Bion-1301: A Novel Fully Blocking APRIL Antibody for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2112-2112 ◽  
Author(s):  
John Dulos ◽  
Driessen Lilian ◽  
Marc Snippert ◽  
Marco Guadagnoli ◽  
Astrid Bertens ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cell ◽  
Board Of Directors ◽  
Clinical Development ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Parental Antibody

Abstract A PRoliferation Inducing Ligand (APRIL, TNFSF13), is a ligand for the receptors BCMA and TACI. APRIL serum levels are enhanced in patients diagnosed with Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), and Colorectal Carcinoma correlated with poor prognosis. Our anti-APRIL antibody blocked CLL survival and inhibited mouse B1 hyperplasia in vivo (Guadagnoli et al., 2011). APRIL is produced by cells in the bone marrow niche, including myeloid-derived cells, osteoclasts and plasmacytoid dendritic cells. APRIL critically triggers BCMA in vitro and in vivoto drive proliferation and survival of human MM cells (Tai et al., 2016). Importantly, APRIL induces resistance to lenalidomide, bortezomib and other standard-of-care drugs. Furthermore, APRIL drives expression of PD-L1, IL-10, VEGF and TGFβ forcing an immunosuppressive phenotype on BCMA+ cells. As MM survival, resistance to treatment and the immunosuppressive phenotype can be blocked by neutralizing APRIL (Tai et al., 2016), development of an antibody blocking APRIL provides a novel avenue for the treatment of MM. A novel mouse anti-human APRIL antibody hAPRIL.01A (Guadagnoli et al., 2011) initially discovered using Aduro's B-Select platform, was humanized and further engineered enhancing its stability (designated as BION-1301). The antibody binds to recombinant human APRIL with a KDof 0.4 ± 0.15 nM determined by BioLayer Interferometry and an EC50 of 0.29 ± 0.05 nM by ELISA. The epitope of BION-1301 was mapped to the BCMA and TACI binding site explaining its fully blocking capacity. Blocking potency (IC50) was 1.61 ± 0.78 nM (BCMA) and 1.29 ± 0.89 nM (TACI) respectively, corroborated by potent and complete blockade of APRIL-induced cytotoxicity of BCMA-Fas and TACI-Fas Jurkat transfectants. In vitro, BION-1301 suppressed APRIL-induced B-cell IgA and IgG class switching in a dose-dependent fashion. In vivo, BION-1301 was shown to suppress human APRIL induced T cell-independent B cell responses to NP-Ficoll. Biophysical and functional experiments indicated that BION-1301 recapitulated all characteristics of the mouse parental antibody hAPRIL.01A. To support the clinical development of BION-1301, quantitative assays were developed using several mouse-anti-human APRIL antibodies and shown to detect free and complexed APRIL in human blood samples. Results obtained with assays demonstrate that APRIL can be quantified reproducibly in human sera and overcome the drawbacks of previous assays, such as requirement of polyclonal sera, Ig adsorption, interference by human serum and reduced sensitivity. In conclusion, we have generated and functionally characterized a novel humanized APRIL neutralizing antibody, designated BION-1301. The mechanism-of-action and anti-tumor activity described for the parental antibody hAPRIL.01A in vitro and in vivo strongly support the development of BION-1301 as a single agent or in combination with lenalidomide, bortezomib, and suggest a rationale for combination with checkpoint inhibitors. BION-1301 is expected to enter clinical development in 2017. References:Guadagnoli M, Kimberley FC, Phan U, Cameron K, Vink PM, Rodermond H, Eldering E, Kater AP, van Eenennaam H, Medema JP. Development and characterization of APRIL antagonistic monoclonal antibodies for treatment of B-cell lymphomas. Blood. 2011 Jun 23;117(25):6856-65Tai YT, Acharya C, An G, Moschetta M, Zhong MY, Feng X, Cea M, Cagnetta A, Wen K, van Eenennaam H, van Elsas A, Qiu L, Richardson P, Munshi N, Anderson KC. APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment. Blood. 2016 Jun 23;127(25):3225-36 Disclosures Dulos: Aduro Biotech Inc.: Equity Ownership. Lilian:Aduro Biotech Inc.: Equity Ownership. Snippert:Aduro Biotech Inc.: Equity Ownership. Guadagnoli:Aduro Biotech Inc.: Equity Ownership. Bertens:Aduro Biotech Inc.: Equity Ownership. David:Aduro Biotech Inc.: Equity Ownership. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Eenennaam:Aduro Biotech Inc.: Equity Ownership. Elsas:Aduro Biotech Inc.: Equity Ownership.

