Antitumor anthracyclines such as doxorubicin and epirubicin are known to cause cardiotoxicity that correlates with anthracycline accumulation in the heart. The anthracycline amrubicin [(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-β-d-erythro-pentopyranosyl)oxy]-7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-napthacenedione hydrochloride] has not shown cardiotoxicity in laboratory animals or patients in approved or investigational settings; therefore, we conducted preclinical work to characterize whether amrubicin attained lower levels than doxorubicin or epirubicin in the heart. Anthracyclines were evaluated in ex vivo human myocardial strips incubated in plasma to which anthracycline concentrations of 3 or 10 μM were added. Four-hour incubations were performed to characterize myocardial anthracycline accumulation derived from anthracycline uptake in equilibrium with anthracycline clearance. Short-term incubations followed by multiple washouts were performed to obtain independent measurements of anthracycline uptake or clearance. In comparison with doxorubicin or epirubicin, amrubicin attained very low levels in the soluble and membrane fractions of human myocardial strips. This occurred at both 3 and 10 μM anthracycline concentrations and was caused primarily by a highly favorable clearance of amrubicin. Amrubicin clearance was facilitated by formation and elimination of sizeable levels of 9-deaminoamrubicin and 9-deaminoamrubicinol. Amrubicin clearance was not mediated by P glycoprotein or other drug efflux pumps, as judged from the lack of effect of verapamil on the partitioning of amrubicin and its deaminated metabolites across myocardial strips and plasma. Limited accumulation of amrubicin in an ex vivo human myocardial strip model may therefore correlate with the improved cardiac tolerability observed with the use of amrubicin in preclinical or clinical settings.

Abstract Abstract 4043 Background: Cereblon (CRBN), a component of the DDB1-CUL4A-Roc1 ubiquitin ligase complex, has been identified as a target of the immunomodulatory agents thalidomide, lenalidomide, and pomalidomide (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011; Ito et al. Science. 2010.). CRBN binding by these agents mediates their anti-proliferative effects in multiple myeloma (MM) cells (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011). However, the role of CRBN quantification as a marker for disease responsiveness or resistance to these drugs remains to be fully defined. Furthermore, it is unclear whether measuring mRNA or protein expression is the best approach for development of a quantitative CRBN expression assay. In order to define the optimal assay approach, we have studied CRBN mRNA and protein expression in MM cell lines (n=20) and MM patient samples. Methods: CRBN isoform mapping was undertaken using a nested PCR approach and Sanger sequencing. Commercially available and newly generated rabbit anti-CRBN antibodies were characterized with recombinant human CRBN protein and MM cell line extracts via western blot analysis. Results: Our data show that in addition to the transcript for full length protein (GenBank Accession NM_016302.3), in MM cells there are at least 6 alternatively spliced isoforms of CRBN as depicted in Figure 1. Five of the 6 CRBN isoforms (CRBN-003, -004, -005, -006, and -007) contain novel splice junctions not previously described. In addition, 3 of the identified transcripts (CRBN-002, -003, and -005) contain in-frame ORFs, suggesting they encode variants of CRBN protein. Of note, exon 10, which contains a portion of the IMiD-binding domain, is not present in CRBN-002. The functional consequence of CRBN-002 remains to be elucidated, but may be a marker of drug resistance. In order to measure CRBN protein levels, we developed and characterized three rabbit monoclonal antibodies to CRBN including antibody CRBN65, which has the potential to discriminate between the different CRBN protein products, including CRBN-002 by western blot analysis. Additionally, we compared 8 commercially available CRBN antibodies. Western blot analysis of cell lines with commercial and newly developed antibodies identified full length protein at 51 kD. Most commercial antibodies also identified multiple bands of other sizes which may represent CRBN protein variants; however, many are likely non-specific bands as they are larger than full-length CRBN. Conclusion: We have identified novel splice variants of CRBN from MM cell lines and primary tumor samples. The structure of the isoforms and their potential ability to be translated into several protein variants of CRBN reflect the complex regulation of the CRBN gene. These data suggest that accurate quantification of CRBN mRNA level in clinical studies may require measurement of both full-length CRBN mRNA as well as other mRNA isoforms. Currently available primers and gene expression arrays are not capable of identifying and/or resolving the complex set of CRBN isoforms present in cells. These data also demonstrate that CRBN65 is a highly specific and sensitive antibody that could be used for detection of CRBN and its key variants. Taken together, our data emphasize the importance for developing standardized reagents and assays for both mRNA and protein level measurement of CRBN before using them as markers for clinical response or resistance. Disclosures: Gandhi: Celgene Corp: Employment, Equity Ownership. Waldman:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Aukerman:Celgene Corp: Employment, Equity Ownership. Chen:Celgene Corp: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Rychak:Celgene Corp: Employment, Equity Ownership. Miller:Celgene Corp: Employment, Equity Ownership. Gaidarova:Celgene Corp: Employment, Equity Ownership. Gonzales:Celgene Corp: Employment, Equity Ownership. Cathers:Celgene Corp: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership.

