Elucidating the Molecular Mechanisms of Action of CC-99282, a Novel Cereblon (CRBN) E3 Ligase Modulator (CELMoD) Agent, Via Genome-Wide CRISPR Screen in a Preclinical Model of Non-Hodgkin Lymphoma (NHL) (2022)

CC-99282 (BMS-986369) is a novel, oral CELMoD® agent under investigation in a phase 1/2 clinical study in patients (pts) with relapsed or refractory (R/R) NHL (NCT03930953), where early CC-99282 monotherapy data show a manageable safety profile and promising efficacy in heavily pretreated pts. Mechanistically, CC-99282 interacts with CRBN, a CRL4CRBN E3 ubiquitin ligase substrate receptor, to induce recruitment and ubiquitin-mediated proteasomal degradation of the transcription factors Ikaros and Aiolos. Compared with immunomodulatory agents such as lenalidomide that also degrade Ikaros/Aiolos, CC-99282 demonstrates a faster, deeper, and more sustained degradation of these transcription factors. This leads to derepression of cyclin-dependent kinase (CDK) inhibitors and interferon-stimulated genes, such as IRF7, and reduction of the critical oncogenic factor, c-Myc, resulting in potent, autonomous cell killing and apoptosis in a large panel of genetically heterogeneous NHL cell lines. CC-99282 also demonstrated in vivo activity in NHL xenograft models, leading to tumor regression and tumor-free animals (Lopez-Girona et al. Hematol Oncol 2021). Antitumor effects of CC-99282 were independent of cell origin (activated B-cell, germinal center B-cell, or primary mediastinal B-cell lymphoma subtypes of diffuse large B-cell lymphoma) or the presence of high-risk chromosomal translocations (MYC, BCL2, and/or BCL6) (Carrancio et al. Blood 2021). To further elucidate the mechanism of action of CC-99282 and identify additional genes and pathways that modulate CC-99282-mediated antiproliferative effects, a genome-wide CRISPR/Cas9 pooled screen was performed in SU-DHL-4, an NHL cell line with moderate sensitivity to CC-99282. Top-ranked CC-99282-sensitizing or resistance genes identified in the screen were then characterized in a panel of NHL cell lines, using genetic tools and orthogonal confirmatory methods. As expected, genetic knockout (KO) of the CC-99282 targets Ikaros or Aiolos sensitized NHL cells to CC-99282-mediated antiproliferation and attenuated cell survival. KO of select epigenetic modifiers, cell-cycle activators, nucleoplasmic transport factors, and anti-apoptotic genes also enhanced the antiproliferative effect of CC-99282, with the anti-apoptotic gene BCL2 among the top-ranked hits. Venetoclax (VEN), a BCL2 inhibitor approved for the treatment of pts with chronic lymphocytic leukemia and acute myeloid leukemia, was tested in aggressive NHL cell lines alone or in combination with CC-99282. Sensitivity to VEN largely correlated with BCL2 expression, and a synergistic effect with CC-99282 was observed in 58.8% of the aggressive NHL cell lines tested. With respect to genes that reduced sensitivity to CC-99282, top-ranked candidates included previously identified components/regulators of the CUL4-DDB1-RBX1-CRBN E3 ubiquitin ligase complex, such as CRBN and UBE2G1, neddylation and deneddylation machinery, and members of key signaling pathways, such as repressors of NF-κB signaling and cell-cycle regulators. KO of NF-κB suppressor genes induced hyperactivation of the canonical and/or noncanonical NF-κB pathway and reduced CC-99282-induced tumor cell apoptosis. Resistance to CC-99282 was also conferred by KO of autophagy and beclin 1 regulator 1 (AMBRA1), an E3 ligase adaptor that regulates the stability of cyclin D, and low AMBRA1 level was reported as a poor prognosis marker for pts with diffuse large B-cell lymphoma (Simoneschi et al. Nature 2021). AMBRA1 KO led to CC-99282 resistance by promoting stabilization of cyclin D3, activation of CDKs, phosphorylation of retinoblastoma, and subsequent cell-cycle progression, thus reducing CC-99282-induced antiproliferation. Lastly, combination of CC-99282 with CDK4/6 inhibitors palbociclib/abemaciclib, which prevent cell-cycle progression, showed strong synergistic effects in NHL cell lines without RB1 loss or CCND3 mutations. CC-99282 is a novel CELMoD agent that demonstrates potent antiproliferative and pro-apoptotic effects in NHL cells. This study revealed genetic alterations that may be associated with clinical response to CC-99282 in pts with NHL. These in vitro data highlight potential biomarkers that could facilitate patient stratification in future clinical trials and provide rationale for combination therapies that may improve clinical outcomes for pts with R/R NHL.