Microenvironment-Dependent Synthetic Lethality: Implications for Tumor Pathophysiology and Anti-Cancer Drug Discovery.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1722-1722
Author(s):  
Douglas McMillin ◽  
Jake Delmore ◽  
Ellen Weisberg ◽  
Joseph M. Negri ◽  
Steffen Klippel ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Stromal Cells ◽  
Research Funding ◽  
Synthetic Lethality ◽  
Cancer Drug ◽  
Advisory Committees ◽  
Accessory Cells

Abstract Abstract 1722 Poster Board I-748 Conventional anti-cancer drug screening in vitro has traditionally been performed in the absence of accessory cells of the tumor microenvironment. These normal cells of the bone marrow milieu can profoundly alter anti-tumor drug activity. To address this major limitation of traditional in vitro models, we developed the tumor cell-specific in vitro bioluminescence imaging (CS-BLI) assay. In this platform, tumor cells (e.g. myeloma, leukemia and solid tumors) stably expressing bioluminescent reporters are co-cultured with non-malignant accessory cells (e.g. stromal cells) to selectively quantify tumor cell viability to treatments in presence vs. absence of accessory cells. We applied CS-BLI to test various chemical libraries and showed that this platform is high-throughput scalable. We also identified stroma-induced chemoresistance in diverse malignancies, including imatinib-resistance in leukemic cells, as well as MM cell resistance to certain investigational agents. The majority of compounds screened in our studies were less active against tumor cells in the presence of stromal cells compared to their absence. Most interestingly, however, we identified a fraction of compounds which were more active against tumor cells in the presence of stromal cells. For example, we identified reversine, a compound exhibiting this stroma-dependent synthetic lethality in vitro, which we further confirmed in vivo, as it is active in an orthotopic model of diffuse MM bone lesions, but not in conventional subcutaneous xenografts. Mechanistically, in vitro kinase activity assays showed that reversine exhibits a distinct pattern of inhibition against targets such as Auroras, JAK2, and SRC, but not against other important kinases for MM survival, such as AKT1, 2, 3, FGFR3, or GSK3. These observations are compatible with the role of SRC and JAK kinases as downstream regulators of IL-6/IL-6R signaling, a key cascade triggered by tumor-stromal interactions in MM. Further mechanistic evaluation of this interaction at the transcriptional level showed that a stromal-induced gene expression signature in MM tumor cells correlates with inferior overall survival in patients with advanced MM (APEX dataset) and includes enhanced amplitude of signatures for activated Akt, Ras, NF-κB, HIF-1á, myc, hTERT, and IRF4; as well as signatures for biological aggressiveness and stem cell self-renewal. This suggests that selective inhibitors which block the activity of these pathways may exhibit tumor specific stromal-dependent synthetic lethality. Historically, synthetic lethality has focused on how tumor cells harboring specific constitutive oncogenetic lesions are responsive to certain agents, but not in absence of these genetic events. Our study introduces the notion that a synthetic lethal phenotype, rather than being exclusively genotype-dependent, can also be driven by the extrinsic influences of the tumor microenvironment. Importantly, the CS-BLI system can probe both genetically- and microenvironment-related synthetic lethality in a high-throughput scalable manner. This allows the testing of a large number of permutations, including multiple candidate therapeutics, cell lines, and non-malignant accessory cells, thus enabling the previously intractable large-scale evaluation of how genetics and microenvironment play a role in modulating cancer cell response to treatment. Importantly, unlike conventional screening, CS-BLI can also identify agents with increased activity against tumor cells when interacting with stroma. These agents, in the past, may have been discarded from further preclinical or clinical development. We now provide a system with which to evaluate the role of the tumor microenvironment and identify novel agents capable of overcoming its protective effects. Disclosures Munshi: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richardson:Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

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

Blood ◽  
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.

