BCL6 Is Critical to Overcome Oncogene-Induced Senescence in RAS-Mediated B Cell Transformation

Abstract Background and Hypothesis: The transcriptional repressor and proto-oncogene BCL6 is a therapeutic target in subtypes of diffuse large B cell lymphoma (DLBCL) and modulates drug-resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL; Duy et al., Nature 2011). BCL6 was shown to be a critical factor that bypasses p53-dependent senescence and thereby enables RAS-driven transformation of mouse embryonic fibroblasts (Shvarts et al., Genes Dev. 2002). Given that ~50% of pediatric ALL cases carry genetic lesions that lead to hyperactivated RAS-ERK signaling (Zhang et la., Blood 2012), we examined the role of BCL6 in RAS-driven pre-B ALL and identified a novel mechanism by which RAS-ERK signaling can mediate BCL6 expression. Results: Using a doxycycline-inducible TetOn- NRASG12D vector system, we found that inducible activation of RAS-ERK signaling strongly upregulated BCL6 expression at both the mRNA (~350-fold) and protein (~50-fold) levels in murine pre-B cells. Increases in BCL6 expression were abrogated upon treatment with a MEK inhibitor (PD325901). In addition, Cre-mediated deletion of Mapk1 suppressed upregulation of BCL6 expression upon imatinib treatment in BCR-ABL1-driven pre-B ALL cells. These findings suggested that elevated expression of BCL6 is a consequence of ERK activation. Previously, we demonstrated that BCL6 expression is negatively regulated by STAT5 in BCR-ABL1 pre-B ALL (Duy et al., Nature 2011). Interestingly, oncogenic NRASG12D inhibited phosphorylation of STAT5-Y694 by activating the inhibitory protein tyrosine phosphatase Ptpn6. Cre-mediated deletion of Ptpn6 induced STAT5 activity. Furthermore, loss of Ptpn6 function abrogated upregulation of BCL6 expression induced by imatinib in BCR-ABL1 pre-B ALL. Taken together, RAS-ERK signaling induces BCL6 expression by suppressing STAT5 activity. To directly test the role of BCL6 in RAS-transformed pre-B ALL, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. Inducible deletion of Bcl6 in NRASG12D-transformed pre-B ALL cells led to rapid depletion from the cell culture and reduced colony forming ability in vitro. These findings suggested that BCL6 is required for maintenance of fully established RAS-transformed ALL. Notably, we found that initiation of NRASG12D-driven leukemia in vivo depends on BCL6 as NRASG12D ALL failed to give rise to leukemia in the absence of Bcl6 in transplant recipient mice. Studying a diagnostic (KRAS wild-type) and a relapsed (KRASG12V) sample from one pre-B ALL patient revealed increased BCL6 expression in KRASG12V relapsed ALL cells. In addition, selective sensitivity to PD325901 and a retro inverso BCL6 peptide inhibitor (RI-BPI) was observed in KRASG12V relapsed ALL cells. Finally, RI-BPI prolonged overall survival of recipient mice transplanted with KRASG12V relapsed ALL cells in vivo. Conclusions: In summary, we demonstrated a novel mechanism by which oncogenic RAS signaling induces expression of BCL6, and showed that BCL6 is critical for RAS-driven transformation in pre-B ALL. Importantly, ALL clones often acquire drug resistance and activating mutations in the RAS pathway (Bhojwani and Pui, Lancet Oncol. 2013). Our findings suggest that pharmacological inhibition of BCL6 may provide a novel therapeutic avenue to overcome drug-resistance and prevent leukemia relapse after initial remission in RAS-driven ALL. Disclosures Melnick: Janssen: Research Funding.