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

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.

Download Full-text

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 ◽

10.1182/blood.v118.21.887.887 ◽

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.

Download Full-text

Silencing The Sialyltransferase Gene ST3GAL6 Inhibits Adhesion and Migration Of Myeloma Cells In Vitro and Reduces The Homing and Proliferation Of Tumor Cells In Vivo

Blood ◽

10.1182/blood.v122.21.275.275 ◽

2013 ◽

Vol 122 (21) ◽

pp. 275-275

Author(s):

Siobhan Glavey ◽

Salomon Manier ◽

Antonio Sacco ◽

Michaela R Reagan ◽

Yuji Mishima ◽

...

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Research Funding ◽

Advisory Board ◽

Myeloma Cells ◽

Sialyl Lewis ◽

And Migration ◽

Rpmi 8226

Abstract Background Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The adhesion and trafficking of multiple myeloma (MM) cells is strongly influenced by glycosylation and multiple myeloma cells express a variety of adhesion molecules, including selectin ligands and integrins, which are typically dependent on glycosylation for their function. We have previously reported that the sialyltransferase ST3GAL6 is up-regulated in plasma cells from MM patients and that increased expression is associated with inferior overall survival (OS) in MM gene expression profiling (GEP) datasets. The functional significance of increased sialylation of MM cells has not previously been reported. Methods MM cell lines MM1s and RPMI-8226 were confirmed to have high expression levels of ST3GAL6 at the gene and protein level compared to healthy controls. Knockdown of ST3GAL6 was confirmed in MM cell lines RPMI-8226 and MM1s using lentiviral shRNAs targeting different regions in the ST3GAL6 mRNA. Specific ST3GAL6 knockdown was confirmed by reduced ST3GAL6 mRNA and protein expression in comparison to a scrambled control. In a calcein-AM fluorescence based adhesion assay we next evaluated the effects of ST3GAL6 knockdown on MM-cell adhesion to bone marrow stromal cells (BMSC’s) and fibronectin coated plates. Migration to 30nM SDF1-α was assessed using transwell plates comparing ST3GAL6 knockdown cells to scrambled controls. The commercially available sialyltransferase inhibitor 3Fax-Neu5Ac was used to pre-treat MM cells in vitro prior to assessment of apoptosis by flow cytometry. shST3GAL6 MM1s cells positive for green fluorescent protein and luciferin (GFP-Luc+) were injected into tail veins of SCID-Bg mice (5x106 cells, n=5/group) and mice were followed weekly using bioluminescent imaging (BLI) for tumor development. Bone marrow homing of tumor cells was assessed using in vivoconfocal imaging of the skull vasculature (n=3/group). Results Knockdown of ST3GAL6 in MM cell lines resulted in a 50% reduction in cell surface staining with the monoclonal antibody HECA-452. This indicated reduced expression of cutaneous lymphocyte associated antigen (CLA), a carbohydrate domain shared by sialyl Lewis X (sLex) and sialyl Lewis a (sLea) antigens, confirming suppression of ST3GAL6 activity. There was a significant reduction in the ability of knockdown cells to adhere to BMSC’s and fibronectin in-vitro compared to scrambled controls (P=0.016, 0.032 respectively). Migration ability of these cells in response to SDF1-α was also reduced (P=0.01). In vivo in a xenograft SCID-Bg mouse model shST3GAL6 cells demonstrated a reduced tumor burden as assessed by weekly BLI (P=0.017 at week 4). A consolidated map of the skull bone marrow niche in mice injected with shST3GAL6 MM1s GFP-Luc+ cells revealed a reduced homing ability of these cells in comparison to mice injected with scrambled control cells. Treatment of the MM cell lines MM1s and RPMI-8226 with a sialyltransferase inhibitor 3Fax-Neu5Ac resulted in almost complete elimination of cell surface sLex and/or sLea expression as determined by HECA-452 staining. Following pre-treatment with 3Fax-Neu5Ac, MM1S cells grown in co-culture with BMSC’s cells showed increased sensitivity to Bortezomib compared to cells treated with bortezomib alone. Conclusions shRNA knockdown of ST3GAL6 in MM cells significantly inhibits adhesion and migration in vitro with reduced homing and proliferation potential in vivo. In conjunction with the results of enzymatic inhibition this indicates that sialylation may play an important role in the malignant behavior of MM cells. Studies are ongoing to address the potential role of altered glycosylation in MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Download Full-text