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

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

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

Emetine Elicits Apoptosis of Intractable B-Cell Lymphoma Cells with MYC Rearrangement through Inhibition of Glycolytic Metabolism

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3019-3019
Author(s):  
Tomohiro Aoki ◽  
Kazuyuki Shimada ◽  
Akihiko Sakamoto ◽  
Keiki Sugimoto ◽  
Takanobu Morishita ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Glucose Metabolism ◽  
Tumor Cells ◽  
Drug Screening ◽  
Stromal Cells ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Lymphoma Cells

Abstract Background: Despite remarkable advances of initial treatment in diffuse large B-cell lymphoma (DLBCL), the prognosis of the disease with MYC rearrangement remains poor with a median overall survival of less than 1 year. The application of intensive or targeting treatment failed to show a benefit for the disease, an innovative approach should be thus required to overcome the obstacle of MYC rearrangement. Recent findings revealed that the close interaction of tumor cells with stromal cells in its microenvironment is involved in resistance to chemotherapy, and that tumor microenvironment has been shed light on a potential attractive therapeutic target. Purpose: To overcome poor prognoses of intractable DLBCL with MYC rearrangement, we explored an effective drug targeting tumor microenvironment through the high-throughput drug screening (Sugimoto et al. Sci Rep. 2015). Material and methods: Allpatient samples were experimentally used with written informed consent. To perform drug screening against primary patient lymphoma cells with intractable clinical course,we firstly developed co-culture system of lymphoma cells and stromal cells, which allowed us to culture them in vitro.For this, isolated stromal cells derived from human lymph node were prepared. Then 3,440 compounds mainly containing known pharmacologically active substance or off-patent drugs were screened to identify effective drugs for patient lymphoma cells. The efficacy and mechanism of action of the drug were confirmed by subsequent in vitro and in vivo analyses. Results: Two patient tumor cells with MYC/BCL2 rearrangement were used for the drug screening. Both patients developed refractory diseases within 1 year after diagnosis. In the screening analyses, primary lymphoma cells obtained from lymph node for patient (Pt) #1 were used, and tumor cells from PDX mouse model for Pt #2 were used to validate the result of Pt #1. The both tumor cells could not survive in in vitro monoculture, while the both lymphoma cells could remarkably survive longer in co-culture with stromal cells. Then we performed drug screening against primary tumor cells from Pt #1. Ninety-nine compounds with the viability of tumor cells less than 0.5 were identified, and we validated cell death of these 99 compounds against the other lymphoma cells from Pt #2. Among 10 compounds identified as potentially effective for the both tumor cells, we picked out emetine, which induced cell death against the both cells with an IC50 of 312 nM and 506 nM, respectively. Regarding the effect of emetine on stromal cells, the proliferation and survival was not affected in the concentration of 2 µM emetine whose concentration was used for the screening. However, stromal cells pretreated 0.5 µM emetine decreased a support potential to tumor cells resulting from decreased ATP production and glutathione in tumor cells. In terms of the effect of emetine on tumor cells, the drug induced a G2/M arrest in tumor cells, which resulted in induction of apoptosis. Based on previous finding that emetine suppresses HIF-1a expression, which is one of key regulators glucose metabolisms, we investigated the expression in tumor cells under the treatment of emetine. HIF-1a expression was suppressed in tumor cells as expected; we subsequently analyzed the status of glucose metabolism in tumor cells. The expression of key enzymes including HK2, PDK1, and LDHA were suppressed and ATP production and GLUT1 expression were also suppressed. The serial cascade of the alteration of glucose metabolism including the decreased mitochondrial membrane potential, the alteration of pentose phosphate pathway, and the reduction of NADPH and glutathione leading to the accrual of reactive oxygen species (ROS) was observed under the presence of emetine. In in vivo analyses, significant growth inhibition was observed under the emetine treatment (Figure A and B). Conclusions: Emetine identified by the drug screening is clearly effective for patient lymphoma cells with intractable clinical course in vitro and in vivo. Subsequent analyses regarding the mechanism of action of emetine revealed that the drug affected the both tumor cells and stromal cells in tumor microenvironment through the inhibition of glucose metabolism. Further investigations of the translation to clinic should be warranted. Disclosures Sugimoto: Otsuka Pharmaceutical Co., Ltd.: Employment. Kiyoi:Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Phizer Japan Inc.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Alexion Pharmaceuticals: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Celgene Corporation: Consultancy; Zenyaku Kogyo Co.LTD.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding.

scholarly journals Duobody-CD3xCD20 Induces Potent Anti-Tumor Activity in Malignant Lymph Node B Cells from Patients with DLBCL, FL and MCL Ex Vivo, Irrespective of Prior Treatment with CD20 Monoclonal Antibodies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4066-4066
Author(s):  
Hilma J Van Der Horst ◽  
A. Vera de Jonge ◽  
Ida H Hiemstra ◽  
Anne T Gelderloos ◽  
Daniella RAI Berry ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Cell ◽  
Cytotoxic Activity ◽  
Board Of Directors ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Research Funding ◽  
Advisory Committees

