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

Abstract

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 knockout 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. Recent comprehensive sequencing studies in a large cohort of DLBCL patients highlight the heterogeneity of alterations including somatic mutations, copy number alterations, and structural variants [2-4]. Among the most frequently rearranged genes are IGH, BCL2, BCL6, and MYC, with 40%, 21%, 19%, and 8% of cases affected, respectively [5-8]. BCL6 is a DNA-binding protein that represses gene transcription in Germinal Center (GC) B-cells through the recruitment of corepressor proteins. In GCs, BCL6 inhibits DNA damage response pathways and thereby prevents cell cycle arrest and apoptosis during class switch recombination and somatic hypermutation required for antibody maturation in B-cells. Subsequent BCL6 downregulation is crucial

Small-molecule-induced polymerization triggers degradation of BCL6

“…BCL6 represses a broad range of genes involved in the DNA damage response 18 , cell cycle checkpoints 19 , and differentiation 20 . As expected, knock-out of BCL6 in lymphoma cells results in tumor stasis 21 . Several peptide and small molecule inhibitors targeting BCL6 have shown efficacy in vivo , but only at high concentrations, which has limited their translation into clinical therapeutic agents 13 , 14 .…”

“…These molecules bind the Broad complex/Tramtrack/Bric-a-brac (BTB) domain, which mediates BCL6 homodimerization and its interactions with co-repressor proteins 22 . BI-3802 induces rapid ubiquitination and degradation of BCL6, resulting in profound de-repression of BCL6 target genes and anti-proliferative effects in DLBCL cell lines, comparable to a genetic knock-out 21 , and superior to non-degrading BCL6 inhibitors such as BI-3812 or heterobifunctional BCL6 degraders 5 , 6 . To uncover the underlying basis of this superior pharmacology, we sought to determine the mechanism by which BCL6 is degraded by BI-3802.…”

High-Grade B-Cell Lymphoma (HGBL) with MYC and BCL2 and/or BCL6 Rearrangements Is Predominantly BCL6-Rearranged and BCL6-Expressing in Taiwan

“…More so, these patients with BCL2 DHL were more advanced in age with median age of 77 ± 10.8 years. Aside these risk factor, consistent with the well documented role of BCL6 in the initiation, therapy evasion, and progression of hematological malignancies, including DLBCL [ 11 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ], it is conceivable that this worse prognosis is associated with the high expression of BCL6 protein in most of these BCL2 DHL cases (62.5% BCL6+ vs. 37.5% BCL6−). Thus, we posit that this observed poor prognosis may be attributable to aberrant BCL6 expression and not because of the BCL2 translocation per se , in the BCL2 DHL cases.…”

“…The predominance of BCL6 rearrangement and associated aberrant expression of BCL6, BCL2, MUM1, and MYC proteins in Taiwanese patients with DHL in contrast to BCL6 DHL rarity in the West informs medical decision making, and does suggest that BCL6 is a promising therapeutic target [ 32 , 33 ]. There are reports demonstrating the druggability of BCL6 and the strong antiproliferative effect of its degradation [ 34 ], as well as evidence that the conditional deletion of BCL6 in DLBCL tumors in vivo induced significant inhibition of tumor growth with initial tumor stasis and subsequent attenuated tumor growth kinetics [ 35 ]. This is of contextual relevance and lends some credence to the findings of our present study which makes a case for the routine screening for BCL6 gene rearrangement by FISH and protein expression by IHC in all newly diagnosed DLBCL cases in Taiwan, and recommends the molecular or pharmacological targeting of BCL6 as an efficacious therapeutic strategy in managing patients with aggressive DHL regardless of COO in Taiwan.…”

High-Yield Human Induced Pluripotent Stem Cell-Derived Monocytes and Macrophages Are Functionally Comparable With Primary Cells

“…Demultiplexing was performed using bcl2fastq v2.20.0.422 from Illumina 1 . Sequencing reads from the RNA-seq experiment were processed with a pipeline building upon the implementation of the ENCODE "Long RNA-seq" pipeline, also described in Schlager et al (2020): filtered reads were mapped against the Homo sapiens (human) genome hg38/GRCh38 (primary assembly, excluding alternate contigs) using the STAR (v2.5.2b) aligner (Dobin et al, 2013), allowing for soft clipping of adapter sequences. For quantification, we used transcript annotation files from Ensembl version 86, which corresponds to GENCODE 25.…”