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M.R. Sapienza et al.
BPDCN patient-derived CAL-1 cell line and identified 1302 non-synonymous single nucleotide variants (SNVs) represented predominantly by missense SNVs (n=1251), nonsense SNVs (n=47), and frameshift insertions/dele- tions (n=7). To verify the robustness of the WES analysis, 2 randomly-selected variants occurring in the ASXL1 and SUZ12 genes, respectively, were validated by Sanger sequencing (Online Supplementary Figure S1). To extend the validation to a higher number of samples and mutations, a targeted sequencing approach was adopted: 21 SNVs were interrogated by MiSeq Illumina technology in the same BPDCN cases analyzed by WES and a concordance of 95.2% (20 out of 21 SNVs) was achieved, underlining a high consistency of data (Online Supplementary Table S7). The 1302 non-synonymous SNVs detected by WES affect- ed 1166 genes, all but 7 known to be related with patho- logical conditions and reported as mutated in the Catalogue of Somatic Mutations in Cancer (COSMIC_v66).
To identify the biological processes that were most altered by the mutational events, we performed a func- tional enrichment analysis of the 9 genes that were recur- rently mutated (≥ 3 samples) and the 45 genes impacted by deleterious (nonsense or frameshift) SNVs. Among the top 10 significantly enriched biological processes, the epigenetic program was the most represented (P=0.0001), followed by hematopoietic stem cell home- ostasis, Rac signaling and gamma-aminobutyric acid (GABA) secretion (Figure 1A and Online Supplementary Table S8). The ASXL1 gene proved to be the most fre- quently mutated (28%, 4 out of 14 samples), followed by TET2 (21%, 3 out of 14 samples); both genes displayed mainly nonsense or frameshift SNVs located within or upstream of the catalytic domain of the proteins, poten- tially leading to their functional disruption (Figure 1B and Online Supplementary Table S9). We consulted the Gene Ontology database28 to identify among the 1166 BPDCN mutated genes those implicated in the epigenetic regula- tion. We found 25 mutated epigenetic modifier genes controlling chromatin accessibility (ARID1a, CHD8, SMARCA1), DNA methylation (TET2, IDH2), or histone post-transcriptional modifications [methylation (ASXL1, SUZ12, MLL family), demethylation (KDM4D), acetyla- tion (EP300, EP400), ubiquitination (PHC1, PHC2), dephosphorylation (EYA2), and exchange (SRCAP)]. Of note, 12 out of 14 BPDCN samples (86%) harbored at least one of the 25 epigenetic regulator genes mutated, and specifically 8 out of 14 patients (57.14%) presented a deleterious lesion (nonsense/frameshift SNV) (Online Supplementary Table S10). Many SNVs clustered in the histone methylation pathway, specifically in genes belonging to the Polycomb-repressive complex 2 (ASXL1, ASXL3, SUZ12) and in histone methyltrans- ferases (ASHL1, SETMAR, MLL), possibly compromising the integrity of the methylation program. Besides genetic lesions targeting epigenetic regulators, we also detected mutations potentially affecting molecular programs com- monly deregulated in myeloid malignancies, such as RAS signaling29 (hot-spot SNVs on KRAS or NRAS, alterna- tively), DNA repair/cycle progression30 (SNVs on BRCA1, ATM, ATR, and RAD52), Wnt-signaling31 (SNVs on WNT3, WNT7B, WNT10 and BCL9L), cell growth32 (SNVs on RUNX2, MAPK1), and splicing machinery33 (an SNV on ZRSR2) (Figure 1C).
Whole-exome sequencing data were also used for
cytogenetic CNV analysis, which highlighted extensive losses along the chromosome 9 and the associated dele- tion of the tumor suppressor CDKN2A gene in 8 out of 14 BPDCN samples (57%) (Online Supplementary Figure S2), as already reported in the literature.12,15,20 In addition, CNV analysis showed that deletions affected six of the nine genes recurrently mutated; deletions were always mutually exclusive with mutations (Online Supplementary Figure S3). However, no significant correlation was found between genetic lesions and the clinical data.
Blastic plasmacytoid dendritic cell neoplasm tran- scriptome profiling confirms the dysregulation of epi- genetic programs
Genetic lesions in key epigenetic modifier genes and in related regulatory networks can induce profound pertur- bations in the transcriptional homeostasis of the cell. To further substantiate the impact of mutations affecting the chromatin remodeling pathway in BPDCN, we per- formed RNA sequencing of 5 BPDCNs, considered as the discovery set, already studied by WES and MiSeq target- ed sequencing. We compared the patients' transcrip- tomes with those of 4 normal plasmacytoid dendritic cell (pDC) samples isolated from the peripheral blood of healthy individuals and used as controls. BPDCN tumor samples and pDCs segregated separately according to their gene expression profiles (Figure 2A). Two thousand and thirty-four genes (2034) were significantly deregulat- ed among patients, and approximately half of them were up-regulated (46%) in the BPDCN setting. Gene set enrichment analysis (GSEA) reported the significant deregulation of two genetic signatures involved in the methylation process, driven by the KDM5B34 histone demethylase and PRMT535 methyltransferase-associat- ed genes, respectively. Of note, GSEA also detected the significant enrichment of a set of genes associated with the response to a DNA demethylating agent,36 namely decitabine (Figure 2B). The GSEA results [normalized enrichment score (NES) ≥2; false discovery rate (FDR) q- value ≤0.0001] were then validated in an extension set of 4 BPDCN samples and in a CAL-1 cell line (Online Supplementary Figures S4 and S5).
Genome-wide ChIP-sequencing substantiates epigenetic dysregulation of cell cycle program in blastic plasmacytoid dendritic cell neoplasms
To investigate if the transcriptional deregulation of BPDCNs could be linked to specific epigenetic features, we analyzed the histone acetylation/methylation pro- files of 2 selected BPDCN patients (BPDCN_25 and BPDCN_37). The trimethylation at lysine 27 of histone 3 (H3K27me3) is closely associated with inactive gene pro- moters, while its acetylation (H3K27ac) closely correlates with gene activation, the two epigenetic modifications being mutually exclusive. Given this, we analyzed the genome-wide distribution of trimethylation and acetyla- tion profiles of H3K27 in BPDCN cases. The analysis of PAT-ChIP sequencing data demonstrated that the 2 patients converged on the same pattern of histone acety- lation, sharing as much as 43.6% of the acetylated pro- moters (Figure 2C and D). PAT-ChIP sequencing results were then integrated into the RNA sequencing data lead- ing to the identification of a signature of 86 genes marked by promoter acetylation and significantly over- expressed in the BPDCN RNA sequencing sets. Gene
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haematologica | 2019; 104(4)