DuoBody-CD3xCD20 (GEN3013) is a novel clinical-stage CD3 bispecific antibody (bsAb) targeting CD20-positive tumor cells. GEN3013 was previously shown to induce potent T cell-mediated cytotoxicity towards B cell Non-Hodgkin lymphoma (B-NHL) cell lines in vitro and in vivo. Here, we investigated the cytotoxic activity of GEN3013 in tumor cells obtained from lymph node (LN) biopsies of B-NHL patients, who were newly diagnosed (ND) or relapsed from/refractory to (RR) treatment regimens containing CD20 monoclonal antibodies. Moreover, we explored whether specific tumor microenvironment characteristics could be associated with sensitivity to GEN3013. To test the intrinsic susceptibility of B-NHL cells to GEN3013, independent of interpatient variation in tumor T cell frequency or activation status, single cell suspensions obtained from LN of B-NHL patients were incubated with GEN3013 in the presence of allogeneic PBMC from a single donor, at an effector to target (E:T) ratio 10:1. GEN3013 (30 ng/mL) induced median tumor cell lysis of 64% in Diffuse Large B Cell Lymphoma (DLBCL, n=14), 69% in Follicular Lymphoma (FL, n=14) and 84% in Mantle Cell Lymphoma (MCL, n=8) samples, with EC50 values ranging from 0.01-3.9 ng/ml. Importantly, cytotoxic activity of GEN3013 was comparable in ND (n=24) and RR (n=12) patients (Figure 1). In these assays considerable heterogeneity in T cell activation, as assessed by expression of CD25, CD69 and granzyme B release, was observed. Furthermore, high expression of T cell activation markers was not always associated with high levels of GEN3013 cytotoxic activity, suggesting tumor-intrinsic resistance mechanisms. In parallel, in all B-NHL samples GEN3013-mediated cytotoxicity was assessed without the addition of allogeneic PBMCs, thus purely relying on T cells present in the LN biopsy. In this setting, median tumor cell lysis was lower; 18% in DLBCL (range 0-46%), 17% in FL (range 0-46%) and 0% in MCL (range 0-11%), but strongly correlated with the number of T cells present in the single cell suspensions. Analysis of the tumor microenvironment by 7 color immunohistopathology of matched FFPE-embedded tumor biopsies (n=24), confirmed that the T cell frequency in the tumor biopsies was the major determinant of GEN3013 cytotoxic activity in DLBCL, FL and MCL. Moreover, experiments using (MACS) purified T cells from 4 DLBCL and 5 FL LN biopsies demonstrated that the intrinsic capacity of tumor LN T cells to induce GEN3013 mediated cytotoxicity was comparable to healthy donor T cells. Detailed tumor microenvironment analysis based on 7 color immunohistopathology staining, including relative frequency and spatial distribution of CD4 and CD8 T cells and macrophages, as well as the T cell activation status, in relation to sensitivity to GEN3013 mediated tumor cell lysis is ongoing and results will be presented. In conclusion, GEN3013 induced potent cytotoxicity in tumor cells of DLBCL, FL and MCL patients ex vivo, irrespective of prior treatment with CD20 monoclonal antibodies. Autologous T-cells at the tumor site were able to mediate GEN3013-induced cytotoxicity, and cytotoxic activity was enhanced in presence of PBMCs suggesting that optimal tumor cell kill by GEN3013 is dependent on T-cells in the tumor microenvironment. The cytotoxic capacity of B-NHL patient T cells within the tumor microenvironment was comparable to healthy donor peripheral blood T cells, emphasizing the therapeutic potential of CD3 bsAb in B-NHL. A First-in-Human trial to assess the safety and preliminary efficacy of GEN3013 in B-NHL patients is currently ongoing (NCT03625037). Figure 1 Cytotoxic activity induced by GEN3013 compared to CD3xcontrol bsAb (both 30ng/ml) towards tumor cells obtained from lymph node (LN) biopsies of newly diagnosed (ND) versus relapse or refractory (RR) DLBCL, FL and MCL patients. GEN3013 achieved comparable lysis in ND versus RR patients (Mann-Whitney U test; not significant). Error bars represent median ± interquartile range. Figure 1 Disclosures Van Der Horst: Genmab: Other: Financial Support. Hiemstra:Genmab: Employment, Equity Ownership, Other: Warrants. de Jong:Genmab: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Chamuleau:Genmab: Research Funding. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Breij:Genmab: Employment, Other: Warrants. Roemer:Genmab: Research Funding. Mutis:Celgene: Research Funding; Janssen Research and Development: Research Funding; Onkimmune: Research Funding; Genmab: Research Funding.

