ALBERT

Description: Background: Cereblon (CRBN) is a substrate receptor of the Cullin 4 E3 ubiquitin ligase complex CRL4CRBN and is the molecular target of the IMiD® immunomodulatory drug lenalidomide. It has been shown that cereblon is required for the anti-proliferative activity of lenalidomide in multiple myeloma (MM) and that reduction of CRBN expression is associated with resistance to IMiD® compounds. Methods: RNA-seq analysis was performed on 12 paired MM patients samples of sorted CD138+ cells obtained prior to lenalidomide treatment initiation and after development of resistance. Transcriptome sequence data was generated on an Ion Torrent Proton sequencer with at least 70 million reads per sample. The STAR aligner was used to align raw reads to the Ensembl74 reference annotation. The HTseq and eXpress algorithms were used to quantify gene and transcript counts, respectively, and the Sailfish algorithm was used to validate eXpress transcript counts. The Deseq2 algorithm was used to determine differential expression at gene and transcript levels between paired samples. Results: Of 272 genes observed to change significantly in expression at relapse (FDR 〈 0.05), a majority (169) were up-regulated. Inter-pathway similarity analysis based on gene set enrichment analysis (GSEA; canonical pathways) suggested 4 distinct processes were down-regulated at relapse, including Notch Signaling, Interferon Signaling and G-coupled protein receptor signaling. Conversely, patients exhibited a single dominant up-regulated process associated with proliferation. Additional GSEA analysis on more specific gene categories revealed up-regulation of the Proliferation gene cluster described in the University of Arkansas for Medical Science (UAMS) classification for newly diagnosed MM (6 of 2599 gene sets tested; FDR

Description: Background: Immunomodulatory drugs exhibit several anti-Multiple Myeloma (MM) activities including: growth arrest, antiangiogenesis, inhibition of tumor necrosis factor-alpha signaling and induction of apoptosis. The mechanism of action of the IMiD® compounds lenalidomide and pomalidomide that are responsible for these effects are starting to become clear. IMiD® compounds directly bind Cereblon (CBRN), a substrate receptor of the cullin ring E3 ligase (CRL4), and induce CRL4CRBN -mediated degradation of the zinc finger transcriptional repressors Aiolos and Ikaros by ubiquitin-mediated proteolysis. However, direct links between Aiolos or Ikaros degradation via the CRBN-Cul4 complex to the anti-MM activities of IMiD® drugs are poorly understood. Methods and Results: IMiD® compound-induced apoptosis in MM cells is associated with extended duration of treatment and increased caspase-8 activity, but the precise mechanism of caspase-8 activation and delayed onset of apoptotic effects in response to treatment are unknown. Here we show that both lenalidomide and pomalidomide induce caspase-8, but not capsase-9 activity, in multiple MM cell lines after 3 days of treatment and apoptosis after 4 days without inducing significant changes in total caspase-8, c-FLIP (a casapse-8 inhibitory molecule) or caspase-3 gene expression. Bioinformatic analysis of public datasets revealed that that the pro-apoptotic protein TRAIL may be induced by IMiD® compound treatment in a CRBN dependent manner. We confirmed in multiple cell lines that IMiD® compound treatment or knockdown of Aiolos or Ikaros induces TRAIL gene expression. Furthermore, IMiD® compound treatment in pomalidomide resistant cells that lack CRBN expression failed to induce TRAIL gene expression suggesting TRAIL induction is regulated through the established IMiD® drug mediated CRBN-Aiolos/Ikaros pathway. ChIP-PCR analysis confirmed that TRAIL is a direct transcriptional target gene of Aiolos and Ikaros in MM cells. Incubation of cells with a TRAIL neutralizing antibody was sufficient to partially attenuate IMiD® induced apoptosis, thus confirming the role of TRAIL in IMiD® drug induced MM cell apoptosis. Furthermore, TRAIL secretion into the media of IMiD® compound treated cells was significantly increased only after 2 days treatment explaining the delayed induction of apoptosis in treated cells. Interestingly, MM cell lines that are intrinsically resistant to the apoptotic effects of IMiD® compounds, despite degrading Aiolos and Ikaros in response to drug, displayed a decrease in TRAIL sensitivity. Conclusions: Taken together this data indicates that IMiD® drugs induce caspase-8 mediated apoptosis by directly inducing TRAIL gene expression via degradation of Aiolos and Ikaros. Additionally, this suggests that therapeutic interventions that can increase MM cell sensitivity to TRAIL might act synergistically with IMiD® compounds. Disclosures Amatangelo: Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene: Employment, Equity Ownership. Klippel:Celgene Corporation: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment. Thakurta:Celgene Corporation: Employment, Equity Ownership.

