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A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).

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.

The cereblon E3 ligase modulator (CELMoD) CC-885 co-opts the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex to trigger the ubiquitination and proteasomal degradation of the translation termination factor G1 to S phase transition 1 (GSPT1), resulting in robust growth inhibition in AML cell lines and primary patient blasts (Matyskiela ME, et al. Nature. 2016;535:252-7). However, CC-885 also triggers off-target degradation of other cereblon (CRBN) neosubstrates, potentially leading to toxicities. Here, we present the identification of a novel GSPT1-selective CELMoD, CC-90009, and the mechanistic rationale for its clinical development in relapsed or refractory (R/R) AML (CC-90009-AML-001; NCT02848001). To identify new CELMoD agents, we performed cell-based phenotypic screening using a panel of 11 human AML cell lines harboring common oncogenic mutations. The screening identified CC-90009, which demonstrated potent antiproliferative and proapoptotic activity with 50% inhibitory concentration (IC50) values ranging from 3 to 75 nM in 10 out of 11 cell lines. Maximal apoptosis was reached between 16 and 48 hours post exposure to CC-90009 in 5 AML cell lines with cells being committed to apoptosis within 8-16 hours of treatment. CC-90009 was then tested in a panel of samples obtained from 30 patients with newly diagnosed or R/R AML. Bone marrow aspirates obtained during patient diagnosis were plated without separation of constituent cells and tested for sensitivity to CC-90009 using the PharmaFlow PM test which was reported to predict clinical response to standard-of-care chemotherapy with 81% accuracy in AML (Martínez-Cuadrón D, et al. Leuk Res. 2019;76:1-10). The efficacy of CC-90009 was concentration dependent in 26 of 30 patient samples, with an average half-maximal response concentration (EC50) of 21 nM. Leukemic cell killing was rapid and highly efficient: > 82% of leukemic cells were eliminated within 24 hours and nearly all were eliminated within 96 hours. In contrast, CC-90009 showed only modest activity against normal lymphocytes in the same patient samples. Knockout of CRBN via CRISPR/Cas9-mediated gene editing completely abrogated the activity of CC-90009 in sensitive AML cell lines, suggesting that the anti-AML activity of CC-90009 is mediated by the degradation of GSPT1. Tandem mass tag quantitative mass spectrometry analysis of AML cells treated with CC-90009 revealed that CC-90009 selectively reduced the abundance of GSPT1 with little to no effect on the rest of the proteome. The degradation of GSPT1 by CC-90009 was blocked by proteasomal inhibition or inactivation of the CRL4CRBN E3 ubiquitinligase complex. Overexpression of a GSPT1-degradation-resistant mutant, G575N, conferred complete resistance to CC-90009, while RNA interference (RNAi)-mediated partial knockdown of GSPT1 gene expression enhanced the response to CC-90009. Mechanistically, GSPT1 degradation induced by CC-90009 triggers the activation of the integrated stress response pathway, which is associated with the induction of apoptosis and inhibition of proliferation. CC-90009 is a CELMoD and first-in-class GSPT1 degrader entering clinical development. The degradation of GSPT1 was confirmed to be essential for CC-90009-induced apoptosis and antiproliferative activity. The profound antiproliferative activity of CC-90009 in > 80% of human AML cell lines and patient blasts strongly supports the ongoing phase 1 study in R/R AML. Disclosures Lopez-Girona: Celgene Corporation: Employment. Lu:Celgene Corporation: Employment, Equity Ownership. Rychak:Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Lu:Celgene Corporation: Employment, Equity Ownership. Rappley:Celgene Corporation: Employment. Fontanillo:Celgene Corporation: Employment. Cathers:Global Blood Therapeutics (GBT): Employment; Celgene Corporation: Equity Ownership. Daniel:Celgene Corporation: Employment. Hansen:Celgene Corporation: Employment.

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.