Preclinical Studies of Salinomycin In Multiple Myeloma (MM) Models: Targeting of Side Population (SP) Cells In the Context of Tumor – Microenvironment Interactions.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1574-1574
Author(s):  
Efstathios Kastritis ◽  
Jana Jakubikova ◽  
Jake Delmore ◽  
Steffen Klippel ◽  
Douglas W. McMillin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cell ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Committees ◽  
Ic50 Values

Abstract Abstract 1574 Cancer cells with stem cell-like features are a topic of intense research because their resistance to existing drugs is considered a culprit for relapses, even in patients with complete remission defined by clinical, biochemical and imaging parameters or by sensitive molecular techniques. Salinomycin, an antibacterial and coccidiodostatic ionophore, is reported (Cell 2009;138(4):645-59) to be >100-fold more potent against breast cancer cells with stem cell-like phenotype after mesenchymal transdifferentiation due to stable transfection with shRNA against CDH1 than against the parental cells. We evaluated whether salinomycin could also exhibit a similar activity against stem cell-like cells in multiple myeloma (MM). To establish a comparative reference for such potential activity, we first tested salinomycin (0-10 uM for up to 72hrs) against a panel of 15 MM cell lines and observed IC50 values <1 uM in 10/15 cell lines tested, including >80% reduction of tumor cell viability in 6/15 cell lines tested at 0.5 uM, i.e. levels lower than the IC50 values for in vitro activity of salinomycin against breast cancer cells with (HMLE-shCDH1, IC50 ∼1 uM) or without (HMLE-shControl, IC50 >>10 uM) stem cell-like features. CD138+ purified primary tumor cells from 3 MM patients responded to salinomycin with IC50 values (105, 332 and 750 nM, respectively) in the same range as MM cell lines. In vitro combinations with bortezomib, doxorubicin, melphalan, and dexamethasone showed overall no antagonism, while evidence of additive or even synergistic effect could be identified in certain dose ranges. Because MM cell lines and primary tumor cells responded concordantly to salinomycin and with higher sensitivity than breast cancer stem cell-like cells, we hypothesized that MM cells may in general be more responsive to salinomycin than other tumors. Since tumor-stromal interactions can increase the expression of transcriptional signatures of “stemness” in MM cells, we embarked on characterizing the anti-MM properties of salinomycin using compartment-specific bioluminescence imaging (CSBLI) assays. These showed that co-culture with stromal cells did not confer resistance to salinomycin in 5 MM cell lines (MM.1S, OCI-My5, KMS-11, KMS-18, NCI-H929) and in fact enhanced its activity against 4 of them. Side population (SP) cells, defined by their ability to efflux Hoechst stain, represent a stem cell-like population which was identified in MM cell lines and could represent the functional equivalent of the mesenchymally transdifferentiated breast cancer stem cell-like cells. We observed that salinomycin reduces the SP fraction of MM cell lines at doses >20 times lower than those required for in vitro effect against the bulk <<main population>> of the respective cell lines. Interestingly, the anti-SP effect of salinomycin was more pronounced in the presence of stroma, similarly to the CSBLI studies on the entire MM cell population and consistent with our prior observation that tumor-stroma interaction enhances transcriptional signatures of ≪stemness≫ in the tumor compartment. However, when we tested the in vivo anti-MM activity of salinomycin in an orthotopic model of i.v. injected Luc+ MM cells, no anti-MM activity (in terms of tumor burden decrease or overall survival prolongation) was observed at the maximum tolerated dose (1 mg/kg i.p. daily, which is consistent with most studies reported thus far in the literature). Ex vivo treatment of KMS-11 cells with salinomycin doses (100 nM for 72 hrs) selectively targeting SP cells was followed by s.c. injection of these cells or vehicle-treated controls in sublethallly irradiated SCID/NOD mice, but no statistically significant improvement in tumor burden or overall survival was observed. Our in vitro results indicate that salinomycin exhibits intriguing in vitro anti-MM activity, not only against SP cells but also against the bulk ≪main≫ MM cell population, even in the presence of stromal support. In contrast, the in vivo activity of salinomycin is compromised by side effects in the orthotopic model of MM lesions, while short term ex vivo exposure of tumor cells is conceivably insufficient to eradicate clonogenic cells and lead to appreciable delay in tumor growth in vivo. Our studies point to intriguing features as well as notable challenges that have to overcome before salinomycin or other more selective agents of this class can be safely tested in clinical trials in MM. Disclosures: McMillin: Axios Biosciences: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

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

Blood ◽  
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.