Targeting Brouton's Tyrosine Kinase with PCI-32765 Blocks Growth and Survival of Multiple Myeloma and Waldenström Macroglobulinemia Via Potent Inhibition of Osteoclastogenesis, Cytokines/Chemokine Secretion, and Myeloma Stem-Like Cells in the Bone Marrow Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 883-883
Author(s):  
Yu-Tzu Tai ◽  
Betty Y Chang ◽  
Sun-Young Kong ◽  
Mariateresa Fulciniti ◽  
Guang Yang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Growth And Survival ◽  
Equity Ownership ◽  
Trap Staining

Abstract Abstract 883 Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in regulating osteoclastogenesis. Although Btk is critical in B cell maturation and myeloid function, it has not been characterized in plasma cell malignancies including multiple myeloma (MM) and Waldenström Macroglobulinemia (WM). We here investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) microenvironment, as well as on MM and WM cancer cells. We further define molecular targets of Btk signaling cascade in OCs and MM in the BM milieu. In CD14+ OC precursor cells, RANKL and M-CSF stimulate phosphorylation of Btk in a time-dependent fashion; conversely, PCI-32765 abrogates RANKL/M-CSF-induced activation of Btk and downstream PLCγ2. Importantly, PCI-32765 decreased number of multinucleated OC (>3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and the secretion of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It increased size of OCs and number of nuclei per OC, with significantly defective bone resorption activity as evidenced by diminished pit formation on dentine slices. Moreover, lack of effect of Dexamethasone on OC activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 significantly reduced cytokine and chemokine secretion from OC cultures, including MIP1α, MIP1β, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1–0.48 nM). It potently decreased IL-6, SDF-1, MIP1α, MIP1β, and M-CSF in CD138-negative cell cultures from active MM patients, associated with decreased TRAP staining in a dose-dependent manner. In MM and WM cells, immunoblotting analysis confirmed a higher Btk expression in CD138+ cells from majority of MM patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated a higher expression of Btk and its downstream signaling components in WM cells than in CD19+ normal bone marrow cells. PCI-32765 significantly inhibits SDF-1-induced adhesion and migration of MM cells. It further blocked cytokine expression (MIP1a, MIP-1β) at mRNA level in MM and WM tumor cells, correlated with inhibition of Btk-mediated pPLCγ2, pERK and NF-kB activation. Importantly, PCI-32765 inhibited growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs in IL-6-dependent INA6 and ANBL6 MM cells. Furthermore, myeloma stem-like cells express Btk and PCI-32765 (10–100 nM) blocks their abilities to form colonies from MM patients (n=5). In contrast, PCI-32765 has no adverse effects on Btk-negative BMSCs and OBs, as well as Btk-expressing dendritic cells. Finally, oral administration of PCI-32765 (12 mg/kg) in mice significantly suppresses MM cell growth (p< 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results provide compelling evidence to target Btk in the BM microenvironment against MM and WM., strongly supporting clinical trials of PCI-32765 to improve patient outcome in MM and WM. Disclosures: Chang: Pharmacyclics Inc: Employment. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership. Elias:Pharmacyclics Inc: Consultancy. Treon:Millennium: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals In Vitro-Selected Nanobody-Based Cellular Therapy Targeting CD72 for Treatment of Refractory B-Cell Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1337-1337
Author(s):  
Matthew Nix ◽  
Yu-Hsiu T. Lin ◽  
Huimin Geng ◽  
Makeba Marcoulis ◽  
Paul Phojanakong ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
B Cell ◽  
Board Of Directors ◽  
B Cell Malignancies ◽  
Advisory Committees ◽  
Car T ◽  
Equity Ownership

