Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a hematologic malignancy in which patients can have leukemic involvement of the blood and bone marrow, and also tumor formation in the skin (in ~90% of cases), lymphoid organs, and in other tissues [1] . Among the unique epidemiologic features of the disease is the extreme male predominance, with a male:female incidence ratio of at least 3:1 in adults [2, 3] . There is no explanation to date for why males are predisposed to the disease, or alternatively, why females are relatively protected. Outcomes for patients with BPDCN are poor, with median survival of 12-24 months from diagnosis [3, 4] , demonstrating the unmet need for additional biological insight.
DNA sequencing of bone marrow or skin involved with BPDCN has identified mutations in genes often affected in other blood cancers, particularly myelodysplastic syndrome (MDS) and related myeloid malignancies. These include point mutations or indel/frameshift mutations in ASXL1, TET2, and TP53, and copy number alterations affecting one or more cell cycle regulators [5] [6] [7] [8] [9] .
However, BPDCN frequently arises in the context of a pre-existing or concurrent myeloid malignancy, such as MDS or chronic myelomonocytic leukemia (CMML) [10] [11] [12] , but most of the prior genetic landscape studies of BPDCN analyzed unsorted bone marrow or skin biopsies with heterogeneous composition. Those biopsies likely included hematopoietic cells, possibly neoplastic, of other lineages, and non-hematopoietic cells. Therefore, one goal of this project was to define the genetic alterations and transcriptional changes present in highly purified BPDCN cells separated from any background clonal hematopoietic disorder and to link these to patient sex. We found that loss-of-function mutations in the RNA splicing factor ZRSR2, encoded on chromosome X, are enriched in BPDCN and are associated with a significant fraction of the male predominance of the disease. nucleotide variant that encodes an IRF8 R404W missense mutation. While the functional consequence of this specific variant is unknown, IRF8 is important for pDC development and function [18, 19] . WES also identified a small number of recurrently mutated genes in this set of BPDCNs that have not been previously identified as mutated in other cancers or in prior BPDCN sequencing (Supplementary Table 2 ). Some of these variants are in homopolymer tracts that are at risk for sequencing errors or are in genes that are not expressed in BPDCN or pDCs, and thus, their contribution to the BPDCN phenotype is not clear.
In the BPDCNs studied by WES, the distribution of variant allele frequency (VAF) for all somatic mutations allowed us to define their clonal structure (Figure 1b) . Most cases harbored known hematologic malignancy associated genes in a single dominant clone (clustered around VAF ~50% for presumed heterozygous mutations), which included all of the variants in the most frequently mutated genes: TET2, ASXL1, and spliceosome components. Some mutations were detected at nearly 100% VAF, including in ZRSR2 and BCORL1, which reside on the X chromosome and were mutated nearly exclusively in males in this cohort, and in ETV6, IDH2, and TP53, which likely represent loss-of-heterozygosity events.
Given that many genes mutated in BPDCN were shared with other myeloid malignancies, we asked if the global mutation pattern might indicate unique BPDCN-specific features. We performed a mutation analysis on all somatic alterations and their surrounding nucleotide context using signatures previously defined in the Catalog of Somatic Mutations in Cancer (COSMIC) [20] . We found that BPDCNs harbored an age-associated pattern ("Signature 1"), which is detected in most cancers in COSMIC (Figure 1c, Supplementary Figure 1) . However, we also detected a strong association with ultraviolet (UV)-induced mutation signatures in the majority of BPDCNs (signature score >0.25 in 6/11 or 55%; Figure 1c ). In contrast, there was no UV signature associated with AML, even though AML shares many recurrently mutated genes with BPDCN and may arise from a similar myeloid progenitor. For comparison, melanoma harbors a uniformly high UV signature in most tumors (Figure 1c) . The BPDCN cells we analyzed were harvested from bone marrow or blood and not directly from UV-exposed tissue (i.e., skin), suggesting that at least some leukemic BPDCN cells maintain a UV signature presumably acquired during a prior skin phase of malignant evolution.
