azab ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Confocal Microscopy ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
In Vivo Confocal Microscopy ◽  
Advisory Committees

Abstract Abstract 887 Background. Stomal-cell-derived factor 1 (SDF-1) is known to be involved in bone marrow (BM) engrafment for malignant tumor cells, including CXCR4 expressing multiple myeloma (MM) cells. We hypothesized that de-adhesion of MM cells from the surrounding BM milieu through SDF-1 inhibition will enhance MM sensitivity to therapeutic agents. We therefore tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vivo and in vitro. Methods. Bone marrow (BM) co-localization of MM tumor cells with SDF-1 expressing BM niches has been tested in vivo by using immunoimaging and in vivo confocal microscopy. MM.1S/GFP+ cells and AlexaFluor633-conjugated anti-SDF-1 monoclonal antibody were used. Detection of mobilized MM-GFP+ cells ex vivo has been performed by flow cytometry. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence detection, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12 followed by bortezomib. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–200nM) treated primary MM BM stromal cells (BMSCs), in presence or absence of bortezomib (2.5–5nM), were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software (combination index: C.I. according to Chou-Talalay method). Results. We first showed that SDF-1 co-localizes in the same bone marrow niches of growth of MM tumor cells in vivo. NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BM stromal cells in vitro. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the PB of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 (50–100 nM) and bortezomib (2.5–5 nM) was observed (C.I.: all between 0.57 and 0.76). These findings were validated in vivo: tumor burden detected by BLI was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 followed by bortezomib was observed as compared to bortezomib alone treated mice (P <.05). Similarly, NOX-A12 + bortezomib combination induced significant inhibition of MM cell homing in vivo, as shown by in vivo confocal microscopy, as compared to bortezomib used as single agent. Conclusion. Our data demonstrate that the SDF-1 inhibiting Spiegelmer NOX-A12 disrupts the interaction of MM cells with the BM milieu both in vitro and in vivo, thus resulting in enhanced sensitivity to bortezomib. Disclosures: Roccaro: Roche:. Kruschinski:Noxxon Pharma AG: Employment. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Advisory Board, Research Funding.

scholarly journals In Vivo Confocal Microscopy Evaluation in Patients with Keratoconus

2022 ◽  
Vol 11 (2) ◽  
pp. 393
Author(s):  
Alvin Wei Jun Teo ◽  
Hassan Mansoor ◽  
Nigel Sim ◽  
Molly Tzu-Yu Lin ◽  
Yu-Chi Liu
Keyword(s):  
Confocal Microscopy ◽  
Nerve Regeneration ◽  
Nerve Fibre ◽  
Ex Vivo ◽  
Fibre Length ◽  
In Vivo Confocal Microscopy ◽  
Corneal Sensitivity ◽  
Mechanical Removal ◽  
Cellular Changes

Keratoconus is the most common primary corneal ectasia characterized by progressive focal thinning. Patients experience increased irregular astigmatism, decreased visual acuity and corneal sensitivity. Corneal collagen crosslinking (CXL), a minimally invasive procedure, is effective in halting disease progression. Historically, keratoconus research was confined to ex vivo settings. In vivo confocal microscopy (IVCM) has been used to examine the corneal microstructure clinically. In this review, we discuss keratoconus cellular changes evaluated by IVCM before and after CXL. Cellular changes before CXL include decreased keratocyte and nerve densities, disorganized subbasal nerves with thickening, increased nerve tortuosity and shortened nerve fibre length. Repopulation of keratocytes occurs up to 1 year post procedure. IVCM also correlates corneal nerve status to functional corneal sensitivity. Immediately after CXL, there is reduced nerve density and keratocyte absence due to mechanical removal of the epithelium and CXL effect. Nerve regeneration begins after 1 month, with nerve fibre densities recovering to pre-operative levels between 6 months to 1 year and remains stable up to 5 years. Nerves remain tortuous and nerve densities are reduced. Corneal sensitivity is reduced immediately postoperatively but recovers with nerve regeneration. Our article provides comprehensive review on the use of IVCM imaging in keratoconus patients.