Introduction: B-cell acute lymphoblastic leukemia (B-ALL) patients that harbor rearrangements of the Mixed-lineage leukemia gene (MLLr; also known as KMT2Ar) have particularly dismal clinical outcomes. Although CAR T immunotherapies targeting CD19 have shown impressive responses treating MLLr B-ALL and other B cell malignancies, relapse, often with loss of relevant CD19 epitope, remains a major clinical concern. The mixed results of CD19 CAR T as a monotherapy underscores the need to pursue additional immunotherapy targets and novel therapeutic modalities for high-risk patients. Results and Methods: Data with existing CAR-T's suggest that increased target antigen density frequently correlates with increased tumor elimination. Therefore, we aimed to define the cell surface proteomic landscape of B-ALL to identify novel, MLLr-enriched candidates for targeted immunotherapy of this poor-prognosis subtype. As an initial screen, using N-glycoprotein capture and mass spectrometry, we quantified differentially abundant cell surface proteins in MLLr (n= 4) versus non-MLLr (n= 5) B-ALL cell lines (Figure 1). Label-free proteomics (n= 3 replicates) quantified &gt;900 high-confidence membrane proteins (FDR=0.05). Principal component analysis identified unique cell surfaceome signatures between B-ALL subtypes, implying different surface landscapes associated with specific genetic alterations. The MLLr B-ALL "surfaceome" is notably characterized by increased expression of adhesion molecules not identified by RNA-sequencing alone. We focused on CD72 as a novel immunotherapy target given significant enrichment on MLLr B-ALL vs. other B-ALL subtypes, near equivalent antigen density to CD19, undetectable expression on HSPCs, T-cells, and other normal tissues, and reported widespread expression on other mature B-cell malignancies. Analysis of transcriptome and ChIP-seq data suggested increased CD72 expression in MLLr B-ALL is not regulated directly by the MLL-AF4 oncoprotein but instead a function of increased CD72 expression at pro-B-cell stage. Flow cytometry and immunohistochemistry on primary samples confirmed high expression of CD72 both in MLLr B-ALL as well as DLBCL. Recombinant CD72 ECD was panned against a fully in vitro nanobody yeast display library (McMahon et al., Nat Struct Mol Biol(2018)) resulting in isolation of multiple unique, highly-specific CD72 nanobody binders with KD's &lt; 5nM. Nanobodies were incorporated into 2nd generation CAR constructs and transduced into normal donor CD8+ T-cells and assessed in vitro for tumor cell lysis, cytokine release, and exhaustion marker expression. Nanobody clone Nb.D4 outperformed others in lysis of B-ALL and DLBCL cells lines displaying a broad range of CD72 expression, had no activity versus CD72 negative cells, and showed similar efficacy to that found with a clinically-used CD19 CAR. To assess in vivo activity, CD72(Nb.D4) CAR-T's at 1:1 CD4:CD8 ratio were injected at an effector:tumor ratio of 5:1 into tumor-bearing NSG mice (luciferase-labeled SEM or MLLr PDX). In vivo results confirmed strong anti-tumor effect of CD72 nanobody CAR-T's, equivalent to clinical CD19 CAR, and significantly increased survival in mice (Figure 2). A CRISPR interference-generated antigen escape model of CD19 was also effectively eliminated by CD72 CAR-T's. We also introduce "antigen escape profiling", where cell surface proteomics of a CRISPRi CD72-knockdown model demonstrated extensive surfaceome rewiring with potential implications for leukemia cell trafficking and adhesion in the setting of acquired resistance. Given CD72's role as a BCR signaling inhibitory receptor, we are currently examining its influence on proximal B-cell receptor signaling and relationship to combination therapies affecting this pathway. Conclusions:By characterizing the surface proteomic landscape of B-ALL, we develop a resource for the research community and identify CD72 as a promising therapeutic target. We demonstrate that a novel, fully recombinant nanobody library can generate potent cellular therapies, which may be extended to other targets in the future. We anticipate that antigen escape profiling will prove broadly useful for anticipating mechanisms of resistance to novel immunotherapies. CD72 CAR-T's are a promising strategy across a range of B-cell malignancies, particularly those refractory to CD19 therapy. Disclosures Nix: UCSF: Patents & Royalties. Wiita:UCSF: Patents & Royalties; Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals 3D-Tissue Engineered Bone Marrow (3DTEBM) Culture Retrospectively Predicts Treatment Clinical Outcomes of Multiple Myeloma Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1987-1987
Author(s):  
Amanda Jeske ◽  
Feda Azab ◽  
Pilar De La Puente ◽  
Barbara Muz ◽  
Justin King ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Ex Vivo ◽  
In Vitro Models ◽  
Advisory Committees ◽  
Treatment Plans ◽  
Equity Ownership