Sequencing purified BPDCN cells rather than bulk marrow or skin samples also allowed us to determine DNA copy number variants (CNVs) that were more confidently BPDCN-associated.
Some of the chromosome and arm level CNVs were similar to those described previously in BPDCN (Figure 2a) [7, 21] . These included loss of 7p (which harbors IKZF1), 9p (CDKN2A, CDKN2B), 12p (CDKN1B, ETV6), 13q (RB1), and 17p (TP53) (Supplementary Figure 2) . Copy number analysis also clarified the disease genetics for patients 2 and 10, who did not have any single nucleotide variants in known blood cancer-associated genes from WES. In those patients, we detected deletions in IKZF1 and RB1 (patient 2) and TET2, RB1, and ZRSR2 (patient 10), which supports that analysis of CNVs in addition to point mutations/indels can detect additional relevant driver events in patients. Integrated analysis of single nucleotide variants/indels and copy loss highlighted four putative BPDCN tumor suppressors targeted by both types of alteration:
ZRSR2, TET2, TP53, and SETD2 (Figure 2b) . PCR confirmed the WES finding that chromosome Xp22.2 copy loss in patient 10, a male, resulted in complete absence of ZRSR2 DNA in tumor but not in germline cells (Figure 2c ).
ZRSR2 mutations in BPDCN are of particular interest for several reasons. First, while other splicing factors are mutated across many solid and blood cancers, ZRSR2 has been associated with myeloid disorders, such as MDS, CMML, and AML. However, in contrast to MDS, where SRSF2, SF3B1, and U2AF1 mutations are more common, ZRSR2 was the most frequently mutated spliceosome gene in this BPDCN cohort. To confirm this finding in a larger and more geographically diverse cohort, we examined BPDCN DNA from three separate groups (from France, Italy, and MD Anderson Cancer Center), and found ZRSR2 mutations in 24 of 93 (26%) (Figure 2d ). This is significantly higher than the incidence of ZRSR2 mutations in MDS, where the gene was mutated in only 13 of 288 patients in one large cohort (4.5%; P<0.0001 by Fisher's exact test) [22] .
Second, nearly all ZRSR2 mutations in our cohort were in males (mutated in 23 of 73 males vs.
1 of 20 females, P=0.019 by Fisher's exact test) (Figure 2e) . The mutational pattern is consistent with loss-of-function, with most variants predicting inactivating events (nonsense, frame shift, deletion), which implicates ZRSR2 as a male-biased X chromosome tumor suppressor gene. In fact, strictly defined inactivating mutations in our cohort were exclusively in males (16 of 73 males vs. 0 of 20 females, P<0.0001 by Fisher's exact test) (Figure 2e ). This is similar to MDS, where loss-of-function mutations in ZRSR2 are almost always restricted to males [22] . ZRSR2 belongs to the minor subset of X chromosome genes that escape X inactivation silencing and is biallelically expressed in female cells [23] . The finding that ZRSR2 is also preferentially mutated in PBDCN and MDS from males strongly suggests that ZRSR2 is an Escape from X Inactivation Tumor Suppressor (EXITS) gene [24]. As we previously described for other EXITS genes across a range of cancers, females are relatively protected from BPDCN because they have two active ZRSR2 alleles and therefore require two mutations to eliminate function, whereas males require only one.
The degree of sex bias in BPDCN incidence linked to ZRSR2 can be expressed as the number of excess ZRSR2 mutations in males per excess case of BPDCN in males [24] . Using that calculation, in this cohort ~42% of the excess male risk of BPDCN is associated with a ZRSR2 mutation.
