Page 133 - Haematologica May 2022

GECPAR, eRNA master regulator of germinal center
levels were similar for the four cell lines (Figure 6A). Further, GECPAR overexpressing SUDHL2 cells were more sensitive to Wnt inhibition than the parental con- trol, in terms of cell cycle perturbation. AZ6102 treat- ment more readily caused G2/M arrest, subG1 accumu- lation and decreased re-entry in G1 in GECPAR overex- pressing cells (Figure 6B).
Discussion
eRNA have recently started to be recognized as potent modulators of coding gene transcription.50,51 Here, we pro- vide the first evidence of a lncRNA, transcribed in a SE specifically active during maturation of GC B cells, which plays an antiproliferative role in DLBCL models and is associated with favorable clinical outcome in GCB- DLBCL patients.
The lncRNA LOC100132078 was previously annotated as an unknown ncRNA, mainly expressed in lymph nodes and testis,52 and reported among p53-induced eRNA in breast cancer.53 Since it mapped inside a SE relevant for GC formation3,23,43 and in a site of recurrent genomic insta- bility in lymphoid tumors,34-36 we elucidated its role in DLBCL, the neoplastic counterpart derived from GC B cells. We defined this lncRNA as eRNA according to the main features of this class of ncRNA: it was encoded within a SE; it was a non-polyA chromatin-associated transcript: its expression, highly cell type specific, was dependent on enhancer activation. We also identified a stabilized 970 nucleotide-long transcript, which, based on its expression pattern, we named GECPAR. It was less expressed in DLBCL samples than in normal tonsil B cells and in vitro experiments showed an inverse correlation with cell proliferation, suggesting an antitumoral func- tion. The latter was further supported by the association between high GECPAR expression and favorable out- come in GCB DLBCL patients. GECPAR did not seem to act by in cis transactivation of the juxtaposed POU2AF1 gene, which is strongly expressed in GC-derived malig- nancies.22 Indeed, although GECPAR and POU2AF1 tran- script levels were correlated in cell lines and in clinical specimens, silencing of the eRNA did not strongly impair expression of the coding gene. This is not uncommon and might be due to redundant functions of multiple enhancers that target a given promoter.54 On the contrary, GECPAR showed in trans activity and directly regulated the expression of several transcripts, mainly involved in cell growth and differentiation. These regulated genes were identified as common GECPAR targets in a GCB- and an ABC- DLBCL cell line, both of which had consti- tutively high GECPAR expression.
GECPAR expression was increased after BCR activa- tion, an event that causes transcriptional reprograming of B cells. The exogenous overexpression of GECPAR in an ABC-DLBCL cell line confirmed its ability to switch the lymphoma cell towards the GCB-DLBCL transcriptional signature.
Nuclear enriched lncRNA regulating transcription in trans have been described and they often modulate cell development.43,55 We propose that GECPAR is used by normal GC B cells to fine-tune the balance between pro- liferation and differentiation by directly repressing MYC and PRDM1 expression. MYC has a stage-specific role in the GC, particularly in light zone B cells, namely centro-
cytes, from which GCB-DLBCL tumor cells derive. After antigen-driven selection, B cells that still need to improve their antigen affinity can re-enter in the dark zone where they undergo additional cycles of somatic hypermuta- tion. This so-called “cyclic re-entry” is critical for main- taining the GC and is induced by the re-expression of MYC via BCR activation through NF-kB and FOXO1.56,57 We propose GECPAR as a key surveillant of this process, as it directly represses MYC in that phase. Termination of the GC reaction is modulated by NF-kB activation down- stream of the BCR. It induces IRF4, master regulator of terminal B-cell differentiation which in turn activates the plasma cell master regulator BLIMP1, encoded by PRDM1.58 GECPAR itself directly represses PRDM1, impeding terminal differentiation into plasma blast. In conclusion, GECPAR, which is induced by BCR activa- tion, would retain B cells in the GC light zone, reducing the tendency to re-enter in the dark zone or to exit and differentiate to plasma cells.
GECPAR also reduces B-cell proliferation rate and the tendency to differentiate, possibly by directly inducing TLE4, a negative repressor of TCF/LEF1. LEF1 is the key mediator of nuclear Wnt signaling and is important in lymphopoiesis. LEF1 is overexpressed in the nucleus of approximately 40% of DLBCL.59 MYC and Wnt pathway are connected in a positive feedback-loop involving LEF1.60 GECPAR, which directly inhibited MYC expres- sion, indirectly enhanced its antiproliferative activity via TLE4 that contributed to the arrest of terminal differenti- ation induced by NF-kB. Indeed, GECPAR expression was inversely correlated with many LEF1 targets, in both DLBCL cell lines and specimens, and some of them were related to NF-kB regulation. Moreover, GECPAR silencing induced upregulation of important NF-kB genes, such as CARD11, REL and IKBKB, supporting the link between GECPAR and Wnt/NF-kB crosstalk. Several bidirectional connections between Wnt and NF-kB pathways45 have been reported in cancer and in particular, in DLBCL.61 We propose GECPAR as an additional layer of control of NF- kB activation in GC B cells, pausing terminal differentia- tion to plasma blasts.
The greater sensitivity of ABC-DLBCL with high GEC- PAR expression to pharmacological inhibition of Wnt fur- ther supports the relationship between GECPAR and Wnt pathway regulation and uncovers alternative therapeutic options for ABC-DLBCL patients.
In conclusion, our work describes a novel mechanism of regulation of GC differentiation, which might con- tribute to DLBCL pathogenesis, and could help in under- standing the heterogeneity of this disease.
Disclosures
LC received a travel grant from HTG. FB received institution- al research funds from Acerta, ADC Therapeutics, Bayer AG, Cellestia, CTI Life Sciences, EMD Serono, Helsinn, ImmunoGen, Menarini Ricerche, NEOMED Therapeutics 1, Nordic Nanovector ASA, Oncology Therapeutic Development and PIQUR Therapeutics AG; received consultancy fees from Helsinn and Menarini; provided expert statements to HTG; received travel grants from Amgen, Astra Zeneca, Jazz Pharmaceuticals and PIQUR Therapeutics. All the other authors have nothing to disclose.
Contributions
SN performed experiments, analyzed and interpreted data,
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