BCL6 Inhibitor Peptide Have Powerful Anti-Lymphoma Activity in Animal Models of Diffuse Large B-Cell Lymphoma and Synergize with Other Anti-Lymphoma Drugs.
Authors: Antun Ana , Cerchietti Leandro , Aparo Santiago , Shaknovich Rita , Melnick Ari 
Published: April 2020

The BCL6 oncogenic transcriptional repressor is constitutively expressed in about 60% of Diffuse Large B-cell Lymphomas (DLBCLs). We previously developed a recombinant inhibitor peptide that specifically blocks the ability of BCL6 to mediate transcriptional repression. Based on this peptide, we developed a novel retro-Inverso peptidomimetic inhibitor called BPI (BCL6 Peptidomimetic Inhibitor) that is far more potent and stable than its prototype. We have treated a large panel of DLBCL cell lines with BPI to determine the spectrum and mechanisms of sensitivity and resistance to this agent. BPI (1 to 20 μM) caused dose dependent killing of 6 of 10 DLBCL cell lines in vitro. Sensitive DLBCL cell lines display a high percentage of necrotic and apoptotic cells as shown by 7-ADD and Annexin-V staining. Additionally, by BrdU incorporation plus PI staining, we demonstrated that cells undergo cell cycle arrest before the death pathway is initiated. Since BCL6 represses genes involved in DNA repair checkpoints, cell cycle and protein ubiquitylation and degradation, we predicted that combination therapy with cytotoxic drugs or drugs that alter cellular protein metabolism might synergize with BPI to kill DLBCL cells. We performed combinatorial therapy studies where additional drugs were administered 24 hours after BPI and cells were evaluated for viability in several different assays. We found an additive to synergistic effect of BPI with several cytotoxic drugs commonly used in lymphoma therapy (such as doxorubicin, alkylating agents, etoposide and dexamethasone), as well as bortezomib (a proteasome inhibitor). In vivo xenotransplantation studies with the BPI sensitive cell lines Ly1, Ly7, SUDHL4 and SUDHL6 showed a marked decrease in tumor size and weight (p=0.03 for control vs BPI, T-test), as well as serum β2-microglobulin (BPI: 6.5 ± 2 μg/ml vs control: 14.3 ± 2.5 μg/ml, p=0.02), even when BPI was administered at very low doses (150 μg per day). The tumor remnants from BPI treated animals showed extensive induction of apoptosis (determined by TUNEL), a lower mitotic index (2 ± 0.8/100 cells vs 4.8 ± 0.9/100 cells, for BPI vs. control respectively, p&lt;0.01), and upregulation of BCL6 target genes such as p53 and p21. Finally, we tested BPI in a series of 30 tissue samples obtained from patients with clinical suspicion of lymphoma. Of these, the 11 patients with BCL6 positive B-cell tumors were highly sensitive to BPI while none of the remaining 19 patients responded (of these 19 = 10 were reactive lymph node, 2 Hodgkin’s disease, 2 T-cell lymphomas and the rest were other non-lymphoid tumors), underlining the specificity of BPI for BCL6 positive tumors. Our data indicates that BPI alone has powerful anti-lymphoma activity and warrants clinical evaluation in patients with DLBCLs. BPI can synergize with other drugs suggesting that combination therapy might provide more potent and less toxic therapeutic regimens for these patients.