Next, we asked how BPDCN transcriptomes relate to their genetics. We performed RNAsequencing on the same sorted BPDCN samples that we had profiled by whole exome sequencing. Peripheral blood pDCs (CD45 + CD123 + BDCA2 + CD3 -) from healthy donors were analyzed in parallel for comparison (Supplementary Table 3 ). Differentially expressed genes between BPDCN and pDCs were similar to those we previously defined that distinguished BPDCNs from normal dendritic cells using single cell RNA-sequencing (Figure 3a ) [14] . We also observed differences in individual oncogenes and tumor suppressors (BCL2, MYB, IRF4, and MAP3K1) known to be dysregulated in BPDCN (Figure 3b) . Furthermore, gene set enrichment analysis (GSEA) identified oncogene-associated signatures in BPDCN, including overexpression of MYC and E2F targets, and of PI3K/AKT/MTORC1 signaling pathway genes (Figure 3c ). In addition to these expected gene expression changes, we also found that BPDCNs had upregulation of RNA splicing machinery and associated genes when compared to normal pDCs ( Figure 3d) . Furthermore, within BPDCN, splicing factor mutant cases showed upregulation of RNA splicing genes and also were enriched for markers of active nonsense mediated decay (NMD) RNA catabolism compared to BPDCNs without ZRSR2, SRSF2, SF3B1, or U2AF1 mutations (Figure 3d) . This was of particular interest given that ZRSR2 loss promotes intron retention [25] , which is known to trigger NMD activation and cause altered myelopoiesis [26].
Next, we analyzed RNA splicing in splicing factor mutated BPDCN compared to normal pDCs and to BPDCN without any splicing factor mutation. BPDCNs with splicing factor mutations showed several types of abnormal splicing, including intron retention (IR), cryptic 3' splice site usage, and exon skipping (Supplementary Figure 3a, Supplementary Table 4) . ZRSR2, SRSF2, SF3B1, and U2AF1 are all involved in U2 intron splicing (99.7% of introns in the human genome). In contrast, ZRSR2 is uniquely necessary for proper splicing of U12 introns (0.3% of human introns), an evolutionarily conserved genomic feature with distinct branch and splice-site sequences [27, 28] . In MDS, ZRSR2 mutations promote aberrant IR with a bias toward retention of U12-type introns [29] . Similarly, we found that BPDCNs with ZRSR2 mutation or loss had increased IR compared to those without spliceosome mutations (Figure 4a ). Both U2-and U12-type introns were affected, but IR was markedly weighted towards U12-type introns in BPDCNs with ZRSR2 mutation (Figure 4b) . We also detected some of the stereotypical mis-splicing patterns associated with specific splicing factor mutations in other cancers [29] . These included preferential exon inclusion or exclusion related to CCNG/GGNG exonic splicing enhancer motifs in cases with SRSF2 mutation [30] and alternative 3' splice sites usage with SF3B1 mutation ( Supplementary Figure 3a- (Figure 4b-c) .
Next, we asked if the splicing abnormalities observed were directly related to loss of ZRSR2. The predominant nonsense and frameshift mutations we detected in BPDCN (Figure 2d -e) are predicted to promote nonsense mediated RNA decay and absence of a mature protein, which is what we observed by western blotting in BPDCN with ZRSR2 mutations (Figure 4d) . Therefore, we used engineered knockdown or knockout cell line models to study ZRSR2 mutations in BPDCN cells. First, we generated BPDCN cell lines harboring doxycycline-inducible shRNAs targeting ZRSR2 to model protein loss and facilitate time-dependent analysis of ZRSR2 depletion effects on splicing (Figure 4e) . Seven days after induction of knockdown, intron retention was the most prominent aberrant splicing event in shZRSR2 compared to shControl cells (Figure 4f) . Both U2-and U12-type intron retention events were increased in ZRSR2 knockdown cells (Figure 4g) and intron retention events detected by RNA-seq could be confirmed by RT-PCR (Figure 4h) .
These experiments suggested that U2-and U12-type intron retention is a direct and immediate consequence of ZRSR2 depletion on RNA splicing in human BPDCN cells.