Anthracycline-related cardiotoxicity correlates with cardiac anthracycline accumulation and bioactivation to secondary alcohol metabolites or reactive oxygen species (ROS), such as superoxide anion (O₂·⁻) and hydrogen peroxide H₂O₂). We reported that in an ex vivo human myocardial strip model, 3 or 10 μM amrubicin [(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-β-D-erythro-pentopyranosyl)oxy]-7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-napthacenedione hydrochloride] accumulated to a lower level compared with equimolar doxorubicin or epirubicin (J Pharmacol Exp Ther 341:464-473, 2012). We have characterized how amrubicin converted to ROS or secondary alcohol metabolite in comparison with doxorubicin (that formed both toxic species) or epirubicin (that lacked ROS formation and showed an impaired conversion to alcohol metabolite). Amrubicin and doxorubicin partitioned to mitochondria and caused similar elevations of H₂O₂, but the mechanisms of H₂O₂ formation were different. Amrubicin produced H₂O₂ by enzymatic reduction-oxidation of its quinone moiety, whereas doxorubicin acted by inducing mitochondrial uncoupling. Moreover, mitochondrial aconitase assays showed that 3 μM amrubicin caused an O₂·⁻-dependent reversible inactivation, whereas doxorubicin always caused an irreversible inactivation. Low concentrations of amrubicin therefore proved similar to epirubicin in sparing mitochondrial aconitase from irreversible inactivation. The soluble fraction of human myocardial strips converted doxorubicin and epirubicin to secondary alcohol metabolites that irreversibly inactivated cytoplasmic aconitase; in contrast, strips exposed to amrubicin failed to generate its secondary alcohol metabolite, amrubicinol, and only occasionally exhibited an irreversible inactivation of cytoplasmic aconitase. This was caused by competing pathways that favored formation and complete or near-to-complete elimination of 9-deaminoamrubicinol. These results characterize amrubicin metabolic advantages over doxorubicin and epirubicin, which may correlate with amrubicin cardiac safety in preclinical or clinical settings.

PURPOSE:Multi-drug resistance and cumulative cardiotoxicity are major limitations for the clinical use of anthracyclines. Here, we evaluated and compared the cross-resistance of amrubicin, a third-generation synthetic anthracycline and potent topoisomerase (topo)-II inhibitor with little or no observed cardiotoxicity to other anthracyclines and the topo-II inhibitor etoposide in drug-resistant tumor models in order to elucidate its potential mechanisms of action.METHODS:Amrubicin activity was assessed in multi-drug-resistant cell lines and human tumor explants using cytotoxicity assays, confocal microscopy, fluorescence time-lapse imaging, flow cytometry, immunoblotting, and gene expression profiling techniques.RESULTS:We demonstrate that both doxorubicin-resistant tumor cell lines and several drug-resistant human ovarian and breast tumor explants retain sensitivity to amrubicin. In addition, we observed similar levels of amrubicin uptake and accumulation in doxorubicin-sensitive versus doxorubicin-resistant cell lines. Although amrubicin is a weak P-glycoprotein substrate, transport and retention of amrubicin were not solely modulated by P-glycoprotein in the resistant cell lines overexpressing drug efflux pumps. The cellular retention of amrubicin is likely to be a result of rapid influx due to its high intrinsic permeability and lipophilic properties, and this may explain why amrubicin overcomes pleiotropic drug resistance. Consistent with drug accumulation studies, amrubicin induced DNA damage, G(2)-M cell cycle arrest, and apoptosis in both doxorubicin-sensitive and doxorubicin-resistant lines. Using gene expression profiling studies, several classes of genes were significantly and uniquely regulated following amrubicin, but not doxorubicin or etoposide, treatment.CONCLUSIONS:Amrubicin appears to have a distinct mode of action that overcomes typical anthracycline resistance mechanisms. Therefore, amrubicin may be useful in the treatment of anthracycline-refractory or anthracycline-resistant tumors.