scholarly journals Systemic Delivery of mLIGHT-Armed Myxoma Virus Is Therapeutic for Later-Stage Syngeneic Murine Lung Metastatic Osteosarcoma

Cancers ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 337
Author(s):  
John D. Christie ◽  
Nicole Appel ◽  
Liqiang Zhang ◽  
Kenneth Lowe ◽  
Jacquelyn Kilbourne ◽  
...  
Keyword(s):  
Lung Metastases ◽  
Ex Vivo ◽  
Tumor Burden ◽  
Myxoma Virus ◽  
Oncolytic Viruses ◽  
Systemic Delivery ◽  
Clinical Cancer Research ◽  
Critical Issues

Cancers that metastasize to the lungs represent a major challenge in both basic and clinical cancer research. Oncolytic viruses are newly emerging options but successful delivery and choice of appropriate therapeutic armings are two critical issues. Using an immunocompetent murine K7M2-luc lung metastases model, the efficacy of MYXV armed with murine LIGHT (TNFSF14/CD258) expressed under virus-specific early/late promoter was tested in an advanced later-stage disease K7M2-luc model. Results in this model show that mLIGHT-armed MYXV, delivered systemically using ex vivo pre-loaded PBMCs as carrier cells, reduced tumor burden and increased median survival time. In vitro, when comparing direct infection of K7M2-luc cancer cells with free MYXV vs. PBMC-loaded virus, vMyx-mLIGHT/PBMCs also demonstrated greater cytotoxic capacity against the K7M2 cancer cell targets. In vivo, systemically delivered vMyx-mLIGHT/PBMCs increased viral reporter transgene expression levels both in the periphery and in lung tumors compared to unarmed MYXV, in a tumor- and transgene-dependent fashion. We conclude that vMyx-mLIGHT, especially when delivered using PBMC carrier cells, represents a new potential therapeutic strategy for solid cancers that metastasize to the lung.

scholarly journals MINDIN secretion by prostate tumors induces premetastatic changes in bone via β-catenin

2020 ◽  
Vol 27 (7) ◽  
pp. 441-456
Author(s):  
Juan A Ardura ◽  
Luis Álvarez-Carrión ◽  
Irene Gutiérrez-Rojas ◽  
Peter A Friedman ◽  
Arancha R Gortázar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Tumor Cell ◽  
Ex Vivo ◽  
Prostate Tumor ◽  
Tumor Cell Adhesion ◽  
Prostate Tumors ◽  
Cell Homing

Bone metastases are common in advanced prostate cancer patients, but mechanisms by which specific pro-metastatic skeletal niches are formed before tumor cell homing are unclear. We aimed to analyze the effects of proteins secreted by primary prostate tumors on the bone microenvironment before the settlement and propagation of metastases. Here, using an in vivo pre-metastatic prostate cancer model based on the implantation of prostate adenocarcinoma TRAMP-C1 cells in immunocompetent C57BL/6 mice, we identify MINDIN as a prostate tumor secreted protein that induces bone microstructural and bone remodeling gene expression changes before tumor cell homing. Associated with these changes, increased tumor cell adhesion to the endosteum ex vivo and to osteoblasts in vitro was observed. Furthermore, MINDIN promoted osteoblast proliferation and mineralization and monocyte expression of osteoclast markers. β-catenin signaling pathway revealed to mediate MINDIN actions on osteoblast gene expression but failed to affect MINDIN-induced adhesion to prostate tumor cells or monocyte differentiation to osteoclasts. Our study evidences that MINDIN secretion by primary prostate tumors creates a favorable bone environment for tumor cell homing before metastatic spread.

scholarly journals ATR addiction in multiple myeloma: synthetic lethal approaches exploiting established therapies