Genes that were misspliced in primary BPDCNs with ZRSR2, SRSF2, or SF3B1 mutations included several that are important in dendritic cell development and/or function, including CSF2RB, IRF4, IRF7, IRF8, and LILRB4 (Supplementary Table 4) . Therefore, to connect missplicing with cellular phenotype we asked how ZRSR2 mutations affect pDC function. ZRSR2 knockdown was inversely correlated with expression of genes normally upregulated in Toll-like receptor 7 (TLR7) agonist (R848, resiquimod) treated dendritic cells (Figure 5a) . This was of particular interest because studies have reported that primary BPDCN cells are less responsive to TLR/infectious stimulation than normal pDCs or have signatures of decreased activation, but the mechanisms are unknown [13, 33] . To test if complete loss of ZRSR2, as we saw in patient samples, was sufficient to impair pDC activation, we generated BPDCN CAL1 cells with CRISPR/Cas9 knockout of ZRSR2 (Figure 5b) . We then stimulated the knockout cells with TLR agonists. Upregulation of the activation marker CD80 after stimulation with lipopolysaccharide (LPS, TLR4 agonist) or R848 (TLR7 agonist) was markedly reduced in lines with knockout of ZRSR2 (Figure 5c ). TLR stimulation of normal pDCs induces secretion of numerous inflammatory cytokines, including type 1 interferons (IFN-a and IFN-b). In contrast, CAL1 cells with ZRSR2 knockout had defective secretion of several cytokines (e.g., IFN-a, IFN-b, IL-6, TNF-a) after LPS or R848 stimulation compared to controls (Figure 5d) . We also found similar defective cytokine production in primary BPDCN cells compared to normal pDCs upon stimulation with R848 ( Figure   5d ). Of note, ZRSR2 loss did not simply cause a complete block in TLR signaling or protein secretion, because other cytokines were produced equivalently in ZRSR2-mutant cells and BPDCN cultures after stimulation (e.g., TRAILR1). Together, these data suggest that ZRSR2 mutations in BPDCN cells cause defective activation and impaired secretion of specific cytokines, including type 1 interferons, in the setting of TLR stimulation.
After activation, normal pDCs undergo apoptotic cell death as part of a negative feedback process that limits inflammation [34] . We hypothesized that ZRSR2 mutations might protect pDCs from apoptosis in the setting of TLR stimulation and this could be a mechanistic link between spliceosome alterations and malignant transformation to BPDCN. We stimulated CAL1 cells with LPS or R848 and found that both initiated apoptosis as measured by activation of caspase 3 and 7 (Figure 6a) . Knockout of ZRSR2 conferred relative protection from LPS or R848-induced apoptosis (Figure 6b) . Interestingly, the growth rate of CAL1 cells at steady state was not changed by ZRSR2 mutation. In contrast, while growth of wild-type cells was inhibited by LPS, ZRSR2 knockout cells were protected from LPS-induced growth arrest (Figure 6c) .
We confirmed the specificity of CRISPR knockout and that loss of ZRSR2 was necessary for protection from apoptosis by demonstrating that reexpression of wild-type ZRSR2 in mutant cells partially rescued the ability of LPS to induce apoptosis and impair growth (Figure 6c-d) . Finally, we confirmed that ZRSR2-mutant CAL1 cells had not simply lost the ability to undergo TLR activation-induced cell death. A downstream consequence of pDC stimulation is production of TRAIL, a TNF-family pro-apoptotic cytokine, which promotes autocrine and paracrine apoptosis [35, 36] . Treatment with exogenous TRAIL promoted apoptosis equivalently in control and ZRSR2 mutant cells, demonstrating that the cell death response downstream of TRAIL was intact ( Figure   6d ).
