CC-220 Is a Potent Cereblon Modulating Agent That Displays Anti-Proliferative, Pro-Apoptotic and Immunomodulatory Activity on Sensitive and Resistant Multiple Myeloma Cell Lines
Authors: Bjorklund Chad C , Kang Jian , Lu Ling , Amatangelo Michael , Chiu Hsiling , Gandhi Anita K. , Pourdehnad Michael , Klippel Anke , Thakurta Anjan 
Published: October 2019

Abstract
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<0.01) for LS, 33% vs. 20% (p<0.01) for ILR, 30% vs. 20% (p<0.01) for ALR, 25% vs.10% (p<0.01) for ALDR, and 23% vs. 8% (ns) for PR cells for CC-220 vs. pomalidomide respectively. Apoptosis was analyzed by flow cytometry and AnnV+/ToPro3+ staining where CC-220 significantly (p<0.01) induced an average of 36% apoptotic cells compared to 30% for pomalidomide in LS cells, and 18% vs. 6% (p<0.5) in PS cells. Importantly, CC-220 showed anti-proliferative and pro-apoptotic activity in PR cells where Cereblon was still expressed. Additionally, both proliferation inhibition and apoptosis were synergistically enhanced across all cell line categories when CC-220 was used in combination with dexamethasone.
We next evaluated the immunmodulatory effects on peripheral blood mononuclear cell (PBMCs)-stimulated killing of MM cells. Following a 72 hr incubation with CD3-stimulated PBMCs, CC-220 significantly induced the death of MM cells (~60%, across all cell type categories) within 4 hr, at concentrations more than 10-fold lower than pomalidomide. The observed CC-220-stimulated PBMC co-culture killing of MM cells closely correlated with dose-dependent increases in IL-2 secretion and Granzyme B release. Notably, CC-220 induced PBMC-mediated death of MM cells lacking observable Cereblon protein expression.
Lastly, we evaluated the mechanism of action of CC-220 in MM cells in vitro. In the absence of Cereblon, as shown by shRNA knockdown or downregulation in a subset of PR cells, there is very little if any cell autonomous activity of CC-220, implicating Cereblon-dependency for its effects. Downstream of Cereblon, CC-220 stimulates the complete proteasomal degradation of both Ikaros and Aiolos in as little as 6 hr. Measurement of the half maximal time for 50% degradation of both Ikaros and Aiolos is kinetically faster from 1.9-2.9 vs. 2.4-6.9 hr depending on the MM cell line at a 10-fold lower dose for CC-220 compared to pomalidomide, respectively. CC-220 is also more efficient than pomalidomide at causing downregulation of the c-Myc/IRF4 axis, which has been shown to be essential for the cytotoxic effect of pomalidomide.(2)
Conclusions: CC-220 is a potent anti-proliferative and pro-apoptotic compound that shows activity in several MM cell line categories with differing sensitivity to lenalidomide, pomalidomide and dexamethasone. Importantly, CC-220 induces PBMC-mediated killing of all MM cell lines regardless of the level of Cereblon expression and cell autonomous sensitivity. Mechanistically CC-220 acts through binding of Cereblon, leading to the degradation of the hematopoietic transcription factors Ikaros and Aiolos, followed by disruption of the MM promoting c-Myc/IRF4 axis. Taken together, these data support the clinical investigation of CC-220 in relapsed/refractory MM patients,who have previously been exposed to pomalidomide.

Disclosures
Bjorklund: Celgene Corporation: Employment, Equity Ownership. Kang:Celgene Corporation: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Amatangelo:Celgene: Employment, Equity Ownership. Chiu:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Klippel:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene: Employment, Equity Ownership.