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Deciphering the Mechanisms of CC-122 Resistance in DLBCL via a Genome-Wide Screen

ABSTRACT: CC-122 is a next-generation cereblon E3 ligase modulating agent that has demonstrated promising clinical efficacy in relapsed or refractory diffuse large B‐cell lymphoma (R/R DLBCL) patients. Mechanistically, CC-122 induces the degradation of IKZF1/3, leading to T cell activation and robust cell-autonomous killing in DLBCL. Here, we report a genome-wide CRISPR/Cas9 positive selection screen for CC-122 in a DLBCL cell line SU-DHL-4 with follow-up mechanistic characterization in 6 DLBCL cell lines to identify genes regulating the response to CC-122. Top-ranked CC-122 resistance genes encode not only well-defined members or regulators of the CUL4-DDB1-RBX1-CRBN E3 ubiquitin ligase complex, but also key components of several signaling and transcriptional networks that have not previously been shown to modulate the response to other cereblon modulators. Ablation of CYLD, NFKBIA, TRAF2, or TRAF3 induces hyper-activation of the canonical and/or non-canonical NF-κB pathways and subsequently diminishes CC-122-induced apoptosis in 5 out of 6 DLBCL cell lines. Depletion of KCTD5, the substrate receptor of the CUL3-RBX1-KCTD5 ubiquitin ligase complex, promotes the stabilization of its cognate substrate, GNG5, resulting in CC-122 resistance in HT, SU-DHL-4, and WSU-DLCL2. Furthermore, knockout of AMBRA1 renders resistance to CC-122 in SU-DHL-4 and U-2932, whereas knockout of RFX7 leads to resistance specifically in SU-DHL-4. The ubiquitous and cell line-specific mechanisms of CC-122 resistance in DLBCL cell lines revealed in this work pinpoint genetic alternations that are potentially associated with clinical resistance in patients, and facilitate the development of biomarker strategies for patient stratification, which may improve clinical outcome of CC-122 for R/R DLBCL.

ORGANISM(S): Homo sapiens

PROVIDER: GSE158303 | GEO | 2020/10/01

REPOSITORIES: GEO

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Project description:We studied transcriptional changes by Affymetrix human microarrays in DLBCL cell lines as a result of treatment with GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 In eukaryotes, epigenetic post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is responsible for repressing target gene expression through methylation of histone H3 on lysine 27 (H3K27). Over-expression of EZH2 is implicated in tumorigenesis and correlates with poor prognosis in multiple tumor types. Recent reports have identified somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The Y641 residue is the most frequently mutated residue, with 22% of GCB (Germinal Cell B-cell) DLBCL and FL harboring mutations at this site. These lymphomas exhibit increased H3K27 tri-methylation (H3K27me3) due to altered substrate preferences of the mutant enzymes. However, it is unknown whether direct inhibition of EZH2 methyltransferase activity alone will be effective in treating lymphomas carrying activating EZH2 mutations. Herein, we demonstrate that GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and dramatically inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma. 10 DLBCL cell lines (7 mutant and 3 wild type EZH2), that were differentially sensitive to GSK126 in proliferation assays, were treated for 72 hours, in duplicate (n=2), with either DMSO (vehicle) or 500nM of GSK126, a potent selective EZH2 inhibitor. EZH2 mutant cell lines are Pfeiffer, KARPAS-422, WSU-DLCL2, SU-DHL-10, SU-DHL-6, DB and SU-DHL-4. EZH2 wildtype cell lines are HT, OCI-LY-19 and Toledo.

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Deciphering the Mechanisms of CC-122 Resistance in DLBCL via a Genome-Wide Screen

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