Description: Abstract 247 Background: β-thalassemia is associated with ineffective erythropoiesis, accelerated erythroid differentiation and apoptosis resulting in anemia and iron overload. The molecular mechanism involved is still incompletely understood. Members of the TGF-β superfamily participate in both proliferation and differentiation of erythroid progenitors. However, the role of these molecules in models of ineffective erythropoiesis has not been addressed so far. RAP-011 is a ligand trap consisting of the extracellular domain of ActRIIA linked to mouse IgG1 Fc domain. We aimed to study the role of ActRIIA signaling in the ineffective erythropoiesis of β-thalassemia and to evaluate the therapeutic impact of RAP-011. Methods: Hbbth1/th1 mice (a model of β-thalassemia intermedia) were subcutaneously treated with RAP-011 (10mg/kg body weight) twice a week for 30–60 days and biological and biochemical parameters were followed. Results: RAP-011 treatment significantly increased hemoglobin levels, red blood cell counts, MCV, MCH and hematocrit with a concomitant decrease in bilirubin levels and reticulocyte counts (since 10 days of treatment and sustained until day 60 of follow up). Flow cytometry analysis showed that RAP-011 significantly decreased late basophilic and polychromatic erythroblast cell numbers in both bone marrow and spleen indicating that RAP-011 corrects ineffective erythropoiesis. We next evaluated the expression of putative ActRIIA ligand(s) in β-thalassemia. Increased expression of Growth Differentiation Factor 11 (GDF11) was observed in cultured erythroblasts and in spleen sections of thalassemic mice. RAP-011 treatment decreased these elevated GDF11 levels in both bone marrow and spleen. We further investigated how BMP/Activin signaling was involved in ineffective erythropoiesis. Anti-GDF11 antibodies, follistatin (activin and GDF11 antagonist) and dorsomorphin (a small molecule inhibitor of SMAD1/5/8 phosphorylation) reduced differentiation, induced FAS-L expression and apoptosis in erythroblasts both in vivo and in vitro whereas noggin (a BMP-2/4 antagonist) had no effect on erythroblast differentiation. Altogether, these data suggest that Activin/BMP signaling controls erythroblast differentiation and targeting BMP type II /activin type II receptors can decrease ineffective erythropoiesis of β-thalassemia. Summary: Sotatercept (a humanized version of RAP-011) is currently in phase II clinical trials for treatment of anemia in patients with Myeloma Bone Disease and End Stage Renal Disease and data from our non-clinical findings support a newly initiated β-thalassemia clinical trial. Our results suggest that sotatercept would be a potential therapeutic tool to improve anemia, increase hemoglobin levels and correct ineffective erythropoiesis and its side effects in β-thalassemic patients. Disclosures: Daniel: Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership. Sung:Celgene: Employment.

