Background and Hypothesis: The transcriptional repressor and proto-oncogene BCL6 was previously identified to be a factor that bypasses oncogene-induced senescence (OIS) caused by activation of oncogenic RAS (Shvarts A. et al. 2002). Recently, we discovered a novel BCL6-dependent mechanism of drug resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL; Duy et al., 2011). Given that activating genetic lesions in the RAS pathway are found in ~50% of high-risk childhood pre-B ALL at diagnosis (Zhang et al., 2012), we tested the concept that BCL6 may facilitate RAS-mediated transformation of pre-B cells by suppressing the OIS-program.
Results: To examine the consequences of acute activation of RAS-ERK signaling on Bcl6 expression, we engineered murine pre-B cells to carry a doxycycline (Dox)-inducible TetOn- NRASG12D vector system. Following induction with Dox, strong upregulation of Bcl6 expression was observed at both the mRNA and protein levels, which were abrogated upon treatment with a MEK inhibitor (PD325901). These findings suggest that increased Bcl6 levels were a consequence of ERK activation. Similarly, inducible expression of BRAF V600E mutant resulted in elevated levels of Bcl6 in parallel with increased phosphorylation of ERK1/2- T202/Y204. Furthermore, studying a diagnostic (KRAS wild-type) and a relapsed (KRASG12V) sample from one pre-B ALL patient revealed hyperactivation of ERK in association with increased BCL6 levels in KRASG12V relapsed ALL cells.
Previously, we showed that BCL6 expression is repressed by STAT5 in pre-B ALL (Duy C. et al. 2011). Interestingly, while overexpression of oncogenic NRASG12D resulted in hyperactivation of ERK in pre-B cells, phosphorylation of STAT5-Y694 was drastically inhibited. Reverse Phase Protein Array (RPPA) measurements revealed that phosphorylation levels of MEK-S217/221 and ERK1/2-T202/Y204 were inversely correlated with phosphorylation of STAT5-Y694 in newly diagnosed cases of adult ALL (MDACC, 1983-2007). In addition, genetic lesions causing activation of the RAS-ERK and STAT5 pathways were found to be mutually exclusive in the majority of high-risk childhood pre-B ALL cases studied (COG P9906). Mechanistically, expression of oncogenic NRASG12D resulted in activation of PTPN6 (SHP1), an inhibitory protein tyrosine phosphatase. Loss of Ptpn6 function resulted in elevated levels of phospho-STAT5-Y694 and suppressed imatinib-mediated induction of Bcl6 expression in BCR-ABL1 -driven pre-B ALL. Taken together, our findings suggest that oncogenic RAS induces BCL6 expression through PTPN6-mediated dephosphorylation of STAT5.
Our observations that RAS-ERK signaling induces BCL6 expression prompted us to examine the role of BCL6 in RAS-mediated malignant transformation of pre-B cells. Activation of oncogenic RAS increased expression of cell cycle checkpoint regulators CDKN1A (p21) and CDKN1B (p27). BCL6 has previously been shown to mediate transcriptional repression of CDKN1A and CDKN1B (Duy C. et al. 2011). Notably, ChIP-seq analysis of patient-derived KRASG12V pre-B ALL cells revealed binding of BCL6 across the loci of cell cycle checkpoint regulators CDKN1A, CDKN1B, CDKN2D and TP53BP1, as well as pro-apoptotic molecules BIM and BMF . Consistent with these findings, Cre-mediated deletion of Bcl6 in pre-B cells expressing NRASG12D led to depletion of cells in competitive-growth assays and impaired the ability to form colonies in semi-solid methylcellulose. Moreover, expression of NRASG12Din Bcl6+/+ pre-B cells resulted in transformation and fatal leukemia in transplant recipient mice. In contrast, Bcl6-/- pre-B cells transduced with NRASG12D failed to initiate fatal disease in transplant recipients.
Conclusions: Our findings suggest that activation of oncogenic RAS enables the switch from STAT5-driven signaling to activation of BCL6, and that RAS-induced dephosphorylation of STAT5 is mediated by PTPN6. Furthermore, we demonstrated that BCL6 is required for RAS -mediated transformation of pre-B cells, suggesting that inhibition of BCL6 may provide a new therapeutic avenue.
Hurtz: Incyte: Research Funding.
Asterisk with author names denotes non-ASH members.