Abstract
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<0.001) in tumor tissue within 6 hrs of administration. To identify proteins and signaling pathways affected by CC-122, tandem-mass tag proteomics was performed on DLBCL cells treated with vehicle or CC-122. While Aiolos and Ikaros levels decreased in a time- and dose-dependent manner, Interferon Stimulated Genes (ISGs) including IRF7, DDX58, and IFIT3 were notably increased (3, 5 and 12.5 fold, respectively). ISG induction with 24 hr CC-122 treatment was further confirmed by Western analysis. Surprisingly, the induction of ISGs occurred in the absence of increased interferon α, β, or γ levels, which was confirmed by qPCR and ELISA. Furthermore, shRNA targeting Aiolos led to inhibition of Aiolos protein expression (2-3 fold) and an increase in the mRNA and protein levels of IRF7, DDX58 and IFIT3 (10, 14 and 22 fold in mRNA expression, respectively). These data indicate that the increase in ISGs is a direct consequence of Aiolos degradation by CC-122 and bypasses the need for interferon production. Exploration of additional downstream functional gene candidates of CC-122 and shAiolos activity revealed a decrease in protein levels of Myc (up to 3 fold decrease with shAiolos), a key driver of DLBCL proliferation and survival associated with poor survival. Importantly, reduction of Aiolos expression and the consequent changes in the molecular pathways described led to inhibition of proliferation (2-4 fold versus control shRNA) and increased apoptosis (3-10 fold vs control shRNA), which paralleled CC-122 effects in DLBCL cells lines. Of note, CC-122 induction of ISGs was comparable in both ABC and GCB lines, whereas Len did not induce ISGs in GCB lines.
The relationship between Aiolos and Ikaros protein expression and DLBCL cell of origin (COO) was further explored in primary DLBCL tissues. A tissue microarray containing 90 DLBCL patient cases immunohistochemically stained for Aiolos, Ikaros and COO markers (Hans) demonstrated Aiolos and Ikaros expression across GCB and non-GCB subtypes, suggesting that CC-122 may have broad-spectrum activity independent of COO. In fact, Aiolos and Ikaros were degraded in lymph node biopsies from both GCB and non-GCB DLBCL patients administered CC-122. The predictive value of Aiolos and Ikaros protein levels for clinical response will be assessed in the Phase I study.
Conclusion: Aiolos degradation-dependent upregulation of ISGs represents a novel mechanism for the activity of CC-122. This mechanism bypasses the interferon-signaling pathway and occurs across cell of origin subtypes, suggesting this is a common mechanism accounting for the broad-spectrum activity of CC-122. Understanding of this mechanism has potential to greatly enhance the clinical development of CC-122 in DLBCL by guiding patient selection strategies and rational combination therapies.
Disclosures
Hagner: Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corp: Employment, Equity Ownership. Couto:Celgene Corporation: Employment, Equity Ownership. Breider:Celgene Corp: Employment. Fontanillo:Celgene Corp: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corp: Employment, Equity Ownership. Havens:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Raymon:Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Barnes:Celgene Corporation: Employment, Equity Ownership. Waldman:Celgene Corporation: Employment, Equity Ownership. Daniel:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene Corp: Employment. Pourdehnad:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership.
The MATH-BTB BPM3 and BPM5 subunits of Cullin3-RING E3 ubiquitin ligases target PP2CA and other clade A PP2Cs for degradation