Identification of BCL6 As Synthetic Lethality in RAS-Driven B-Cell Transformation
Authors: Chan Lai N. , Hurtz Christian , Leveille Etienne , Kume Kohei , Robinson Mark E. , Geng Huimin , Cosgun Kadriye Nehir , Müschen Markus 
Published: November 2021

Abstract
Background: Genetic lesions in the RAS-ERK pathway (NRAS, KRAS, RAF1, MAP2K1, PTPN11, NF1) are oncogenic drivers in ~40% of B-cell acute lymphoblastic leukemia (B-ALL) cases and are associated with chemoresistance and relapse. During normal B-cell development, persistent activation of RAS-ERK-signaling induces negative B-cell selection and cell death (Yasuda et al., 2011). The deleterious effect of persistent RAS-ERK signaling is mainly caused by PRDM1-mediated repression of BCL6 (Setz et al., 2018). Hence, the tumor suppressor PRDM1 and the proto-oncogene BCL6 are reciprocal antagonists during B-cell development (Shaffer et al., 2002). Genetic lesions that cause permanent activation of the RAS-ERK pathway in B-ALL would be expected to cell death as in normal B-cell development. Here we examined the mechanistic basis of how oncogenic activation of RAS-ERK signaling in B-ALL not only avoids cell death but also promotes malignant transformation.
Results: Strikingly, doxycycline-inducible expression of NRAS G12D in murine B-cell precursors, resulted on dramatic increases of BCL6 mRNA (~390-fold) and protein (~50-fold) levels, which came at the expense of PRDM1. For these reasons, we explored mechanisms underpinning transactivation of BCL6 and tested whether RAS-dependent induction of BCL6 represents a mechanism to oppose PRDM1-mediated cell death.
Consistent with transactivation of BCL6 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. Highlighting an essential role of Erk-signaling in RAS-mediated upregulation of Bcl6 expression, pharmacological ERK-inhibition abrogated NRAS G12D-mediated Bcl6 induction. Likewise, Erk2-deletion abolished the ability of B-ALL cells to induce expression of Bcl6. Furthermore, pharmacological activation of ERK (BCI-215) markedly induced BCL6 expression. Together, these findings reveal a new pathway of BCL6-activation in B-ALL cells that depends on oncogenic RAS-ERK-signaling.
To address the mechanistic role of BCL6 in RAS-driven B-ALL, we established a genetic model for Cre-mediated deletion of Bcl6 in NRAS G12D B-ALL cells. Genetic ablation of Bcl6 resulted in rapid depletion of B-ALL cells and compromised colony formation. Notably, Bcl6-deletion prevented leukemia-initiation in transplant recipient mice (P=0.007). Furthermore, patient-derived RAS-driven B-ALL cells were highly sensitive to pharmacological inhibition of BCL6 using peptide (RI-BPI) and small molecule (FX1) inhibitors. Importantly, treatment with RI-BPI delayed onset of fatal disease and prolonged survival of transplant recipient mice (P=0.009). We then tested whether targeting BCL6 can be leveraged in combination with existing treatment regimen and found that RI-BPI potentiated the effects of vincristine on killing patient-derived RAS-driven B-ALL cells. Hence, BCL6 represents a previously unrecognized therapeutic target in RAS-driven B-ALL that can be leveraged to sensitize to conventional chemotherapy.
Mechanistically, genetic ablation or pharmacological inhibition of BCL6 increased expression of PRDM1 in RAS-driven B-ALL cells. shRNA-mediated knockdown of Prdm1 rescued Bcl6-deficiency in RAS-driven B-ALL cells. While Bcl6-deletion resulted in cell death of B-ALL cells, this was largely reversed by loss of Prdm1. Altogether, our findings suggest that compromised leukemogenesis was a result of aberrant PRDM1 expression in BCL6-deficient RAS-driven B-ALL cells.
Conclusions: While permanent activation of the RAS-ERK pathway induces negative selection and cell death in normal B-cell precursors owing to excessive activation of the PRDM1 tumor suppressor, B-ALL cells carrying RAS-activating lesions have evolved a mechanism to evade PRDM1-mediated cell death by massive (&gt;300-fold) upregulation of BCL6. In RAS-driven B-ALL, oncogenic expression of BCL6 suppresses PRDM1 and enables malignant transformation. Importantly, BCL6 expression in RAS-driven B-ALL represents a previously unrecognized synthetic lethality. Hence, peptide and small molecule inhibition of BCL6 de-represses PRDM1, reconstitutes PRDM1-dependent tumor suppression and represents a selective vulnerability in RAS-driven B-ALL cells that can be leveraged to overcome conventional mechanisms of drug-resistance in refractory B-ALL.

Disclosures
No relevant conflicts of interest to declare.