Background The cytidine nucleoside analogs azacitidine (AZA) and decitabine (DAC) are used for the treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Few non-clinical studies have directly compared the mechanisms of action of these agents in a head-to-head fashion, and the agents are often viewed as mechanistically similar DNA hypomethylating agents. To better understand the similarities and differences in mechanisms of these drugs, we compared their in vitro effects on several end points in human AML cell lines. Methodology/Principal Findings Both drugs effected DNA methyltransferase 1 depletion, DNA hypomethylation, and DNA damage induction, with DAC showing equivalent activity at concentrations 2- to 10-fold lower than AZA. At concentrations above 1 µM, AZA had a greater effect than DAC on reducing cell viability. Both drugs increased the sub-G1 fraction and apoptosis markers, with AZA decreasing all cell cycle phases and DAC causing an increase in G2-M. Total protein synthesis was reduced only by AZA, and drug-modulated gene expression profiles were largely non-overlapping. Conclusions/Significance These data demonstrate shared mechanisms of action of AZA and DAC on DNA-mediated markers of activity, but distinctly different effects in their actions on cell viability, protein synthesis, cell cycle, and gene expression. The differential effects of AZA may be mediated by RNA incorporation, as the distribution of AZA in nucleic acid of KG-1a cells was 65∶35, RNA∶DNA.

Non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) account for about 9% of new cancer cases annually or 64,000 cases per year in the United States. Although the survival rate has significantly improved recently due to new combination therapy regimens, an unmet medical need remains for refractory or resistant patients. HCD122 is a fully human antagonistic anti-CD40 therapeutic monoclonal antibody (mAb) with a dual mechanism of action: blocking CD40 and CD40 ligand (CD40L) interactions and mediating antibody-dependent cellular cytotoxicity (ADCC). CD40 is expressed in all human B-cell malignancies, and the CD40/CD40L interaction is important for tumor cell proliferation and survival. Previously HCD122 was shown to potently inhibit CD40L-induced human B-cell and follicular NHL cell proliferation, mediate ADCC against CD40-positive human malignant B-cell lines and inhibit tumor growth in Burkitt's lymphoma and multiple myeloma xenograft models. In this study the antitumor activity of HCD122 was assessed in preclinical models of HD and other subtypes of human NHL, such as Mantle cell and Follicular lymphoma. CD40 was expressed in 5 of 7 established human HD and 11 of 12 NHL tumor cell lines tested, including Hs445, HDLM-2, KM-H2, L428, L1236, Jeko-1 and WSU-NHL. Using purified human NK cells as effector cells, HCD122 mediated potent ADCC against these cell lines in vitro with a picomolar EC50. When human macrophages were used as effector cells, HCD122 also induced antibody-dependent cellular phagocytosis (ADCP) against the NHL Daudi cell line and the HD cell line Hs445. The antitumor activity of HCD122 was further evaluated in vivo in EBV-negative NHL and HD xenograft models. When tested in a staged human Mantle cell lymphoma Jeko-1 s.c. xenograft model in which treatment was initiated when the mean tumor volume reached 100 mm3, HCD122 was highly efficacious and induced complete tumor regression in 70% (7/10) of treated animals when administered intraperitoneally at 1 mg/kg weekly for 4 weeks. In a staged human HD L428 s.c. xenograft model, which expresses CD20 as well as CD40, the antitumor activity of HCD122 was compared to rituximab. HCD122 was highly efficacious and induced a mean 74 % tumor growth inhibition (TGI) when administered at 0.1 mg/kg weekly for 3 weeks (p<0.001). At the same dose and schedule, rituximab achieved only 40% TGI (HCD122 vs. rituximab: p<0.001). These data combined with our previous studies in multiple myeloma and EBV-positive Burkitt's lymphoma models show that HCD122 is a potent anti-CD40 antibody with pronounced antitumor activity in both EBV-positive and EBV-negative malignant B cell preclinical models. HCD122 is currently in Phase I clinical trials in B-cell malignancies.

