CC-122 Has Robust Anti-Proliferative Activity in a Primary Chronic Lymphocytic Leukemia (CLL) Co-Culture Model and Is Superior to Lenalidomide
Authors: Blocksidge Jemma , Glenn Mark , Gandhi Anita K. , Klippel Anke , Pourdehnad Michael , Chopra Rajesh , Kalakonda Nagesh 
Published: October 2019

Abstract
Background: The Ikaros family zinc finger transcription factor, Aiolos, is upregulated in more than 80% of CLL patients with data suggesting that it may be an important driver of CLL survival and hence a bona fide target for therapeutic intervention. To date, transcription factors such as Aiolos have not been amenable to drug therapy. We and others have shown that the IMiDs™ immunomodulatory drugs such as Lenalidomide (LEN) bind Cereblon (CRBN), a substrate receptor of the cullin ring E3 ubiquitin ligase complex (CRL4CRBN) and promote ubiquitination and subsequent proteosomal degradation of lymphoid transcription factors Ikaros and Aiolos. Such insights have been critical for the design and development of next generation IMiD analogues. CC-122 is a novel non-phthalimide, first-in-class, PPM™ pleiotropic pathway modifier drug with anti-proliferative, anti-angiogenic, and immunomodulatory properties. CC-122 specifically targets CRBN and promotes degradation of Aiolos and Ikaros. We have now investigated the effects of LEN and CC-122 on CLL proliferation, and Aiolos and Ikaros degradation, in a cohort of primary CLL cases using an in vitro co-culture system based on CD154 expressing mouse fibroblasts and exogenous IL-21 that recapitulates the in vivo CLL microenvironment.
Results: Forty CLL primary patient PBMC samples, selected to represent diverse prognostic subgroups, were cultured in media supplemented with LEN, CC-122, or control and assessed in a BrdU incorporation assay. Among these analyzed samples, four distinct ‘response’ subsets were identified. In subset 1 (n=10, 25% of cases), we found that CC-122 is superior to LEN in inhibiting proliferation (mean IC50: LEN 5.7µM; CC-122 1.2µM). Subset 2 (n=23; 57% of cases) was sensitive to CC-122, but unresponsive to LEN. In these cases, CC-122 inhibited proliferation by at least 50% at a concentration of ≤3µM despite the failure of LEN (IC50: >10 µM) to inhibit proliferation, highlighting a qualitative difference between the two agents. In subset 3, the anti-proliferative activities of CC-122 and LEN were comparable (n=2, 5% of cases). Finally, both CC-122 and LEN were inactive (up to 10µM) in a 4th subset (n=5; 12% of cases). We subsequently explored associations between CC-122 activity and known CLL prognostic risk factors in these samples. In 5 out of 6 cases with 17p deletion CC-122 inhibited proliferation (IC50: <0.3-0.8 mM). Similarly, in 4 out 6 cases harbouring an 11q- defect CC-122 was active (IC50: 0.8-3 µM). In general, the efficacy of either drug at inhibiting CLL cell proliferation did not segregate with established prognostic variables (e.g. IgVH mutation status, cytogenetic abnormalities, etc.), or previous treatment exposure (e.g. chlorambucil, fludarabine, alemtuzumab, etc.). Preliminary investigations of the CLL cells demonstrate wide variation in baseline Ikaros, Aiolos, and CRBN levels between individual cases as measured by either flow cytometry and/or western blot. We show that treatment of CLL cells with CC-122 or LEN results in variable degradation of Ikaros (LEN 0-100%; CC-122 55-100%) and Aiolos (LEN 72-100%; CC-122 97-100%) compared to untreated controls. Of note, CC-122 was consistently superior to LEN in promoting Aiolos and Ikaros degradation. In contrast, levels of CRBN, the molecular target of CC-122 and LEN, were unaffected by either compound. The predictive value of baseline Aiolos, Ikaros and CRBN expression, as well as their degradation, by CC-122 in relation to cellular response is currently being assessed.
Conclusions: Taken together, these data demonstrate that CC-122 has robust anti-proliferative activity in primary CLL cells through a unique mechanism of selective degradation of Aiolos and Ikaros transcription factors. CC-122 is superior to LEN and is active across several prognostic backgrounds. Furthermore, we show significant activity of CC-122 even in cases that are non-responsive to LEN suggesting that CC-122 targets a novel pathway in CLL. These data suggest that further evaluations of CC-122 in pre-clinical and clinical studies, as a single agent or in combination with established or novel compounds (such as Btk and PI3Kd inhibitors), are warranted.

Disclosures
Blocksidge: Celgene: Research Funding. Glenn:Celgene: Research Funding. Gandhi:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership. Kalakonda:Celgene: Research Funding.