Description: Abstract 1055 Background: Cereblon (CRBN) is a component of the E3 ubiquitin ligase complex including CUL4A, DDB1, and ROC-1, and was found to be the molecular binding target of thalidomide (Thalomid®), lenalidomide (Revlimid®), and pomalidomide. CC-220 is a novel immunomodulatory compound developed with increased potency and is currently in development for the treatment of immune conditions. The effect of CC-220 on CRBN binding, ubiquitination, and cell proliferation was profiled. Methods: Binding studies to CRBN were conducted using thalidomide analog-conjugated beads in a competitive assay. Endogenous CRBN from human U266 multiple myeloma (MM) cells was measured by incubating cell extracts with varying concentrations of either CC-220 or pomalidomide as a positive control. Affinity beads coupled to a thalidomide acid analog were incubated with the U266 extracts and, after extensive washing of the beads, the bound proteins were eluted. CRBN binding to the thalidomide-coupled affinity beads was determined by quantitative CRBN immunoblot determination. CRBN ubiquitination was measured in HEK293T cells, which were transfected with an amino-terminal His-biotin-tagged CRBN construct, then preincubated with compounds for one hour followed by treatment with the MG132 proteasome inhibitor (to arrest degradation of ubiquitinated proteins). Cells were lysed and processed to measure CRBN ubiquitination by SDS-PAGE and immunoblot analysis using an anti-ubiquitin antibody. Cell proliferation studies were conducted in lenalidomide-sensitive and -refractory multiple myeloma cells. Lenalidomide-resistant or -sensitive H929 MM cell lines were treated with CC-220 for 5 days, and then cell proliferation and viability were assessed by 7-aminoactinomycin D (7-AAD) staining. T-cell costimulation was measured in purified primary human T cells stimulated using immobilized anti-CD3 antibody in cell culture for 2 days, and cytokine secretion was measured by ELISA. Immunoglobulin M and G (IgG and IgM) production was measured from normal donor peripheral blood mononuclear cells by culturing in the presence of the B cell differentiation factors recombinant human IL-2 (20 U/mL), IL-10 (50 ng/mL), IL-15 (10 ng/mL), His-tagged CD40 Ligand (50 ng/mL), polyHistidine mouse IgG1 antibody (5 μg/mL), and ODN 2006-Human TLR9 ligand (10 μg/mL) for 4 days, followed by IL-2, IL-10, IL-15, and IL-6 (50 ng/mL) for an additional 3 days. IgM and IgG were measured by ELISA. Results: In the competitive CRBN binding studies, preincubation with pomalidomide at a concentration of 3 μM resulted in approximately 50% less CRBN bound to the affinity beads, while CC-220 at a concentration of 0.1 μM resulted in similar CRBN binding. CRBN ubiquitination studies in the transfected HEK293T cells resulted in the following potencies: CC-220 IC50 = 0.19 μM; lenalidomide IC50 = 12.9 μM; and pomalidomide IC50 = 21.6 μM. The IC50 value for inhibition of proliferation by CC-220 shifted from 0.01 μM in the parental H929 cell line and 0.04 μM in the DMSO-treated subclone to 0.51–1.58 μM in the lenalidomide-resistant subclones. A 50% decrease in cell cycle (S-phase) was evident after 24 hours of treatment of H929 cells with CC-220. At 48 hours, CC-220 decreased expression of survivin and retinoblastoma protein (pRB) and increased expression of the cyclin-dependent kinase inhibitor p27. CC-220 costimulated IL-2 production by T cells with an EC50 of approximately 0.29 nM, compared with 10 nM for pomalidomide. CC-220 inhibited IgM and IgG production with an IC50 of 0.35 and 2.1 nM, respectively, compared to 17 nM and 63 nM for pomalidomide. Conclusions: The results indicate that CC-220 binds to CRBN with approximately 30-fold higher affinity than pomalidomide, and inhibits CRBN ubiquitination with approximately 110-fold greater potency than pomalidomide in this system. CC-220 is approximately 34-fold more potent than pomalidomide for costimulating IL-2 production by T cells, and is 30- to 48-fold more potent than pomalidomide for inhibiting immunoglobulin production. In summary, CC-220 is a novel high affinity CRBN ligand with cellular potencies 1 or 2 orders of magnitude greater than that of pomalidomide, and is currently in development for the treatment of immune conditions, including those involving B cell dyscrasias. Disclosures: Schafer: Celgene: Employment, Equity Ownership. Rychak:Celgene: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Parton:Celgene Corp: Employment, Equity Ownership. Capone:Celgene Corp: Employment, Equity Ownership. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership.

