SU-DHL-4

7-DMAG Inducing SU-DHL-4 Cell Apoptosis

17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) is a water-soluble, semisynthetic derivative of endotoxin that has anticancer effects. Cells were treated with different concentrations of 17-DMAG for 48 h, and then cell viability was analyzed using an MTT assay. The results revealed that 17-DMAG inhibited the proliferation of SU-DHL-4 in a dose-dependent manner (Fig. 1). Cells were cultured with 17-DMAG at different concentrations for 24 h and stained with PI, following which the proportion of PI-positive cells was measured using flow cytometry. The results revealed that the apoptosis of SU-DHL-4 cells was induced by 17-DMAG in a dose-dependent manner (Fig. 2).

To determine whether 17-DMAG induces apoptosis through the activation of the mitochondrial apoptotic pathway, the expression levels of several proteins involved in the mitochondrial apoptotic pathway were measured using western blot analysis. The expression levels of the anti-apoptotic protein Bcl-2 were increased and HSPA5 was reduced following treatment with 17-DMAG, whereas levels of the proapoptotic protein Bax were constant (Fig. 3). These results indicated that 17-DMAG may be an effective novel agent for the treatment of diffuse large B-cell lymphoma.

Fig. 1 Effects of 17-DMAG on the viability of the diffuse large B-cell lymphoma cell line SU-DHL-4. (Li JJ, et al., 2017)

Fig. 2 17-DMAG treatment induces apoptosis in diffuse large B-cell lymphoma cells line SU-DHL-4. (Li JJ, et al., 2017)

Fig. 3 Western blot analysis of apoptosis-related proteins with β-actin as an equal loading control. (Li JJ, et al., 2017)

Genome-Wide CRISPR/Cas9 Screening for CC-122 in SU-DHL-4 Cell Line

CC-122 is a next-generation cereblon E3 ligase-modulating agent that has demonstrated promising clinical efficacy in patients with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). To delineate genetic alterations in patients with DLBCL that might attenuate the response to CC-122, a genome-wide CRISPR/Cas9 screening was performed to identify genes and pathways that affect the response to CC-122 in the DLBCL cell line SU-DHL-4.

SU-DHL-4 cells stably expressing Cas9 were transduced with a pooled lentiviral library targeting 19 011 protein-encoding genes with 4 to 8 different sgRNAs for each gene. At day 3 after transduction (time 0; T₀), cells were treated with 10 µM CC-122 or dimethyl sulfoxide (DMSO) vehicle control for an additional 11 days to allow for 10 more doublings of the DMSO-treated cells (time 10; T₁₀), followed by amplification of sgRNA coding regions from genomic DNA collected from viable cells at T₀ and T₁₀, and next-generation sequencing to identify and quantify the abundance of sgRNAs (Fig. 4A).

Among the 52 top-ranked genes with a more than twofold enrichment at the gene level (log2FC >1), many genes encoding well-characterized proteins were identified that mediate the degradation of IKZF1 and IKZF3 induced by cereblon modulators. These include the CRL4^CRBN subunits CRBN and CUL-4A, the ubiquitin conjugation enzymes UBE2G1 and UBE2D3, and multiple members of the COP-9 signalosome (Fig. 4B-C). In addition, pathway enrichment analysis of top-ranked resistant hits identified 4 genes encoding well-known repressors of the canonical or noncanonical NF-κB signaling pathways: CYLD, NFKBIA, TRAF2, and TRAF3 (Fig. 4B-D). Moreover, a few CC-122-resistant genes were identified such as KCTD5, AMBRA1, and RFX7 that had not been reported to modulate the response to other cereblon modulators (Fig. 4B-C).

Fig. 4 A Genome-wide CRISPR screening reveals candidate CC-122 resistance genes in DLBCL of SU-DHL-4 cells. (Mo Z, et al., 2021)