Targeting BCL6-Mediated Drug-Resistance in High-Risk Childhood ALL
Authors: Hurtz Christian , Ramezani-Rad Parham , Geng Huimin , Kharabi Behzad , Carroll William L. , Willman Cheryl L , Armstrong Scott A. , Melnick Ari , Muschen Markus 
Published: October 2019

Abstract
Abstract 776

Background:
BCL6 is known as a protooncogene and transcriptional repressor in Diffuse Large B cell lymphoma (DLBCL), where BCL6 is frequently involved in chromosomal rearrangements. We recently found that BCL6 mediates a novel form of drug resistance to tyrosine kinase inhibitors (TKI) in Ph+ ALL and CML (Duy et al., Nature 2011; Hurtz et al., J Exp Med 2011). This was based on the finding that Ph+ ALL and CML cells respond to TKI-mediated inhibition of BCR-ABL1 kinase activity by upregulation of BCL6, which protects from p53-mediated apoptosis. Our current study was prompted by the finding that high expression levels of BCL6 represent a predictor of poor clinical outcome in children with Non-Ph ALL. We therefore performed detailed studies of expression and function of BCL6 in various subtypes of Non-Ph childhood ALL.

Results:
In a gene expression analysis of 207 children with high-risk B cell precursor ALL (COG P9906 trial), we found that high expression levels of BCL6 at the time of diagnosis represents a strong predictor of poor overall (OS; p=0.007) and relapse-free (RFS; p=0.02) survival. Also, high expression levels of BCL6 are predictive of positive minimal residual disease (MRD+) status on day 29 after the onset of chemotherapy. For 49 patients in this clinical trial, matched sample pairs at diagnosis and relapse were available. In these patients, BCL6 expression was significantly higher at relapse compared to diagnosis (p=0.003). We next studied BCL6 mRNA (n=122) and protein (n=21) levels in a larger cohort of primary childhood ALL patient samples and found that BCL6 expression levels are particularly high in MLL-AF4 infant ALL. This finding was unexpected and we tested if the MLL-AF4 oncogene drives aberrant BCL6 expression in these cells. First, ChIP-analysis revealed that the chimeric oncoprotein MLL-AF4 directly binds to the BCL6 promoter, suggesting that MLL-AF4 may indeed drive BCL6 expression. In support of this hypothesis, BCL6 Western blot analyses of inducible MLL-AF4-transgenic pro-B cells demonstrated 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 investigate the potential function of BCL6, we used a genetic mouse model of childhood ALL based on bone marrow precursor cells from BCL6−/− mice. Since mutations in the RAS pathway are found in about 30% of childhood ALL cases, we transformed B cell progenitor cells from BCL6−/− and wildtype mice with oncogenic NRASG12D, which represents the most frequent RAS lesion in B cell lineage leukemia. Surprisingly, BCL6-deficiency results in a failure of NRASG12D ALL cells to initiate leukemia, while NOD/SCID mice that were transplanted with BCL6 wildtype NRASG12DALL succumbed to the disease.

Clinical relevance:
To verify if these observations are relevant to human disease, we transduced primary human childhood ALL xenografts with MLL-AF4 gene rearrangement with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell cycle arrest and cell death. To test if BCL6 inhibition is of potential use for children with MLL-AF4 leukemia, we used a recently developed retro-inverso BCL6 peptide inhibitor (RI-BPI, Cerchietti et al., Blood 2009). RI-BPI is able to inhibit BCL6 function and is currently under clinical development for the treatment of BCL6-dependent DLBCL. Treatment of the primary human MLL-AF4 ALL xenograft cells with RI-BPI compromised colony formation in methylcellulose and leukemia-initiation in transplant recipient mice and had a strong synergistic effect when combined with Vincristine, which represents the backbone for most high risk regimen in pediatric ALL.

Conclusions:
These findings identify BCL6 as a therapeutic target in high-risk childhood leukemia and its pharmacological inhibition as a novel strategy as therapeutic adjuvant. Interestingly, MLL-AF4 ALL cells exhibit constitutively high expression levels of BCL6. 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 standard chemotherapy as potential approach to reduce the risk of ALL relapse and improve overall outcome.

Disclosures:
No relevant conflicts of interest to declare.