ALBERT

Description: Introduction: CC-92480 is a novel cereblon E3 ligase modulator (CELMoD) with enhanced autonomous cell-killing and immunomodulatory activity against multiple myeloma (MM) cells. CC-92480 is currently in phase 1 development in a late-line myeloma patient population (NCT03374085). Here, we sought to characterize the antitumor activity of CC-92480 in combination with dexamethasone (DEX), bortezomib (BORT), or daratumumab (DARA) in MM cell lines in vitro and xenograft mouse models in vivo. Methods: CC-92480 activity in combination with DEX was evaluated in MM cell lines. Apoptosis was measured by quantification of caspase-3 activation. The effect of BORT on CC-92480-induced Ikaros and Aiolos degradation was determined by concurrent treatment of MM cells with BORT and CC-92480. β5-site proteasome activity was also determined in the same experiment. The in vitro activity of CC-92480 in combination with BORT was characterized using washout experiments to more faithfully model the short in vivo exposure but more prolonged, gradually diminishing proteasome inhibitory activity of BORT. Apoptosis and cell viability of CC-92480 with BORT were analyzed by flow cytometry. The effect of CC-92480 on CD38 expression was also evaluated across a panel of MM cell lines. The effect of CC-92480 in combination with DARA was characterized with antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) assays. CC-92480 in combination with DEX or BORT was tested in a lenalidomide-resistant (H929-1051) xenograft mouse model. Female SCID mice were inoculated with H929-1051 cells in the right hind leg. For the DEX combination, groups of tumor-bearing mice (n = 9-10) were dosed with vehicle, DEX, or CC-92480 once daily (QD), or CC-92480 in combination with DEX throughout the study, starting when the tumor volumes reached approximately 115 mm3. For combination with BORT, mice (n = 9-10/group) were dosed with vehicle, CC-92480, or BORT, or the CC-92480 and BORT combination starting when the tumor volumes reached approximately 500 mm3. CC-92480 was administered orally QD for 3 days and BORT as a single intravenous dose. Tumor volumes were measured twice a week for the duration of the studies. Results: CC-92480 synergized with DEX in reducing cell viability and potentiated DEX-induced apoptosis in a concentration-dependent manner in MM cell lines. Of note, the combination showed activity at concentrations of both DEX and CC-92480 that had minimal activity as single agents. In the xenograft model with H929-1051 cells, the combination of CC-92480 and DEX significantly inhibited tumor growth (−84%) when compared with either agent alone (−34% and −20% for CC-92480 and DEX, respectively) and was classified as a synergistic effect using the fractional product method. Although proteasome activity is required for CC-92480-induced degradation of Ikaros and Aiolos, CC-92480 nevertheless maintained its ability to efficiently degrade Ikaros and Aiolos in the presence of doses of BORT that cause clinically relevant levels of proteasome inhibition. The in vitro combination of CC-92480 with BORT resulted in greater cytotoxic activity on MM cells than either single agent alone. The in vivo efficacy of CC-92480 and BORT, administered concurrently, showed a strongly synergistic effect with a near complete or complete tumor regression in every animal, and 6 of 9 animals remained tumor-free through an observation period extending 157 days after the control group was terminated. Anti-CD38 therapies, including DARA and isatuxumab, target CD38-expressing MM cells for killing by immune cells through cytotoxic and phagocytic mechanisms. In a panel of MM cell lines, CC-92480 treatment caused increased cell surface expression of CD38 (2-3 times that of control). Pretreatment of MM cells with CC-92480 resulted in increased DARA-mediated ADCC and ADCP compared with DMSO-treated controls. Conclusions: The strong preclinical synergy in MM cell killing exhibited by CC-92480 in combination with DEX, BORT, and with an anti-CD38 antibody (DARA), highlights its potential to bring clinical benefit to patients with MM in combination with these agents and supports the rationale for testing these combinations in clinical studies. Disclosures Wong: Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Leisten:Celgene Corporation: Employment. Bauer:Celgene Corporation: Employment, Equity Ownership. Groza:Celgene Corporation: Employment, Equity Ownership. Gaffney:Celgene: Employment. Havens:Celgene: Equity Ownership; Pfizer: Employment, Equity Ownership. Choi:AnaptysBio Inc: Employment, Equity Ownership; Celgene Corporation: Equity Ownership, Other: Formerly Employed. Lopez-Girona:Celgene Corporation: Employment. Hansen:Celgene Corporation: Employment. Cathers:Celgene Corporation: Equity Ownership; Global Blood Therapeutics (GBT): Employment. Carmichael:Celgene plc: Employment, Equity Ownership. Pierce:Celgene Corporation: Employment, Equity Ownership.

