Global and MYC-Specific Translation Is Enhanced in Activated Chronic Lymphocytic Leukemia Cells Carrying NOTCH1 C.7541_7542delct Mutations

Introduction: mRNA translation is increased in activated tumor cells of the aggressive form of Chronic Lymphocytic Leukemia (CLL), typically unmutated (U) immunoglobulin gene heavy-chain variable region (IGHV) with a strong sIgM signaling capacity (Yeomans et al, Blood 2016). C-MYC protein is a master regulator of cell performance and its expression is controlled at both transcriptional and translational levels. C-MYC protein is over-expressed in the proliferation centers of CLL and high c-MYC mRNA expression is associated with poor prognosis. In leukemic cell lines, c-MYC is an essential mediator and direct target of NOTCH1. Pro-activating c.7541_7542delCTmutations in NOTCH1 PEST domain of chromosome 9 exon 34 (NOTCH1_ΔCT) are enriched in U-CLL with high sIgM levels/signaling capacity and associate with poorer prognosis in CLL (D'Avola et al, Blood, 2016), likely due to accumulation of more stable NOTCH1 protein and enhanced signaling in tissue activated CLL cells (Arruga et al, Leukemia, 2014). Aims and Methods: We investigated the consequences of NOTCH1_ΔCT on global mRNA and c-MYC translation using a novel flow cytometry-based O-propargyl-puromycin (OPP) incorporation assay ('Click-iT' assay) and by c-MYC-specific immunoblotting in U-CLL. Since prolonged culture of CLL cells in vitro in the absence of stimuli led to spontaneous inactivation of NOTCH1 pathway, CpG-mediated TLR9 induction was used as a tool for activation of CLL cells in vitro. Cycloheximide (CHX) was used as a negative control for mRNA translation. For this study, 2 cohorts were investigated: i) a test "CLL_ΔCT cohort" of U-CLL with NOTCH1_ΔCT (variant allele frequency [VAF] by droplet digital PCR, range 42.6-48.9%, median 47% of the CD19⁺CD5⁺ CLL cell population), but no additional genetic lesion other than 13q deletion, and ii) a control "CLL_WT cohort" of U-CLL with no NOTCH1_ΔCT (VAF<1% in all cases) or additional genetic lesion other than 13q deletion. CLL cells were incubated with 7.5 μg/ml CpG-ODN 2006 for 24 hours and assays were performed at baseline, 3 and 24 hours. NOTCH1 pathway γ-secretase inhibition was performed with DAPT GSi. Results: The CLL_ΔCT cohort had higher sIgM levels (range 31-372 MFI, median 81 MFI) and signaling capacity (Fab'2 anti-IgM induced intracellular calcium mobilization sIgM [iCa²⁺] range 47-54%, median 51) than the CLL_WT cohort (sIgM levels range 19-288 MFI, median 47 MFI; IgM iCa²⁺ range 2-78%, median 25%). Following TLR9-mediated cell activation, the CLL_ΔCT cohort had sustained NICD (NOTCH1-intracellular cleaved domain) protein accumulation for up to 24 hours and expressed higher NOTCH1 target gene HES1 (hairy enhancer of split) transcript levels than in the CLL_WT cohort. These data indicated NOTCH1 canonical pathway sustainment in the CLL_ΔCT upon activation. Global mRNA translation after 24 hours in the presence of CpG was 11.5 fold higher than that without CpG in the CLL_ΔCT cohort and only 4 fold higher in the CLL_WT cohort, revealing significantly higher levels of translation in CLL_ΔCT than in CLL_WT (p=0.03). CpG-induced global mRNA translation in the CLL_WT cohort was similar to that in the CLL_ΔCT cohort treated with CHX. By using CpG-induced global mRNA translation in the presence of CHX inhibitor as background levels for each group, DAPT GSiat 2.5 to 10 μM showed from 47% to 63% inhibition of the residual CpG-induced global translation in CLL_ΔCT (p<0.05), but no effect in CLL_WT. Remarkably, c-MYC mRNA translation after 3 hour culture with CpG was higher in CLL_ΔCT than in CLL_WT (p= 0.02), and a similar trend was maintained in the cases investigated at 24 hour. Treatment of CLL_ΔCT cells with DAPT GSi decreased expression of c-MYC in a dose-dependent manner. Conclusion: NOTCH1_ΔCT mutations associate with a very aggressive clinical behavior in CLL. These results now indicate that pro-activating mutations of NOTCH1 pathway associate with increased global mRNA translation and c-MYC expression. They highlight a mechanism by which NOTCH1 pathway may induce c-MYC overexpression in CLL, likely leading to increased proliferation and survival. The association of increased NOTCH1 variant allele frequency with sIgM levels and signaling capacity indicate that these mechanisms are predominant in the less anergic subgroup of U-CLL and make NOTCH1 mediated c-MYC translation an attractive target for therapeutic inhibition.