ABSTRACT The lack of a robust small-animal model for hepatitis C virus (HCV) has hindered the discovery and development of novel drug treatments for HCV infections. We developed a reproducible and easily accessible xenograft mouse efficacy model in which HCV RNA replication is accurately monitored in vivo by real-time, noninvasive whole-body imaging of gamma-irradiated SCID mice implanted with a mouse-adapted luciferase replicon-containing Huh-7 cell line (T7-11). The model was validated by demonstrating that both a small-molecule NS3/4A protease inhibitor (BILN 2061) and human alpha interferon (IFN-α) decreased HCV RNA replication and that treatment withdrawal resulted in a rebound in replication, which paralleled clinical outcomes in humans. We further showed that protease inhibitor and IFN-α combination therapy was more effective in reducing HCV RNA replication than treatment with each compound alone and supports testing in humans. This robust mouse efficacy model provides a powerful tool for rapid evaluation of potential anti-HCV compounds in vivo as part of aggressive drug discovery efforts.

Recombinant interleukin-2 (rIL-2) is a pleiotropic cytokine that activates select immune effector cell responses associated with antitumor activity, including antibody-dependent cellular cytotoxicity (ADCC). Rituximab is an anti-CD20 monoclonal antibody that activates ADCC in non-Hodgkin lymphoma (NHL). The ability of rIL-2 to augment rituximab-dependent tumor responses was investigated. The efficacy of rIL-2 in combination with rituximab was evaluated in 2 NHL tumor xenograft models: the CD20hi, rituximab-sensitive, low-grade Daudi model and the CD20lo, aggressive, rituximab-resistant Namalwa model. Combination of rIL-2 plus rituximab was synergistic in a rituximab-sensitive Daudi tumor model, as evidenced by significant tumor regressions and increased time to tumor progression, compared with rIL-2 and rituximab single agents. In contrast, rituximab-resistant Namalwa tumors were responsive to single-agent rIL-2 and showed an increased response when combined with rituximab. Using in vitro killing assays, rIL-2 was shown to enhance activity of rituximab by activating ADCC and lymphokine-activated killer activity. Additionally, the activity of rIL-2 plus rituximab F(ab')2 was similar to that of rIL-2 alone, indicating a critical role for immunoglobulin G1 Fc-FcgammaR-effector responses in mediating ADCC. Antiproliferative and apoptotic tumor responses, along with an influx of immune effector cells, were observed by immunohistochemistry. Collectively, the data suggest that rIL-2 mediates potent tumoricidal activity against NHL tumors, in part, through activation and trafficking of monocytes and natural killer cells to tumors. These data support the mechanistic and therapeutic rationale for combination of rIL-2 with rituximab in NHL clinical trials and for single-agent rIL-2 in rituximab-resistant NHL patients.

CD40, a member of the tumor necrosis factor receptor family, is expressed in all human B-cell malignancies and engagement by the CD40 ligand (CD40L) is important for both cell proliferation and cell survival. CD40L has been shown to be co-expressed with CD40 in neoplastic B-cells from Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkins Lymphoma (NHL), suggesting the importance of an autocrine CD40/CD40L loop in these malignancies. HCD122 (formerly known as CHIR-12.12) is a fully human, highly potent, IgG1 antagonist anti-CD40 monoclonal antibody (mAb) that blocks CD40/CD40L interactions in vitro and also mediates ADCC. Previous studies showed that HCD122 can mediate ADCC in vitro and has anti-proliferative and anti-tumor activities as a single agent in CLL, MM, and Burkitts Lymphoma in vitro and in vivo. In this study, the activity of HCD122 on a subtype of NHL, Diffuse Large B-Cell Lymphoma (DLBCL) was examined. The DLBCL derived cell lines, RL and SU-DHL-4, were selected for this study based upon in vivo characterization as well as their sensitivity to Rituximab as reported in the literature. These cell lines were subsequently confirmed for the expression of CD40 and CD20 by flow cytometry. The in vivo anti-tumor effects of HCD122 as single agent was demonstrated in these two xenograft models and was compared to Rituximab, an anti-CD20 antibody therapeutic currently approved for the treatment of relapsed or refractory, low-grade or follicular, NHL. HCD122 when administered intraperitoneally weekly at 1 mg/kg significantly reduced tumor growth with a tumor growth inhibition (TGI) of 85.5% (P<0.01) in the RL model. At the same dose and schedule in the RL model, TGI achieved with Rituximab was 31.7% (P>0.05). In the SU-DHL-4 model, an 85% TGI (P<0.01) was observed at the 1 mg/kg dose of HCD122. In comparison, Rituximab at this dose elicited a 57.6% TGI (P<0.05). Additionally, the downstream CD40/CD40L signal transduction pathways were also examined in order to elucidate the molecular mechanism underlying the HCD122-mediated effects in DLBCL. Taken together, these results support the clinical development of HCD122 for the treatment of DLBCL. Currently HCD122 is in Phase I trials for treatment of CLL and MM.

