We demonstrate the usefulness of synthetic lethal screening of a conditionally BCL6-deficient Burkitt lymphoma cell line, DG75-AB7, with a library of small molecules to determine survival pathways suppressed by BCL6 and suggest mechanismbased treatments for lymphoma. Lestaurtinib, a JAK2 inhibitor and one of the hits from the screen, repressed survival of BCL6-deficient cells in vitro and reduced growth and proliferation of xenografts in vivo. BCL6 deficiency in DG75-AB7 induced JAK2 mRNA and protein expression and STAT3 phosphorylation. Surface IL10RA was elevated by BCL6 deficiency, and blockade of IL10RA repressed STAT3 phosphorylation. Therefore, we define an IL10RA/JAK2/STAT3 pathway each component of which is repressed by BCL6. We also show for the first time that JAK2 is a direct BCL6 target gene; BCL6 bound to the JAK2 promoter in vitro and was enriched by ChIP-seq. The place of JAK2 inhibitors in the treatment of diffuse large B-cell lymphoma has not been defined; we suggest that JAK2 inhibitors might be most effective in poor prognosis ABC-DLBCL, which shows higher levels of IL10RA, JAK2, and STAT3 but lower levels of BCL6 than GC-DLBCL and might be usefully combined with novel approaches such as inhibition of IL10RA.There is a need for new treatments for poor-prognosis activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), 6 which continues to have a cure rate Ͻ40% with conventional chemotherapy (1).The majority of ABC-DLBCL, in contrast to germinal center B-cell-like DLBCL, have low level expression of BCL6 mRNA and protein (2). JAK/STAT3 signaling is active in ABC-DLBCL and is enhanced by constitutive activity of the NF-B pathway (3), which in turn is driven by oncogenic CARD11 mutations (4), chronic active B-cell receptor signaling (5), and MYD88 mutations (6). However, other factors are also likely to be important in determining the overall activity of JAK/STAT3 signaling. BCL6 directly represses both STAT3 (7) and NF-B p105/p50 (8) transcription, and levels of BCL6 might, therefore, be a factor independent of the known oncogenic mutations, which determines the activity of signaling pathways required by ABC-DLBCL. In this report we develop a novel B-cell line to pursue the hypothesis that genes repressed by BCL6 are components of survival signaling pathways in lymphomas with low level expression of this transcription factor.BCL6 is a zinc finger transcription factor that is highly expressed in normal germinal center B-cells (9) and is required for high affinity antibody production (10, 11). It is also constitutively expressed in ϳ40% of cases of the high grade B-cell lymphoma DLBCL due to either chromosomal translocations, mutations of a negative regulatory site in the promoter region (12-14), or abnormalities of post-translational regulation (15)(16)(17).The N-terminal POZ domain of BCL6 associates with co-repressors NCOR1, BCOR, and SMRT (NCOR2), which in turn recruit histone deacetylases to accomplish transcriptional repression. Work largely carried out with human Burkitt lymphoma cel...

Increased STAT3 signalling is a factor in driving ~50% of diffuse large B-cell lymphoma. In some cases increased cytokine production by the lymphoma is responsible for activation of JAK2 and STAT3 but the regulation of signalling through this pathway is not clear. We constructed a conditional BCL6 deficient cell line through disruption of the endogenous BCL6 loci of a genetically tractable human B-cell lymphoma by homologous recombination, and insertion of a tetracycline regulatable BCL6 transgene. On induction of BCL6 deficiency growth of the cell line slowed by 3 to 4-fold. A synthetic lethal screen employing a library of small molecule inhibitors in genetically BCL6 deficient lymphoma cells showed that lestaurtinib, a JAK2 inhibitor, enhanced loss of viability. We investigated the hypothesis that JAK2 is a direct BCL6 target gene. JAK2 mRNA and protein expression were induced by BCL6 deficiency. Inspection of the JAK2 proximal promoter region demonstrated a potential BCL6 binding site. BCL6 bound to this sequence in vitro and mutagenesis of the binding site relieved BCL6 mediated transcriptional repression in luciferase reporter assays. Analysis of ChIP-seq data showed a binding peak in the JAK2 proximal promoter region confirming in vivo BCL6 binding. Data from a large and publicly available gene expression dataset confirmed an inverse correlation between BCL6 and JAK2 mRNA whilst there was a positive correlation between JAK2 and STAT3 mRNA. STAT3 is a known target of BCL6 transcriptional repression. We suggest that BCL6 represses both JAK2 and STAT3 and that relatively high BCL6 levels will tend to reduce JAK2-STAT3 signalling whereas lower BCL6 levels will amplify this pathway. Mouse xenografts utilising our conditional BCL6 deficient cell line showed that lestaurtinib alone caused minimum reduction to tumour size but the combination of BCL6 deficiency and JAK2 inhibitor caused growth reduction with central necrosis. In summary we identify JAK2 as a BCL6 target gene and propose that BCL6 has an important role in regulation of JAK2-STAT3 signalling in DLBCL. BCL6 mRNA expression may be a biomarker to enable the rational use of JAK2 inhibitors in DLBCL. Disclosures No relevant conflicts of interest to declare.