BCL6-Mediated Survival Signaling Promotes Drug-Resistance in BCRABL1- Driven Acute Lymphoblastic Leukemia

During B cell development, the transcriptional repressor BCL6 is almost exclusively expressed in germinal center B cells and promotes germinal center B cell survival by suppression of Arf and p53 signaling. Compared to germinal center B cells, BCL6 is expressed at extremely low levels in pro- and pre-B cells. Also in acute lymphoblastic leukemia (ALL) derived from BCR-ABL1-transformed pre-B cells, BCL6 expression is hardly detectable at both the mRNA and protein levels. Upon BCR-ABL1 kinase inhibition by Imatinib-treatment, however, BCL6 expression levels increased by 30 to 70- fold at the mRNA and protein levels, respectively. To test whether upregulation of BCL6 in response to Imatinib-treatment has a function we transformed bone marrow pre-B cells from Bcl6−/− mice and wildtype littermates with a retrovirus encoding BCR-ABL1. After two weeks, both Bcl6−/− and wildtype bone marrow gave rise to BCR-ABL1-driven ALL. Compared to BCR-ABL1-induced ALL from wildtype bone marrow, Bcl6−/− ALL cells grow at a significantly lower proliferation rate, which suggests that even low levels of BCL6 expression in wildtype leukemia cells has some biological significance. We next treated Bcl6−/− and wildtype BCR-ABL1 ALL cells with low dose (1 μmol/l) Imatinib. About 50% of wildtype BCR-ABL1 ALL cells were still viable after four days in culture. In contrast, induction of apoptosis in Bcl6−/−BCR-ABL1 ALL cells was greatly accelerated and no viable cells could be detected after four days of Imatinib-treatment. In addition, Bcl6-deficiency significantly delayed leukemia cell engraftment in a transplantation experiment: Upon tail vein injection of 3 × 106 firefly luciferase-labeled Bcl6−/− or wildtype BCR-ABL1 ALL cells in each ten sublethally irradiated NOD/SCID mice, engraftment of Bcl6−/−BCR-ABL1 ALL cells was delayed by approximately one week as compared to wildtype ALL cells by luciferase bioimaging. Whereas most recipient mice that were injected with wildtype BCR-ABL1 ALL cells died within three weeks despite treatment with 100 mg/kg/day Imatinib i.p., all mice injected with Bcl6−/−BCR-ABL1 ALL cells are currently still alive. We conclude that BCL6 decreases sensitivity of BCR-ABL1 ALL cells to BCR-ABL1 kinase inhibition by Imatinib both in vitro and in vivo. We next investigated whether a peptidomimetic inhibitor of BCL6 (RI-BPI, Cerchietti et al., 2008) was active to sensitize human BCR-ABL1 ALL cells to Imatinib-treatment. At 20 μmol/l but not 5 μmol/l, the RI-BPI induced apoptosis in untreated human BCR-ABL1 ALL cells, so we decided to study 5 μmol/l RI-BPI in combination with Imatinib: Whereas about 30% of human BCR-ABL1 ALL cells survived treatment with 10 μmol/l Imatinib for four days, viability of human BCR-ABL1 ALL cells was decreased to less than 5% if Imatinib was combined with 5 μmol/l RI-BPI. To mechanistically address the function of BCL6 in Imatinib-treated BCR-ABL1 ALL cells, we investigated whether the cytotoxic effect of the RI-BPI requires Arf and p53. Previous work had shown that BCL6 negatively regulates Arf and p53-dependent apoptosis in germinal center B cells. To this end, we transduced bone marrow from Arf−/−, Tp53−/− and wildtype mice with BCR-ABL1. After establishment of growth factor-independent ALL, leukemia cells were treated with Imatinib and with or without RI-BPI at various concentrations. Arf−/−, Tp53−/− and wildtype BCR-ABL1 ALL cells showed a similar degree of sensitivity to treatment with Imatinib alone. A combination of Imatinib and R-BPI at 20 μmol/l was toxic for all BCR-ABL1 ALL cells, regardless of genotype. In contrast, Imatinib in combination with RI-BPI at 5 μmol/l induced apoptosis selectively in wildtype but not in Arf−/− and Tp53−/− BCR-ABL1 ALL cells. We conclude that BCL6 mediates survival signaling in Imatinib-treated BCR-ABL1 ALL by suppression of Arf/p53 signaling. Inhibition of BCL6 using a peptidomimetic inhibitor represents a novel treatment concept to prevent drug-resistance in BCR-ABL1-driven ALL.