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: CC-122 modulates the CRL4 E3 ligase complex, which results in recruitment and degradation of Aiolos and Ikaros. This results in cell autonomous anti-lymphoma and immunomodulatory effects on T and NK-cell function; such as increased expression of activation markers and cytokine production such as IFN-γ and TNF-α (Gandhi, ASH 2012). Single agent CC-122 is currently in a Phase I clinical trial for diffuse large B-cell lymphoma (DLBCL), multiple myeloma, and solid tumors (NCT01421524) and in combination with obinutuzumab (GA101) in DLBCL and indolent non-Hodgkin lymphoma (iNHL) (NCT02417285). We sought to understand the effects of combining CC-122 with anti-CD20 monoclonal antibody obinutuzumab (GA101) in in vitro and in vivo models of DLBCL and follicular lymphoma (FL). Methods: Apoptosis and cell toxicity was measured by Annexin V/ToPro-3 flow cytometry and CellTiter Glo luciferase assays, respectively. Antibody dependent cellular cytotoxicity (ADCC) was measured by anti-CD3 stimulated peripheral blood mononuclear cells (PBMCs) treated with vehicle or 100 nM CC-122 for 3 days. Prior to co-culture, target tumor cells were incubated with vehicle or GA101 for 1 hour, washed and incubated with treated PBMCs for an additional 4 hours followed by an apoptosis assay. DOHH2 and OCI-LY10 xenograft models were analyzed in C.B-17/Icr-Prkdcscid/IcoIcr mice, which retain NK cells, and CC-122 was dosed at 30 mg/kg, qd days (days 1-5 out of a 7 day schedule). Results: In the DOHH2 FL cell line, the percentage of Annexin V and ToPro-3 positive DOHH2 cells treated with 40 nΜ CC-122 or 1000 ng/mL GA101 was 35% and 40%, respectively. The combination of CC-122 and GA101 resulted in a synergistic enhancement with 82% cell death (theoretical additivity = 61%). Treatment of RL FL cells with 40 nΜ CC-122 or 2000 ng/mL GA101 resulted in 14% and 12% apoptotic cells, respectively. CC-122 combined with GA101 showed a synergistic enhancement of 44% cell death (theoretical additivity = 24.2%). In ADCC assays, DOHH2 cells incubated with vehicle treated PBMCs demonstrated 14% apoptosis. DOHH2 cells treated with 1000 ng/ml GA101 and vehicle treated PBMCs exhibited 42% apoptosis. The addition of CC-122 (100 nM) treated PBMCs to GA101 treated DOHH2 cells increased the level of ADCC to 57%. Similarly, CC-122 treatment of PBMCs co-cultured with GA101 treated RL cells led to increased levels of apoptosis by ADCC (75.7%) compared to DMSO treated PBMC with GA101 treated RL cells (62.8%) and vehicle treated co-cultures (33.9%). In a DOHH2 xenograft model, the mean tumor volume (MTV) for vehicle treated controls was 2818 mm3 at the conclusion of the study, compared to CC-122 MTV of 1568 mm3 (44% tumor growth inhibition (TGI)). GA101 monotherapies at 0.5 and 3 mg/kg once weekly yielded dose-related MTVs of 1576 mm3 (44% TGI) and 826 mm3 (71% TGI), respectively. CC-122 in combination with GA101 at 0.5 mg/kg resulted in a MTV of 694 mm3 (75% TGI), whereas the combination of CC-122 with GA101 at 3 mg/kg yielded a MTV of 493 mm3 (83% TGI). All regimens produced TGI that was statistically different from vehicle treated controls (p≤0.001). In 2 DLBCL cell lines (OCI-LY10, ABC-DLBCL and WSU-DLCL2, GCB-DLBCL), CC-122 treatment induced apoptosis with an IC50 ranging from 100 to 300 nΜ. Cell toxicity of either single agent CC-122, GA101 or a combination of the two was measured. OCI-LY10 and WSU-DLCL2 cells treated with 40 nΜ CC-122 reduced viability by 20% and 15%, respectively. OCI-LY10 and WSU-DLCL2 cells treated with 2000 ng/mL GA101 reduced viability by 46% and 62%, respectively. The combination of CC-122 with GA101 in either cell line resulted in additive effects of 58% and 68%, respectively. In the OCI-LY10 xenograft model, CC-122 demonstrated no significant reduction in MTV compared to vehicle. Single agent GA101 at 0.5 and 2.5 mg/kg once weekly yielded dose-related reduction in TGI's of 30.5% and 49.2%, respectively. CC-122 in combination with GA101 at 0.5 mg/kg resulted in 47% TGI), whereas the combination of CC-122 with GA101 at 2.5 mg/kg yielded a 94.5% TGI. Conclusion: Together, these data demonstrate that CC-122 has potent cell autonomous and immunomodulatory activities and that the addition of GA101 provides a synergistic effect in FL and is additive in DLBCL when compared to either as single agents. These data provide pre-clinical proof of concept that a combination of CC-122 and GA101 in the treatment of DLBCL and iNHL may be clinically beneficial. Disclosures Chiu: Celgene: Employment, Equity Ownership. Hagner:Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment, Equity Ownership. Daniel:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Klippel:Celgene Corporation: Employment, Equity Ownership. Klein:Roche: Employment. Thakurta:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene: Employment, Equity Ownership.