LNA Anti-MicroRNA-155: A Novel Therapeutic Strategy in Waldenstrom Macroglobulinemia and Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.2728.2728 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2728-2728

Author(s):

Yong Zhang ◽

Christopher P. Rombaoa ◽

Aldo M Roccaro ◽

Susanna Obad ◽

Oliver Broom ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Tumor Cells ◽

Stromal Cells ◽

Research Funding ◽

Mrna Levels ◽

Advisory Committees ◽

Qrt Pcr

Abstract Abstract 2728 Background. We and others have previously demonstrated that primary Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) cells show increased expression of microRNA-155 (miR-155), suggesting a role in regulating pathogenesis and tumor progression of these diseases. However, developing therapeutic agents that specifically target miRNAs has been hampered by the lack of appropriate delivery of small RNA inhibitors into tumor cells. We tested the effect of a novel LNA (locked nucleic acid)-modified anti-miR-155 in WM and CLL. Methods. WM and CLL cells, both cell lines (BCWM.1; MEC.1) and primary tumor cells; BCWM.1 Luc+ cells; and primary WM bone marrow (BM) stromal cells were used. WM and CLL cells were treated with antisense LNA anti-miR-155 or LNA scramble oligonucleotide. Efficiency of delivering FAM-labeled LNA into cells was determined by flow cytometry. Survival and cell proliferation were assessed by MTT and thymidine uptake assay, respectively. Synergistic effects of LNA with bortezomib were detected on BCWM.1 or MEC1 cells. Co-culture of BCWM.1 or MEC1 cells with WM bone marrow stromal cells was performed to better define the effect of the LNA-anti-miR155 in the context of the bone marrow microenvironment. miR-155 levels were detected in stromal cells from WM patients by qPCR. Co-culture of BCWM.1 or MEC1 cells with either wild-type or miR155−/− mice BM stromal cells was examined after LNA treatment. Gene expression profiling analysis was performed on BCWM.1 cells treated with either LNA anti-miR-155 or scramble control. miR-155 target gene candidates were predicted by TargetScan software. mRNA levels of miR-155, and its known target genes or gene candidates were detected by qRT-PCR. A microRNA luciferase reporter assay was used to determine whether miR-155 target candidates could be directly regulated by miR-155. mRNA levels of miR-155 targets were detected by qRT-PCR from primary WM or CLL cells treated with LNA. The activity of the LNA-anti-miR-155 was also detected in vivo using bioluminescence imaging and mRNA levels of miR-155 targets were detected by qRT-PCR ex vivo. Efficiency of introducing the FAM-labeled LNA into mice BM cells was determined by flow cytometry 1 week or 2 weeks after intravenous injection. Results. The efficiency of delivering LNA oligos into both WM and CLL-derived cell lines and primary samples was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines by 30–50%, as compared to LNA scramble control. In contrast, LNA antimiR-155 didn't exert significant cytotoxicity in BCWM.1 or MEC.1. LNA synergistically decreased BCWM.1 or MEC1 cell growth co-treated with bortezomib and decreased BCWM.1 or MEC1 cell growth co-cultured with WM BM stromal cells in vitro. A higher level of miR-155 was found in WM BM stromal cells compared to normal ones. LNA decreased BCWM.1 or MEC1 cell growth when co-cultured with BM stromal cells from miR155−/− mice compared with wild-type. We demonstrated increased expression of miR-155-known targeted genes, including CEBPβ, SOCS1, SMAD5, and several novel target candidates including MAFB, SH3PXD2A, and SHANK2, in WM cells upon LNA anti-miR-155 treatment. These target candidates were confirmed to be directly regulated by miR-155 using a luciferase reporter assay. mRNA levels of miR-155 targets were upregulated by 1.5–2 fold at 48 hr after direct incubation of the LNA with primary WM or CLL samples, indicating efficient delivery and biologic effect of the LNA in cells. Moreover, this LNA showed significant in vivo activity by inhibiting WM cell proliferation in a disseminated xenograft mouse model. Upregulation of miR-155 targeted genes were confirmed ex vivo, in WM cells isolated from the BM of treated mice compared to control. Mice BM cells were FAM positive 1 or 2 weeks after injection indicating efficient delivery of FAM-labeled LNA into cells in vivo. Summary. A novel LNA (locked nucleic acid)-modified anti-miR against miR-155 could be highly efficiently delivered into tumor cells in vivo in the bone marrow microenvironment. Anti-WM activity of LNA anti-miR-155 was confirmed both in vitro and in vivo and anti-CLL activity was confirmed in vitro. Novel miR-155 direct target genes including MAFB, SH3PXD2A, and SHANK2 were identified. These findings will help to design individualized clinical trials for WM and CLL patients with elevated levels of miR-155 in their tumor cells. Disclosures: Roccaro: Roche:. Obad:Santaris Pharma: Employment. Broom:Electroporation: Employment. Kauppinen:Santaris Pharma: Employment. Brown:Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Ghobrial: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; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Immunophenotypic Analysis of Myeloma Precursors: Antigens for Therapeutic Targeting