Thalidomide and the immunomodulatory drug, lenalidomide, are therapeutically active in hematological malignancies. The ubiquitously expressed E3 ligase protein cereblon (CRBN) has been identified as the primary teratogenic target of thalidomide. Our studies demonstrate that thalidomide, lenalidomide and another immunomodulatory drug, pomalidomide, bound endogenous CRBN and recombinant CRBN-DNA damage binding protein-1 (DDB1) complexes. CRBN mediated antiproliferative activities of lenalidomide and pomalidomide in myeloma cells, as well as lenalidomide- and pomalidomide-induced cytokine production in T cells. Lenalidomide and pomalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not thalidomide-binding defective CRBN(YW/AA). Overexpression of CRBN wild-type protein, but not CRBN(YW/AA) mutant protein, in KMS12 myeloma cells, amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21(WAF-1) expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to both pomalidomide and lenalidomide, CRBN protein was undetectable. Our biophysical, biochemical and gene silencing studies show that CRBN is a proximate, therapeutically important molecular target of lenalidomide and pomalidomide.

Overexpression of the transcription factor interferon regulatory factor-4 (IRF4), which is common in multiple myeloma (MM), is associated with poor prognosis. Patients with higher IRF4 expression have significantly poorer overall survival than those with low IRF4 expression. Lenalidomide is an IMiD immunomodulatory compound that has both tumouricidal and immunomodulatory activity in MM. This study showed that lenalidomide downregulated IRF4 levels in MM cell lines and bone marrow samples within 8 h of drug exposure. This was associated with a decrease in MYC levels, as well as an initial G1 cell cycle arrest, decreased cell proliferation, and cell death by day 5 of treatment. In eight MM cell lines, high IRF4 levels correlated with increased lenalidomide sensitivity. The clinical significance of this observation was investigated in 154 patients with MM. Among MM patients with high levels of IRF4 expression, treatment with lenalidomide led to a significantly longer overall survival than other therapies in a retrospective analysis. These data confirm the central role of IRF4 in MM pathogenesis; indicate that this is an important mechanism by which lenalidomide exerts its antitumour effects; and may provide a mechanistic biomarker to predict response to lenalidomide.

Thalidomide and the IMiD immunomodulatory drugs, lenalidomide and pomalidomide, are widely used in the treatment of multiple myeloma (MM), del(5q) myelodysplastic syndromes and other hematologic malignancies, including mantle cell lymphoma. Ito et al.1 recently identified cereblon as a key target of thalidomide. Subsequent studies confirmed cereblon to be a common target for lenalidomide and pomalidomide, and established its essential role in mediating anticancer and immunomodulatory effects of these drugs.2, 3 Cereblon is encoded by the CRBN gene on chromosome 3 containing 11 exons, and the fully spliced transcript produces a 51-kDa protein. Cereblon is a component of the cullin ring E3 ubiquitin ligase complex (CRL4CRBN) that also contains DNA damage-binding protein 1 (DDB1), cullin (Cul) 4a and regulator of cullins (Roc) 1.1 E3 ligases attach ubiquitin moieties to specific substrate proteins in the cell that can mark them for proteasomal degradation. The putative role of cereblon within the E3 ligase complex is that of a substrate receptor.

Decreased p27Kip1 levels are a poor prognostic factor in many malignancies, and can occur through up-regulation of SCFSkp2 E3 ligase function, resulting in enhanced p27 ubiquitination and proteasome-mediated degradation. While proteasome inhibitors stabilize p27Kip1, agents inhibiting SCFSkp2 may represent more directly targeted drugs with the promise of enhanced efficacy and reduced toxicity. Using high-throughput screening, we identified Compound A (CpdA), which interfered with SCFSkp2 ligase function in vitro, and induced specific accumulation of p21 and other SCFSkp2 substrates in cells without activating a heat-shock protein response. CpdA prevented incorporation of Skp2 into the SCFSkp2 ligase, and induced G1/S cell-cycle arrest as well as SCFSkp2- and p27-dependent cell killing. This programmed cell death was caspase-independent, and instead occurred through activation of autophagy. In models of multiple myeloma, CpdA overcame resistance to dexamethasone, doxorubicin, and melphalan, as well as to bortezomib, and also acted synergistically with this proteasome inhibitor. Importantly, CpdA was active against patient-derived plasma cells and both myeloid and lymphoblastoid leukemia blasts, and showed preferential activity against neoplastic cells while relatively sparing other marrow components. These findings provide a rational framework for further development of SCFSkp2 inhibitors as a novel class of antitumor agents.