Description: Background: CC-220 is a Cereblon (CRBN) binding compound currently under clinical investigation for systemic lupus erythematosus. Comparable to other Cereblon-binding agents, ex vivo treatment of CC-220 on B-cells, T-cells and monocytes leads to the degradation of the hematopoietic transcription factors Ikaros (IKZF1) and Aiolos (IKZF3).(1) Currently, CC-220 is being investigated in a phase Ib/IIa study CC-220-MM-001 (clintrial.gov trial #NCT02773030) as a single agent, or in combination with dexamethasone in relapsed/refractory multiple myeloma (RRMM) in patients who may have previously been exposed to pomalidomide. Here, we provide pre-clinical data and mechanistic rationale for the clinical development of CC-220 in heavily pre-treated RRMM. Results: In order to evaluate the ability of CC-220 effects on MM cells in vitro, we generated a large panel of MM cell lines (~69) that consist of 5 categories, including lenalidomide-sensitive (LS; n=26), intrinsically lenalidomide-resistant (ILR; n=7), acquired lenalidomide-resistant (ALR; n=12), acquired lenalidomide/dexamethasone-dual-resistant (ALDR; n=12), and acquired-pomalidomide-resistant (APR; n=12). Cell proliferation by 3H-thymidine incorporation at concentration between 0.01-100 μM was assessed by the area under the curve (AUC) for both CC-220 and pomalidomide. The average AUC was significantly reduced by 65% vs. 52% (p

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: Background: The zinc finger transcription factors, Aiolos (IKZF3) and Ikaros (IKZF1) were identified as lenalidomide (LEN) and pomalidomide (POM)-induced substrates of the cereblon (CRBN)-dependent Culin4 E3-ligase complex. While recent studies suggest that the anti-proliferative activity of LEN and POM in multiple myeloma (MM) cell lines in vitro is due in part to the targeted ubiquitination and subsequent proteasomal degradation of Aiolos and Ikaros, the downstream molecular mechanisms remain unknown. Using inducible shRNA-mediated knockdown combined with kinetic analyses, we systematically investigated the biological mechanisms associated with the degradation of Ikaros and Aiolos in MM cell lines that are sensitive to or have acquired resistance to LEN and POM. Results: In MM1.S and U266 MM cell lines stably engineered with doxycycline (DOXY)-inducible shRNAs, knockdown of either Ikaros or Aiolos showed a reduction in cell proliferation (80%-90%) as measured by 3H-thymidine incorporation after a 4 day treatment with DOXY. We demonstrated that this anti-proliferative effect is inherently tied to and precedes the induction of apoptosis, which was maximized (60%-80% AnnV+/ToPro3+) 5 days following Aiolos or Ikaros knockdown compared with a control shRNA. shRNA-mediated knockdown of Aiolos or Ikaros was furthermore associated with decreases in both c-Myc and IRF4 protein expression levels (70%-90% and 60%-80%, respectively) that were maximized by day 4. In turn, shRNA knockdown of either c-Myc or IRF4 elicited anti-proliferative (〉 80% inhibition) and pro-apoptotic (50%-80%) responses as early as 48hrs after shRNA induction. These data suggest that the reduction of c-Myc and IRF4 protein levels downstream of Aiolos and Ikaros degradation account for the apoptotic effect and marks the onset of the cytotoxic response induced by LEN and POM in MM cells. To define the temporal order of events involving Aiolos, Ikaros, c-Myc and IRF4 in more detail, kinetic experiments following shRNA-mediated knockdown in parallel with drug treatments were performed. Data from these experiments showed that there is a distinct kinetic order of both LEN- and POM-mediated effects, initiated by immediate targeted degradation of Aiolos and Ikaros (within 90 min), followed by a decrease in c-Myc levels (24-48 hrs) with subsequent IRF4 downregulation (48-72 hrs), and finally, resulting in programmed cell death (3-5 days). Importantly, DOXY washout