Mesenchymal Stromal Cells Derived From Chronic Lymphocytic Leukemic Patients Express Different Genes and Produce Different Cytokines Compared to MSC Derived From Normal Subjects,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3872-3872
Author(s):  
Wei Ding ◽  
Traci Sassoon ◽  
Esteban Braggio ◽  
Charla Secreto ◽  
Neil Kay
Keyword(s):  
Board Of Directors ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Research Funding ◽  
Cytokine Production ◽  
Fold Increase ◽  
Normal Subjects ◽  
Leukemic Cells ◽  
Advisory Committees

Abstract Abstract 3872 Background: Mesenchymal stromal cells are one of the components in the CLL microenvironment known to support leukemic cells survival and growth. Crosstalk between CLL B-cells and marrow-derived mesenchymal stromal cells (MSC) can modulate the activation of both cell types. Indeed we have shown that soluble factors secreted from CLL B cells can activate MSC with increased production of angiogenic cytokines (Blood. 2010. 116:2984). We hypothesize that MSC derived from CLL patients are modified by their interaction with leukemic cells in their gene expression profiles (GEP) and cytokine production (CP) capabilities. Given this, intrinsic differences should be present amongst MSC derived from CLL patients versus normal subjects. Method: MSC collected from CLL patients with Rai stage 1–4 before any therapy or who have not had therapies for at least 2 years as well as normal MSC from healthy controls were cultured and expanded one time in vitro. Subsequently, MSC after one in vitro passage were sorted with CD45 negative selection to eliminate any contaminating non-stromal cells. Approximately 100,000 to 300,000 cells were then stored at −70C and used for RNA purification. Nine CLL MSC and five normal MSC cell samples had RNA purified from each sample and then the RNA preparations were used for comparative analysis of GEP using affymetrix U133A plus B chip. Data were transformed using two different methods: MAS5 or Plier and only probes that were present in at least 50% of cases were included in the analysis. We have recently observed that MSC derived from bone marrow of CLL patients promotes Th17 cell differentiation (Blood. 2010. 116: Abstract 2414). Since IL-17 is the signature cytokine produced by TH17 cells, we decided to test if IL-17 could alter the cytokine production from CLL and normal MSC. Cytokine production from both CLL MSC and normal MSC as secreted into culture medium (CM) spontaneously or with recombinant IL-17 during a 72h-culture were tested using a commercial cytokine array (R&D). Result: Both MAS5 and Plier method of transformation yielded similar results for GEPs of the MSC. The preliminary data from these analyses showed that multiple transcription factors including PITX2, HOXB7, SIM1, ZFY, EMX2, HOXB3, HOXB4, and IRF6 were significantly up-regulated (more than 2 fold) in CLL MSC compared to normal MSC. In contrast, the genes up-regulated in normal MSC compared to CLL MSC include genes involved in the immune response (IGH, IGK, IGL, BST2, PPBP, RGS1, etc.), antigen processing and presentation (HLA genes, IFI30, etc.) and inflammatory response (CD163, LYZ, TNFAIP6, etc.). We are currently in the process of performing further analysis using additional MSC samples to validate these findings with further GEP assays and also by validating the dysregulated genes detected by our GEP studies using real-time PCR analysis. We performed the analysis of multiple cytokines produced by CLL (n=16) and normal MSC (n=12) with and without stimulation with recombinant IL-17 (8 ng/ml) over a 72 hour culture period. Without stimulation, there is approximately two-fold increase of MCP-1 production in CLL MSC compared to normal MSC (CLL vs. normal, 164.2 ± 26.1 vs. 82.7 ± 15.4 pg/ml, p = 0.02). After IL-17 stimulation, an approximately four-fold increase of MCP-1 production (CLL vs. normal, 1200.1 ± 258.6 vs. 334.1 ± 68.2 pg/ml, p = 0.005), three-fold increase of IL-8 production (CLL vs. normal, 2073.1 ± 504.1 vs. 708.7 ± 218.9 pg/ml, p = 0.02), two-fold increase of IL-6 production (CLL vs. normal, 1400.6 ± 214.0 vs. 705.6 ± 98.2 pg/ml, p = 0.008) were detected in CLL MSC compared to normal MSC. Conclusion: We have detected the presence of intrinsic mRNA expression differences for both transcription factors and immune response genes between CLL and normal MSC. The expression differences of these genes could be facilitating the differential cytokine responses we observed in CLL MSC compared to normal MSC. Given these findings it is possible that the dynamic interactions between the CLL leukemic cell and MSC influence the transcription profiles and cytokine production from MSC. This is of biologic and clinical interest in that the GEP dysregulation in CLL MSC may be a primary mechanism for the differential cytokine production seen in CLL MSC. This outcome in turn has the potential to modify the immune and non-immune environment in favor of tumor proliferation and progression. Disclosures: Kay: Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.

