Rationale for targeting BCL6 inMLL-rearranged acute lymphoblastic leukemia

Abstract

Chromosomal rearrangements of the mixed lineage leukemia (MLL) gene occur in ∼10% of B-cell acute lymphoblastic leukemia (B-ALL) and define a group of patients with dismal outcomes. Immunohistochemical staining of bone marrow biopsies from most of these patients revealed aberrant expression of BCL6, a transcription factor that promotes oncogenic B-cell transformation and drug resistance in B-ALL. Our genetic and ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analyses showed that MLL-AF4 and MLL-ENL fusions directly bound to the BCL6 promoter and up-regulated BCL6 expression. While oncogenic MLL fusions strongly induced aberrant BCL6 expression in B-ALL cells, germline MLL was required to up-regulate Bcl6 in response to physiological stimuli during normal B-cell development. Inducible expression of Bcl6 increased MLL mRNA levels, which was reversed by genetic deletion and pharmacological inhibition of Bcl6, suggesting a positive feedback loop between MLL and BCL6. Highlighting the central role of BCL6 in MLL-rearranged B-ALL, conditional deletion and pharmacological inhibition of BCL6 compromised leukemogenesis in transplant recipient mice and restored sensitivity to vincristine chemotherapy in MLL-rearranged B-ALL patient samples. Oncogenic MLL fusions strongly induced transcriptional activation of the proapoptotic BH3-only molecule BIM, while BCL6 was required to curb MLL-induced expression of BIM. Notably, peptide (RI-BPI) and small molecule (FX1) BCL6 inhibitors derepressed BIM and synergized with the BH3-mimetic ABT-199 in eradicating MLL-rearranged B-ALL cells. These findings uncover MLL-dependent transcriptional activation of BCL6 as a previously unrecognized requirement of malignant transformation by oncogenic MLL fusions and identified BCL6 as a novel target for the treatment of MLLrearranged B-ALL.

Precursor B-ALL Cell Lines Differentially Respond to SYK Inhibition by Entospletinib

“…Moreover, it was shown that BCL-6 is able to bind the BCL2L11 locus (BIM) and consequently regulates BIM expression. The deletion of BCL-6 induced an increased BIM level [ 49 ]. Altogether, a high BCL-6 expression keeps pro-apoptotic BIM under control, which in turn prevents apoptosis induction in MLL -rearranged B-ALL.…”

The MLL/SET family and haematopoiesis

“… Methyltransferase activity required for haematopoiesis? HSPC transcriptional response following deletion MLL1 Vav-Cre No change in ST-HSC or LT-HSC numbers at E13.5 in FL [ 79 ] No change in LSK CD48 − numbers by E15.5 in FL [ 82 ] Very low short term and no long term repopulation capacity [ 82 ] No change in LSK CD48 − numbers by E15.5 in FL (Vav-Cre) [ 82 ] 3 weeks [ 82 ] Not quantified Significantly defective myeloid output in CFU [ 79 , 82 ] No, foetal haematopoiesis proceeds normally (Vav-Cre); no change in global H3K4 [ 84 ] In ESC, promotes mesodermal and haematopoietic cell fate [ 113 ] Loss of HoxA cluster in HSPC [ 76 , 78 , 79 , 80 ] HoxB cluster in ESC [ 125 ] MLL2 Germline KO only – No indication for requirement in haematopoiesis [ 88 ] No analysis No analysis No analysis No analysis No analysis No SET mutants No ChiP in null HSPCs No analysis MLL3 Germline KO mice die shortly after birth, no obvious defects [ 94 ] Note - All experimental data from p53 null E13.5.-E14.5 FL HSPCs transplanted into adult mice [ 102 ] shRNA - competitive transplants – increased number of LT-HSC, loss of ST-HSC [ 102 ] No analysis No Vav-Cre mice Reduced MPP, CMP, GMP, no change to MEP [ 102 ] Reduced myeloid CFU output [ 102 ] No SET mutants in blood context Loss of H3K3me3 at some observed loci [ 102 ] Downregulation of early haematopoietic progenitor differentiation, correlation to 7q MDS [ 102 ] MLL4 Germline KO...…”

“…Further implicated was a network of various mRNAs and microRNAs, and no single candidate gene could explain the observed phenotype, suggesting that these RNA candidates act between Mll1 and RAS-MEK-ERK signalling. Further insight into Mll1 function during normal B-cell development has been derived from the analysis of MLL-AF4 patient data, where an increased MLL1 and BCL6 expression signature was identified [ 84 ]. Virally induced Cre-mediated deletion of Mll1 in murine pre-B and mature splenic B-cells resulted in a loss of Bcl6 upregulation from 2 weeks.…”

“…Interestingly, doxycycline-mediated induction of Bcl6 expression in murine pre-B cells increased Mll1 expression through an indirect mechanism, where Bcl6 represses the expression of Bmi1, Ctbp2 and Kdm2B, which in turn are transcriptional repressors of Mll1. This suggests that Mll1 and Bcl6 are connected through a positive feedback loop, which appears to be conserved in leukaemic cells [ 84 ].…”

“…Furthermore, conditional Mll1 deletion has demonstrated a need for Mll1 during adult haematopoiesis [ 79 , 81 , 82 ]. In further support, aberrantly increased expression of BCL6 correlates with increased MLL1 expression in both paediatric and adult MLL-rearranged B-cell acute lymphoblastic leukaemia (B-ALL) and defines a specific subset of patients with poor clinical outcome [ 84 ]. Specific to this subset of B-ALL is a dependency on BCL6, which can operate through a positive feedback loop with MLL1 to upregulate BCL6 (discussed above) and repress the proapoptotic BH3-only molecule BIM.…”