Collectively, these data suggested that ZRSR2 mutation may perturb specific pathways downstream of TLRs that result in impaired activation-induced cell death. While it is likely that multiple effects of splicing mutations contribute to BPDCN, we asked if any common targets across BPDCNs might be involved in the hypo-activation phenotype. When we analyzed aberrant splicing across BPDCNs harboring any splicing factor mutation, IRF7 was among the overlapping candidates. The IRF7 gene encodes a transcription factor, interferon regulatory factor 7, that is activated by TLR signaling and is important for induction of downstream genes, including type 1 interferons and TRAIL [37, 38] . Interestingly, the IRF7 mRNA transcript is not classified as having U12-type introns but it does contain a so-called "weak intron" (intron 4) that is known to be subject to intron retention and NMD in normal dendritic cells during activation [39] . Rate-limiting splicing is a feature of U12-type introns and also more generally of a group of genes (with either U2 or U12 introns) that participate in "programmed delayed splicing" associated with inflammatory response regulation at the level of RNA processing [40, 41] . IRF7 intron 4 was aberrantly retained in ZRSR2 mutant BPDCNs (2 of 2) and the same intron-exon region was also misspliced in SRSF2 (4 of 4) and SF3B1-mutated (1 of 1) cases but not in splicing factor wild-type BPDCNs or in normal pDCs (Supplementary Table 4 ).
Therefore, we hypothesized that TLR stimulation of BPDCN with a ZRSR2 mutation would result in impairment of IRF7 induction and downstream gene activation including pro-apoptotic pathways. Indeed, whereas the baseline IRF7 protein levels were minimally affected by ZRSR2 mutation, mutant cells had severely impaired ability to increase IRF7 protein after LPS stimulation (Supplementary Figure 4b) . This suggested that loss of ZRSR2 affected the induction of IRF7 protein after LPS stimulation, but activity downstream of activated IRF7 remained intact. Together, these data support a model in which ZRSR2 mutation in BPDCN inhibits apoptosis and promotes a growth advantage compared to non-mutated cells in the setting of TLR activation.
To evaluate the contribution of BPDCN-associated mutations to pDC development and function in vivo, we generated bone marrow chimeras with hematopoietic cells harboring mutations in Zrsr2, Tet2, or both. Tet2 was chosen because it is the most frequently mutated gene in BPDCN and is the most commonly co-occurring mutated gene in BPDCNs with mutated ZRSR2. We harvested c-kit + bone marrow cells from Rosa-Cas9 knock-in animals, transduced with lentiviruses encoding sgRNAs targeting Zrsr2, Tet2, or control non-targeting guides in pairwise fashion, each co-expressing GFP or tagRFP. Eight weeks after transplantation of transduced marrow into lethally irradiated wild-type recipients, we observed approximately equivalent singleand double-positive GFP/tagRFP cells in the bone marrow across genotypes (Figure 7a) . We confirmed CRISPR insertion-deletion events at sgRNA target sites using a T7 endonuclease assay (Supplementary Figure 5a) .
We compared the GFP/tagRFP double positive populations in each of the four recipient groups (sgControl/sgControl, sgZrsr2/sgControl, sgControl/sgTet2, and sgZrsr2/sgTet2). We focused on dendritic cells and their precursors, but we also analyzed other hematopoietic stem and progenitor (HSPC) populations. Zrsr2 and Tet2 targeting was associated with relative expansion of lineagenegative, Sca1+, cKit+ (LSK) HSPCs, common myeloid progenitors (CMP), and megakaryocyteerythroid progenitors (MEPs) in the bone marrow (Supplementary Figures 5b-c, 6 ). In dendritic cell development, common dendritic progenitors (CDP) differentiate into conventional DCs (cDCs) and pDCs. In recipients of transduced and transplanted bone marrow, CDP and cDC percentage and absolute number were not different in Zrsr2, Tet2, or Zrsr2/Tet2 double mutants compared to controls. In contrast, pDCs were modestly expanded, particularly in Tet2 and Zrsr2/Tet2 double mutant bone marrow (Figure 7b) .
We assessed the transcriptome of mutant cells by sorting pDCs from bone marrow and performing RNA-seq. GSEA in mutant pDCs was negatively correlated with gene sets associated with DC activation and positively correlated with signatures of interferon alpha receptor knockout DCs exposed to viral infection (Figure 7c ). This suggests that mutant mouse pDCs, similar to ZRSR2-mutant BPDCN cells, harbored signatures of decreased pDC activation and impaired type 1 interferon-dependent signaling. We also detected a higher frequency of mis-splicing events in the presence of Zrsr2, Tet2, and Zrsr2/Tet2 mutation. Similar to what we observed in BPDCN, pDCs harboring these mutations accumulated several types of aberrant splicing (Figure 7d ).
