ALBERT

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