Over expression of the PI3 kinase/mTOR/AKT pathway has been well documented in MM patient biopsies and human MM cell lines, suggesting this pathway plays a key role in the survival and proliferation of malignant plasma cells. Rapamycin and the rapalogs are allosteric inhibitors of the mTORC1 complex (consisting of mTOR, raptor, mLST8 and PRAS40), inducing mainly cytostatic effects but not cell death. Inhibition of mTORC1 prevents a negative feedback loop to the mTORC2 complex (consisting of mTOR, Rictor, mLST8 and Sin 1) leading to the phosphorylation of AKT. Phosphorylated AKT is a key inducer of anti-apoptosis mechanisms and cell cycle progression, which may explain the limited results of the rapalogs in the clinic. Recently developed mTOR kinase inhibitors (i.e., CC-223) target both mTORC1 and mTORC2 complexes in order to inhibit tumor growth and importantly, induce cell death. Here we evaluate the effects of CC-223 on a panel of MM cell lines, in combination with current standard of care agents in MM (the corticosteroid, dexamethasone [DEX] and the IMiD® immunomodulatory drugs, lenalidomide [LEN] and pomalidomide [POM]), as well as in the context of LEN resistance. Single agent CC-223 was shown to inhibit cell proliferation in a panel of 10 MM cell lines achieving IC50 values between 0.1-1 µM following 5 days of treatment. CC-223 also reduced cell viability reaching IC50 values between 0.4-1 µM in 5 out of 10 MM cell lines tested. CC-223 induced concentration-dependent G1 phase arrest within 24h of treatment followed by an induction of cell death by 48h. The anti-MM tumor activity of CC-223 (0.3-10 mg/kg) was further tested in SCID mice with xenotransplants of NCI-H929 grown to approximately 100-150 mm3 in size. A dose-dependent tumor growth inhibition and tumor growth delay was seen with once daily dosing of CC-223. Combination of CC-223 with standard of care therapy compounds was also evaluated in vitro. The combination of CC-223 and DEX demonstrated synergistic effects on the inhibition of cell proliferation in 6 MM cell lines (combination index: 0.0002-0.38) tested over 5 days. CC-223 also had synergistic effects on the same panel of MM cell lines when combined with LEN (combination index: 0.05-0.8). Acquisition of drug resistance in patients receiving standard of care therapies is still one of the major clinical problems in MM. POM, the next generation of IMiD® immunomodulatory agents, has shown clinically meaningful results in patients that are resistant or have relapsed to their drug regimens, including LEN. We have recently developed in vitro cellular models of LEN-resistance using the H929 MM cell line. H929 cells with acquired resistance to LEN (H929 R10-1, R10-2, R10-3 and R10-4) were shown to have one copy number loss of cereblon compared to their matched LEN-sensitive control (H929 D1). In addition to this, protein expression analysis identified that these resistant cell lines also gained the activation of signaling pathways such as PI3K/AKT/mTOR, MEK/MAPK as well as anti-apoptotic factors. For example, S473 AKT phosphorylation was highly elevated in LEN-resistant cell lines which correlated with loss of PTEN protein expression (H929 R10-3 and R10-4). Interestingly, regardless of PI3K/AKT/mTOR pathway status, all LEN-sensitive and resistant H929 cells responded to CC-223 treatment with a strong inhibition of cell proliferation (H929 D1 IC50 0.2 µM, and H929 R10 1-4 IC50 0.2-0.35 µM) and to a lesser effect, induction of cell death, over a 5 day period. Similar to the panel of MM cell lines, G1 arrest occurred after 24h treatment and cell death (Sub-G1) was increased by 72h of treatment. CC-223 treatment reduced S473 pAKT and p-4EBP1 after 1h while total AKT and 4EBP1 remained unchanged in both the sensitive and resistant MM cell lines. Combination treatment of LEN-sensitive and resistant H929 cells with CC-223 and POM had synergistic inhibitory effects on cell proliferation (combination index: 0.35-0.7) and cell viability (combination index: 0.15-0.42). In conclusion, the mTOR kinase inhibitor, CC-223 potently inhibited MM cell proliferation by inducing G1 arrest and cell death in a panel of MM cell lines and reduction of tumor volume in vivo. The combination of LEN, POM or DEX with CC-223 had synergistic effects on MM cell proliferation and viability. Therefore, CC-223 in combination with other standard of care agents could become an important clinical tool for the treatment of MM in the future. Disclosures: Rychak: Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene: Employment, Equity Ownership. Miller:Celgene Corporation: Employment, Equity Ownership. Leisten:Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Raymon:Celgene Corporation: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Lopez-Girona:Celgene: Employment, Equity Ownership.

