Résumé
Introduction: Large B-Cell Lymphoma (LBCL) is an aggressive and the most common non-Hodgkin lymphoma, accounting for 30% of cases. The first-line treatment consists of a R-CHOP-like regimen, where 20-40% of patients are refractory to the treatment or will relapse (R/R). Aiming to develop new therapies and identify new biomarkers, we analyzed a recently studied biological process: epitranscriptomic modifications. These chemical modifications affect RNA nucleosides, including methylation like the N6-methyladenoside (m6A), or isomerization of the uridine (Ψ, pseudouridine). These modifications are realized by epitranscriptomic enzymes called “writers” and “erasers”, and processed by “readers”.
Methods: The first part of the project was exploratory, aiming to determine whether epitranscriptomic modifications play a role in LBCL, using primary tumor cells. We included 62 patients from the Hemodiag cohort (malignant hemopathies at the CHU of Montpellier), with available clinical and biological data, as well as RNA sequencing data. Mass spectrometry was performed on the RNA of the 62 samples to analyze epitranscriptomic modifications, and we correlated these findings with overall survival via MaxStat analysis. Then we worked on cell lines to validate our findings and investigate the underlying mechanisms. We used CellTiter-Glo® (CTG) and Annexin-V/7-AAD with flow cytometry for viability assays and dynamic BH3-profiling, as well as Western Blots to evaluate apoptosis and DNA damage.
Results: Twelve epitranscriptomic marks were found associated with a good or a bad prognosis, including m1G, m1A (bad overall survival = OS), and i6A (good OS). We then analyzed 52 genes that code for epitranscriptomic enzymes using RNA-seq. The expression of 27 of these was significantly correlated with good OS, including TRIT1 (writer of i6A), and 4 with a bad OS, including TRMT5 (writer of m1G) and TRMT61A (writer of m1A). Based on these results, we then tested in vitro the effect of inhibiting the epitranscriptomic enzymes METTL1-WDR4, METTL3, ALKBH5, and YTHDC1 on 3 DLBCL cell lines. Using CTG, the IC50 values for YTHDC1 were 3.75, 2.63, and 2.183 µM for OCI-LY1, NUDHL1, and RI-1, respectively. Interestingly, METTL3i's IC50 on OCI-LY1 was high (32.97 µM) and non-reached (NR) on RI-1, but low on NUDHL1 (2.74 µM), showing cell specificity. IC50 were NR for METTL1i and ALKBH5i. At a dose of 3 µM and after 24 h of treatment, apoptosis was induced by the inhibition of YTHDC1, as evidenced by the cleavage of caspase 3 and PARP, confirmed by the analysis of apoptosis by Annexin-V/7-AAD. The 4 inhibitors did not induce DNA damage at a dose of 3 µM (no γH2AX and S15P53). Finally, we performed dynamic BH3-profiling to evaluate the combination effect of YTHDC1i with BH3 inhibitors on cell viability by CTG and on BH3 dependencies by flow cytometry. The results showed a synergic effect between the anti-apoptotic protein inhibitors tested and YTHDC1i, particularly with at a low dose of venetoclax (0.01 µM, BCL-2 inhibitor), with a mean of 17% of viability of OCI-LY1 cells, in comparison to venetoclax alone (mean of 35% viability). Interestingly, when the MCL-1 inhibitor (0.1 µM, AZD-5991) was combined with YTHDC1i, we reached a mean of 8% of viability in comparison to MCL-1i alone (mean of 62%). No synergic effect was found between YTHDC1i and the BCL-xL inhibitor (A-1155463). Nevertheless, no increased BH3 dependencies were found. These results will be updated if presented at the ASH meeting.
Conclusion: Our study reveals that specific epitranscriptomic modifications and the expression of their regulatory enzymes are significantly associated with overall survival in patients with LBCL. Among these, i6A and its writer, TRIT1, correlate with a better prognosis, while m1G and m1A, along with their associated enzymes, predict poorer outcomes. Functional assays identified YTHDC1 as a promising therapeutic target, with its inhibition inducing apoptosis and demonstrating strong synergy with BCL-2 and MCL-1 inhibitors in DLBCL cell lines. These findings establish a novel epitranscriptomic landscape in LBCL and support the development of targeted therapies based on RNA modification profiles.