To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon cancer, a series of angiogenesis-related gene promoters was analyzed for beta-catenin/TCF binding sites. Strikingly, the gene promoter of human vascular endothelial growth factor (VEGF, or VEGF-A) contains seven consensus binding sites for beta-catenin/TCF. Analysis of laser capture microdissected human colon cancer tissue indicated a direct correlation between up-regulation of VEGF-A expression and adenomatous polyposis coli (APC) mutational status (activation of beta-catenin signaling) in primary tumors. In metastases, this correlation was not observed. Analysis by immunohistochemistry of intestinal polyps in mice heterozygous for the multiple intestinal neoplasia gene (Min/+) at 5 months revealed an increase and redistribution of VEGF-A in proximity to those cells expressing nuclear beta-catenin with a corresponding increase in vessel density. Transfection of normal colon epithelial cells with activated beta-catenin up-regulated levels of VEGF-A mRNA and protein by 250-300%. When colon cancer cells with elevated beta-catenin levels were treated with beta-catenin antisense oligodeoxynucleotides, VEGF-A expression was reduced by more than 50%. Taken together, our observations indicate a close link between beta-catenin signaling and the regulation of VEGF-A expression in colon cancer.

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine that is selective in its recruitment of naive T cells and dendritic cells (DCs). In the lymph node, SLC is believed to play an important role in the initiation of an immune response by colocalizing naive T cells with DC-presenting antigen. Here, we used SLC as a treatment for tumors established from the poorly immunogenic B16 melanoma. Intratumoral injections of SLC inhibited tumor growth in a CD8+, T cell-dependent manner. SLC elicited a substantial infiltration of DCs and T cells into the tumor, coincident with the antitumor response. We next used SLC gene-modified DCs as a treatment of established tumors. Intratumoral injections of SLC-expressing DCs resulted in tumor growth inhibition that was significantly better than either control DCs or SLC alone. Distal site immunization of tumor-bearing mice with SLC gene-modified DCs pulsed with tumor lysate elicited an antitumor response whereas control DCs did not. We also found that s.c. injection of lysate-pulsed DCs expressing SLC promoted the migration of T cells to the immunization site. This report demonstrates that SLC can both induce antitumor responses and enhance the antitumor immunity elicited by DCs.

We examined the effects of human recombinant basic fibroblast growth factor (bFGF) on the proliferation and migration of cultured dog smooth muscle cells (SMCs) and endothelial cells (ECs) and the effect of continuous local boundary layer infusion of bFGF on intimal hyperplasia in endarterectomized dog artery.In vitroproliferation and migration of dog SMCs or ECs were performed using direct counting and Boyden's chamber, respectively. At a dose of 10 ng/mL, bFGF significantly promoted both SMC and EC proliferation (7- and 4-fold, respectively) and migration (2.3- and 1.9-fold, respectively). Six dogs underwent bilateral carotid endarterectomies. A newly designed local infusion device with an osmotic pump continuously delivered bFGF to one artery or vehicle solution to the contralateral artery for 14 days. The intimal thickness and area in the bFGF-treated vessels were increased by 72 and 81%, respectively, compared with control arteries (P< 0.05). As assessed by the bromodeoxyuridine index, the proliferative activity was increased by 73% in bFGF-treated arteries (P= 0.03). Furthermore, cell proliferation at the distal anastomoses of local infusion device was significantly increased in the bFGF-infused grafts compared with distal anastomoses in the control grafts (13.24 ± 1.24% versus 5.24 ± 1.01%,P< 0.01). These data demonstrate that human recombinant bFGF has a potent effect on dog SMC and EC proliferation and migration, and that local infusion of exogenous bFGF significantly enhances the intimal hyperplasia formation and cell proliferation to vascular injury. We conclude that the bFGF pathway may contribute to the development of intimal hyperplastic lesions.