Comparison of each group revealed genotype-specific patterns, such as Zrsr2 mutation associated with a bias toward U12-type intron retention events, as we observed in human BPDCN cells with ZRSR2 mutations (Supplementary Figure 5d) . Mutation of Tet2 alone was associated with splicing abnormalities and, in some genes, we observed additivity between Tet2 and Zrsr2 mutation to promote increased intron retention (Figure 7e) . These data are consistent with existing evidence that mutations in TET2 and other epigenetic modifiers can themselves promote splicing abnormalities and cooperate with splicing factor mutations to produce cooperative effects on splicing and hematopoiesis [31, 43] . Together, these in vivo data supported our findings in human BPDCN cells that ZRSR2 loss promotes specific transcriptome changes that provide a clonal advantage to pDCs with impaired activation.
Our findings contribute to understanding how acquired mutations in hematopoietic cells drive dendritic cell transformation. By sorting tumor cells, we are confident that the somatic mutations reported are not simply passengers of concomitant myeloid malignancies, but indeed are present in the BPDCN cells. Furthermore, the findings presented here provide evidence that these mutations, particularly in RNA splicing factors and TET2, may have cell-type specific effects on pDCs, impairing their activation and apoptosis in response to TLR stimulation. We propose that innate immune signaling via IRF7 functions as a tumor suppressor in the pDC lineage to prevent malignant transformation. Further work will be required to determine if and how other mutations in BPDCN target this pathway and contribute to similar phenotypes.
The high frequency of ZRSR2 loss-of-function mutations in BPDCN exclusively in male patients, nominate it as a lineage-specific Escape from X Inactivation Tumor Suppressor (EXITS) gene. By conservative estimation including only unambiguously inactivating alterations, ZRSR2 mutations are associated with nearly half the male bias of BPDCN. We did not identify ZRSR2 as an EXITS gene in our prior study [24] because it is most often mutated in BPDCN, MDS, and other myeloid neoplasms such as CMML, cancers that were not included in large-scale sequencing efforts like
The Cancer Genome Atlas (TCGA). Therefore, this suggests that additional sex-biased cancer genes remain discoverable in understudied diseases. These data also illustrate how identifying functional consequences of a sex-biased mutation may uncover more generalized tumor biology that is relevant even in cases without the specific mutation of interest.
The effect of ZRSR2 loss was much more dramatic in TLR-stimulated cells than at baseline. The fact that IRF7 is regulated by delayed splicing during inflammation [40] may help explain how mutations in multiple splicing factors can contribute to a similar phenotype in BPDCN. We previously found that IRF7 was among the key pDC genes downregulated in BPDCN using single cell RNA-seq, suggesting that suppression of this pathway might be a common feature of pDC transformation even in the absence of a splicing factor mutation [14] . We would be surprised if all the activation defects observed in BPDCN are solely explainable by IRF7 splicing abnormalities.
However, it is plausible that distinct acquired alterations in pDCs converge on TLR signaling more generally to confer a clonal advantage by evasion of activation-induced apoptosis. Thus, optimal modeling of transformation by BPDCN-associated mutations may need to be performed in the setting of inflammation and/or in vivo.
