Identification Of BCL6 As a Therapeutic Target In MLL-Rearranged ALL
Authors: Hurtz Christian , Geng Huimin , Ballabio Erica , Xiao Gang , Ng Carina , Masouleh Behzad Kharabi , Willman Cheryl L. , Armstrong Scott A. , Milne Thomas , Melnick Ari , Muschen Markus 
Published: October 2019

Abstract

Background
BCL6 has been identified in diffuse large B cell lymphoma (DLBCL), where it is frequently translocated to immunoglobulin loci and acts as a protoncogene and transcriptional repressor. We found in Ph+ acute lymphoblastic leukemia (ALL) that BCL6 is involved in a novel form of drug resistance to tyrosine kinase inhibitors (TKI) by protecting cells from p53-mediated apoptosis (Duy et al., Nature 2011). Our current study is focusing on the function of BCL6 in Non-Ph+ALL based on the finding that high BCL6 levels represent a predictor of poor clinical outcome.

Results
We analyzed the gene expression data from 207 children with high-risk B cell precursor ALL enrolled in the pediatric clinical trial (COG P9906) and found that high expression levels of BCL6 at the time of diagnosis correlated with a poor overall and relapse-free survival (OS p=0.007; RFS p=0.02). Furthermore, multivariate analyses showed that patients with high BCL6 expression levels and established predictors of poor clinical outcome such as high white blood counts (WBC), a positive minimal residual disease (MRD) or mutations in the tumor suppressor IKZF1 had a significantly poor OS and RFS. Matched sample pairs from 49 patients at diagnosis and relapse showed that BCL6 levels were increased at relapse compared to diagnosis (p=0.003). We next studied BCL6 protein levels in Non-Ph+ cell lines and childhood ALL patient samples and found that BCL6 levels are particularly high in MLL-AF4 ALL (n=19). Surprisingly, the patients from the clinical trial that had high BCL6 levels and MLLR rearrangements were those with the worst clinical outcome (OS p= 0.0009 and RFS p= 0.000208). We next tested if the MLL-AF4 oncogene drives aberrant BCL6 expression. First, we performed a ChIP-analysis with the oncoprotein MLL-AF4 and found that the BCL6 promoter is directly bound by MLL-AF4, suggesting that MLL-AF4 may indeed drive BCL6 expression. We then performed a BCL6 Western Blot of inducible MLL-AF4-transgenic pro-B cells, demonstrating that activation of the MLL-AF4 transgene is sufficient to induce ∼10-fold upregulation of BCL6 protein levels. We conclude that aberrant expression of BCL6 in childhood ALL can be the direct consequence of MLL-AF4 activity. To further elucidate the BCL6 signaling pathway, we performed a ChIP-analysis with human MLL-AF4 leukemia cells and found that tumor suppressor genes (e.g. CDKN1B and BACH2) and B cell linage specific genes like PAX5 and CD19 are repressed by BCL6, potentially explaining the mixed lineage phenotype of MLLR driven B-ALL. We used a genetic mouse model of childhood ALL based on bone marrow precursor cells from BCL6-/- mice to decipher the function of BCL6. Since mutations in the RAS pathway are found in about 30% of childhood ALL cases, we transduced B cell progenitor cells from BCL6-/- and BCL6+/+ mice with Nras using an inducible oncogenic TET-NRASG12D system. Similar to MLL-AF4 driven cells, activation of NRASG12D results in higher BCL6 protein expression. Strikingly, BCL6-deficiency results in a failure of NRASG12DALL cells to initiate leukemia.

Clinical relevance
To verify if the high BCL6 expression levels in MLL-AF4 patients are important for the disease progression, we transduced primary human childhood 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 children with MLL-AF4 leukemia, we treated human MLL-AF4 driven primary human xenograft cells with a recently developed retro-inverso BCL6 peptide inhibitor (RI-BPI). 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 an important factor in leukemia initiation and survival and its pharmacological inhibition as a novel strategy to treat childhood ALL. Aberrant expression of BCL6 in MLL-AF4 ALL is the direct consequence of MLL-AF4 activity in these cells. Based on these findings, we propose combinations of BCL6 inhibitors (e.g. RI-BPI) with currently used chemotherapeutics as potential approach to reduce the risk of ALL relapse and improve overall outcome.

Disclosures:
No relevant conflicts of interest to declare.