Haematologica ◽  
2019 ◽  
Vol 105 (10) ◽  
pp. 2440-2447 ◽  
Author(s):  
Oronza A. Botrugno ◽  
Silvia Bianchessi ◽  
Desirée Zambroni ◽  
Michela Frenquelli ◽  
Daniela Belloni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Synthetic Lethality ◽  
Ex Vivo ◽  
Myeloma Cell ◽  
Tumor Burden ◽  
In Vivo Models ◽  
Cancer Multiple

Therapeutic strategies designed to tinker with cancer cell DNA damage response have led to the widespread use of PARP inhibitors for BRCA1/2-mutated cancers. In the haematological cancer multiple myeloma, we sought to identify analogous synthetic lethality mechanisms that could be leveraged upon established cancer treatments. The combination of ATR inhibition using the compound VX-970 with a drug eliciting interstrand cross-links, melphalan, was tested in in vitro, ex vivo, and most notably in vivo models. Cell proliferation, induction of apoptosis, tumor growth and animal survival were assessed. The combination of ATM inhibition with a drug triggering double strand breaks, doxorucibin, was also probed. We found that ATR inhibition is strongly synergistic with melphalan, even in resistant cells. The combination was dramatically effective in targeting myeloma primary patient cells and cell lines reducing cell proliferation and inducing apoptosis. The combination therapy significantly reduced tumor burden and prolonged survival in animal models. Conversely, ATM inhibition only marginally impacted on myeloma cell survival, even in combination with doxorucibin at high doses. These results indicate that myeloma cells extensively rely on ATR, but not on ATM, for DNA repair. Our findings posit that adding an ATR inhibitor such as VX-970 to established therapeutic regimens may provide a remarkably broad benefit to myeloma patients.

Stroma-Derived Exosomes Mediate Oncogenesis in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 625-625 ◽  
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Abdel Kareem Azab ◽  
Yu-Tzu Tai ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Confocal Microscopy ◽  
Tumor Growth ◽  
Cell Lines ◽  
Advisory Board ◽  
Immunogold Labeling ◽  
Qrt Pcr ◽  
Mirna Expression Profiling