Blood ◽

10.1182/blood.v116.21.sci-5.sci-5 ◽

2010 ◽

Vol 116 (21) ◽

pp. SCI-5-SCI-5

Author(s):

Martin Perez-Andres ◽

Bruno Paiva ◽

Leandro Thiago ◽

Nico A Bos ◽

Dirk Hose ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Tumor Cells ◽

Research Funding ◽

Plasma Cells ◽

Somatic Hypermutation ◽

Ex Vivo ◽

Multidrug Resistant

Abstract Abstract SCI-5 Multiple myeloma (MM) is a malignant disorder characterized by the (mono)clonal expansion of terminally-differentiated plasma cells (M-PC) in the bone marrow (BM) that produce and secrete a monoclonal immunoglobulin (Ig), detectable in the serum and/or urine. Infiltration by the expanded M-PC is easily identified in the involved tissues, through conventional morphology and immunophenotyping. However, the possibility exists that rather than M-PC, a less differentiated B-cell that represents a minor fraction of all tumor cells and retains self-renewal properties, is responsible for the outgrowth of the more differentiated M-PC compartment. In B-cell disorders, the idiotypic Ig produced by tumor cells and defined by its CDR3 sequence, acts as a genetic fingerprint for clonally-related B-cells. In MM, the specificity of the idiotype is further enhanced through modification of V genes by somatic hypermutation (SHM) at the germinal center (GC), since M-PC display extensively mutated VH genes which are stable throughout the disease. This suggests that in MM, malignant transformation could occur in a post-GC B-cell. Based on the CDR3 sequences of the Ig genes of M-PC, preliminary studies have identified tumor-associated circulating peripheral blood (PB) CD19+ B-cells, whose malignant/clonogenic potential remained to be demonstrated. More recently, the Matsui group has reported that while in MM cell lines both CD138− and CD138+ cells retain clonogenic capacity after in vitro serial plating clonogenic assays, in primary MM samples, such (ex vivo and in vivo) ability would be restricted to the CD34−/CD138− compartment. Of note, the clonogenic growth of these later cells significantly decreased after depletion by CD19, CD22, CD20 and CD45 antibodies, and it was associated with an in vitro multidrug-resistant functional phenotype (restricted to CD19+/CD27+, CD138− cells but not CD138+ cells) and the Hedgehog (Hh) stem cell-associated signaling pathway. These results point out the potential existence of a CD19+, CD20+, CD138− pre-PC compartment responsible for the expansion of M-PC in MM. In turn, evidence also exists in both the SCID-Hu model and in Rituximab treated MM patients, which suggests that plasmablasts/PC -but not pre-plasmablasts-, could act as MM “stem” cells, the precise characteristics of such cells remaining to be precisely defined. Alternatively, it could also be possible that both cell cellular components coexist and are relevant to MM progression through appropriate interaction with the BM stroma. Independently of all the above, trafficking of such cells through PB to BM niches could also play a key role in the spread of the tumor and its malignant behavior. In this regard, we recently confirmed that a relatively high percentage of MM patients (and a substantial fraction of all MGUS cases) show circulating PB PC with i) tumor-related clonal VH gene rearrangements and ii) an aberrant immunophenotypic profile which largely overlaps with that of BM M-PC from the same subjects; the only minor differences consisted of a significantly lower expression of CD38 and CD138, smaller size and internal complexity, features that indicate a slightly more immature plasmablastic/PC profile. Noteworthy, this was the only PB B-cell compartment for which clonally-related B-cells were detected with a sensitivity of <1 cell/50μ L in all cases investigated. In summary, accumulating evidence suggest the existence of a clonal hierarchy in MM but uncertainties remain as regards the precise immunophenotypic features of those cells responsible for tumor growth in primary patient samples, that could be of help in developing new targeted therapies. Disclosures: Sonneveld: Millennium: Consultancy; Celgene: Consultancy. Orfao:Becton/Dickinson Biosciences Europe: Patents & Royalties, Research Funding; Cytognos SL: Patents & Royalties; Alexion: Membership on an entity's Board of Directors or advisory committees; Vivia Biotech: Research Funding; Mundipharma: Research Funding.