Description: Lenalidomide- and pomalidomide-based therapies are effective drugs in the treatment of patients with multiple myeloma (MM), however most patients with MM eventually relapse or become resistant. CC-92480, a novel cereblon (CRBN) E3 ligase modulator (CELMoD) with multiple activities including potent immunomodulation and single-agent antiproliferative effects, is being investigated in a phase 1 clinical trial (CC-92480-MM-001; NCT03374085) for patients with relapsed/refractory MM (RRMM). The present study investigates the preclinical data and mechanism of action of CC-92480 in MM models. CELMoD agents bound to CRBN confer differentiated substrate-degradation specificity on the CRL4CRBN E3 ubiquitin ligase. CRBN-modulator agents mediate destruction of Ikaros and Aiolos, transcription factors that contribute to myeloma cell survival. CC-92480 was found to produce rapid, deep, and sustained degradation of Ikaros and Aiolos, with superior antimyeloma activity. Accordingly, in a CRBN protein competitive binding assay, CC-92480 displaced a Cy-5-labeled CELMoD analog from CRBN with a 50% inhibitory concentration (IC50) value of 0.03 μM, whereas lenalidomide competed with an IC50 value of 1.27 μM in the same assay, demonstrating a higher binding affinity of CC-92480 for CRBN. Additionally, CC-92480 promoted the recruitment of Ikaros to the CRBN E3 ligase complex more effectively than pomalidomide in 2 orthogonal CRBN/Ikaros binding assays; it also triggered a more extensive cellular ubiquitination of Ikaros, and a faster, more efficient depletion of cellular Ikaros and Aiolos than pomalidomide. In various MM cell lines, including those with acquired resistance to lenalidomide or pomalidomide and low levels of CRBN, CC-92480 produced robust degradation of Ikaros and Aiolos followed by strong reduction of 2 additional and highly critical transcription factors, c-Myc and interferon regulatory factor 4, which are linked to the induction of apoptosis as measured by cleaved caspase-3. The tumoricidal activity of CC-92480 was shown to be CRBN dependent, since the effect was prevented by complete loss of CRBN or by the stabilization of Ikaros and Aiolos. CC-92480 displayed broad and potent antiproliferative activity across a panel of 20 MM cell lines that are either sensitive, have acquired resistance, or are refractory to lenalidomide or pomalidomide; the cell lines also contained diverse chromosomal translocations and oncogenic drivers typically found in MM patients. Approximately half of the MM cell lines evaluated were highly sensitive to CC-92480, with IC50 values for antiproliferative activity ranging from 0.04 to 5 nM; only 2 cell lines had IC50 values 〉 100 nM. CC-92480 inhibits cell proliferation and induces apoptosis in MM cell lines that are not sensitive to lenalidomide or pomalidomide. This panel of cell lines includes both refractory cell lines and resistant cell lines generated through continuous exposure to lenalidomide and pomalidomide that acquired low levels of CRBN protein or mutations in the CRBN gene. CC-92480 also induced deep destruction of Ikaros and Aiolos in cultures of peripheral blood mononuclear cells (PBMCs), which led to the activation of T cells and increased production of the cytokines interleukin-2 and interferon gamma. These responses occurred at the range of CC-92480 concentrations that show potent tumoricidal effect against MM cells. The T cell activation and enhanced cytokine production by CC-92480 led to the potent and effective immune-mediated killing of MM cells in co-cultures with PBMCs. CC-92480 is a potent antiproliferative and proapoptotic novel CELMoD with enhanced autonomous cell-killing activity in MM cells that are either sensitive, resistant, or