Description: Abstract 738 Thalidomide, lenalidomide and pomalidomide are therapeutically active in a number of hematological malignant and premalignant conditions including myelodysplastic syndromes, multiple myeloma, and lymphomas. Clinical efficacy is ascribed to a complement of overlapping activities including direct antitumor effects, immune system activation and inhibition of stromal support of tumor growth. Thalidomide has previously been shown to bind cereblon (CRBN) a protein required for the teratogenic effects of thalidomide in zebrafish and chicken embryos (Ito et al). CRBN forms an ubiquitin E3 ligase complex with DNA damage-binding protein 1 (DDB1), cullin 4 (CUL4) and protein Rbx1 and thalidomide treatment has been shown to inhibit the ubiquitin ligase activity of the complex (Ito et al). Using two independent biophysical methods, we demonstrate that lenalidomide and pomalidomide bind to CRBN-DDB1 complex. Fluorescence-based thermal shift analysis was carried out using purified ZZ-CRBN-DDB1. Phthalimide showed no appreciable binding to the CRBN-DDB1 complex while dose-dependent interaction of thalidomide, lenalidomide and pomalidomide was observed. Thalidomide binding showed approximately ten-fold less affinity (∼ 30 μM) than lenalidomide and pomalidomide (each ∼ 3 μM IC50). Following the procedure of Ito et al, we used thalidomide analog-coupled beads (Thal-beads) and demonstrated binding of CRBN in complex with DDB1 from U266B1 myeloma cell extracts. Preincubation of the extracts with lenalidomide (100 μM) prevented CRBN binding to Thal-beads. Furthermore, the binding of CRBN was dose-dependently inhibited by preincubation with either lenalidomide or pomalidomide with IC50s of 2.3 and 2.1 μM, respectively. We then investigated whether CRBN was required for lenalidomide and pomalidomide responses associated with efficacy. First, CRBN expression was reduced in activated human T cells using CRBN siRNAs. After T cell activation, incubation with lenalidomide (1 μM) or pomalidomide (1 μM) resulted in an 11 to 14- fold-increase in IL-2 and a 5 to 10-fold increase in TNF-α. This increase was reduced ∼60% in the presence of siCRBN. Since IL-2 and TNF-α are important cytokines for tumor surveillance by activated T cells, our results indicate that some of the immunomodulatory effects of lenalidomide and pomalidomide are mediated via CRBN. We next determined if CRBN was required for the antiproliferative effect of lenalidomide and pomalidomide in myeloma cells. Multiple siRNAs were used to silence the expression of CRBN in U266B1 cells resulting in the absence of CRBN protein as determined by immunoblot analysis. Propidium-iodide staining showed that depletion of CRBN affected neither cell cycle nor proliferation of U266B1 cells. However, knockdown of CRBN markedly abrogated lenalidomide- and pomalidomide-induced delay of cell cycle progression. In addition, using lentiviral vectors we produced U266B1 cell lines with either 60% or 75% less expression of CRBN and showed that relative to the parental cell line these cells were gene dose-dependently less responsive to inhibition of proliferation by lenalidomide. The U266B1 cells in which CRBN had been decreased were also less responsive to inhibition by pomalidomide, but this compound maintained greater inhibition of proliferation than lenalidomide in the context of decreasing CRBN. Moreover, gene profile changes by lenalidomide and pomalidomide were reversed in the presence of siCRBN. In particular, induction of p21WAF1 cyclin –dependent kinase inhibitor protein was prevented in the absence of the expression of CRBN. Similar results on different myeloma cell lines and using multiple CRBN siRNAs, confirmed the same critical role of CRBN in the antiproliferative response to lenalidomide and pomalidomide of myeloma cells. Finally, we demonstrated that CRBN expression decreases concomitantly with the acquisition of lenalidomide resistance in H929 myeloma cells. Lenalidomide-resistant H929 cells remain responsive to inhibition by pomalidomide despite relatively reduced expression of CRBN. However, in pomalidomide-resistant DF15R myeloma cells, the complete absence of CRBN confers resistance to both lenalidomide and pomalidomide. Our data demonstrate that CRBN is a direct target of lenalidomide and pomalidomide and plays a crucial role in the antitumor efficacy of lenalidomide and pomalidomide. Disclosures: Lopez-Girona: Celgene Corp: Employment, Equity Ownership. Mendy:Celgene Corp: Employment, Equity Ownership. Miller:Celgene Corp: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership. Kang:Celgene Corp: Employment, Equity Ownership. Carmel:Celgene Corp: Employment, Equity Ownership. Abbasian:Celgene Corp: Employment, Equity Ownership. Mahmoudi:Celgene Corporation: Employment, Equity Ownership. Jackson:Celgene Corporation: Employment, Equity Ownership. Cathers:Celgene Corporation: Employment, Equity Ownership. Rychak:Celgene Corporation: Employment, Equity Ownership. Richard:Celgene Corporation: Employment, Equity Ownership. Brady:Celgene Corporation: Employment, Equity Ownership. Schafer:Celgene Corporation: Employment, Equity Ownership. Evans:Celgene Corporation: Consultancy. Daniel:Celgene Corporation: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership.