Download Full-text

Evaluation Of Acadesine, a Drug Stimulating Cell Autophagy, In Azacitidine(AZA)-Resistant Myelodysplastic Syndromes (MDS)

Blood ◽

10.1182/blood.v122.21.1568.1568 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1568-1568

Author(s):

Thomas Cluzeau ◽

Guillaume Robert ◽

Jean Michel Karsenti ◽

Frederic Luciano ◽

Nicolas Mounier ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Myelodysplastic Syndromes ◽

Research Funding ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Cell Metabolism ◽

Vitro Effect

Abstract Background AZA is currently the first line treatment for intermediate-2 and high-risk IPSS myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with 20 to 30% of marrow blasts. We have previously reported that cells from AZA-resistant patients exhibit impaired mitochondrial apoptosis but maintain functional autophagy (Cluzeau et al. Cell Cycle 2011, Oncotarget 2012). Acadesine (ACA), also known as AICAR or Aica-Riboside is a nucleoside analogue that has been shown to trigger autophagy in AZA resistant cells. Methods In vitro effect: We used an AZA-resistant MDS/AML cell line (SKM1-R) as a tool to decipher AZA resistance. Cells were treated with increasing doses of ACA (0.5-2mM) or with a maximally efficient dose of AZA (1µM) and induction of cell death was assessed by cell metabolism and Propidium Iodide (PI) assays. In vivo effect: The effect of ACA was also assessed in a mouse xenograft model of SKM1-R cells. When tumors reached 100 mm3, mice were treated daily with an intra-peritoneal injection of the vehicle alone, 5 mg/kg AZA, or 200 or 400 mg/kg ACA. Ex vivo effect: We finally used primary bone marrow cells from AZA-resistant MDS or AML-resistant patients (n=12) (clinicalTrials.gov identifier: NCT01210274) to perform cell metabolism assays. Results In vitro effect: Only a slight decrease of cell metabolism and a moderate increase of PI staining were detected following stimulation with 1µM AZA confirming the resistance of SKM1-R cells to AZA. In identical conditions, ACA induced a robust increase of cell death in AZA-resistant cells with a maximal effect at 2mM. Induction of cell death by ACA was independent of apoptosis but relied on autophagy induction, as shown by the conversion of LC3-I to LC3-II and an increase of cathepsin B activity, that are respectively early and late markers of autophagy. In vivo effect: As expected, AZA failed to trigger tumor regression of AZA-resistant SKM1-R cells in vivo compared to vehicle alone, whereas ACA was found to induce a statistically significant inhibition of tumor growth at both tested concentrations. Ex vivo effect:Bonferroni’s Multiple Comparison Test performed in 6 AZA-resistant MDS patients showed significant reduction of cell metabolism between ACA and untreated cells (66% and 78% at 1 and 2 mM of ACA) and between ACA and AZA-treated cells (60% and 72% at 1 and 2mM of ACA,). Identical results were found in 6 AML AZA-resistant AML patients with a significant reduction of cell metabolism between ACA and untreated cells (63% and 81% at 1 and 2mM of ACA) and ACA and AZA-treated cells (56% and 75% at 1 and 2 mM of ACA). Conclusion Our results show the high efficacy of Acadesine in vitro, in vivo and ex vivo in AZA-resistant MDS and AML cell lines and patient’s bone marrow cells. Induction of cell death by autophagy seems to be the main mechanism by which ACA circumvents AZA resistance in MDS and AML cells. These encouraging results prompted us to initiate a multicenter phase I/II clinical trial with the French MDS Group (GFM) to assess the safety and efficacy of ACA in MDS and AML patients with 20 to 30% of marrow blasts not responding or relapsing after AZA treatment (clinicalTrials.gov identifier: NCT01813838). Disclosures: Cluzeau: Advancell: Research Funding. Robert:Advancell: Research Funding. Auberger:Advancell: Research Funding.