TET2, ASXL1, and RNA splicing factor mutations are associated with clonal hematopoiesis (CH) and myeloid malignancies, such as MDS and CMML, that can pre-date BPDCN [44-46]. The pathogenesis model proposed here suggests that specific mutations in CH/MDS/CMML contribute to a pDC pool that is poised for transformation. A testable hypothesis for future research is that patients with MDS who develop BPDCN are enriched for having a history of abnormal inflammation. These findings also suggest that mutations in patients with myeloid malignancies or in individuals with age-related clonal hematopoiesis (ARCH or clonal hematopoiesis of indeterminate potential, CHIP) may affect pDC response to TLR stimulation and thereby contribute to impaired viral or tumor immunity. In support of this hypothesis, TET2 regulates Irf7 expression via DNA methylation and is important for pDC type 1 interferon production and survival during viral infection in a mouse model [47] . Similarly, defective type 1 interferon production by pDCs is linked to deleterious IRF7 variants in patients with inferior outcomes during severe COVID-19 / SARS-CoV-2 infection, a subgroup that is also male-biased and older than the general population [48]. Our data suggest that age-associated clonal hematopoiesis mutations, such as in TET2 or ZRSR2, might also contribute to these hypoactive pDC phenotypes.
Finally, there may be therapeutic implications of these data. Splicing modulator drugs are in development and malignancies with splicing factor mutations are more sensitive to these agents do not yet know if this is related to splicing mutations and impaired activation-induced apoptosis, but the data presented here provide additional rationale for evaluating BCL2 inhibition in BPDCN.
Genes mutated in BPDCN are similar to those in MDS and AML. However, many clinical characteristics of BPDCN and AML are distinct, including epidemiology, clinical presentation, pathology, and response to certain therapies. These data suggest a mechanism by which the consequences of the same gene mutation (the 'seed'; e.g., ZRSR2), could have lineage-specific effects based on cell context (the dendritic cell 'soil'). The TLR-IRF7-type 1 interferon-apoptosis axis that is dysfunctional in BPDCN may not be the target of the same splicing mutations in other cancers. This highlights the need to study BPDCN and other rare cancers as unique entities and to consider the importance of cellular milieu when evaluating the function of cancer genes. 
293T packaging cells were obtained from ATCC. CAL1 cells were provided by T. Maeda 
Genomic DNA from mouse bone marrow cells was extracted by the DNeasy Blood and Tissue 
All patients were consented to a local institutional review board (IRB) approved protocol. Tumor and paired germline DNA samples were collected and prepared for whole-exome sequencing using the Agilent SureSelect Human All Exon V5(50M) capture. Sequencing was performed on Hiseq4000 platform with a 150 base pair paired-end protocol targeted to 100x mean coverage.
The sequences were generated as 90/100 base paired-end reads using Illumina base calling Software (ver. 1.7). The adapter sequences and low-quality reads were filtered from the raw sequencing data and the "clean data" was aligned by 
Analysis results were visualized using R 3.
Python (https://www.python.org). Co-mutation plot was created using the "GenVisR" package in R [66]. RNA sequencing data will be deposited in GEO, accession number pending.
a Figure 1 b TET2   TET2   ZRSR2  ETV6   SRSF2   TET2   BCL11B   TP53   TET2   ETV6   GATA3   SRSF2   TET2  TET2   BCORL1   DNMT3A   FLT3   RUNX1   SF3B1   WHSC1   SRSF2  TET2   ASXL1   IDH2   TP53  ASXL1  NRAS   TET2 [14] in BPDCN (n=11) compared to normal pDCs (n=4) from the current cohort. ES, enrichment score; NES, normalized enrichment score; FDR, false discovery rate. Heatmaps of the same genes plotted as low (blue) to high (red) relative expression. b. RNA expression of the indicated genes in BPDCN (n=11) compared to normal pDCs (n=4), groups compared by t test. IFNa  IFNb  IFNL1  IP10  IL6  TNFa  TNFb  TNFRI  TRAILR1  CCL2  TWEAK  APRIL   IFNa  IFNb  IFNL1  IP10  IL6  TNFa  TNFb  TNFRI  TRAILR1  TRAILR2  CCL2  IL10  PTX3 DNA copy ratio of tumor to normal calculated from whole exome sequencing is plotted for 11 BPDCN tumor/normal pairs by chromosome, where a copy ratio of 1 means no difference between tumor and normal, <1 is copy loss and >1 is copy gain. Chromosome positions of genes of interest are indicated. 