Description: Abstract 2845 Lenalidomide is an immunomodulatory agent that has both direct tumoricidal and immunomodulatory activities which are critical for its clinical activity in the treatment of various hematologic malignancies. This activity is at least in part mediated by enhanced T-cell and NK-cell effector function to eliminate tumor B cells, attributed to restoration of impaired T-cell activity and formation of immunologic synapses. Rituximab is an anti-CD20 monoclonal antibody that is active in the treatment of B-cell lymphomas through a variety of mechanisms, including antibody-dependent cellular cytotoxicity (ADCC). Preclinical studies and early clinical trials have shown an enhancement, and potentially synergy, in antitumor activity when lenalidomide is combined with rituximab. In order to further explore the mechanistic basis of this enhancement we investigated the impact of lenalidomide and rituximab on immune synapse formation and ADCC. We have previously shown that the combined use of lenalidomide and rituximab enhances NK cell-mediated immune synapse formation and the resultant cytotoxicity, versus each agent alone. Here we evaluate the molecular events that take place on the cell surface upon exposure of JeKo-1 cells (mantle cell lymphoma) and primary B-CLL cells to lenalidomide alone or lenalidomide plus rituximab. Change in CD20 expression resulting from exposure to vehicle control (0.1% DMSO) or 1 μM lenalidomide for 30 min or 24, 48, 72 hrs was assessed using immunocytochemistry, flow cytometry and isolation of cell membrane-associated proteins followed by Western blotting. At all time points evaluated, levels of cell surface and cell membrane-associated CD20 expression were unchanged in JeKo-1 cells. However, the distribution of CD20 was dramatically altered within 30 minutes after addition of lenalidomide. CD20 redistribution was accompanied by F-actin polymerization and lipid raft aggregation associated with the polarized localization (capping) of a number of proteins including CD20, CD19 and cytoskeleton signaling molecules Rac1 and Vav1, critical regulators of immune synapse formation in effector cells. Of note, other surface proteins involved in signaling such as CD45 were not part of this capping mechanism. By 48 hours of lenalidomide treatment, the majority of JeKo-1 cells (〉80%) showed continued capping of CD20. These responses were also seen in primary B-CLL cells, although the effects were variable. In addition, CD20, F-actin and lipid rafts co-localized at the immune synapses formed between JeKo-1 and NK cells treated with either 1 μM lenalidomide for 24 hrs, 0.1% DMSO for 24 hrs followed by 10 μg/ml rituximab for 30 min, or treated sequentially with 1μM lenalidomide for 24 hrs followed by 10 ug/ml rituximab for 30 min. Lenalidomide and rituximab induced similar effects on B-CLL cells and the immune synapses formed between B-CLL and NK cells. We also determined whether formation of lipid rafts and actin cytoskeleton modifications were a prerequisite for CD20 capping. Cholesterol extraction from JeKo-1cells by 5 mM methyl-β-cyclodextrin (MCD) treatment for 30 min led to complete abrogation of lenalidomide-induced capping. The polymerization of the F-actin cytoskeleton and capping of CD20 was also affected, with no impact on cell viability. In addition, MCD treatment inhibited the formation of immunologic synapses between JeKo-1 cells and NK cells treated with 1 μM lenalidomide alone and in cells co-treated with 1 μM lenalidomide and 10 μg/ml rituximab. These data are consistent with a requirement for the integrity of lipid rafts to maintain the capping of CD20 and to potentially mediate lenalidomide enhancement of ADCC by rituximab. Our results further demonstrate that lenalidomide does not down-regulate CD20, but rather induces its polarized localization at the cell surface. The capping of CD20 is accompanied by redistribution of proteins such as Vav1 and Rac1 that become part of the immune synapse complex. Therefore the capping process induced by lenalidomide appears integral to immune synapse formation and may coordinately enhance the clustering of both the CD20 antigen and the attached rituximab, potentially further enhancing its activity, which would support the clinical combination of these agents. Ongoing studies are currently examining the role of the capping process and intracellular signaling cascades in the direct tumoricidal activity of lenalidomide. Disclosures: Gaidarova: Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Heise:Celgene Corporation: Employment. Aukerman:Celgene Corporation: Employment. Daniel:Celgene Corporation: Employment. Chopra:Celgene Corporation: Employment. Lopez-Girona:Celgene Corporation: Employment, Equity Ownership.