Download Full-text

Antimyeloma Effect of the Simultaneous Inhibition of MCL-1 (with S63845) and BCL-2 (with Venetoclax) in the Presence of the Microenvironment

Blood ◽

10.1182/blood-2018-99-116075 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 954-954

Author(s):

Esperanza M Algarín ◽

Andrea Díaz-Tejedor ◽

Pedro Mogollón ◽

Susana Hernández-García ◽

Luis Corchete ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Microenvironment ◽

Board Of Directors ◽

Research Funding ◽

Ex Vivo ◽

Advisory Committees ◽

Stromal Microenvironment ◽

Castilla Y Leon

Abstract Background: Venetoclax is a BCL-2 inhibitor particularly effective in patients with multiple myeloma (MM) harboring the t(11;14). However, resistance to venetoclax has been linked to MCL-1 overexpression. On the other hand, it is wellknown that MM cells depend on MCL-1 rather than BCL-2 for survival, and this dependence has recently been reported to be enhanced by the tumor-associated microenvironment. Therefore, the combination of venetoclax with the potent MCL-1 inhibitor S63845 arises as a promising and novel approach for the treatment of MM. Aims: To evaluate the efficacy and mechanism of action of S63845 alone and in combination with venetoclax in absence and presence of the bone marrow tumor microenvironment in preclinical in vitro, ex vivo and in vivo models of MM. Methods: S63845 was provided by an agreement with Servier and Novartis. In vitro activity of S63845 and venetoclax alone and in combination was evaluated by bioluminescence on a MM cell line expressing luciferase (MM.1S-luc) in absence and presence of mesenchymal stromal cells isolated from bone marrow aspirates of MM patients (pMSCs). MM.1S cells cultured in absence or presence of pMSCs were analyzed for MCL-1 and BCL-2 protein levels by Western blot. Interactions between these anti-apoptotic proteins with the pro-apoptotic protein BIM were assessed by immunoprecipitation assays. The efficacy of S63845 and venetoclax alone and in combination was also evaluated ex vivo in MM cells and normal lymphocytes from MM patients. Finally, a disseminated MM model in BRG mice was used for in vivo studies. Results: S63845 and venetoclax showed a strong antimyeloma dose-dependent effect on MM.1S-luc cells co-cultured with pMSCs. However, whereas the presence of tumor-associated MSCs increased the IC50 value of venetoclax in MM.1S-luc cells from 6.2 to 9.8 mM, it reduced that of S63845 from 94.1 to 81 nM, suggesting a mild sensitization to this drug in the context of the microenvironment. Neither S63845 nor venetoclax affected pMSC viability even at high concentrations by MTT assay. The co-culture with the BM stromal microenvironment increased MCL-1 expression on untreated MM.1S cells in two out of four experiments performed with MSCs from different MM patients, whereas it surprisingly induced a decrease on BCL-2 levels in all of them. Treatment with S63845 completely blocked