Abstract Background: Multiple Myeloma (MM) is the second most common hematological malignancy, and continues to be a fatal disease even with the development of novel therapies. Despite promising preclinical data in standard tissue culture models, most drugs fail in clinical trials and show lower efficacy in patients. This highlights the discrepancy between the current in vitro models, the pathophysiology of the disease in the patients, and the urgent need for better in vitro models for drug development and improved prediction of efficacy in patients. We have previously developed a patient-derived 3D-Tissue Engineered Bone Marrow (3DTEBM) culture model, which showed superior properties for proliferation of primary MM cells ex vivo, and better recapitulated drug resistance. The long-term goal of this study is to use the 3DTEBM model as a tool to perform drug screens on BM aspirates of MM patients and prospectively predict the efficacy of different therapies in individual patients, and help treatment providers develop personalized treatment plans for each individual patient. In the current study, we used the 3DTEBM model to, retrospectively, predict clinical responses of MM patients to therapy, as a proof of concept. Methods: We used whole-BM, viably frozen tissue banked samples from 20 MM patients with clear clinical response patterns of complete remission, and either very good partial response (sensitive) or progressive disease (non-sensitive). The BM aspirates were used to develop a 3DTEBM that represents each individual patient. The patient-derived 3DTEBM cultures were treated ex vivo with the same therapeutic regimen that the patient received in the clinic for 3 days. The treatment ex vivo was based on combinations at different concentrations which mimic the steady state concentrations (Css) of each drug. The efficacy of the treatment ex vivo was evaluated by digestion of the 3DTEBM matrix, extraction of the cells, and analysis for prevalence of MM cells in the treatment groups compared to the non-treated controls. Patients were defined "sensitive" if the effect reached 50% killing in the range of 10xCss. The ex vivo sensitivity data was then correlated with the clinical response outcomes. Results: We found that the 3DTEBM was predictive in approximately 80% of the cases (in about 85% of the combination therapy cases, and in about 70% of the single therapy cases). Broken down by individual drug, it was predictive in 80% of the cases treated with Bortezomib, 78% Lenalidomide, 84% Dexamethasone, 100% Daratumumab, 50% Carfilzomib, 50% Pomalidomide, and 100% Doxorubicin. Conclusions: The 3DTEBM is a more pathophysiologically relevant model which predicts clinical efficacy of drugs in multiple myeloma patients, retrospectively. This data provides the bases for future studies which will examine the ability of the 3DTEBM model to predict treatment efficacy, prospectively, for development of personalized treatment plans in individual multiple myeloma patients. Disclosures Jeske: Cellatrix LLC: Employment. Azab:Cellatrix LLC: Employment. De La Puente:Cellatrix LLC: Other: Co-founder. Vij:Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Azab:Ach Oncology: Research Funding; Cellatrix LLC: Equity Ownership, Other: Founder and owner; Glycomimetics: Research Funding; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner.

Re-Evaluation of CD37 As Target for Radioimmunotherapy of Non-Hodgkin Lymphoma,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3732-3732
Author(s):  
Jostein Dahle ◽  
Ada Repetto ◽  
Camilla Sivertsen Mollatt ◽  
Katrine Brustad Melhus ◽  
Øyvind Sverre Bruland ◽  
...  
Keyword(s):  
B Cell ◽  
Board Of Directors ◽  
Clinical Results ◽  
Antigenic Drift ◽  
Antibody Concentration ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Anti Cd20

Abstract Abstract 3732 The monoclonal anti-CD20 antibody rituximab alone or as part of combination therapy, is considered standard therapy for non-Hodgkin's B-cell lymphomas. However, significantly better clinical results have been obtained for beta-emitting anti –CD20 radioimmunoconjugates (RICs) than for rituximab. RICs targeting CD20 may be problematic because of antigenic drift and antigen blocking caused by previous treatments with rituximab. Therefore, novel therapeutic approaches targeting other B cell antigens might be more effective after rituximab treatment failure than a new anti-CD20 treatment. In the present study, we have compared the therapeutic effect of the novel anti-CD37 RIC 177Lu-DOTA-HH1 with the anti-CD20 RIC 177Lu-DOTA-rituximab against Daudi human lymphoma cells in vitro and in vivo. At the same antibody concentration 177Lu-DOTA-HH1 was significantly more effective in inhibiting cell growth in vitro than 177Lu-DOTA-rituximab. SCID mice were intravenously injected with 10 million Daudi cells one week before RIC treatment. A significantly increased survival of mice treated with 177Lu-DOTA-HH1 as compared with 177Lu-DOTA-rituximab treatment was observed. Furthermore, the toxicity of the 177Lu-DOTA-HH1 treatment was significantly lower than for 177Lu-DOTA-rituximab. In addition, we have compared binding properties and biodistribution of HH1 with rituximab. The affinity of HH1 to CD37 was similar to the affinity of rituximab to CD20. The CD37-HH1 complex was internalized 10 times faster than CD20-rituximab. Both antibodies had a relevant biodistribution and low uptake in bone. This work warrants further preclinical and clinical studies with 177Lu-HH1. Disclosures: Dahle: Nordic Nanovector: Employment. Bruland:Nordic Nanovector: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Larsen:Nordic Nanovector: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Selectin Inhibition Disrupts Multiple Myeloma Cells Interaction with the Bone Marrow Microenvironment and Sensitizes Them to Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 453-453
Author(s):  
Abdel Kareem A. Azab ◽  
Phong Quang ◽  
Feda Azab ◽  
John Magnani ◽  
John Patton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Bioluminescence Imaging ◽  
Plasma Cells ◽  
Tumor Burden ◽  
Advisory Committees ◽  
Normal Plasma