have acquired resistance to lenalidomide and pomalidomide. CC-92480 has a unique and rapid degradation profile stemming from the enhanced efficiency to drive the formation of a protein-protein interaction between Ikaros and Aiolos and CRBN, inducing cytotoxic effects in a CRL4CRBN-dependent fashion that leads ultimately to the induction of apoptosis, even in the context of low or mutated CRBN protein. Additionally, similar to lenalidomide, CC-92480 conserves immunomodulatory activity against MM cells. These data support the clinical investigation of CC-92480 in patients with RRMM. Disclosures Lopez-Girona: Celgene Corporation: Employment. Havens:Pfizer: Employment, Equity Ownership; Celgene: Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Rychak:Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Gaffney:Celgene: Employment. Surka:Celgene: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Matyskiela:Celgene corporation: Employment. Khambatta:Celgene: Employment. Wong:Celgene Corporation: Employment, Equity Ownership. Hansen:Celgene Corporation: Employment. Pierce:Celgene Corporation: Employment, Equity Ownership. Cathers:Global Blood Therapeutics (GBT): Employment; Celgene Corporation: Equity Ownership. Carmichael:Celgene plc: 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: Key Points Lenalidomide inhibits CLL proliferation in a cereblon/p21-dependent manner. Treatment with lenalidomide induces p21 in CLL independent of p53.

Description: Background: Cereblon (CRBN) is required for the antiproliferative activity of the IMiDs® immunomodulatory drugs lenalidomide (LEN) and pomalidomide (POM) in multiple myeloma (MM), but its value as a biomarker of IMiD response remains unknown. We have shown a disconnect between CRBN mRNA and protein regulation, indicating that measurement of CRBN protein is critical. However, accurate quantification of CRBN protein levels is challenging due to scarcity of standardized and validated reagents and assays. Several publications have suggested that CRBN mRNA and/or protein levels detected with commercial reagents and assays correlate with LEN and/or POM treatment outcomes. In an effort to establish a standardized and validated approach for CRBN protein measurement in MM biopsies, we developed a dual bright-field immunohistochemistry (IHC) assay to measure CRBN levels in CD138+ MM cells. Methods: Using the highly specific CRBN65 rabbit monoclonal antibody against amino acids 65-76 of human cereblon, a CRBN/CD138 dual IHC assay scored using the H-score method was developed and validated. H-scores range from 0 to 300 and are a result of the sum of the products for the percentage of tumor cells (0%-100%) and the intensity of staining (0-3). Since CRBN is a nuclear and cytoplasmic protein, both nuclear and cytoplasmic H-scores were recorded for each sample evaluated with the assay. The dual IHC assay with CRBN65 antibody was used to evaluate the high CRBN–expressing DF15 MM cell line and the low CRBN–expressing DF15R MM cell line (made resistant to POM), as well as bone marrow core biopsies or aspirate clots from 22 patients with MM. For comparison, the same set of samples were stained using a commercially available CRBN antibody in a single IHC assay format. To evaluate interpathologist score variation, H-scores were recorded by 3 pathologists examining the samples independently. Interassay variation was evaluated by staining serial sections of biopsies from the same patient in 3 different experiments and calculating the coefficient of variation (CV) among scores produced for each patient by the same pathologist. Results: The assay was specific and able to detect high CRBN levels in DF15 cells (cytoplasmic score = 190; nuclear score = 150) and low CRBN