MCL-1 binding to BIM, both in the absence or presence of pMSCs but did not induce the compensatory increase of BCL-2/BIM complexes observed in MM.1S cells in monoculture. Venetoclax also completely blocked the binding of BCL-2 to BIM in MM.1S alone or in co-culture, and induced a similar compensatory increase of MCL-1/BIM complexes in both situations. Importantly, the double combination S63845 + venetoclax was significantly superior to both drugs in monotherapy in killing MM.1S-luc cells co-cultured in the presence of the stromal microenvironment. BIM immunoprecipitation assays showed that the double combination was able to counteract the compensatory upregulation of MCL-1 bound to BIM observed on MM.1S cells treated with venetoclax and to entirely disrupt BCL-2/BIM complexes, both in the absence and presence of pMSCs. Furthermore, S63845 + venetoclax increased the percentage of apoptotic MM plasma cells from three MM patients with respect to single treatments with moderate toxicity detected on normal lymphocytes, suggesting the existence of a therapeutic window for the double combination. Finally, the combination of S63845 + venetoclax clearly delayed tumor growth as compared with the agents in monotherapy in a disseminated model of MM with statistically significant differences from day 19 of treatment. This in vivo effect translated into a significatively improved survival for mice treated with the double combination (median 60 days) vs control mice (median 32 days; log-rank test P=0.045). Conclusion: Our preclinical data demonstrate the potent activity of the combination of venetoclax with S63845 in MM even in presence of the stromal associated-tumor microenvironment, and provides the rationale for the clinical development of this combination in relapsed or refractory MM patients. This project was supported by Novartis Pharmaceuticals and by the Spanish , ISCIII-FIS PI15/00067 and PI15/02156, GRS 1604/A/17 and CRMRTC de Castilla y León. Predoctoral grant to EMA by Consejería de Educación de Castilla y León. Disclosures Schoumacher: Servier: Employment. Banquet:Servier: Employment. Kraus-Berthier:servier: Employment. Kloos:Servier: Employment; Novartis: Other: Partnership. Halilovic:Novartis: Employment, Equity Ownership. Maacke:Novartis: Employment. Mateos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ocio:AbbVie: Consultancy; Novartis: Consultancy, Honoraria; BMS: Consultancy; Seattle Genetics: Consultancy; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Pharmamar: Consultancy; Sanofi: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding.

Download Full-text

Citron Rho-Interacting Serine/Threonine kinase (CIT) Is a Novel Therapeutic Target in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v124.21.3430.3430 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3430-3430

Author(s):

Yawara Kawano ◽

Ilyas Sahin ◽

Michele Moschetta ◽

Jinhua Wang ◽

Salomon Manier ◽

...

Keyword(s):

