MiR-146a up-Regulation Is Associated with Anti-Tumor Activity of Pan-Histone Deacetylase Inhibitor ITF2357 (Givinostat®) in Human Burkitt's Lymphoma
Authors: Zappasodi Roberta , Cavanè Alessandra , Iorio Marilena V , Tortoreto Monica , Ruggiero Giusi , Magni Michele , Carlo-Stella Carmelo , Tagliabue Elda , Zunino Franco , Croce Carlo M. , Gianni Alessandro M. , Di Nicola Massimo 
Published: October 2019

Abstract
Abstract 2729
Histone deacetylase inhibitors (HDACis) are among the most promising novel classes of anti-cancer compounds thanks to their ability of simultaneously targeting multiple pro-survival and anti-apoptotic pathways, while retaining a relatively good safety profile. Through the promotion of histone and non-histone protein acetylation, they can modulate not only expression and activation of key signaling proteins but also non-coding small RNAs (microRNAs) that post-transcriptionally control gene expression. Increasing evidence is pointing to an altered pattern of microRNA expression at the basis of the genesis and the maintenance of lymphoid malignancies and in particular Burkitt's lymphoma (BL).
To better elucidate the mechanism accounting for the potent anti-lymphoma activity of the new-generation pan-HDACi ITF2357 (Givinostat®, Italfarmaco S.p.A.), we studied its ability to modulate the microRNA expression profile in human BL cell lines.
We performed microRNA expression profiling of Raji cells exposed to 200 nM ITF2357 for 24, 48, 72 hours or untreated. At any time points, we found an average of 6 and 4 microRNAs that displayed a more than 1.8 fold increase and decrease respectively (p<0.01) in relative transcript analysis when compared with untreated cells. Interestingly, miR-146a, whose anti-cancer properties have very recently started to be uncovered, was among the most consistently induced miRNAs. Quantitative real time PCR analyses confirmed these results also in the Namalwa BL cell line, and showed that the most significant induction of miR-146a was reached 48 hours after treatment in both cell lines (Raji p<0.001, Namalwa p<0.01).
Notably, nuclear factor kappa B (NF-kB) has been demonstrated to directly activate the translation of miR-146, and represents the best-described mechanism of its induction. Indeed, by confocal microscopy, we observed an augmented nuclear localization of the NF-kB p50/p65 complex in treated compared to untreated Namalwa and Raji cells, which peaked 48 hours after ITF2357 exposure. NF-kB functions are widely known to sustain lymphoma growth. However, in keeping with recent reports showing that NF-kB activation sensitizes lymphoma cells to Fas-mediated apoptosis, we found that treated Namalwa cells expressed an higher amount of Fas on their surface compared to control samples, and its expression gradually increased over the 72 hours of ITF2357 exposure (p<0.01). In Raji cells, which constitutively express very high level of Fas and are insensitive to its stimulation due to c-FLIP overexpression, Fas was not affected by ITF2357 treatment. By contrast, both treated Raji and Namalwa cells down-regulated the expression of the extrinsic apoptosis pathway inhibitor c-FLIP short isoform (c-FLIPs), as demonstrated by western blot analysis. Therefore, in the presence of high level of miR146a, which keeps the NF-kB signaling pathway in check, the pro-apoptotic activity of NF-kB may be exacerbated.
The administration of ITF2357 to SCID mice subcutaneously xenotransplanted with Raji cells significantly delayed tumor growth (p<0.001) without any sign of aspecific toxicity based on macroscopic criteria as well as microscopic examination of the various tissues upon necropsy. Consistent with the in vitro findings, treated tumor biopsies showed a significant up-regulation of miR-146a compared to the untreated ones (p<0.01). This effect was associated with c-FLIPs down-modulation and significant caspase-8 cleavage, indicating the activation of the extrinsic apoptosis pathway.
To definitely verify the anti-lymphoma properties of miR-146a in human BL cell lines, we transiently transfected Raji and Namalwa cells with pre-miR-146a, and, after 48 and 72 hours, apoptosis induction and cell-cycle progression were analyzed. Interestingly, we found a significant reduction of the S phase in miR-146a-overexpressing Raji cells (p<0.05), and the increase of the sub-G1 peak in mir146a-overexpressing Namalwa cells, indicating the occurrence of apoptosis.
Taken as a whole, these results point to miR-146a as a crucial mediator of the anti-lymphoma activity of ITF2357 and strongly support its candidacy as a novel lymphoma suppressor microRNA.

Disclosures:
No relevant conflicts of interest to declare.