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.

Role of TORC1 and TORC2 in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1815-1815
Author(s):  
Patricia Maiso ◽  
Yi Liu ◽  
Abdel Kareem Azab ◽  
Brittany Morgan ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Stromal Cells ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Committees ◽  
Cycle Arrest

Abstract Abstract 1815 Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 (Raptor) and TORC2 (Rictor). TORC1 leads to the phosphorylation of p70S6 kinase and 4E- BP1, while TORC2 regulates phosphorylation of Akt and other 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 and its analogues have not shown significant activity in MM, likely due to the lack of inhibition of TORC2. In this study, we dissected the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. Methods: Eight 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, immunochemistry, 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. In vivo homing was checked by in vivo flow. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: Raptor (TORC1) and Rictor (TORC2) knockdowns led to significant inhibition of proliferation of MM cells even in the presence of bone marrow stromal cells, this effect was also accompanied by inactivation of p-Akt, p-rS6 and p-4EBP1. We used INK128, a dual and selective TORC1/2 kinase inhibitor with similar effects to Raptor plus Rictor knockdown. We examined the protein expression levels of both mTOR complex and their downstream effectors in MM plasma cells from patients and cell lines. mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all samples. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin) even in the presence of cytokines or stromal cells. INK128 induced cell cycle arrest, autophagy and apoptosis in cell lines and primary plasma cells even in the presence of bone marrow stromal cells (BMSCs). INK128 also showed a significant effect inhibiting cell adhesion in our in vivo homing model. Oral daily treatment with INK128 highly decreased the percentage of CD138+ tumor plasma cells in mice implanted with MM cells and reduced the levels of p-Akt and p-4EBP. These results suggest that potent and complete blockade of mTOR as part of TORC1 and TORC2 is potential therapeutic strategy to induce cell cycle arrest, apoptosis and disruption of MM cells interaction with the BM microenvironment. 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: Liu: Intellikine: Employment. Roccaro:Roche: Research Funding. Rommel:Intellikine: Employment. Ghobrial:Celgene: Consultancy; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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.

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 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 Interactions with a "Humanized" Mesenchymal Bone Marrow Stromal Niche In Vivo Modify the Patterns of Essential Genes for Myeloma Cells: Therapeutic Implications

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 40-40
Author(s):  
Ryosuke Shirasaki ◽  
Sondra L. Downey-Kopyscinski ◽  
Ricardo De Matos Simoes ◽  
Olga Dashevsky ◽  
Sara Gandolfi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Broad Spectrum ◽  
Research Funding ◽  
Advisory Committees ◽  
Stromal Compartment ◽  
Oncology Research

Background: The biology and treatment response of human multiple myeloma (MM) cells in vivo is influenced by interactions with mesenchymal bone marrow stromal cells (BMSCs). For several key BMSC-derived cytokines (including IL-6) only the human, not murine, form optimally interacts with their respective receptor(s) on human MM cells. To better simulate the treatment responses of human MM cells in the BM, "humanized" BM-like niches in vivo have been engineered with biocompatible ceramic scaffolds "functionalized" via osteogenic differentiation of human mesenchymal BMSCs and implanted subcutaneously in immunocompromised mice. Aim: To determine if the patterns of genetic dependencies elucidated through in vitro CRISPR-based functional genomic studies are recapitulated when human MM cells are grown in mice within BM-like scaffolds with "humanized" mesenchymal stromal compartment. Methods: Cas9+ MM cell lines KMS11 and XG7 were transduced with a library of 1372 single-guide RNAs (sgRNAs) targeting 184 genes of interest (4 sgRNAs/gene), including 89 genes preferentially essential for MM cell lines compared to other neoplasms in vitro; broad-spectrum oncogenic targets (e.g. KRAS, MYC); tumor suppressor genes (e.g. PTEN); genes with limited in vitro essentiality in MM cells, but significantly higher expression vs. non-MM lines (e.g. ZFP91, ZBP1); and 155 olfactory receptor (OR) genes (typically not expressed or biologically inactive in tumor cells) as "DNA cutting" control sgRNAs. MM cells transduced with this focused CRISPR knockout (KO) library were injected into "humanized" scaffolds implanted subcutaneously (s.c.) in NSG mice. Tumors were collected when mice reached criteria for euthanasia and next-generation sequencing quantified the changes in sgRNA distribution at the end vs. start of experiment. Results: A large majority of genes defined by in vitro CRISPR KO screens as MM-preferential dependencies were also essential for MM cells in BM-like scaffolds. Among 57 MM-preferential dependencies in vitro which were plausible dependencies for KMS11 cells (e.g. CERES scores &lt;-0.4), 50 genes were essential for KMS11 cells in BM-like scaffolds in vivo (average log2fold change&lt;-1.0 and depletion of 3+ of 4 sgRNAs relative to the 99% confidence interval for control sgRNAs). These genes included key transcription factors/cofactors (e.g. IRF4, PRDM1, POU2AF1, RELB, MAF); epigenetic regulators (e.g. CARM1, MTA2, DOT1L); kinases upstream of NFkappaB (CHUK, IKBKB); ER regulators (e.g. SYVN1). Most "core-essential" or broad-spectrum oncogenic dependencies (e.g. MYC, CFLAR, CDK7 on both lines; KRAS in XG7) of this sgRNA library remained essential in vivo; while PTEN KO cells were enriched. Notably, several genes had more pronounced essentiality in vivo vs. invitro (e.g. BCL2, PIM2); or converted from non-essential in vitro to essential in vivo. For instance, among 95 genes of this library which are not likely dependencies in vitro (CERES scores &gt;-0.4) for KMS11 cells, 29 genes exhibited in vivo essentiality for both KMS11 and XG7 cells: several of these latter "in vivo dependencies" are recurrently essential for other MM lines in vitro (e.g. ZBTB38, UBE2J1, TCF3, DNAJB11), while also others have limited if any in vitro essentiality (e.g. ZBP1, PYGO2) across MM despite significantly higher transcript levels vs. other neoplasias. Notably, several genes with increased essentiality in the "humanized" BM scaffolds vs. in vitro also had stronger in vivo dependency in the BM scaffolds vs. when growth of the same MM cells as s.c. plasmacytomas (e.g. BCL2, PIM2, UBE2J1, SYVN1, ALG9, AMFR). Co-culture with BMSCs or IL-6 treatment induces several of these transcripts in MM cells suggesting that increased dependency of MM on these genes in the "humanized" BM model is due at least partly to its human cytokines. Conclusions: This study provides evidence for significant impact of the "humanized" BM-like niche on the patterns of genetic dependencies for human MM cells. Most genes preferentially essential for MM cells in vitro remain essential for their in vivo growth in the "humanized" BM model. Notably, several genes that do not meet criteria for dependency in vitro show such metrics in "humanized" BM scaffolds, but not sc plasmacytomas. These observations highlight important implications of the "humanized" BM-like in vivo model for current and future efforts to define and prioritize therapeutic targets for MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Tango Therapeutics: Consultancy; Cedilla Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Janssen/Johnson & Johnson: Research Funding; Arch Oncology: Research Funding; TEVA: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; EMD Serono: Research Funding; Abbvie: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.

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.

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.

A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1841-1841
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Arghya Ray ◽  
Teru Hideshima ◽  
Paul G. Richardson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Advisory Committees ◽  
Acid Analog

Abstract Abstract 1841 Introduction: The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results: We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions: We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures: Hideshima: Acetylon: Consultancy. 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. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

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