Intravenous apoptotic spleen cell infusion induces a TGF-beta-dependent regulatory T-cell expansion.: Apoptosis and regulatory T cells

International audienceApoptotic leukocytes are endowed with immunomodulatory properties that can be used to enhance hematopoietic engraftment and prevent graft-versus-host disease (GvHD). This apoptotic cell-induced tolerogenic effect is mediated by host macrophages and not recipient dendritic cells or donor phagocytes present in the bone marrow graft as evidenced by selective cell depletion and trafficking experiments. Furthermore, apoptotic cell infusion is associated with TGF-beta-dependent donor CD4+CD25+ T-cell expansion. Such cells have a regulatory phenotype (CD62L(high) and intracellular CTLA-4+), express high levels of forkhead-box transcription factor p3 (Foxp3) mRNA and exert ex vivo suppressive activity through a cell-to-cell contact mechanism. In vivo CD25 depletion after apoptotic cell infusion prevents the apoptotic cell-induced beneficial effects on engraftment and GvHD occurrence. This highlights the role of regulatory T cells in the tolerogenic effect of apoptotic cell infusion. This novel association between apoptosis and regulatory T-cell expansion may also contribute to preventing deleterious autoimmune responses during normal turnover

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Harbors Frequent Splicesosome Mutations That Cause Aberrant RNA Splicing Affecting Genes Critical in pDC Differentiation and Function

Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive malignancy thought to result from transformation of plasmacytoid dendritic cells (pDCs). Clinical outcomes are poor and pathogenesis is unclear. To better understand BPDCN genomics and disease mechanisms, we performed whole exome- (12 BPDCNs), targeted DNA- (additional 12 BPDCNs), bulk whole transcriptome RNA- (12 BPDCNs and 6 BPDCN patient-derived xenografts [PDXs]), and single cell RNA-sequencing (scRNA-seq) compared to normal DCs. We observed RNA splicing factor mutations in 16/24 cases (7 ZRSR2, 6 SRSF2, 1 each SF3B1, U2AF1, SF3A2, SF3B4). Additional recurrent alterations were in genes known to be mutated in other blood cancers: TET2, ASXL1, TP53, GNB1, NRAS, IDH2, ETV6, DNMT3A, and RUNX1. From exome sequencing we also discovered recurrent mutations in CRIPAK (6/12 cases), NEFH (4/12), HNF1A (2/12), PAX3 (2/12), and SSC5D (2/12) that may be unique to BPDCN. ZRSR2 is notable among the recurrently mutated splicing factors in hematologic malignancies in that all mutations are loss-of-function (e.g., nonsense, frameshift). Of note, BPDCN is very male predominant, ZRSR2 is located on chrX and all mutations are in males. ZRSR2 plays a critical role in "minor" or U12-type intron splicing (only 0.3% of all introns). Thus, we hypothesized that mis-splicing, possibly of U12 genes, contributes to BPDCN pathogenesis. Using RNA-seq, we measured aberrant splicing in BPDCN. Intron retention was the most frequent abnormality in ZRSR2 mutant BPDCNs and PDXs compared to non-mutant cases. ZRSR2 mutant intron retention predominantly affected U12 introns (patients: 29.4% of retained introns, P<0.0001; PDX: 94%, P<0.0001). To test if ZRSR2 loss directly causes U12 intron retention in otherwise isogenic cells, we performed ZRSR2 knockdown using doxycycline-inducible shRNAs in the BPDCN cell line, CAL1, which has no known splicing factor mutation. RNA-seq was performed 0, 2, and 7 days after addition of doxycycline in 3 independent clones each of control or ZRSR2 knockdown. Consistent with what we observed in primary BPDCN, intron retention events were higher in ZRSR2 compared to control shRNA cells after 7 days of doxycycline (mean 885.7 vs 122.7 events, P=0.041). Aberrant intron retention after ZRSR2 knockdown largely involved U12 introns (30/732 U12 vs 37/207,344 U2 introns, P<0.0001). SRSF2 and SF3B1 mutations in BPDCN were at hotspots seen in other cancers: SRSF2 P95H/L/R and SF3B1 K666N, mutants that induce specific types of aberrant splicing (Kim, Ca Cell 2015; Darman, Cell Rep 2015). Mutant BPDCNs demonstrated the same aberrations: SRSF2, exon inclusion/exclusion based on CCNG/GGNG exonic splicing enhancer motifs; SF3B1, aberrant 3' splice site recognition. We hypothesized that aberrant splicing may affect RNAs important for pDC development or function. To further define genes uniquely important in BPDCN, we performed scRNA-seq on 4 BPDCNs and on DCs from healthy donors. By principal component analysis, BPDCNs were more similar to pDCs than to conventional DCs (cDCs) or other HLA-DR+ cells. However, several critical genes for pDC function had markedly lower expression in BPDCN including the transcription factors IRF4 and IRF7. Next we determined which genes were commonly mis-spliced in splicing factor mutant BPDCNs. Strikingly, this list included genes already known to be important in driving DC biology or identified in our scRNA-seq as being differentially expressed between BPDCN and healthy pDCs, including IRF7, IRF8, IKZF1, FLT3, and DERL3. To determine if splicing factor mutations affect DC function, we stimulated ZRSR2 knockdown or control CAL1 cells with Toll-like receptor (TLR) 7, 8, and 9 agonists (R848 or CpG oligo). ZRSR2 knockdown inhibited upregulation of the CD80 costimulatory molecule and aggregation of CAL1 cells, suggesting impairment in activation. Using mouse conditional knock-in bone marrow in ex vivo multipotent progenitor assays, DC differentiation induced by FLT3 ligand was biased toward pDCs and away from cDCs in SRSF2 P95H mutant compared to wild-type cells. However, cDC and monocyte differentiation in the presence of GM-CSF was not affected. In conclusion, splicing factors are frequently mutated in BPDCN and lead to specific splicing defects. Splicing factor mutations may promote BPDCN by affecting pathways important in DC maturation or activation, which could contribute to transformation. Disclosures Seiler: H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Lane:Stemline Therapeutics: Research Funding; N-of-1: Consultancy