Abstract Title Submitted by Hollie Pegram to the 2012 ASH Annual Meeting: Expansion and Modification of Umbilical Cord Blood T Cells with a Chimeric Antigen Receptor and IL-12

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1907-1907
Author(s):  
Hollie J. Pegram ◽  
Juliet Barker ◽  
Sergio Giralt ◽  
Renier J Brentjens
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Umbilical Cord Blood ◽  
Cell Expansion ◽  
Research Funding ◽  
Advisory Committees

Abstract Abstract 1907 Transplantation of unrelated umbilical cord blood (CB) derived stem cells is often used to treat adult patients with B-cell acute lymphoblastic leukemia (B-ALL). However, patients can relapse post transplant and prognosis of those with advanced or refractory disease is poor. We hypothesize that additional therapy involving adoptive transfer of CB derived T cells modified to express a CD19-specific chimeric antigen receptor (CAR) could improve patient outcome following CB transplantation in this population. We have previously demonstrated that human T cells which express the anti-CD19 1928z CAR, containing the signaling domains of the co-stimulatory CD28 receptor and CD3z chain, effectively eradicate CD19+ tumors in SCID-Beige mice, and have shown promise in early clinical trials. In this study, we demonstrate the ability to effectively isolate and expand T cells from CB, necessary for application of adoptive CB T cell therapy. We compared T cell expansion in in vitro cultures with the addition of exogenous stimulatory cytokines, including IL-2, IL-7, IL-12, IL-15 or combinations thereof. We demonstrate that in vitro culture in the context of exogenous IL-12 combined with IL-15 resulted in optimal expansion of CB T cells (over 150-fold). In addition, expansion of T cells in the context of exogenous IL-12 and IL-15 resulted in a favorable phenotype, with maximal expression of memory T cell markers, CD62L and CCR7. Furthermore, these cells were shown to produce high levels of IFNγ and express high levels of CD107a following stimulation with PHA. CB T cells expanded in IL-12 and IL-15 were therefore shown to have a unique memory cell phenotype combined with effector T cell function. Previous studies in our laboratory have demonstrated that expression of IL-12 in tumor targeted T cells resulted in increased anti-tumor function. Given these studies and the favorable effect of IL-12 on CB T cell expansion we used retroviral modification to express both 1928z CAR and IL-12 in CB T cells. CB T cells modified with both IL-12 and 1928z were shown to have increased cytokine secretion when cultured with CD19+ tumor cells, compared to UCB T cells modified with a CAR alone. Additionally, these cells were demonstrated to have increased lytic function, resulting in increased specific lysis of CD19+ tumor cells in a 51Cr release assay. Our data suggest that CB T cells modified to express both CAR and IL-12 will have improved anti-tumor function, and could therefore decrease relapse and improve overall survival following CB transplant for the treatment of B-ALL. Disclosures: Giralt: Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals FT596: Translation of First-of-Kind Multi-Antigen Targeted Off-the-Shelf CAR-NK Cell with Engineered Persistence for the Treatment of B Cell Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 301-301 ◽  
Author(s):  
Jode P Goodridge ◽  
Sajid Mahmood ◽  
Huang Zhu ◽  
Svetlana Gaidarova ◽  
Robert Blum ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Advisory Committees