experiments, resulting in re-accumulation of Aiolos or Ikaros at early time points (24 hrs) partially overcame the antiproliferative effects of the shRNA-mediated knockdown of either target. Interestingly, upon the onset of c-Myc downregulation (24-48 hrs), the commitment to cell death could no longer be reversed in our experiments. Further, we generated MM1.S and U266 cells with acquired resistance to POM (10 µM; also cross-resistant to LEN) (MM1.S/PomR and U266/PomR , respectively), in which CRBN protein expression is substantially decreased (〉 90%). Consequently, in these resistant cell lines, neither Aiolos nor Ikaros are degraded in the presence of LEN or POM. However, bypass of CRBN-dependent Aiolos degradation by DOXY-induced knockdown rescued c-Myc and IRF4 downregulation and concomitant inhibition of growth (90% and 60%, respectively), suggesting that resistant MM cells with acquired CRBN loss remain dependent on Aiolos and Ikaros. Conclusions: For the first time, our studies showed that degradation of Aiolos and Ikaros sets up a molecular sequence of events culminating in programmed cell death in MM cells. Our mechanistic studies showed that c-Myc is a key intermediate factor whose downregulation is a rate-limiting step for the transcriptional downregulation of IRF4 as well as for the commitment to cell death. Taken together, our results demonstrate a molecular sequence of events underlying the mechanism of action of cytotoxicity of LEN or POM in MM cells. Quantitative measurements of Aiolos and Ikaros degradation, and c-Myc and IRF4 downregulation in clinical samples would help validate these findings. Disclosures Bjorklund: Celgene Corp: Employment, Equity Ownership. Havens:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corp: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corp: Employment, Equity Ownership. Amatangelo:Celgene Corp: Employment, Equity Ownership. Lu:Celgene Corp: Employment. Wang:Celgene Corp: Consultancy. Breider:Celgene Corp: Employment. Ren:Celgene Corp: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene Corp: Employment. Chopra:Celgene Corp: Employment, Equity Ownership.

Description: Background: CC-122 is a Cereblon (CRBN)-dependent Cul4 E3-ligase complex modulating compound currently being investigated in several hematologic malignancies, including relapsed/refractory multiple myeloma (MM). CC-122 binding to Cereblon results in the selective ubiquitination and subsequent proteasomal degradation of the hematopoietic transcription factors Ikaros (IKZF1) and Aiolos (IKZF3), accounting for its cytotoxic and immunomodulatory effects in diffuse large B-cell lymphoma.(1) In a phase I study CC-122-ST-001 (clintrial.gov trial #NCT01421524), CC-122 showed single agent overall response rates of ~18% in heavily pre-treated (median 6 lines prior therapy) relapsed/refractory MM. CC-122 is currently being explored as a single agent or in combination with dexamethasone in patients who previously failed lenalidomide.(2) Here, we examine the effects of CC-122 in pre-clinical models of MM with varying degrees of sensitivity to lenalidomide and dexamethasone. Results: We established lenalidomide-resistant and lenalidomide/dexamethasone dual-resistant cell lines according to a previously reported protocol.(3) A broad panel of MM cell lines (57) consisting of lenalidomide-sensitive (LS; n=26), intrinsically lenalidomide-resistant (ILR; n=7), acquired lenalidomide-resistant (ALR; n=12), and acquired lenalidomide/dexamethasone-dual-resistant (ALDR; n=12) MM cell lines were investigated for the effects of either lenalidomide or CC-122, with and without dexamethasone. Proliferation analysis by 3H-thymidine incorporation at similar concentrations (0.01-100 μM), showed a greater reduction over the averaged area under the curve (AUC) for CC-122 compared to lenalidomide of 53% to 34% (p

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.