Cell Proliferation ◽

Research Funding ◽

Plasma Cells ◽

Advisory Board ◽

Thymidine Uptake ◽

Serine Threonine Kinase ◽

Data Set ◽

Threonine Kinase

Abstract Introduction: Citron rho-interacting serine/threonine kinase (CIT) is a serine/threonine kinase which is a key component of the midbody and is essential for cytokinesis. CIT localizes to the central spindle and midbody and functions to promote efficient cytokinesis. CIT knockdown may disrupt cytokinesis and therefore cell growth. CIT has been reported to be upregulated and important for growth of several cancers. However, the significance of CIT has not been investigated in the field of multiple myeloma (MM). We therefore dissected the role of CIT in MM growth in vitro and in vivo. Materials and methods: CIT gene expression in MM cells was compared to normal plasma cells using public-available gene expression profile (GEP) data set (GSE6477). Kaplan-Meier curve for MM patient survival between high and low CIT expressing patients were examined by using the GEP data set (GSE4581). Protein expression of CIT in MM cells was confirmed by proteomic analysis and immunohistochemistry. Knockdown of CIT was performed in MM cell lines MM1s and OPM2 using lentiviral shRNAs. CIT knockdown was confirmed by reduced CIT mRNA in comparison to a scrambled control. Differences in cell proliferation and cell cycle between CIT knockdown cells and scramble control were analyzed by using thymidine uptake and PI staining, respectively. Cytokinesis failure was analyzed by immunofluorescence using alpha-tubulin antibody and DAPI. shCIT OPM2 (n=7) and the scrambled control cells (n=8) were injected subcutaneously into SCID-Bg mice (5x106 cells/mouse) and were followed for tumor development and survival. Results: CIT expression was significantly higher in MM patients’ plasma cells compared to healthy donors in GEP (p=0.02), proteomic analysis and immunohistochemistry. Also CIT expression was higher in relapsed patients compared to newly diagnosed patients by GEP. MM patients with high CIT expression had significantly worse overall survival compared to low CIT expressing patients (p=0.04). CIT knockdown MM cell lines showed reduced cell proliferation and G2 cell cycle arrest by thymidine uptake and PI staining compared to the scrambled control. Significantly, large amount of multinucleated cells, which indicates cytokinesis failure, were observed in the CIT knockdown cells compared to scrambled control. Reduced tumor growth (p<0.001) and prolonged survival (p<0.001) was observed in CIT knockdown MM cell line injected mice. Conclusions: shRNA knockdown of CIT in MM cells induces G2 arrest leading to cytokinesis failure in vitro with reduced cell proliferation in vivo. Since MM cells have significantly higher expression of CIT compared to normal plasma cells, CIT represents a novel therapeutic target for MM. Studies are ongoing to develop drugs to target CIT for MM treatment. Disclosures Anderson: Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy. Ghobrial:Onyx: Advisory board Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Download Full-text

The MEMIC: An ex vivo system to model the complexity of the tumor microenvironment

Disease Models & Mechanisms ◽

10.1242/dmm.048942 ◽

2021 ◽

Author(s):

Libuše Janská ◽

Libi Anandi ◽

Nell C. Kirchberger ◽

Zoran S. Marinkovic ◽

Logan T. Schachtner ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Cells ◽

Ex Vivo ◽

Tumor Stroma ◽

Stem Cell Differentiation ◽

Blood Stream ◽

Direct Access ◽

Ex Vivo Model

There is an urgent need for accurate, scalable, and cost-efficient experimental systems to model the complexity of the tumor microenvironment. Here, we detail how to fabricate and use the Metabolic Microenvironment Chamber (MEMIC) – a 3D-printed ex vivo model of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is the accessibility to the blood stream that provides key resources such as oxygen and nutrients. While some tumor cells have direct access to these resources, many others must survive under progressively more ischemic environments as they reside further from the vasculature. The MEMIC is designed to simulate the differential access to nutrients and allows co-culturing different cell types, such as tumor and immune cells. This system is optimized for live imaging and other microscopy-based approaches, and it is a powerful tool to study tumor features such as the effect of nutrient scarcity on tumor-stroma interactions. Due to its adaptable design and full experimental control, the MEMIC provide insights into the tumor microenvironment that would be difficult to obtain via other methods. As a proof of principle, we show that cells sense gradual changes in metabolite concentration resulting in multicellular spatial patterns of signal activation and cell proliferation. To illustrate the ease of studying cell-cell interactions in the MEMIC, we show that ischemic macrophages reduce epithelial features in neighboring tumor cells. We propose the MEMIC as a complement to standard in vitro and in vivo experiments, diversifying the tools available to accurately model, perturb, and monitor the tumor microenvironment, as well as to understand how extracellular metabolites affect other processes such as wound healing and stem cell differentiation.

Download Full-text

A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo

Leukemia ◽

10.1038/leu.2016.388 ◽

2016 ◽

Vol 31 (8) ◽

pp. 1743-1751 ◽

Cited By ~ 86

Author(s):

S Hipp ◽

Y-T Tai ◽

D Blanset ◽

P Deegen ◽

J Wahl ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cell ◽

Plasma Cells ◽

Ex Vivo ◽

Xenograft Model ◽

Selective Lysis ◽

Bispecific T Cell Engager

Abstract B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.

Download Full-text