Induced pluripotent stem cell (iPSC)-derived effector cells offer distinct advantages for immune therapy over existing patient- or donor- derived platforms, both in terms of scalable manufacturing from a renewable starting cellular material and precision genetic engineering that is performed at the single-cell level. iPSC derived natural killer (iNK) cells offer the further advantage of innate reactivity to stress ligands and MHC downregulation and the potential to recruit downstream adaptive responses. These unique features form the basis of our multi-antigen targeted chimeric antigen receptor (CAR) CAR-iNK cell product candidate, termed FT596, which is further combined with additional functionality to enhance effector function. FT596 is consistently manufactured from a master iPSC line engineered to uniformly express an NK cell-calibrated CD19-targeting CAR (CD19-CAR), an enhanced functioning high-affinity, non-cleavable CD16 (hnCD16) and a recombinant fusion of IL-15 and IL-15 receptor alpha (IL-15RF) for cytokine-autonomous persistence. The design of the CD19-CAR involved exploiting the intrinsic polyfunctionality of NK cells, which function by engaging multiple signaling pathways activated through combinations of distinct germline encoded receptors. Using this approach, the transmembrane region of activating receptor NKG2D, combined with the intracellular signaling domains of SLAM co-receptor 2B4 and CD3ζ, proved the most effective in triggering antigen specific functional responses in NK cells. Chimerization of an anti-CD19 scFv onto this NKG2D-2B4-CD3ζ signaling platform produced specific in vitro recognition of CD19+ B cell lymphoma cells in short-term and long-term NK cytotoxicity assays (&gt;80% and &lt;40% clearance of tumor cells at 60H, p&lt;0.001 respectively). The functionality of the CD19-CAR was further enhanced in combination with autonomous IL-15 signaling. Introduction of the IL-15RF enabled expansion of iNK cells without addition of soluble cytokine and greatly improved longevity and functional persistence of iNK cells both in vitro and in animal models. Moreover, iNK cells modified with IL-15RF showed enhanced functional maturation, including upregulated expression of effector molecules such as granzyme B. iNK cells with both CD19-CAR and IL-15RF resulted in enhanced CAR functionality in vitro, and mouse models for B cell malignancy demonstrated that treatment with iNK cells engineered with CD19-CAR and IL-15RF were curative against B cell lymphoma (p&lt;0.002), when compared with iNK cells alone or iNK cells modified with CD19-CAR alone. In combination with hnCD16, co-expression of CD19-CAR and IL15-RF culminates in iNK cells capable of dual-specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. In long term killing assays, FT596 alone demonstrated equivalent levels of CD19 targeted anti-tumor activity as primary CD19-targeted CAR (CAR19) T cells when tested against CD19+ CD20+ B lymphoblast target cells and demonstrated enhanced levels of activity when used in combination with anti-CD20 (rituximab). When targeting CD19- CD20+ B lymphoblast target cells and used in combination with rituximab, only FT596 was able to effectively eliminate the CD19 antigen escaped target cell (64% vs 30% clearance of tumor cells at 36H vs rituximab alone). In vivo FT596 showed equivalent levels of tumor cell clearance as primary CAR19 T cells against the CD19+ acute lymphoblastic leukemia cell line NALM6 and CD19+CD20+ Burkitts lymphoma cell line RAJI, and enhanced clearance of RAJI tumor cells in combination with rituximab (p=0.0002). Furthermore, utilizing an allogenic human CD34 engrafted NSG mouse model, FT596 demonstrated improved survival and safety over primary CAR19 T cells, either as a monotherapy or as a combination therapy with rituximab versus RAJI tumor cells. Together, these studies demonstrate FT596 provides a multi-antigen targeting, potent and persistent engineered immune cell that is derived from a master iPSC line which utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy in a single, standardized, scalable, off-the-shelf platform and supports the rational for a first-of-kind Phase I Study as a monotherapy and in combination with CD20-targeted mAbs including rituximab in subjects with relapsed/refractory B-cell lymphoma and leukemia. Figure Disclosures Goodridge: FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Bjordahl:Fate Therapeutics, Inc.: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Chu:FATE THERAPEUTICS: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Groff:FATE THERAPEUTICS: Employment. Meza:FATE THERAPEUTICS: Employment. Malmberg:Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Consultancy, Research Funding. Miller:Moderna: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; CytoSen: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

Inhibition of the Proteasome in Bone Marrow-Derived CD138+ Tumor Cells Following Carfilzomib Administration in Relapsed or Refractory Myeloma Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1845-1845
Author(s):  
Suzanne Trudel ◽  
Susan Lee ◽  
Christopher J. Kirk ◽  
Nashat Gabrail ◽  
Sagar Lonial ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Whole Blood ◽  
Research Funding ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Advisory Committees ◽  
Post Dose ◽  
Blood Samples

