BCL6 Is Required for the Maintenance of Leukemia-Initiating Cells In Chronic Myeloid Leukemia
Authors: Hurtz Christian , Duy Cihangir , Cerchietti Leandro , Chatzi Katerina , Park Eugene , Klemm Lars , Kim Yong-mi , Kahn Michael , Braig Melanie , Muller Martin C. , Hochhaus Andreas , Ye B. Hilda , Melnick Ari M. , Muschen Markus 
Published: October 2019

Abstract
Abstract 202

Background:
Chronic myeloid leukemia (CML) can be effectively treated for many years with tyrosine kinase inhibitors (TKI). However, unless CML patients take TKI-treatment life-long, leukemia will eventually recur, which is attributed to the failure of TKI-treatment to eradicate leukemia stem cells in CML. Relapse from leukemia stem cells in CML often results in TKI-resistant blast crisis, which is fatal within months.

Approach:
Recent work demonstrated that FoxO3A is critical for maintenance of leukemia stem cells in chronic myeloid leukemia (CML). The mechanism of FoxO3A-dependent maintenance of leukemia stem cells remained unclear. Here we identified the BCL6 protooncogene downstream of FoxO3A as a critical effector molecule of self-renewal signaling in CML-initiating cells. BCL6 is known as a proto-oncogene in Diffuse Large B cell Lymphoma (DLBCL), where it functions as transcriptional repressor of p53.

BCL6-Null CML cells fail to initiate leukemia:
Studying gene expression changes of CML cells in 6 patients before and after treatment with Imatinib, we found that BCL6 mRNA levels were increased by >15-fold in response to Imatinib-treatment. Studying CD34+ CD38- CML cells from leukapheresis samples of two patients and CML cell lines, we found that overnight incubation with Imatinib resulted in a >12-fold increase of BCL6 expression at the mRNA and protein level. Previous studies showed that FoxO factors are required for transcriptional activation of BCL6 and FoxO3A was recently identified as a critical factor of leukemia stem cell maintenance in CML. Here we showed that inducible activation of FoxO3A indeed leads to de novo expression of BCL6 in human CML cells. We next tested the functional significance of BCL6 expression in CML cells in a genetic experiment. To this end, we used a classical mouse model for CML-like leukemia and transformed Lin− Sca-1+ c-kit+ (LSK) cells from BCL6+/+ and BCL6-Null mice with BCR-ABL1. While CML transformation efficiency was similar for BCL6+/+ and BCL6-Null LSK cells, the LSK phenotype was rapidly lost in BCL6-Null CML cells. To elucidate the mechanism of progressive loss of LSK cells in BCL6-Null CML, we performed a systematic analysis of gene expression changes in BCL6+/+ and BCL6-Null CML cells: The ABCG2 transporter, which is required for the side population (SP+) phenotype in LSK cells was reduced by >7-fold in the absence of BCL6. SP+ LSK cells represent a highly drug resistant CML subpopulation with leukemia-initiation capacity. In addition, BCL6-Null CML cells express excessively high levels of p53 at the protein level. A genome-wide mapping approach of BCL6-DNA interactions using ChIP-seq showed that BCL6 strongly binds to and represses the p53 promoter. Importantly, BCL6 expression represents a critical requirement for CML cells to form colonies in semisolid agar. Compared to BCL6+/+ CML cells (>400 colonies/10,000 cells), colony formation by BCL6-Null CML cells was reduced by ≂f100-fold (<5 colonies). After an initially successful engraftment of BCL6-Null CML cells in NOD/SCID mice (as visualized by luciferase bioimaging), BCL6-Null CML cells failed to initiate leukemia, whereas NOD/SCID mice injected with BCL6+/+ CML cells succumbed to the disease.

Targeted inhibition of BCL6 for leukemia stem cell eradication in CML:
A retro-inverso BCL6 peptide inhibitor (RI-BPI) was recently developed for targeted therapy of DLBCL. Therefore, we tested the potential therapeutic usefulness of RI-BPI for eradication of BCL6-dependent leukemia-initiating cells. Human CML cells were incubated overnight in the presence of 5 μ mol/l RI-BPI or vehicle control. While Hoechst 33342 staining revealed a distinct side population of ≂f2.8% CML cells, overnight incubation with RI-BPI reduced the frequency of ABCG2+ side population cells by more than 30-fold. Likewise, RI-BPI incubation decreased colony formation by >5-fold in human CML cells. Importantly, RI-BPI treatment of CML cells resulted in significantly prolonged overall survival of xenografted NOD/SCID mice and decreased penetrance of leukemia (10 mice/group; p=0.032),

Conclusion:
Pharmacological inhibition of BCL6 represents a powerful strategy to eradicate leukemia-initiating cells in CML. Clinical validation of this concept could limit the duration of TKI-treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation.

Disclosures:
Hochhaus: Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.