Oncogenic Feedback Activation Between BCL6 and MLL Promotes Malignant Transformation in MLL-RearrangedAcute Lymphoblastic Leukemia
Authors: Hurtz Christian , Chan Lai N , Ballabio Erica , Willman Cheryl L. , Carroll William L. , Armstrong Scott A. , Ernst Patricia , Melnick Ari , Milne Thomas , Müschen Markus 
Published: October 2019

Abstract
Background: BCL6 is known as a protooncogene and transcriptional repressor in diffuse large B cell lymphoma, where it is frequently involved in chromosomal rearrangements. We recently identified BCL6 as a novel mediator of drug resistance to tyrosine kinase inhibitors (TKI) in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myeloid leukemia (Duy et al., Nature 2011; Hurtz et al., J Exp Med 2011). In addition,BCL6 directly competes with the tumor suppressor BACH2 for p53 promotor binding to protecting cells from p53-mediated apoptosis in multiple ALL subgroups (Swaminathan et al., Nature Medicine 2013). Based on this, our current study is focusing on the function of BCL6 in different subtypes of human ALL.
Results: Analysis of gene expression data from 207 children with high-risk B cell precursor ALL (COG P9906) showed that high expression levels of BCL6 at the time of diagnosis correlated with a poor overall and relapse-free survival (p=0.007). Furthermore, 49 matched sample pairs from patients at diagnosis and relapse showed increased BCL6 levels at relapse compared to diagnosis (p=0.003). To test whether or not there are specific subtypes of leukemia with high BCL6 expression levels, we studied BCL6 expression via western blot and immunohistochemistry staining in Non-Ph+ cell lines and ALL patient samples (n=76). Interestingly, BCL6 levels are particularly elevated in MLL-rearranged (MLLr) ALL cases. In addition, patients from the clinical trial that had high BCL6 levels and had MLL rearrangements had the worst clinical outcome (p=0.0009). We next tested if MLLr oncogenes drive aberrant BCL6 expression. First, we performed a ChIP-analysis using antibodies against MLL, AF4, and ENL, which provided evidence for direct binding to the BCL6 promoter. We then performed a BCL6 Western blot analysis of inducible MLL-AF4-transgenic and retrovirally transduced MLL-ENL pre-B cells, demonstrating that both oncogenes are sufficient to induce ~10-fold upregulation of BCL6 protein levels. Additionally, we used a newly developed conditional BCL6 knock out/reporter mouse model to decipher the function of BCL6. We transduced B cell progenitor cells from BCL6fl/fl mice with MLL-ENL and either with a control or Cre-expression vector. Using the BCL6 reporter capability of the mouse we found that BCL6 is significantly higher expressed in MLL-ENL transduced cells. To test if MLL is required for BCL6 upregulation, we used a conditional MLL knock out mouse and found that after Cre-mediated deletion of MLL, pro-B, mature-B, and MLL-AF4 transduced ALL cells almost lost the ability to upregulate BCL6. Interestingly, using inducible BCL6 transgenic and knockout as well as retrovirally transduced pre-B and ALL cells showed that overexpression of BCL6 leads to higher expression levels of MLL and deletion of BCL6 results in lower expression levels of MLL. Therefore, we conclude that MLL and BCL6 both cooperate and activate each other's expression in an activating feedback loop. Strikingly, Cre-mediated BCL6- deficiency results in apoptosis of MLL-ENL transduced cells.
Clinical relevance: To verify if the high BCL6 expression levels in MLL-AF4 patients are important for the disease progression, we transduced primary human ALL xenografts with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell cycle arrest and cell death. To test if pharmacological inhibition of BCL6 is of potential use for patients with MLLr leukemia, we treated multiple MLL-AF4 rearranged human xenograft cases with a RI-BPI a BCL6 peptide inhibitor. Strikingly, treatment with RI-BPI not only compromised colony formation in methylcellulose it also prevents leukemia-initiation in transplant recipient mice. RI-BPI also had a strong synergistic effect when combined with the chemotherapy drug Vincristine, which represents the backbone for most high risk regimen in pediatric ALL.
Conclusions: These findings identify BCL6 as a central factor in leukemia initiation and survival and its pharmacological inhibition as a novel strategy to treat MLL-rearranged ALL. Aberrant expression of BCL6 in MLLr ALL is the direct consequence of the MLLr oncogenic activity in these cells. Based on these findings, we propose combinations of BCL6 inhibitors with currently used chemotherapeutics as potential approach to reduce the risk of ALL relapse and improve overall outcome.

Disclosures
Armstrong: Epizyme, Inc: Consultancy. Ernst:Amgen: Other: stocks. Melnick:Janssen: Research Funding.