levels in DF15R cells (cytoplasmic score = 20; nuclear score = 30). The dual IHC assay was able to detect different CRBN staining levels in biopsies and clots from patients with MM, with average cytoplasmic H-scores ranging from 67 to 240 and nuclear H-scores ranging from 17 to 250. Compared with our dual assay, the single IHC assay performed with a commercial CRBN antibody demonstrated lack of specificity, did not detect nuclear CRBN, and showed similar immunoreactivity in the DF15 and DF15R cells. In addition, the commercial CRBN antibody had a narrow range of detection for CRBN (estimated) in patients with MM (Table). Interpathologist comparison of MM biopsy H-scores from 3 pathologists demonstrated high concordance (r2 = 0.73). Assay precision was shown for both cytoplasmic and nuclear CRBN in MM bone marrow biopsies, with CV values of 2% and 5%, respectively. Conclusion: A dual CD138/CRBN IHC assay was developed to provide a reliable semiquantitative method to evaluate CRBN protein levels in both bone marrow core biopsies and clots from patients with MM that is specific, precise, and reproducible. The data highlight the requirement for standardization in the evaluation of CRBN expression. This assay will be used in future trials to assess the value of CRBN as a biomarker in MM and other hematologic malignancies. Table: Estimated CRBN detection in patients with MM Samples Cytoplasmic H-Score Nuclear H-Score Dual IHC Single IHCa Dual IHC Single IHCa Cell lines DF15 (CRBN high) 190 120 150 0 DF15R (CRBN low) 20 120 30 0 Patient biopsies MM16 (CRBN high) 270 200b 270 0 MM20 (CRBN low) 90 100b 10 0 a Commercially available antibody for CRBN. b Values are estimated because tumor cell cannot be reliably identified. Disclosures Ren: Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corp: Employment, Equity Ownership. Couta:Celgene Corp: Employment, Equity Ownership. Hansel:Celgene Corp: Consultancy. Miller:Celgene Corp: Employment, Equity Ownership. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Bjorklund:Celgene Corp: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership. Breider:Celgene Corp: Employment, Equity Ownership.

Description: Numerous cellular effects have been attributed to IMiDs lenalidomide (LEN) and pomalidomide (POM) over the years. However, the zinc finger transcription factors Aiolos (IKZF3) and Ikaros (IKZF1) were identified only recently as substrates targeted for destruction in the presence of IMiDs compounds. LEN and POM bind directly to cereblon (CRBN), a substrate receptor of the cullin ring E3 ligase 4 (CRL4). Binding of these compounds to CRBN triggers the activation of CRL4CRBN, ubiquitylation and destruction of Aiolos and Ikaros. We systematically compared growth, gene expression, and signal transduction responses elicited by doxycycline-inducible, stable Aiolos knockdown or POM treatment in MM cell lines, and their POM-resistant counterparts. Upon exposure to POM, U266 MM cells undergo rapid degradation of Aiolos and Ikaros (90 min) and subsequent downregulation of IRF4 and Myc at 72 hr. At 72-96 hr, a decrease in proliferation and an increase in apoptosis are also observed. U266 MM cells selected in vitro for resistance to POM have lost CRBN expression and consequently no longer downregulate Aiolos and Ikaros after POM administration. Knockdown of Aiolos in these POM-resistant U266 MM cells is sufficient to inhibit their proliferative capacity by 50%, while Aiolos knockdown in POM-sensitive cells caused a greater inhibition of proliferation (90%). This suggests that MM cells with acquired resistance are still dependent on Aiolos for growth, but that a second mechanism may contribute to the antitumorigenic effect of Aiolos downregulation in POM-sensitive