Abstract Abstract 453 Introduction: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment, which includes stromal (BMSCs) and endothelial cells (ECs), plays a crucial role in MM pathogenesis and drug resistance. We have previously shown that the chemokine stromal cell-derived factor-1 (SDF-1), its receptor-CXCR4, and GTPases in the downstream signaling of the receptor regulate this interaction. Selectins are adhesion molecules which are involved in the primary interaction of lymphocytes with the endothelium. In this study, we have tested the expression of selectins and their ligands in MM, and explored their role in the interaction with the BM milieu and its potential therapeutic applications. Methods AND RESULTS: Flow cytometry and immunohistochemical (IHC) staining of tissue microarrays revealed that P-selectin glycoprotein ligand-1 (PSGL-1, CD162) was over expressed in MM cells from patients (n=20) and cell lines (MM1s, H929, RPMI, OPM1 and OPM2) compared to normal plasma cells (n=3). Gene expression profiling (GEP) analysis showed that the expression of PSGL-1 was directly correlated with MM stage of progression (normal plasma cells, n=15 < MGUS, n=20 < smoldering MM, n=23 < MM, n=68 p<0.01). Moreover, it was shown that both BMSCs (isolated from MM patients and HS5 cell line) and ECs (isolated from MM patients and HUVECs) had high expression of P-selectin. SDF1 treatment increased the expression of P-selectin on ECs but it had no effect on the expression of PSGL on MM cells. The interaction of PSGL and P-selectin played a major role in the adhesion of MM cell to both BMSCs and ECs, and the inhibition of this interaction either by the pan-selectin inhibitor GMI-1070 (500uM, 3hrs) or by knock-down of P-selectin expression significantly decreased (50-60%) the adhesion of MM cells to BMSCs and ECs. The CXCR4 inhibitor AMD3100 (25uM, 3hrs) similarly induced similar inhibition of adhesion, and the combination of AMD3100 and GM1070 had more profound inhibition of MM adhesion to BMSCs and ECs (p = 0.006). Both AMD3100 and GMI1070 induced MM cell de-adhesion from BMSCs and ECs, but the combination of both drugs was not additive. AMD3100, GMI1070 or their combination prevented BMSCs or ECs mediated induction of proliferation of MM cells. Moreover, it was shown that the co-culture of MM cells with BMSCs and ECs reduced their sensitivity to bortezomib (5nM, 24hrs) and dexamethasone (25nm, 24hrs) compared to MM cells cultured alone. Importantly, GMI1070 restored the sensitivity of MM cells to bortezomib and dexamethasone to the level observed without co-culture with BMSCs or ECs. These effects were next tested in vivo using an orthotopic xenograft model of MM. SCID-beige mice were injected with luciferase-expressing MM1S cells, and tumor burden was assessed bioluminescence imaging. Mice with established disease were divided into treatment groups (n=10 per group) treated with vehicle, GMI1070 by osmotic pump, velcade at 1.5 mg/kg IP weekly, or a combination of GMI1070 and bortezomib. Tumor burden was determined by bioluminescence imaging. Treatment with GMI1070 alone was not different from vehicle treated control mice. While treatment with bortezomib alone had a minimal delay in tumor progression, the combined treatment of bortezomib and GMI1070 resulted in synergistic anti-tumor efficacy (p=0.0017). Conclusion: We have shown that PSGL-1 is highly expressed in MM cells as compared to normal plasma cells, and that it plays a major role in the interaction of MM cells with the BM microenvironment in relation with the SDF1/CXCR4 axis in vitro, an effect which was inhibited by the pan-selectin inhibitor GMI1070. We also demonstrated that selectin inhibition by GMI1070 reduced MM cell proliferation induced by BMSCs and ECs sensitized MM cells to bortezomib and dexamethasone in vitro, and significantly increased the sensitivity of MM tumors to bortezomib in vivo. This information provides the rationale for future clinical trials for increasing efficacy of existing therapies through a combination with selectin inhibitors for the treatment of myeloma. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Overcoming Drug Resistance in Myeloma By Synchronized Delivery of Therapeutic and Bone Marrow Disrupting Agents By Nanoparticles Targeting Tumor-Associated Endothelium

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1931-1931
Author(s):  
Cinzia Federico ◽  
Barbara Muz ◽  
Jennifer Sun ◽  
Kinan Alhallak ◽  
Justin King ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Tumor Progression ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Dual Drug

