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Epigenetic matrix and targeted therapy of BPDCN
Ontology analysis of the 86 selected genes highlighted the enrichment in biological processes involved in cell cycle progression (FDR q-value <0.001) (Figure 2E and Online Supplementary Table S11).
In vivo blastic plasmacytoid dendritic cell neoplasm modeling demonstrates combined epigenetic therapy as effective in controlling disease progression
The integration of results obtained from WES, RNA sequencing and PAT-ChIP-sequencing experiments clear- ly pointed to a fundamental role for epigenetic dysregu- lation in BPDCN and allowed us to hypothesize that this malignancy could display susceptibility to drugs active on the epigenetic regulation. Following the demonstra- tion that the CAL-1 cell line, like primary BPDCN sam- ples, had mutations clustering in chromatin remodeling pathway (Figure 1C) and enrichment in the same epige- netic programs (Online Supplementary Figure S5), we developed an in vivo CAL-1 xenograft BPDCN-like model to explore the effects of treatments targeting the acetyla- tion, methylation, and also the NF-kB pathways, accord- ing to previous results.17,18 To this end, we focused on four different FDA-approved compounds: 5’-azacytidine, decitabine, romidepsin and bortezomib.
NSG mice intravenously injected with 5x103 CAL-1 cells rapidly developed a systemic BPDCN-like progres- sive disease, which was defined by the flow cytometry identification of human CD56+CD38+ malignant cells in the peripheral blood, bone marrow, spleen and liver, as evaluated at 39 days after injection (Figure 3A). The pathological infiltration by malignant BPDCN cells in the mouse model was also confirmed at the same time point by the histopathological analysis of the bone marrow and spleen samples, which showed the presence of atyp- ical cells with blastic morphology and expressing the human CD303/BDCA2 pDC marker (Figure 3B). Xenografted mice were divided into 11 treatment groups (n=110 mice) one day after CAL-1 injection and treated with either saline or with the hypo-methylating agents 5’-azacitidine or decitabine, the proteasome inhibitor bortezomib, and the histone deacetylase inhibitor romidepsin, used as single agents or in combination, according to the treatment schedule summarized in Figure 3C. The administration of 5’-azacytidine and decitabine used as single agents significantly prolonged OS of the mice when compared with saline (median sur- vival 43.6 days vs. 32 days, P<0.01 for 5’-azacytidine; median survival 44.7 days vs. 32 days, P<0.05 for decitabine) while neither bortezomib nor romidepsin alone showed beneficial effects on disease outcome. When the same agents were associated in combined treatment experiments, three different combinations were seen to significantly prolong mouse survival: i) the association of romidepsin and decitabine (median sur- vival 42.8 days vs. 32 days, P<0.05); ii) the combination of romidepsin, decitabine, and 5’-azacytidine (median survival 41.8 days vs. 32 days, P<0.01); and iii) the asso- ciation of decitabine and 5’-azacytidine (median survival 52.8 days vs. 32 days, P<0.01), which achieved the best result in terms of survival (Figure 3D). Consistently, 5’- azacytdine and decitabine administered alone reduced the CAL-1-induced splenomegaly as evaluated at day 39 post injection and their combination proved to be even more effective (Figure 3E).
Discussion
Blastic plasmacytoid dendritic cell neoplasm is a rare myeloid malignancy with dismal prognosis and no stan- dard therapy. In the present study, we performed WES on the largest series of BPDCNs that, to the best of our knowledge, has so far been reported in the literature. Thanks to the integration of WES with RNA and PAT- ChIP sequencing, we provide new insights into BPDCN pathobiology by highlighting the dysregulation of the epi- genetic program as a hallmark of the disease and suggest possible novel therapeutic interventions.
We found BPDCN patients extensively affected by mutations of genes involved in the epigenetic regulation: 25 epigenetic modifiers were mutated in almost all BPDCN patients (13 out of 14) and the CAL-1 cell line. In more than half of the patients (8 out of 14), the mutations heralded damaging functional alterations (Figure 1C). Some of the mutated genes have already been reported in previous studies (e.g. ASXL1, RAS, ATM, ARID1A, and IDH2), although, at times, at different rates than in our series (see ASXL1 and TET2, which were found to be mutated in 28.6% and 21.4% of our samples vs. 32% and 36% of those of Menezes et al.19). In this respect, it should be remembered that the aim of our study was not only to extensively explore the mutational landscape of BPDCN, but also to possibly translate molecular notions into a pre- clinical approach. In any case, thanks to the employment of a WES approach, which did not limit our investigation to a priori-selected genes, we recognized additional mutat- ed epigenetic factors that have never been described before but which are potentially relevant in the context of BPDCN, like PHF2 histone demethylase, that enhances the TP53-tumor suppressor activity,37 and the CHD8 Chromodomain helicase DNA-binding protein-8, that promotes the E2F-dependent transcription and cell cycle progression.38 Besides the epigenetic pathway, we also detected mutations affecting programs common to other myeloid malignancies, such as DNA repair process,30 Wnt/β-catenin signaling,31 and the differentiation path- way.32
Importantly, the functional enrichment analysis of WES data showed that among all genes/pathways explored the epigenetic program was the most deregulated (P<0.0001).
To evaluate the impact of the identified epigenetic mutations at gene expression level, we analyzed the tran- scriptome of samples studied by WES. Among up-regulat- ed genes, GSEA revealed the significant enrichment of two methylation pathways, driven respectively by the KDM5B histone-demethylase34 and by the PRMT5 argi- nine methyltransferase-5;35 these two epigenetic modifiers are reported to be over-expressed in several cancer types and also represent promising therapeutic targets.39 Blockade of the PRMT5 activity reduces cell survival in chronic myelogenous leukemia40 and inhibition of KDM5B demethylation correlates with cell growth arrest in hepatocellular carcinoma and breast cancers.41,42 We also identified the overexpression of one gene signature36 specifically responsive to the administration of the hypomethylating agent decitabine; a molecular finding bearing important therapeutic implications (FDR q=1.85E- 5). To gain a functional insight into the epigenetic land- scape of BPDCN samples, we performed PAT-ChIP sequencing of H3K27-acetylation/trymethylation signals of 2 BPDCN patients. The trimethylation of H3K27 marks
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