U266 cells. We have found that treatment with LEN or POM induces expression of antiviral response genes in MM cells. The induction of interferon (IFN)-stimulated genes (ISGs) such as DDX58, IFIT1, IFIT3, XAF1, ISG15, IFI44, and IFI27 are seen by qPCR in 8 hr of compound treatment (1.8- to 5-fold increase in transcript level) and this effect is further enhanced at 24 and 72 hr. Of note, this response is not accompanied by an increase in β IFN production. The IMiD compound-induced upregulation of the antiviral response correlates with CRL4-CRBN-mediated destruction of the lymphoid restricted transcription factor, Aiolos. In agreement with this, Aiolos knockdown by shRNA is sufficient to trigger a similar effect. These data suggest that Aiolos functions as a transcriptional repressor of ISGs, regulating the antiviral response. Consequently, Aiolos chromatin immunoprecipitation and sequencing (ChIP-Seq) experiments were performed, demonstrating that Aiolos binds near the transcription start site of numerous ISGs, including DDX58, IFIT1, ISG15, XAF1, IFI44, and IFI35. In addition, our data suggest that Aiolos co-binds with STAT and IRF family transcription factors and thereby co-regulates expression of these genes. STAT1 is part of the ISGF3 complex that drives ISG transcription upon viral infection. POM-resistant MM cells lacking CRBN expression do not have STAT1 activity and do not upregulate ISGs upon Aiolos knockdown, even though Myc and IRF4 are still being downregulated. In order to elucidate the relevance of the ISG expression in patients receiving IMiD treatment, we compared the gene expression profile of 12 patients after relapse or disease progression. Paired pre- and posttreatment samples from bone marrow-isolated CD-138 cells were evaluated with RNAseq and gene set enrichment analysis. We found an overall decrease in expression of ISGs, with significant negative enrichment of genes involved in IFN α, β, and γ signaling in relapsed patients. These data from clinical samples confirmed the importance and relevance of the ISGs in the response to IMiDs. In conclusion, our results indicate that Aiolos is a substrate of consequence in IMiD-sensitive MM cells, based on at least 2 pathways: driving the Myc-IRF4 feedback loop and repressing the antiviral pathway. Both in vitro and in vivo patient data suggest that one mechanism of IMiD resistance may be the abrogation of the STAT1 pathway resulting in subsequent blunting of the ISG induction. Finally, while upregulation of ISGs by IMiD treatment may serve as a relevant diagnostic marker of patient responsiveness to these drugs, these data highlight how response and resistance of the IMiD drugs are regulated by the interplay between complex pathway networks, suggesting that the measurement of only one component will not necessarily define the clinical course and outcomes for an individual patient. Disclosures Havens: Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corp: Employment, Equity Ownership. Kang:Celgene Corp: Employment, Equity Ownership. Ortiz:Celgene Corp: Employment, Equity Ownership. Fontanillo:Celgene Corp: Employment, Equity Ownership. Amatangelo:Celgene Corporation: Employment, Equity Ownership. Lu:Celgene Corp: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Bahlis:Celgene Corp: Honoraria, Research Funding. Thakurta:Celgene Corp: Employment, Equity Ownership. Trotter:Celgene Corp: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene Corp: Employment. Chopra:Celgene Corp: Employment, Equity Ownership.

Description: Key Points CC-122 is a novel agent for DLBCL with antitumor and immunomodulatory activity. CC-122 binds CRBN and degrades Aiolos and Ikaros resulting in a mimicry of IFN signaling and apoptosis in DLBCL.