Description: Sotatercept, a recombinant human fusion protein containing the extracellular domain of ActRIIA binds to and inhibits activin and other members of the TGFß superfamily to induce a rapid increase in red cell number and hemoglobin. We sought to investigate if RAP-011, the murine ortholog of sotatercept, might regulate iron availability, which is important for erythropoiesis, through modulating hepcidin levels. Hepcidin is a circulating peptide hormone that negatively regulates iron transport into the plasma membrane by binding the iron transporter, ferroportin, and causing degradation of the complex. Hepcidin is primarily secreted by hepatocytes and also by hematopoietic cells, macrophages, kidney, heart, pancreas, and adipose cells (Gantz, T., Blood, 2003). The hepcidin promoter can be activated by both SMAD-4 and STAT-3, downstream targets of BMP-6 and IL-6 respectively (Andriopoulos Jr, B., Nature Genetics, 2009). BMP-6 binds its receptor, triggering downstream SMAD-1/5/8 signaling leading to SMAD-4 activation and initiation of HAMP (the gene encoding hepcidin) transcription. SMAD-4 signaling is also implicated in STAT-3 activation of HAMP transcription downstream of inflammatory signals such as IL-6 (Wang, R.H., Cell Metabolism, 2005). In the current study, we evaluated the effect of RAP-011 on hepcidin expression and regulation in primary human hepatocytes. Consistent with its role as an activin receptor ligand trap, RAP-011 reduced baseline p-SMAD 1/5/8 expression and subsequently decreased HAMP expression. Next, we asked whether RAP-011 could also decrease HAMP expression under conditions of exogenous BMP-6 stimulation. When hepatocytes were treated with 50 ng BMP-6, an approximately 50-fold induction of HAMP was observed; interestingly, RAP-011 treatment decreased this induction by half. Similarly, treatment of the hepatocytes with the inflammatory cytokine IL-6 led to nearly a 7-fold induction of HAMP which was reduced to baseline levels following the addition of RAP-011. Hepcidin protein levels, although much more variable, mirrored that of HAMP expression following BMP-6 and combined BMP-6 + RAP-011 treatment. Protein changes following IL-6 stimulation were less apparent; this may be due to the fact that the peak of IL-6-stimulated HAMP expression occurred earlier than the 48 hours post-treatment at which the protein was assayed. Our data demonstrate that RAP-011 can modulate SMAD signaling and HAMP expression in primary human hepatocytes and suggests that RAP-011 may allow for increased iron availability for erythropoiesis. Importantly, RAP-011 was able to rescue IL-6-induced HAMP expression, suggesting that the drug might be able to restore iron availability in antinflammatory environment. Anemia of inflammation is associated with diseases such as renal, Castleman’s, and Rheumatoid arthritis. Hepcidin has been shown to play a role in restricting iron and promoting anemia in these pathologies (Steinbicker, A.U., Blood, 2011). In summary, RAP-011, through a yet unknown mechanism, may help to regulate iron availability by regulating hepcidin in pathological situations such as inflammation, iron restriction, or stress erythropoiesis. Disclosures: Malek: Celgene Corp.: Employment. Heise:Celgene: Employment, Equity Ownership. Chopra:Celgene Corp.: Employment, Equity Ownership. Daniel:Celgene Corp.: Employment, Equity Ownership. Sung:Celgene Corp.: Employment, Equity Ownership.

