BCL6 Enables RAS-Mediated Pre-B Cell Transformation in Childhood Acute Lymphoblastic Leukemia

Background & Hypothesis: The transcriptional repressor and proto-oncogene BCL6 has recently been identified as a therapeutic target in subtypes of diffuse large B cell lymphoma (DLBCL) and as mediator of a novel for of drug-resistance in Ph⁺ acute lymphoblastic leukemia (ALL; Duy et al., Nature 2011). A previous senescence rescue screen identified BCL6 as a key factor that bypassed senescence and thereby enabled RAS-mediated transformation of mouse embryonic fibroblasts (Shvarts et al., Genes Dev 2002). Since ~50% of pediatric cases of ALL carry genetic lesions that result in hyperactivation of the Ras-Erk pathway (Zhang et al., Blood 2012), we tested the role of Bcl6 in this large subgroup of childhood leukemia.

Results: Mutations leading to hyperactivation of the Ras-Erk pathway are found in about 50% of childhood ALL cases (Zhang et al., 2012). Among 26 ALL xenografts, we found 9 cases with constitutive Erk-T²⁰²/Y²⁰⁴ phosphorylation, which was paralleled by elevated expression levels of BCL6 in these cases. Studying mouse pre-B cells that were engineered with a doxycycline-inducible NRAS^G12D mutant, we were able to directly measure the consequences of acute activation of the Ras-Erk pathway on BCL6 expression levels. First we incubated the cells with doxycycline to induce the expression of NRAS^G12D and then harvested the cells at different times point to test for BCL6 protein and mRNA expression levels. Interestingly, after 24h NRAS^G12D-TetO pre-B cells cells showed strong upregulation of BCL6 at the mRNA (352-fold) and protein level (15-fold). Upregulation of BCL6 in response to NRAS^G12D-activation was sensitive to treatment with the MEK kinase inhibitor PD325901, upstream of Erk, suggesting that BCL6 expression is a consequence of Erk activation in pre-B cells. Likewise, treatment of patient-derived pre-B ALL cells with the MEK inhibitor PD325901 reversed BCL6 expression, as demonstrated by quantitative RT-PCR and Western blot. From one patient, a diagnostic (KRAS wildtype) and a relapse sample with an acquired KRAS^G12V mutation were available. Consistent with specific expression of BCL6 in the KRAS^G12V relapse ALL sample, only KRAS^G12V ALL cells from the relapse but not wildtype cells from the diagnostic sample were sensitive to a retro inversoBCL6 peptide inhibitor (RI-BPI; Cerchietti et al., 2009).

Development of a genetic mouse model for inducible ablation of Bcl6. These findings suggest an important role of BCL6 as a cofactor of RAS-driven pre-B cell transformation, comparable to previous findings in mouse embryonic fibroblasts. To directly test a mechanistic role of Bcl6 in RAS-mediated pre-B cell transformation, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. For lineage-specific deletion in vivo, we crossed these mice with an Mb1-Cre deleter strain, in which Bcl6 was deleted in pro-B cells, resulting in a differentiation block at the pre-B cell stage. Interestingly, Mb1-Cre x Bcl6^fl/fl B cell lineage cells could be transduced with NRAS^G12D retroviral vectors, however these cells did not give rise to leukemia when injected into congenic recipients, whereas NRAS^G12D-transduced Bcl6^fl/fl pro-B cells that retained Bcl6 function developed B cell lineage leukemia in all transplant recipients. In a second experiment, we transformed Bcl6^fl/fl pro-B cells with NRAS^G12D and induced Cre with a second, tamoxifen-inducible vector in full-blown leukemia cells. Acute ablation of Bcl6 in NRAS^G12D ALL cells completely abrogated the ability of NRAS^G12DALL cells to form colonies in methylcellulose and resulted in rapid apoptosis and depletion from the cell culture. We conclude that BCL6 is not only required for the initiation of RAS-driven ALL in vivo but also for the maintenance of fully established RAS-driven leukemia.

Conclusion: These findings provide genetic evidence for BCL6 function as a critical cofactor of RAS-mediated transformation in childhood ALL. Inhibition of BCL6 in RAS-driven ALL may be useful to prevent leukemia relapse after initial remission (Bcl6-dependent leukemia-initiation) and also to achieve profound remission by combining conventional cytotoxic therapies with BCL6 inhibition (e.g. RI-BPI).