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M.R. Sapienza et al.
inactive gene promoters and enhancers, while its acetyla- tion correlates with gene activation.43 PAT-ChIP sequenc- ing data showed that the 2 patients converged on the same epigenetic pattern sharing approximately half of the identified H3K27-acetylated promoters. Interestingly, the common acetylated regions comprised 10 super- enhancers (SE) bound by the Bromodomain-containing protein 4 (BRD4), as described by Ceribelli et al. in a recent work on BPDCN (data not shown).24
The integration of PAT-ChIP and the RNA sequencing data highlighted a set of 86 genes involved in the cell-cycle progression aberrantly over-expressed and marked by H3K27-promoter acetylation. This finding suggests that the cell-cycle deregulation could be driven by H3K27- acetylation signals, a hypothesis meriting future ad hoc studies that could help to clarify the mechanism of prolif- eration of this largely obscure disease.
The rarity of the disease (with an incidence of 0.000045%) and its extremely aggressive behavior (OS 10- 19 months) limits the number of available patients includ- ed in biological and/or clinical studies. For these reasons, not surprisingly, BPDCN is still an orphan tumor lacking a standardized and effective therapeutic approach. In the last few years, new molecular studies have opened the way to innovative target therapies (e.g. bortezomib,17,18 venetoclax,22 BET-inhibitors,24 SL-40125) being used in clinical trials. Some of these are showing promising results, although still concerns remain regarding their safe- ty. Of note, all the treatments proposed are mainly the result of investigation into the RNA transcriptome, while the DNA features of BPDCN patients have barely been evaluated.
We therefore decided to tackle this yet incurable dis- ease by designing the first therapeutic strategy modeled on the DNA mutational status of BPDCN patients, ana- lyzed by WES. The WES mutational findings enhanced by the RNA and PAT-ChIP sequencing results clearly evi- denced the prominent role of the epigenetic program dys- regulation among BPDCN patients and guided our thera- peutic approach towards the use of epigenetic agents. In particular, we tested in vivo the efficacy of US Food and Drug Administration-approved epigenetic drugs which could be considered for potential repositioning in clinical
trials: two hypomethylating agents such as decitabine and 5’-azacytidine, and the histone deacetylase inhibitor romidepsin. We hypothesized that these drugs could impact on tumor progression because: i) BPDCN patients displayed potential sensitivity to hypomethylating agents, particularly to decitabine, as detected by GSEA analysis; ii) both 5’-azacytidine and decitabine are currently used for the treatment of myelodysplastic syndromes,44,45 which are myeloid neoplasms sharing many epigenetic mutated genes with BPDCN; iii) preclinical studies on several malignancies demonstrated that the action of decitabine is synergized by romidepsin.46 In the light of this, our exper- imental design focused on epigenetic drugs with a large- scale activity, aiming to explore whether we might induce cell death by perturbation of the malignant epigenetic pro- gramme. In addition to the epigenetic drugs, we also veri- fied the efficacy of bortezomib, a proteasome inhibitor, which had previously been shown to significantly induce in vitro and in vivo BPDCN cell death.17,18 Our experiments revealed that the treatment with 5’-azacytidine in combi- nation with decitabine significantly inhibits disease pro- gression and extends survival (P<0.01) in a preclinical mouse model. In the past, two reports experimented the use of 5’-azacytidine in elderly BPDCN patients, though this therapeutic choice was not yet sustained by a molec- ular rationale.47,48 Here we demonstrate that 5’-azacytidine is more effective in tumor eradication when combined with decitabine. Further studies are ongoing to elucidate the synergistic mechanisms between the two drugs.
In conclusion, we have identified the deregulation of the epigenetic program as a genetic hallmark of BPDCN and suggest a novel therapeutic approach based on the combi- nation of two hypomethylating agents, 5’-azacytidine and decitabine, to be tested in future clinical trials.
Funding
The present work was supported by the AIRC grants IG 15762 and 5x1000 10007 “Genetics-driven targeted manage- ment of lymphoid malignancies” and the Grant “Innovative approaches to the diagnosis and pharmacogenetic-based thera- pies of primary hepatic tumours, peripheral B and T-cell lym- phomas and lymphoblastic leukaemias” Strategic Programme 2010-2012 Regione Emilia Romagna - Università (all to SAP).
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