Description: Introduction: Mantle cell lymphoma (MCL) has had one of the poorest prognoses of all NHL subtypes, and is characterized by a relatively short duration of response, progression-free survival, and time-to-progression, with most patients relapsing. The immunomodulatory drug lenalidomide has shown promising activity in MCL and currently is being investigated further in the clinic for treatment of this tumor. In previous experiments, lenalidomide stimulated proliferation as well as activation of γδ T cells in vitro. Potential mechanisms of lenalidomide’s anti-tumor activity in MCL were investigated here. Methods: As γδ T lymphocytes mediate anti-tumor effects through antigen recognition and intracellular signaling, we assessed the effect of lenalidomide on tumor cell expression of co-stimulatory and antigen-presenting molecules, as well as other immune response molecules, using microarray analysis. We then assessed the impact of these tumor cell changes on tumor-cell recognition by γδ T lymphocytes. We treated peripheral blood mononuclear cells (PBMCs) from healthy donors with vehicle or lenalidomide. We next purified γδ T cells from control or lenalidomide treated PBMCs and co-cultured them with MCL cells. Results: Lenalidomide upregulated the expression of several genes involved in the immune response, cell adhesion, cytokine-cytokine receptor interaction, cell cycle and apoptosis by at least 2-fold. In the immune response category, co-stimulatory and antigen-presenting molecules were strongly up regulated including CD86, CD40, CD58, and CD1c. The lenalidomide-induced up-regulation of CD1c was confirmed in MCL lines by quantitative RT-PCR as well as flow cytometry and was also observed in primary B-CLL cells and normal B cells treated with lenalidomide ex-vivo. CD1c, a member of the CD1 family of MHC-like molecules, is expressed mainly in professional antigen-presenting cells, such as B cells, and mediates the presentation of lipid antigens to γδ T cells. Lenalidomide strongly promoted expansion of the Vδ1 subpopulation of γδ T cells and enhanced production of interferon-g. Treatment of MCL cells and γδ T cells with lenalidomide induced changes in the organization of the actin cytoskeleton, re-localization of surface markers and enhanced the number of γδ T-MCL synapses. Furthermore, gd T cells, expanded in the presence of lenalidomide, had enhanced killing activity against MCL cells. Conclusion: Our studies show that lenalidomide might provide dual activity against MCL cells by inducing expression of CD1c in the tumor cells and by enhancing γδ T cell-mediated cytotoxicity. The therapeutic implications, as well as the prospects for novel biomarker development will be discussed further.

Description: Introduction: Lenalidomide is approved in the US for the treatment of transfusion-dependent patients with anemia due to Low- or Intermediate-1-risk myelodysplastic syndromes associated with a del (5q) cytogenetic abnormality, with or without additional cytogenetic abnormalities. Lenalidomide is also approved for use in the US and Europe in combination with dexamethasone in previously treated multiple myeloma patients. In vitro and ex vivo studies have shown that lenalidomide has a direct antiproliferative effect against tumor cells and induces antiangiogenesis. It has also been shown to have immunomodulatory activity, including co-stimulatory effects on T and NK cells. Using the yeast 3-hybrid system, based on a leukemia cDNA library and biotinylated lenalidomide analog, we identified 16 putative lenalidomide-binding proteins from 700,000 clones screened. These included CD3-epsilon-associated protein (CAST) and GDP-mannose pyrophosphorylase A (GMPPA). CAST binds to the T cell receptor and is also part of the RNA polymerase 1 complex. GMPPA is a nucleotidyl transferase that converts mannose-1-phosphate and GTP to GDP-mannose, which is involved in the production of N-linked oligosaccharides. To determine whether these proteins are required for lenalidomide-induced immunomodulatory activity, we evaluated the lenalidomide-induced upregulation of interleukin-2 (IL-2) production in primary T cells transfected with siRNA against CAST and GMPPA. Methods: Primary human peripheral blood T cells were transfected with CAST or GMPPA siRNA, or mock siRNA as a control. Reduction of CAST and GMPPA expression was confirmed by qRT-PCR. After 24 hours, transfected cells were stimulated with anti-CD3 mAb in the presence or absence of 0.1 and 1 μM lenalidomide 48 hours. Cells were lysed after 48 hours, and IL-2 mRNA and mature protein levels were quantified using qPCR and ELISA, respectively. Results: CAST gene expression in T cells was reduced by 60% (p