Abstract Abstract 1845 Poster Board I-871 Background: Proteasome inhibition is an effective strategy for the treatment of multiple myeloma. In patients, proteasome inhibition has primarily been measured in peripheral blood samples (whole blood or mononuclear cells). However, it is unknown whether myeloma cells in the bone marrow (BM) are equally sensitive to proteasome inhibitors such as bortezomib (BTZ) and carfilzomib (CFZ). Aim: To measure proteasome inhibition in purified tumor cells from BM samples taken from patients enrolled in two ongoing Phase 2 trials of single agent CFZ in relapsed or refractory myeloma: PX-171-003 (003) and PX-171-004 (004). Methods: CFZ was administered as an IV bolus of 20 mg/m2 on Days 1, 2, 8, 9, 15 and 16 of a 28-day cycle on both trials. Bone marrow samples, from an optional sub-study of both trials, were taken during screening and Day 2 (post-treatment) and sorted into CD138+ and CD138− cells. Proteasome activity was measured by an enzymatic assay using a fluorogenic substrate (LLVY-AMC) for the chymotrypsin-like (CT-L) activity and an active site ELISA (ProCISE) to quantitate levels of the CT-L subunits of the constitutive proteasome (Beta5) and immunoproteasome (LMP7) and the immunoproteasome subunit MECL1. Results: Whole blood samples from patients treated with CFZ showed inhibition of CT-L activity of ∼80+, similar to values obtained in Phase 1 studies. A total of 10 CD138+ screening samples, 6 from 004 and 4 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed for proteasome levels and activity. In addition, 15 CD138−screening samples, 7 from 004 and 8 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed. When compared to the average base-line activity, CFZ treatment resulted in 88% CT-L inhibition in CD-138+tumor cells from 004 patients (P = 0.0212 by unpaired t-test) and 59% CT-L inhibition in CD-138+ tumor cells from 003 patients (P = 0.25). Baseline CT-L activity in CD138+ tumor cells was 3-fold higher in 004 than 003, which includes a more heavily pre-treated patient population with greater prior exposure to BTZ. Higher specific enzymatic activity was due to increased levels of both constitutive and immunoproteasomes in tumor cells, where immunoproteasomes account for >75% of total cellular proteasomes. No differences between trials were seen in baseline CT-L activity from non-tumor (CD138−) cells. Inhibition in CD138− cells was 84% (P = 0.0380 and 42% (P = 0.38) in 004 and 003, respectively. Using ProCISE, we measured inhibition of LMP7 (66%), beta5 (48%) and MECL1 (64%) in CD138+ tumor cells from 004 patients. Three patients from 004 and one from 003 had both a screening and post-dose tumor cell samples available for analysis. Inhibition of CT-L activity was >80% in two of the 3 patients on 004; the third patient showed no proteasome inhibition by ProCISE and was unavailable for analysis by CT-L. CT-L activity in the CD138+ tumor cells in the 003 patient was not inhibited, however, inhibition was seen in non-tumor cells. Conclusions: CFZ inhibits the proteasome activity of myeloma cells in the bone marrow of relapsed and refractory myeloma patients. The levels of inhibition were similar to those measured in whole blood samples, supporting the use of the blood-based assay as a surrogate marker for proteasome inhibition in tumor cells. CFZ treatment resulted in inhibition of both CT-L subunits as well as additional subunits of the immunoproteasome in tumor cells. Reduced baseline activity in the more heavily pretreated 003 patients may reflect reduced tumor-dependency on the proteasome and may be related to prior treatment with BTZ in these patients. More samples are needed in order to make correlations between levels of proteasome inhibition in bone marrow tumor cells and prior therapies or response. These observations support further evaluation of proteasome activity and the effects of this promising new agent in primary tumors cells from myeloma patients. Disclosures: Trudel: Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria. Lee:Proteolix, Inc.: Employment. Kirk:Proteolix, Inc.: Employment. Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Wang:Proteolix, Inc.: Research Funding. Kukreti:Celgene: Honoraria. Stewart:Genzyme, Celgene, Millenium, Proteolix: Honoraria; Takeda, Millenium: Research Funding; Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. McDonagh:Proteolix: Research Funding. Zonder:Celgene: Speakers Bureau; Pfizer: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen: Consultancy; Millennium: Research Funding. Bennett:Proteolix: Employment.

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