Description: Erythropoiesis, the process of cell proliferation and differentiation that produces erythrocytes, is a tightly regulated process, but apart from early progenitor development and the EPO-dependent response, very little is known about other molecular signals which control cellular fate during RBC production. Members of the transforming growth factor beta (TGFβ) superfamily have been studied as potential regulators of erythropoiesis, iron regulation and globin expression. Sotatercept, an ActRIIA ligand trap, binds to and inhibits activin and other members of the TGFβ superfamily to induce a rapid increase in red cell number and hemoglobin in healthy volunteers. Pharmacological findings demonstrate that RAP-011, a murine ortholog of sotatercept, stimulates RBC parameters in mice through a mechanism distinct from EPO. We conducted the current study to evaluate if RAP-011 may stimulate expansion of a late-stage erythroblast population that is not normally expanded and/or may induce faster differentiation of erythroid precursors. In order to determine if RAP-011 promotes proliferation or differentiation during erythropoiesis, the number of cell divisions was quantified by CFSE staining. During in vitro erythroid differentiation, RAP-011 did not appear to alter the number of cell divisions; however, the percentage of cells that underwent the last division was higher in cultures treated with RAP-011, suggesting that the drug induced faster cellular maturation/differentiation. We also analyzed cell viability of GPA+ cells at the end of the differentiation process and observed that the percentage of apoptotic death was higher in control vs. RAP-011-treated cells. This suggests that RAP-011 may promote survival of late-stage precursors. To assess potential candidates which may mediate the erythropoietic effects of RAP-011, we selected three high affinity RAP-011 ligands, Activin A, Activin B and GDF-11, and proceeded to evaluate their effects on Smad signaling and on erythroid differentiation of human bone marrow progenitors. First, we observed that RAP-011 blocked ligand-induced Smad2/3 phosphorylation in the bone marrow-derived cells. Secondly, RAP-011 rescued activin A-induced inhibition of BFU-E colony formation. Finally, when mature CD36+ cells were differentiated in liquid media containing each of the three ligands, RAP-011 was able to reverse GDF-11- and Activin A-induced inhibition of of erythroid cell proliferation. GDF-11 and Activin A also significantly decreased the percentage of GPA-positive cells in culture, while significantly increasing the percentage of CD45-positive cells. Consistent with proliferation results, RAP-011 blocked these ligand effects. Treatment of CD36+ cells with Activin B did not alter growth or differentiation. These data suggest that GDF-11 and Activin A may contribute, in part, to the erythropoietic stimulatory effects of RAP-011. Several members of the TGFβ superfamily of ligands have been implicated as negative growth regulators, or “chalones”, functioning in homeostasis to maintain specific, mature tissue size. The results from our studies using the ActRIIA-Fc ligand trap, RAP-011, suggest that GDF-11 and Activin A, as well as other sotatercept ligands, may also be “chalones” for the blood, specifically regulating homeostasis of mature RBCs. We suggest that sotatercept increases red blood cell maturation and survival by blocking the negative growth regulation by TGFβ members. In pathologic states such as ineffective erythropoiesis, sotatercept may have an even greater impact than in the healthy, homeostatically-balanced environment. Disclosures: Carrancio: Celgene Corp.: Employment. Markovics:Celgene Corp.: Employment. Wong:Celgene Corp.: Employment. Heise:Celgene: Employment, Equity Ownership. Daniel:Celgene Corp.: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Sung:Celgene Corp.: Employment.

Description: Background: IMiDs® Immunomodulatory agent lenalidomide (Len) is active in a range of hematologic cancers including diffuse large B-cell lymphoma (DLBCL). The molecular target of Len is cereblon (CRBN), a substrate receptor of the cullin 4 RING E3 ubiquitin ligase complex. In a CRBN-dependent manner, Len promotes ubiquitination of lymphoid transcription factors Ikaros and Aiolos leading to their subsequent degradation. This was shown to contribute to Len’s anti-Multiple Myeloma and T-cell activation properties. CC-122 is a non-phthalimide analog of thalidomide and has potent anti-proliferative, immunomodulatory and anti-angiogenic activities giving it a potentially broader range of activity than Len. CC-122, a first-in-class pleiotropic pathway modifier (PPM®), is currently in a Phase I clinical trial for DLBCL, multiple myeloma, and solid tumors (ClinicalTrials.gov NCT01421524) and showing encouraging clinical activity. The present study investigated the mechanism of action of CC-122 in pre-clinical models of DLBCL and in clinical samples from the ongoing Phase I study. Results: CC-122 inhibited proliferation (IC50=0.01-1.5μΜ) and induced apoptosis (6.5-12 fold) in 4 ABC and 3 GCB DLBCL cell lines in a dose-dependent manner. CC-122 induced rapid degradation of Aiolos and Ikaros in a CRBN-dependent manner; an effect blocked by MLN4924, a small molecule inhibitor of Cullin-RING E3 ligase complexes. In vivo anti-tumor activity of CC-122 was demonstrated in 2 xenograft models of human DLBCL, and was associated with significant reduction in Aiolos and Ikaros expression (94% and 69%, respectively, p