Abstract
Ras-pathway lesions are oncogenic drivers in ~45% of B-cell acute lymphoblastic leukemia (B-ALL) cases. Activating mutations of NRAS and KRAS are oncogenic drivers in B-ALL while the BRAFV600E mutation occurs in almost all cases of B-cell hairy cell leukemia. Less frequent lesions resulting in increased ERK-signaling in B-cell malignancies include activating mutations of RAF1, MAP2K1 and the PTPN11 phosphatase as well as deleterious mutations of the Ras-GTPase activator NF1. Interestingly, increased immunoglobulin light chain gene expression was observed in B-ALL cases with RAS-pathway lesions (COG P9906), reflecting engagement of pre-B cell receptor (pre-BCR) downstream signaling. Here we tested the hypothesis that oncogenic RAS-signaling in B-ALL mimics pre-BCR-induced developmental rewiring of signal transduction at the pro-B to pre-B cell transition and identified PTPN6 and BCL6 as therapeutic targets in RAS-driven B-ALL.
During early B-cell development, pro-B cells transition from cytokine- to pre-B cell receptor (pre-BCR)-dependent survival and proliferation signals. Inducible activation of immunoglobulin (Ig) µ heavy chain (µHC) expression induced developmental progression and surface expression of Ig κ light chains. Notably, inducible activation of oncogenic NRASG12D had the same effect and resulted in increased surface expression of Ig κ light chains. Furthermore, studying genetic models for this transition revealed that both pre-BCR signaling and RAS-oncogenes suppressed cytokine receptor/STAT5-signaling and induced massive de novo expression of the proto-oncogene and transcriptional repressor BCL6. Our genetic studies revealed that the SH2-domain containing protein tyrosine phosphatase PTPN6 was activated by oncogenic RAS-signaling and essential for the switch from STAT5 to BCL6-activation. Given that oncogenic RAS activated PTPN6, we tested the role of PTPN6 in RAS-driven leukemogenesis. To this end, ablation of Ptpn6 in NRASG12D-driven B-ALL resulted in depletion of cells from cell culture in competitive-growth assays and reduced the number of colonies formed in semi-solid methylcellulose. Collectively, these findings suggest that PTPN6 represents a potential therapeutic intervention point in RAS-driven B-ALL.
In addition to PTPN6, we investigated the role of BCL6 in RAS-driven B-ALL. Aberrant activation of oncogenic RAS results in oncogene-induced senescence (OIS) characterized by induction of ARF/p53 and irreversible cell cycle arrest in the G1 phase. For oncogenic Ras-signaling, BCL6 was required to oppose ERK-mediated activation of p21, p27 and p53 checkpoint molecules in B-ALL. Here we tested the hypothesis that BCL6 bypasses the RAS-mediated OIS program to facilitate transformation. To this end, increases in number of colonies formed in semi-solid methylcellulose were observed upon inducible activation of Bcl6 in NRASG12D B-ALL cells. Furthermore, loss of Bcl6 function in NRASG12D B-ALL cells resulted in depletion of cells from cell culture in competitive growth assays and reduced colony forming ability. Importantly, expression of NRASG12D in Bcl6+/+ pre-B cells resulted in transformation and fatal leukemia in transplant recipient mice. In striking contrast, Bcl6-/- pre-B cells transduced with NRASG12D failed to initiate fatal disease in vivo. Furthermore, pharmacological inhibition of BCL6 restored sensitivity to chemotherapy in patient-derived KRASG12V B-ALL cells.
In conclusion, we identified oncogenic RAS-signaling as functional mimics of pre-BCR signaling. Oncogenic RAS induced expression of BCL6 at the expense of cytokine receptor/STAT5-signaling. Our genetic studies identified PTPN6 as a critical effector molecule of the switch from cytokine receptor to pre-BCR signaling. Importantly, we identified PTPN6 and BCL6 as potential therapeutic intervention points in RAS-driven B-ALL.
No relevant conflicts of interest to declare.
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