Description: Abstract 2963 Background: The immunomodulatory agents thalidomide (THAL), lenalidomide (LEN), and pomalidomide (POM) have significant activity in a wide range of hematologic cancers. THAL is primarily a potent anti-angiogenic agent with minimal immunomodulatory activity. LEN and POM both demonstrate significant immunomodulatory activity. Additionally, POM displays anti-myeloma activity in patients with LEN-resistant multiple myeloma (MM). Recently, modulation of cereblon (CRBN)-bound E3 ubiquitin ligase complexes has been implicated in the mechanisms of action of THAL, LEN, and POM. This has enabled rational development of a next generation of compounds. CC-122 is a non-phthalimide analog of the immunomodulatory drugs and a first in class PPM™ pleiotropic pathway modulator that binds the CRBN-DDB1-Cul4-Roc1 E3 ubiquitin ligase complex. This study investigated the anti-proliferative, immunomodulatory, and anti-angiogenic activity of CC-122 in MM and lymphoma cells. Results: CC-122 inhibited proliferation of H929 MM cells in a CRBN-dependent and dose-dependent manner (IC50 = 43 nM). CC-122 induced cell cycle arrest at G0/G1 stage, which was associated with reduced retinoblastoma protein phosphorylation, and increased CDK inhibitor p27 protein expression. CC-122 also inhibited the growth of LEN-resistant H929 cells, although the proliferation IC50 for CC-122 was relatively higher in LEN-resistant cells vs H929 control cells (Table). CC-122 has significant anti-myeloma activity, and has greater activity in LEN-resistant H929 cells vs LEN and POM. Compared with LEN, CC-122 had a greater anti-proliferative effect in diffuse-large B-cell lymphoma (DLBCL). Furthermore, CC-122 had greater anti-proliferative effects in ABC- and PBML-subtypes compared with GCB subtype lines. In ABC-subtype U2932 and OCI-Ly10-DLBCL cell lines, 10 μM CC-122 treatment significantly inhibited DNA-binding of NF-κB p65 (P 〈 .001), p50 subunits (P 〈 .05), and IRF4 in a CRBN-dependent manner. In vivo anticancer activity of CC-122 was demonstrated in xenograft models of human lymphoma and MM. CC-122 exhibits potent immunomodulatory activity in whole blood, T, and natural killer (NK) cells. Additionally, CC-122 enhanced the anti-CD3 mAb-stimulated T-cell production of IL-5, IL-13, GM-CSF, IFN-γ, RANTES, and TNF-α. The immunomodulatory activity of CC-122 was 10-fold more potent vs LEN. We investigated the anti-angiogenic properties of CC-122. In a human umbilical artery sprout outgrowth assay, CC-122 inhibited new vessel growth as well as endothelial cell migration and invasion. It also inhibited endothelial cell sprout formation and migration in a growth factor-induced endothelial cell migration and invasion assay. CC-122 has significantly greater anti-angiogenic activity compared with the LEN and POM in human angiogenesis assays (Table). In contrast, it has less of an anti-platelet effect as measured by megakaryocyte proliferation vs LEN and POM. CRBN binding competition assays were conducted with THAL-binding beads. As demonstrated by the higher IC50 concentration, CC-122 has less potency with regard to CRBN binding compared with LEN or POM. Conclusion: Together, these data demonstrate that the first-in-class PPM™ CC-122 has anti-proliferative, immunomodulatory, and anti-angiogenic properties that may have clinical significance in the treatment of advanced refractory lymphoproliferative disorders and is currently in Phase I studies. Furthermore the data suggest that the potency of binding to CRBN per se does not explain the broad pleiotropic activity of CC-122. Disclosures: Gandhi: Celgene Corp: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Parton:Celgene Corp: Employment, Equity Ownership. Wu:Celgene Corp: Employment, Equity Ownership. Kosek:Celgene Corp: Employment, Equity Ownership. Zhang:Celgene Corp: Employment, Equity Ownership. Capone:Celgene Corp: Employment, Equity Ownership. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership.