BI-3812 and BI-3802 were obtained by opnMe, Boehringer Ingelheim. Where indicated, the following drugs were used for the indicated times and concentrations: cycloheximide (Sigma), doxycycline hyclate (Sigma), puromycin (Santa Cruz Biotechnology, Dallas, TX), blasticidin (Calbiochem), doxorubicin (Selleckchem), and FX1 (Selleckchem). Cell lines with stable Omomyc expression or FBXO11 deletion/depletion were generated by lentiviral transduction and puromycin selection (as detailed in “Virus preparation and cell transduction”). All cell lines were grown at 37☌ in a humidified atmosphere with 5% CO 2. Cells were grown in RPMI 1640 supplemented with 15% FBS, penicillin-streptomycin (100 U/mL), and l-glutamine (2 mM) and cultured for ≥4 weeks before proceeding with further experiments. Single-cell suspensions were prepared from fresh tumoral lymph nodes using mechanical disaggregation and isolated with a 40-μm nylon cell strainer (BD Biosystems). Briefly, at the humane end point, mice were euthanized, and tumoral lymph nodes were resected. Murine lymphoma cell lines were obtained from Eμ-myc transgenic mice with the corresponding genotype. Thus, by validating the functional role of FBXO11 mutations in BL, we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches, such as BCL6 degraders and direct MYC inhibition, could be exploited as a targeted therapy for BL.
Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. In wild-type and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degraders or inhibitors partially impaired lymphoma growth in vitro and in vivo. Conditional deletion of 1 or 2 copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B-cell lymphoma onset, providing experimental evidence that FBXO11 is a haploinsufficient oncosuppressor in B-cell lymphoma. FBXO11 mutations impaired BCL6 degradation, and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Here, we show that FBXO11 mutations are found in 23% of patients with Burkitt lymphoma (BL). FBXO11 was recently identified as a ubiquitin ligase that is involved in the degradation of BCL6, and it is frequently inactivated in lymphoma or other tumors.
The expression of BCL6 in B-cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions, or reduced proteasome-mediated degradation.
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