Abstract Proteasome inhibitors (PIs) have improved the treatment of multiple myeloma (MM) and prolonged patient survival, but several challenges remain to overcome drug-resistance and toxicity. Bone marrow microenvironment (BMM) drives tumor progression and PIs-resistance in MM; and agents that inhibit the interaction between MM and BMM have been shown to re-sensitize MM cells to therapy. However, the synchronized in vivo delivery of BMM-targeting agents with PIs has been a challenge so far. Nanoparticles offer a valuable platform to encapsulate drugs, and if functionalized, they can facilitate specific delivery to tumor, thus improving treatment efficacy and reducing off-target effects. Within the BMM, the endothelium plays a relevant tumor promoting role. By analyzing the expression of an array of markers in normal and in MM-related endothelium, we found high levels of P-selectin expression on MM-activated endothelial cells (ECs) than normal cells and on ECs collected from the BM of either MM patients or MM-bearing mice compared to their respectively healthy BMMNCs. We next sought to develop lipid nanoparticles (LNPs) targeting the MM-related endothelium, loaded with both PI and BMM-targeting agent for synchronized delivery and reversal of the BMM-induced drug resistance. At this aim, we developed targeted LNPs towards P-selectin by decorating their surface with P-selectin-glycoprotein-ligand-1 (PSGL-1). PSGL-1-targeted LNPs showed specific binding to recombinant P-selectin than identically non-targeted particles, and to MM-associated endothelium compared to healthy endothelium, both in vitro and in vivo. To reverse BMM-induced resistance, LNPs were loaded with bortezomib (BTZ) together with a BMM disrupting agent, ROCK-inhibitor (Y-27632) that inhibits the downstream signaling of the RhoA GTPase pathway, known to be instrumental to the interaction of MM cells with BMM. Consequently, we tested the effect of synchronized delivery of BTZ and Y-27632 in the same LNP on MM cell survival in co-culture with the BMM in vitro. While Y-27632-loaded LNPs did not affect cell proliferation, LNPs loaded with both Y-27632 and BTZ enhanced responsiveness of MM cells to BTZ, compared to BTZ-loaded LNPs, thus overcoming the BMM-induced resistance. Mechanistically, we observed more significant inhibition of PI3K and MAPK signaling, decrease of pRb and up-regulation of p21 and induction of pro-apoptotic pathway (caspase-3, caspase-9 and PARP) by drug-loaded LNPs, compared to free drugs. In addition, drug-loaded LNPs were able to decrease adhesion and impair the migration of MM cells to ECs. We also investigated the in vivo efficacy of BTZ/Y-27632-loaded PSGL-1-targeted LNPs in a humanized murine model of MM. The synchronized delivery of both agents using dual drug-loaded PSGL-1-targeted LNPs delayed the MM tumor progression and prolonged survival significantly more than all the controls. The synchronized delivery of both agents using dual drug-loaded PSGL-1-targeted LNPs delayed the MM tumor progression and prolonged survival significantly more than all the controls (vehicle, BTZ and Y-27632 alone or in combination as free drugs, or encapsulated in non-targeted or in PSGL-1-targeted LNPs) demonstrating that both P-selectin targeting and combination of Y-27632 with BTZ reverses the BMM-induced drug resistance and enhances the efficacy of therapy in vivo. Altogether, our data demonstrate the ability of PSGL-1-decorated LNPs to specifically target MM-BMM; to efficiently encapsulate and deliver drugs to tumor tissue; to overcome BMM-induced drug resistance in vitro and in vivo, to reduce tumor growth and prolong overall survival. This study provides the preclinical basis for future clinical trials using MM-BMM-targeted nanomedicine able to enhance the effect of PIs or other drugs for the treatment of MM. Disclosures Roccaro: GILEAD: Research Funding; AMGEN: Other: Advisory Board. Vij:Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Azab:Cellatrix LLC: Equity Ownership, Other: Founder and owner; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner; Ach Oncology: Research Funding; Glycomimetics: Research Funding.

scholarly journals Biodistribution and Efficacy of L-Annamycin in a Novel Imageable AML Mouse Model

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5150-5150
Author(s):  
Rafal Zielinski ◽  
Krzysztof Grela ◽  
Stanislaw Skora ◽  
Rodrigo Jacamo ◽  
Izabela Fokt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Residual Disease ◽  
Advisory Committees ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Equity Ownership

