Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations. Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients. An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL. However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors. Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and in vivo. We show that BCL6 knock-out in SU-DHL-4 cells in vitro results in an anti-proliferative response 4-7 days after Cas9 induction that was characterized by cell cycle (G1) arrest. Conditional BCL6 deletion in established DLBCL tumors in vivo induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics. Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/Cas9 systems for probing oncogene addiction. For the generation of dox-inducible cell lines, SU-DHL-4 cells were modified to express the rtTA3 using lentiviral transduction of MSCV-rtTA3-IRES-EcoR-PGK-Puro followed by selection with Puromycin. The resulting cell line was transduced with a TRE3G promoter-driven Cas9-GFP cassette (pRRL-TRE3G-Cas9-P2A-GFP). Single cell clones were picked, expanded and tested for Cas9 induction and promoter leakiness as described. A selected SU-DHL-4 Cas9 clone was infected with a vector allowing for U6 promoter driven constitutive expression of negative control and BCL6 targeting sgRNAs and in addition contains an EF-1alpha short (EF1as) promoter driven mCherry fluorescence marker. Editing efficiency was confirmed in a bulk depletion assay as described before. mCherry+ cells (sgRNA expressing cells) were single cell sorted using a Sony Sorter SH800. For treatment of SU-DHL-4 cells to induce Cas9 expression, doxycycline (Sigma #D9891) was dissolved in sterile water and was added to the cell culture medium at a final concentration of 100 ng/ml if not indicated differently. Subsequently cells were harvested for total RNA isolation using TriZol (Ambion) and further purified using miRNAeasy columns (Qiagen). Approximately 500ng of total RNA were subjected to library preparation using the TruSeq RNA Library Prep Kit v2 with polyA selection as recommended by the manufacturer (Illumina) without generating stranded information. Library were multiplexed and sequenced on a NextSeq 500 system using paired-end sequencing with 76 cycles.