CC-90009, a novel cereblon E3 ligase modulator, targets acute myeloid leukemia blasts and leukemia stem cells.

A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).

CC-122, a pleiotropic pathway modifier, mimics an interferon response and has antitumor activity in DLBCL.

Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of 2 common substrates, transcription factors Aiolos and Ikaros. Here we report that CC-122, a new chemical entity termed pleiotropic pathway modifier, binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo, and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or short hairpin RNA-mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon (IFN)-stimulated genes independent of IFN-α, -ß, and -γ production and/or secretion and results in apoptosis in both activated B-cell (ABC) and germinal center B-cell DLBCL cell lines. Our results provide mechanistic insight into the cell-of-origin independent antilymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide.

Pomalidomide in combination with dexamethasone results in synergistic anti-tumour responses in pre-clinical models of lenalidomide-resistant multiple myeloma.

Pomalidomide is an IMiD(®) immunomodulatory agent, which has shown clinically significant benefits in relapsed and/or refractory multiple myeloma (rrMM) patients when combined with dexamethasone, regardless of refractory status to lenalidomide or bortezomib. (Schey et al, ; San Miguel et al, 2013; Richardson et al, 2014; Scott, ) In this work, we present preclinical data showing that the combination of pomalidomide with dexamethasone (PomDex) demonstrates potent anti-proliferative and pro-apoptotic activity in both lenalidomide-sensitive and lenalidomide-resistant MM cell lines. PomDex also synergistically inhibited tumour growth compared with single-agent treatment in xenografts of lenalidomide-resistant H929 R10-1 cells. Typical hallmarks of IMiD compound activity, including IKZF3 (Aiolos) degradation, and the downregulation of interferon regulatory factor (IRF) 4 and MYC, seen in lenalidomide-sensitive H929 MM cell lines, were also observed in PomDex-treated lenalidomide-resistant H929 MM cells. Remarkably, this resulted in strong, synergistic effects on the induction of apoptosis in both lenalidomide-sensitive and resistant MM cells. Furthermore, gene expression profiling revealed a unique differential gene expression pattern in PomDex-treated samples, highlighted by the modulation of pro-apoptotic pathways in lenalidomide-resistant cells. These results provide key insights into molecular mechanisms of PomDex in the lenalidomide-resistant setting.

Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity.

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.

Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs.

The Cul4-Rbx1-DDB1-Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.