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.

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.

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.

Introduction: The proteasome inhibitor bortezomib (VELCADE®; formerly PS-341) has been approved for therapy of relapsed/refractory multiple myeloma but may be limited in its effectiveness by activation of an anti-apoptotic heat shock protein response. In contrast, targeting a unique E3 ubiquitin ligase could selectively stabilize specific cellular proteins whose ubiquitination is regulated by this E3, reducing the likelihood of any unwanted pro-survival effects on other pathways. Methods: To this end, we sought to identify a small molecule that preferentially stabilized the tumor suppressor proteins p21Cip1 and p27Kip1. Results: Here we report the identification of CC-125007, a small molecule that stabilized p21Cip1, p27Kip1, and p57 in models of multiple myeloma. Unlike bortezomib, CC-125007 did not induce accumulation of other cell cycle regulatory proteins such as p53. CC-125007 reduced viability in a time- and concentration-dependent fashion and, again unlike bortezomib, which induced arrest at G2/M, CC-125007 induced cell cycle arrest at the G1/S phase. Decreased viability due to CC-125007 was not accompanied by caspase-mediated apoptosis, however, since it could not be blocked by pre-incubation with a pan-caspase inhibitor. Instead, a puncate monodansylcadaverine staining pattern was observed, demonstrating the formation of autophagic vacuoles consistent with activation of autophagy, or type II programmed cell death. Further supporting this possibility, combination of the autophagy inhibitor bafilomycin A1 and CC-125007 diminished autophagic vacuole formation. Furthermore, Western blotting demonstrated that CC-125007 induced conversion of microtubule-associated protein light chain 3 form I to II, a biochemical marker for autophagy. Meanwhile, Western blotting also showed that CC-125007 did not activate the anti-apoptotic heat shock response proteins 27, 70, and 90, which would be typical of bortezomib. CC-125007 overcame melphalan and bortezomib resistance in multiple myeloma cell lines, and a combination of low dose bortezomib and CC-125007 triggered synergistic anti-myeloma activity. Importantly, in freshly isolated bone marrow mononuclear cells purified from multiple myeloma patients, CC-125007 decreased the viability of the CD138+ population to a much greater extant than that of the CD138− cells. CC-125007 also stabilized p27Kip1 in samples from patients with acute myeloid leukemia, and enhanced bortezomib-induced apoptosis. Studies to elucidate the mechanism by which CC-125007 stabilized p27Kip1 indicate direct targeting of the E3 ligase. Conclusions: Together, these data suggest that p27Kip1 E3 ligase inhibition with agents such as CC-125007 is a rational strategy for the treatment of multiple myeloma and other hematological malignancies, providing a framework for further development of this novel class of agents.