Abstract Abstract 625 Background. Bone marrow (BM)-derived mesenchymal stem cells (MSCs) support multiple myeloma (MM) cell growth, but little is know about the putative mechanisms that may regulate the interaction between clonal MM plasma cells and the surrounding BM milieu. It is known that cell-to-cell communication is partially mediated by exosomes. We therefore characterized the role BM-MSCs-derived exosomes as key regulators of MM pathogenesis in vivo and in vitro. Methods. MSCs were collected from BM of either healthy subjects or relapsed/refractory MM patients. MM cell lines (MM.1S; RPMI.8226) and normal BM stromal cell line (HS-5) were used. Purity of BM-MSCs was evaluated by flow cytometry (CD34−,14−, 45−, 19−, 138−; CD73+, 90+, 105+, 106+). Exosomes were collected from conditioned medium of either normal and MM BM-MSCs, or HS-5 cells; and studied using electron microscopy, immunogold labeling, and western blot for the detection of CD63 and CD81. Transfer of PKH67-fluorescently labeled exosomes to MM cells was evaluated by confocal microscopy and fluorescence plate reader. Transfer of murine-derived miRNA-containing exosomes into human MM cell lines 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 has been tested in vivo by using in vivo confocal microscopy and bioluminescence (BLI) imaging, respectively. Normal and MM BM-MSCs, as well as HS-5 cells, were transfected with either anti- or pre-miRNA-15a or scramble probe; and evaluated for their ability to modulate MM cell proliferation and adhesion in vitro. Results. Primary normal and MM BM-MSCs release 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. This transfer was further confirmed in human MM cell lines incubated with murine (C57BL/6 miRNA-15a/16-1−/− and wild type) BM-MSCs-derived exosomes: qRT-PCR showed presence of murine miRNAs in human MM cell lines. The impact of normal and MM BM-MSCs-derived exosomes on MM cell behavior in vivo was next evaluated. MM cells co-cultured with exosomes derived from MM BM-MSCs induced rapid tumor growth at the site of the TEB scaffold, as well as rapid dissemination in the BM niches. In contrast, MM cells co-cultured with exosomes derived from normal BM-MSCs led to minimal tumor growth and minimal dissemination at distant BM niches. These results indicate that MM BM-MSCs-derived exosomes contribute to tumor growth and dissemination of MM. To further explore the mechanisms by which exosomes induce tumor growth, we performed miRNA expression profiling on exosomes isolated from both normal and MM BM-MSCs: supervised hierarchical clustering analysis showed increased expression of 24 miRNAs and reduced expression of 3 miRNAs in MM BM-MSCs-derived exosomes versus normal (1.5 fold change; P<0.05). Notably, we found that miRNA15a is significantly lower in exosomes derived from BM-MSCs of MM patients. We previously showed that miRNA15a shows lower expression in primary MM cells. We therefore sought to examine whether genetic transfer of miRNAs or lack of transfer of tumor suppressor miRNAs (such as miRNA15a) can lead to the significant change in tumor growth and dissemination in MM that we observed in vivo. We therefore transfected HS-5 stromal cells and primary normal BM-MSCs with pre-miRNA15a, and found that by over-expressing miRNA-15a in BM-MSCs 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−/: miRNA15a-deficient BM-MSCs significantly induced MM cell proliferation (P<0.05). Moreover, exosomes isolated from HS-5 pre-miRNA15a-transfected cells both inhibited MM cell proliferation and reduced their adhesion properties. Conclusions. These findings demonstrate the existence of exosome-driven interactions between the BM milieu and MM cells, and suggest that exosomes might constitute a novel mechanism for intercellular transfer of genetic information in the form of miRNAs in clonal plasma cell disorders, such as MM. Disclosures: Roccaro: Roche: Advisory Board. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Ghobrial:Novartis: Advisory Board; Celgene: Advisory Board; Millennium: Advisory Board; Noxxon: Advisory board; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4009-4009
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Michelle R. Kuhne ◽  
Abdel Kareem Azab ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Advisory Board ◽  
Dependent Manner ◽  
In Vivo Confocal Microscopy ◽  
Transfected Cells ◽  
Cell Dissemination ◽  
Dose Dependent ◽  
Bone Marrow Niches

Abstract Abstract 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.

A Novel Activating Mutation Of CXCR4 Plays a Crucial Role In Waldenstrom Macroglobulinemia Biology

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 272-272 ◽  
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Cristina Jimenez ◽  
Patricia Maiso ◽  
Michele Moschetta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Tumor Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
Advisory Board ◽  
Beta Catenin ◽  
Activating Mutation

Abstract Background The C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in modulating the biology of B-cell lymphoproliferative disorders. Recent whole genome sequencing studies have identified unique CXCR4 mutations in 29% of the 55 evaluated patients with Waldenstrom Macroglobulinemia (WM). In this study, we sought to better define the mutation status of CXCR4 in B-cell malignancies and define the functional role of this mutation in the progression of WM in vivo. Methods Allele-specific(AS) PCR has been performed on bone marrow (BM)-derived tumor cells of patients with WM (n: 131); IgM monoclonal gammopathy of undetermined significance (MGUS; n: 40); as well as in patients with diffuse large cell lymphomas (DLBCL; n: 75), splenic marginal zone lymphoma (SMZL; n: 14), B-chronic lymphocytic leukemia (B-CLL; n: 37), hairy cell leukemia (HCL; n: 35), multiple myeloma (MM; n: 36), IgA/IgG MGUS (n: 22), lymphoplasmacytic lymphoma without WM criteria (n: 13), and amyloidosis (n: 6). CXCR4-loss and -gain of function studies have been performed on WM cells stably expressing either shRNA-CXCR4, CXCR4-ORF-GFP-tagged or scramble-RFP-tagged (generated via lentivirus-based infection). A mutagenesis kit has been used to generate the C1013GCXCR4 mutant protein (C1013GCXCR4) in WM cells, via lentivirus-based infection. CXCR4-knock-in or C1013GCXCR4-mutated cells and the corresponding controls have been injected i.v. into SCID/Bg mice and tumor dissemination has been evaluated ex vivo by immunohistochemistry IHC (human-CD20; -CXCR4). C1013GCXCR4-mutated cells have been characterized at mRNA levels (U133 plus2) using GSEA. A novel human anti-CXCR4 mAb (BMS-936564/MDX-1338; Bristol Myers Squibb, NY) has been tested in vitro (cell proliferation, MTT, adhesion and migration to primary WM BM mesenchymal stromal cells) and in vivo (10mg/kg i.p. x3-4/week). Tumor growth has been evaluated by IHC ex vivo (hCD20; hCXCR4) and by immunofluorescence. Results We examined the mutational status of C1013GCXCR4 and confirmed the presence of this specific mutation in 28% of the 131 cases evaluated. The mutation was also detected at the stage of IgM-MGUS (20%); while it was present in a minority of patients with DLBCL (1%) and SMZL (7%). Remarkably, it was absent in all MM (n=36) and IgA/IgG MGUS patients (n=22), and it was not detected in healthy subjects (n=32). The functional relevance of the C1013G-CXCR4 variant was next examined in vivo. Mice injected with C1013GCXCR4-cells presented with a significant dissemination of tumor cells, demonstrating involvement of liver, bone marrow, lymph nodes, kidney and lung. IHC showed the presence of CXCR4+ and CD20+ cells in all the tissues examined; and quantification of CXCR4 and CD20 positivity was higher in C1013GCXCR4-cells-, compared to parental(p)-WM cell-injected mice (NIS Elements software, Nikon, Melville, NY; P<0.05). In addition, C1013GCXCR4-cells were further characterized in vitro, showing increased adhesion and cell proliferation in the presence of primary WM BM-MSCs. These findings were also confirmed using CXCR4-overexpressing cells. In contrast CXCR4-knock-down cells presented the opposite behavior, where reduced adhesion and proliferation in presence of primary WM BM-MSCs were observed. By performing GSEA we demonstrated that genes related to invasiveness, cell proliferation, anti-apoptosis, and oncogenesis were all enriched in C1013GCXCR4-cells compared to the parental-WM cells. These findings let us hypothesize that C1013GCXCR4 may act as an activating mutation in WM cells. Indeed, in a different mouse model, CXCR4 over-expressing cells and scramble infected cells were injected into mice, showing similar phenotype to the one observed upon C1013GCXCR4-WM cell-injected-mice. Finally, the novel antibody BMS-936564/MDX-1338 exerted anti-WM activity both in vitro and in vivo, with anti-tumor effects observed also against the mutated variant. This was supported by inhibition of pro-survival pathways (p-ERK; pAKT); induction of pro-apototic proteins (cleaved-PARP and -caspase-9); up-regulation of p-GSK3beta, p-beta catenin and relative beta catenin degradation. Conclusion These findings demonstrate that C1013GCXCR4 acts as an activating mutation in WM; and it is targetable by using MDX936564/1338 thus providing the basis for translating these observations into clinical trials for WM patients. Disclosures: Kuhne: BMS: Employment. Cardarelli:BMS: Employment. Ghobrial:BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisoryboard Other; Noxxon: Research Funding; Sanofi: Research Funding.

scholarly journals Dissecting the Mechanisms of Activity of SLAMF7 and the Targeting Antibody Elotuzumab in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3431-3431 ◽  
Author(s):  
Siobhan Glavey ◽  
Michaela Reagan ◽  
Salomon Manier ◽  
Michele Moschetta ◽  
Yawara Kawano ◽  
...  
Keyword(s):  
Research Funding ◽  
Xenograft Model ◽  
Myeloma Cell ◽  
Tumor Burden ◽  
Advisory Board ◽  
Xenograft Models ◽  
Human Igg1

Abstract Background Signaling lymphocytic activation molecule family 7 (SLAMF7) is a cell surface receptor which is expressed at high levels in multiple myeloma (MM) cells and serves as a therapeutic target in this disease. Elotuzumab, a humanized monoclonal antibody targeting SLAMF7, has shown promising activity, particularly in combination with lenalidomide, in MM patients in the clinical trial setting. While the primary mechanism of action of elotuzumab in pre-clinical in vitro studies has been shown to be antibody-dependent cellular cytotoxicity (ADCC) mediated via NK-cell CD16, other possible mechanisms of activity of elotuzumab in MM are not well defined. Specifically, the downstream signaling effects of SLAMF7 have not been delineated in myeloma cells. We set out to examine the effect of stable SLAMF7 knockdown (KD) on MM tumor development in vivo and evaluated the ability of elotuzumab to exert an anti-myeloma effect within the bone marrow niche in MM mouse models. Methods: In-vitro: KD of SLAMF7 was performed in myeloma cell line MM1s using lentiviral shRNAs targeting different regions in the SLAMF7 mRNA. Specific SLAMF7 knockdown was confirmed by reduced SLAMF7 mRNA and protein expression in comparison to cells receiving scrambled control shRNA lentivirus. Survival of SLAMF7 KD cells in comparison to scrambled control cells was assessed by MTT assay. The effect of elotuzumab treatment on the survival of MM1s cells was also assessed. In vivo: Tumor Xenograft models SLAMF7 KD; MM1S-GFP-Luc+ cells (5X106) were injected intravenously (IV) into SCID-Bg mice (n=7/group) and animals underwent bioluminescent imaging (BLI) weekly following injection. Xenograft models elotuzumab; Forty eight hours after IV injection of MM1S-GFP-Luc+ cells (5X106) elotuzumab or human IgG control antibody (both 10mg/kg) was administered via intraperitoneal (IP) injection twice weekly and mice (n=7/group) were followed for tumor burden and survival outcomes. A follow up study was performed (n=5/group) using a modified form of elotuzumab (IgG1.1mAb 10mg/kg IP) harboring mutations in the IgG1 CH2 domain which decreases ADCC via decreased Fc-γ receptor binding allowing evaluation of the CD16 mediated effect in this model. Results SLAMF7 KD resulted in a significant reduction in the survival of MM1s cells at 24 hours as assessed by MTT assay (P = 0.001). No significant reduction in survival was noted for the MM1s myeloma cell line following elotuzumab treatment at 24 hours in the absence of effector cells. Interestingly, knockdown of SLAMF7 resulted in a reduction in tumor burden in the SCID-Bg xenograft model as assessed by weekly BLI signal (P = 0.005 at week 6) with a consequent increase in survival for mice receiving SLAMF7 KD cells. (average 49 vs. 110 days Log Rank P<0.0001). Treatment of mice that had been administered MM1s cells and subsequently treated with elotuzumab resulted in a significant reduction in tumor burden (P = 0.001 at 5 weeks) and prolonged survival (Log Rank P = 0.0001) compared to human IgG1 control antibody treated mice (n=7/group). A follow up 3 arm study comparing IgG1.1f mAb to elotuzumab or human IgG1 control antibody (n=5/group) demonstrated no significant difference in tumor burden between the group receiving IgG1.1 mAb (no CD16 binding) and human IgG1 control antibody (P =0.8 at week 3). The difference in tumor burden between IgG1.1 mAb and elotuzumab was significant (P = 0.019 at 3 weeks) indicating that the effect on tumor burden seen in these mice was indeed CD16 dependent. Conclusions Knockdown of SLAMF7 in MM cells results in reduced tumor burden and survival in xenograft mice, indicating that SLAMF7 potentially plays a role in tumor biology in this disease. In addition elotuzumab treatment reduces tumor burden in a xenograft model of MM with defective NK cells in a CD16 dependent manner indicating that additional mechanisms beyond ADCC may be important in mediating the anti-myeloma effect of elotuzumab. Disclosures Glavey: BMS: Consultancy, Research Funding. Robbins:Bristol-Myers Squibb: Employment; Bristol-Myers Squibb: Equity Ownership. Ghobrial:Onyx: Advisory board Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

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