Annamycin (Ann) is an anti-tumoral anthracycline whose anti-leukemia activity is relatively unaffected by P-glycoprotein-related multidrug resistance. Unlike for the related doxorubicin (DOX), Ann accumulates in multidrug resistant cell lines, which is accompanied by DNA damage and apoptosis. In preclinical toxicology studies, in contrast to DOX, free Ann displayed a greatly reduced cardiotoxicity, while L-Ann appeared to be non-cardiotoxic. A liposomal formulation of Ann, termed L-Annamycin (L-Ann), is currently evaluated in patients with acute myeloid leukemia (AML). Anti-leukemia activity of Ann was demonstrated in several leukemia models as judged by circulating blast cytoreduction and extension of overall survival. However, the efficacy of L-Ann in the microenvironment of the bone marrow and other organ tissues remains unclear. In the current study, we assessed the anti-AML efficacy of Ann in a novel AML model that allows visualizing the dynamics of individual AML cells in vivo by two-photon microscopy. In this model, mouse AML cells bearing the MLL/ENL-FLT3/ITD[p53-/-] mutations co-express high levels of the cyan fluorescent protein mTurquoise2. Upon intravenous infusion of several tens of thousands cells into syngeneic immunocompetent C57BL6 mice, lethal AML disease reliably develops within 2 weeks. Using host mice expressing appropriate fluorescence reporter genes, the bright cyan fluorescence enables sensitive intravital imaging of individual AML cells in the context of organ architecture. Using this model in Thy1-RFP reporter mice expressing red fluorescence in all organ tissues with the blood flow marked by BSA-AF647 fluorescence, we evaluated AML cellularity reduction in the bone marrow and other organs after a single dose of L-Ann as well as in response to chronic treatment. In addition, we assessed the localization of the surviving AML cells at a high spatial resolution. We evaluated the in vivo organ biodistribution of intravenously infused L-Ann in C57BL6 mice by flow cytometry and two-photon microscopy based on the intrinsic fluorescence of the drug. In addition, we visualized the intracellular compartmentalization of L-Ann using confocal microscopy. Consistent with in vitro findings, we observed a rapid and deep reduction of AML blasts in the peripheral blood after a single dose of L-Ann in a dose-dependent manner (1-4 mg/kg). This reduction was strongly correlated with prolongation of animal survival from 14 days (vehicle) to 37 days (L-Ann 4 mg/kg once weekly started on day 10). In vitro and intravital microscopy revealed a distinct pattern of L-Ann distribution in organ tissues, which correlated in part with the local index of AML cellularity reduction and residual disease localization. Interestingly, in addition to the expected uptake of Ann in the cell's nucleus, Ann was also accumulated in the cytosol of the cells. This bi-compartmental intracellular distribution pattern contrasted with the nuclear-only localization of DOX. Administration of L-Ann early in the course of AML resulted in occasional complete responses some of which associated with resistance to AML re-challenge, suggesting capacity for anti-AML immune memory induction. This study confirms the efficacy of the drug in the model setting of syngeneic, immune-competent AML. Besides reinforcing the rationale for further development of Annamycin in AML, this study demonstrates a highly advantageous AML mouse model that is highly informative in studies of AML pharmacology, minimum residual disease (MRD), microenvironment and immunology. Disclosures Fokt: Moleculin Biotech, Inc.: Equity Ownership, Research Funding. Andreeff:Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees. Priebe:Moleculin Biotech, Inc.: Consultancy, Equity Ownership, Research Funding. Zal:VueBio.com: Equity Ownership; BioLineRx: Research Funding; Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding; NIH-CTEP: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding.

scholarly journals Phase II Trial of the Combination of Ixazomib, Lenalidomide, and Dexamethasone in High-Risk Smoldering Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 804-804 ◽  
Author(s):  
Mark Bustoros ◽  
Chia-jen Liu ◽  
Kaitlen Reyes ◽  
Kalvis Hornburg ◽  
Kathleen Guimond ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
High Risk ◽  
Disease Progression ◽  
Board Of Directors ◽  
Rna Sequencing ◽  
Research Funding ◽  
Response Rate ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Background. This study aimed to determine the progression-free survival and response rate using early therapeutic intervention in patients with high-risk smoldering multiple myeloma (SMM) using the combination of ixazomib, lenalidomide, and dexamethasone. Methods. Patients enrolled on study met eligibility for high-risk SMM based on the newly defined criteria proposed by Rajkumar et al., Blood 2014. The treatment plan was designed to be administered on an outpatient basis where patients receive 9 cycles of induction therapy of ixazomib (4mg) at days 1, 8, and 15, in combination with lenalidomide (25mg) at days 1-21 and Dexamethasone at days 1, 8, 15, and 22. This induction phase is followed by ixazomib (4mg) and lenalidomide (15mg) maintenance for another 15 cycles. A treatment cycle is defined as 28 consecutive days, and therapy is administered for a total of 24 cycles total. Bone marrow samples from all patients were obtained before starting therapy for baseline assessment, whole exome sequencing (WES), and RNA sequencing of plasma and bone marrow microenvironment cells. Moreover, blood samples were obtained at screening and before each cycle to isolate cell-free DNA (cfDNA) and circulating tumor cells (CTCs). Stem cell collection is planned for all eligible patients. Results. In total, 26 of the planned 56 patients were enrolled in this study from February 2017 to April 2018. The median age of the patients enrolled was 63 years (range, 41 to 73) with 12 males (46.2%). Interphase fluorescence in situ hybridization (iFISH) was successful in 18 patients. High-risk cytogenetics (defined as the presence of t(4;14), 17p deletion, and 1q gain) were found in 11 patients (61.1%). The median number of cycles completed was 8 cycles (3-15). The most common toxicities were fatigue (69.6%), followed by rash (56.5%), and neutropenia (56.5%). The most common grade 3 adverse events were hypophosphatemia (13%), leukopenia (13%), and neutropenia (8.7%). One patient had grade 4 neutropenia during treatment. Additionally, grade 4 hyperglycemia occurred in another patient. As of this abstract date, the overall response rate (partial response or better) in participants who had at least 3 cycles of treatment was 89% (23/26), with 5 Complete Responses (CR, 19.2%), 9 very good partial responses (VGPR, 34.6%), 9 partial responses (34.6%), and 3 Minimal Responses (MR, 11.5%). None of the patients have shown progression to overt MM to date. Correlative studies including WES of plasma cells and single-cell RNA sequencing of the bone microenvironment cells are ongoing to identify the genomic and transcriptomic predictors for the differential response to therapy as well as for disease evolution. Furthermore, we are analyzing the cfDNA and CTCs of the patients at different time points to investigate their use in monitoring minimal residual disease and disease progression. Conclusion. The combination of ixazomib, lenalidomide, and dexamethasone is an effective and well-tolerated intervention in high-risk smoldering myeloma. The high response rate, convenient schedule with minimal toxicity observed to date are promising in this patient population at high risk of progression to symptomatic disease. Further studies and longer follow up for disease progression are warranted. Disclosures Bustoros: Dava Oncology: Honoraria. Munshi:OncoPep: Other: Board of director. Anderson:C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; BMS: Consultancy.

