BCL6-Mediated Escape from Negative Selection Enables RAS-Driven B-Cell Transformation

Background: In B-cell acute lymphoblastic leukemia (B-ALL), RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) are found in ~40% of cases (Irving et al., 2014; Jerchel et al., 2018; Zhang et al., 2011). Evidence suggests that RAS-pathway mutations in B-ALL subclones are selected for during therapy and are associated with relapse, which has a poor prognosis and remains one of the leading causes of death in children with malignancy (Irving et al., 2014; Jerchel et al., 2018). Beyond its role as a driver of B-ALL, ERK-activation can induce expression of PRDM1, which functions as a tumor suppressor and triggers negative B-cell selection and cell death (Yasuda et al., 2011; Hug et al., 2014; Setz et al., 2018). During B-cell development, PRDM1 and the proto-oncogene BCL6 are reciprocal antagonists (Shaffer et al., 2002). Here, we examined the mechanism by which oncogenic RAS-ERK signaling in B-ALL avoids negative selection and cell death to promote leukemogenesis.

Results: Mimicking survival signals from pre-B cell receptor (pre-BCR), constitutive Erk activation upon oncogenic RAS (NRAS^G12D) expression in mouse B-cell precursors massively induced the mRNA (~390-fold) and protein (~50-fold) levels of the proto-oncogene Bcl6. Highlighting an essential role of Erk-signaling in upregulation of Bcl6 expression downstream of oncogenic RAS, pharmacological Erk-inhibition markedly reduced NRAS^G12D-mediated Bcl6 induction. Both the pre-BCR linker molecule Blnk and the surrogate light chains (SLCs) of the pre-BCR are required for Erk-mediated induction of Bcl6. Genetic ablation of Blnk or the SLC component λ5 abrogated RAS-mediated Bcl6-induction. Hence, oncogenic expression of Bcl6 in RAS-driven B-ALL cells depended on structural elements of the pre-BCR.

Additionally, ERK-activation in patient-derived B-ALL cells had the same effect on BCL6 expression. Pharmacological reactivation of ERK (BCI-215) markedly induced BCL6 expression, whereas small molecule inhibition of ERK-activity (trametinib) reduced BCL6 levels in patient-derived B-ALL cells. Consistent with BCL6-transactivation downstream of ERK-activation, ChIP-seq analysis revealed binding of ERK-dependent transcription factors (CREB1, ELK1, EGR1, JUND and C-JUN) to the BCL6 promoter in human B cells. Altogether, our findings illuminated a critical role of ERK-signaling in positive regulation of BCL6 expression.

Using a genetic model for Cre-mediated deletion of Bcl6 in NRAS^G12D B-ALL cells, we found that Bcl6 was required for leukemia-initiation in transplant recipient mice (P=0.007). Patient-derived RAS-driven B-ALL cells were highly sensitive to pharmacological inhibition of BCL6 using peptide (RI-BPI) and small molecule (FX1) inhibitors. Furthermore, pharmacological inhibition of BCL6 delayed onset of fatal disease and prolonged survival of transplant recipient mice bearing patient-derived RAS-driven B-ALL cells (P=0.009). Taken together, our findings demonstrated that suppressing BCL6 function is a promising option for targeted therapeutics in RAS-driven B-ALL.

Mechanistically, genetic ablation or pharmacological inhibition of BCL6 increased PRDM1 expression. Gene expression analysis revealed that ectopic expression of Prdm1 in B-cell precursors promoted gene expression programs of apoptosis and cell cycle arrest (GSE111692). While Bcl6-deletion resulted in cell death of B-ALL cells, this was largely reversed by shRNA-mediated silencing of Prdm1. Altogether, our results showed that compromised leukemogenesis was a result of aberrant PRDM1 expression in BCL6-deficient RAS-driven B-ALL cells.

Conclusions: We previously investigated targeted engagement of negative selection in B-ALL and showed that hyperactivation of SYK or AKT functionally mimics autoreactive pre-BCR signaling and results in negative selection and cell death (Chen et al., 2015; Shojaee et al., 2016). Here, we reinforced this concept by providing a mechanistic basis on how negative selection is downregulated in RAS-driven B-ALL and how it can be reactivated for therapeutic benefit - namely, BCL6 curbs ERK-mediated induction of PRDM1 expression to promote leukemogenesis and this mechanism can be exploited as synthetic lethality in the treatment of this disease.

No relevant conflicts of interest to declare.