scholarly journals TTI-281, an Orally Bioavailable Bromodomain and Extraterminal Domain (BET) Inhibitor, Is a Promising Candidate for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2095-2095
Author(s):  
Zezhou Wang ◽  
Jaehyun Choi ◽  
Peter Dove ◽  
Chunlei Wang ◽  
Aaron D. Schimmer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Therapeutic Agents ◽  
Vehicle Control ◽  
Advisory Committees ◽  
Bet Inhibitor ◽  
Low Toxicity ◽  
Orally Bioavailable

Abstract Although recent advances in the development of multiple myeloma (MM) therapies such as proteasome inhibitors and immunomodulatory agents have improved patient outcomes, MM remains incurable. Additional therapeutic agents with high efficacy, low toxicity and the convenience of oral administration are in high demand. BET inhibitors, such as JQ-1, have been considered as potential therapeutic agents for MM. In the present study, we report that TTI-281, an orally bioavailable BET inhibitor, displays anti-MM activity with a low toxicity profile in preclinical studies. First, TTI-281 was tested for binding and anti-tumor activity in vitro. BROMOscan and AlphaScreen assays demonstrated that TTI-281 bound to bromodomains of BRD2/BRD3/BRD4 with Kd values less than 10 nM. In MTS assays, TTI-281 inhibited the growth of MM cell lines (MM.1s, NCIH929, and RPMI-8826) with cell growth-inhibition (IC50) values less than 300 nM. Next, in vitro ADME screening and in vivo PK studies were conducted. Permeability assays using murine gastrointestinal epithelial cells indicated that TTI-281 had good permeability with little efflux liability (efflux ratio <1), suggesting favorable properties for oral absorption. Indeed, TTI-281 displayed excellent oral bioavailability in both mice and rats (93.1% and 91.8%, respectively). In addition, TTI-281 did not interfere with the metabolism of representative CYP isozyme substrates at concentrations up to 50 μM in pooled human liver microsomes. Data also suggested minimal potential for drug-drug interactions, allowing for the possible combination with first-line therapy to improve therapeutic and survival outcomes. Finally, TTI-281 was tested for anti-myeloma efficacy and tolerability in vivo. NOD-SCID mice (n=10/group) subcutaneously engrafted with the human myeloma cell line MM.1S were treated orally once daily for 21 days with different doses of TTI-281, vehicle control or the benchmark drug carfilzomib. TTI-281 reduced tumor growth in a dose-dependent manner in this MM xenograft model. At 30 mg/kg/day, TTI-281 led to a statistically significant decrease in tumor growth compared with the vehicle control and carfilzomib (reduced tumor volume: 67% after TTI-281 treatment vs 33% after carfilzomib treatment, p<0.0003). Furthermore, TTI-281 treatment was well tolerated, with no effect on body weight or other obvious toxicity. In summary, our preclinical data suggest that the orally available BET inhibitor TTI-281 has an excellent efficacy and safety profile, highlighting its potential as a promising drug candidate for myeloma therapy. Disclosures Wang: Trillium Therapeutics: Employment, Patents & Royalties. Choi:Trillium Therapeutics: Employment. Dove:Trillium Therapeutics: Employment, Patents & Royalties. Wang:Trillium Therapeutics: Employment. Schimmer:Novartis: Honoraria. Petrova:Trillium Therapeutics Inc: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Uger:Trillium Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Slassi:Trillium Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 133-133 ◽  
Author(s):  
Patricia Maiso ◽  
AbdelKareem Azab ◽  
Yang Liu ◽  
Yong Zhang ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Plasma Cells ◽  
Bone Marrow Microenvironment ◽  
Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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