Male-Biased Spliceosome Mutations in Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Impair pDC Activation and Apoptosis

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive, male-biased (>3:1 M:F) hematologic malignancy in which some patients have bone marrow involvement at diagnosis (50%) and most have tumor formation in the skin (~90%), often preceding marrow disease. The prognosis is poor (median survival of 12-24 months) and there is unmet need for biological insight. TET2, ASXL1, and RNA splicing genes (SRSF2, SF3B1, and ZRSR2) are recurrently mutated in BPDCN. The X chromosome gene ZRSR2 was the most frequently mutated spliceosome gene reported in a prior BPDCN cohort (7 of 24, 29.2%; Taylor, ASH 2013). Our goal was to define the functional consequences of ZRSR2 mutations in BPDCN. First, we confirmed the frequency of ZRSR2 mutations in a larger cohort from the US and Europe; we found ZRSR2 mutations in 24 of 93 (25.8%). Notably, ZRSR2 mutations were almost exclusively in males (23/73 males vs 1/20 females, P=0.019). Next, we compared the global mutation pattern to 30 predefined signatures from >7000 cancers in COSMIC. Analysis of all somatic single nucleotide variants in 11 tumor-normal pairs using whole exome sequencing (tumor was sorted BPDCN cells from marrow) revealed that BPDCN had an ultraviolet (UV)-induced mutation signature (score >0.25 in 6/11 or 55%; Figure 1A). For comparison, we detected the UV signature in melanoma but not in AML from The Cancer Genome Atlas. These data suggest that mutations acquired in the skin stage of BPDCN evolution are retained in subsequent leukemic disease. Next, we performed RNA-sequencing from sorted BPDCN and normal plasmacytoid dendritic cells (pDCs). Differentially expressed genes between BPDCN and pDCs (BCL2, MYB, IRF4, CEP70, IGLL1, GZMB) were similar to those that distinguish BPDCNs from pDCs by bulk and single cell RNA-sequencing. By gene set enrichment analysis (GSEA), BPDCNs were enriched for overexpression of MYC/E2F targets and PI3K/AKT/MTORC1 signaling pathway-associated genes. BPDCNs transcriptomes were also enriched for gene sets associated with RNA splicing machinery and RNA nonsense mediated decay (NMD). To link RNA splicing with functional consequences of ZRSR2 mutations, we generated ZRSR2-knockout BPDCN cells (CAL1) using CRISPR/Cas9. This models primary tumors because ZRSR2-mutant BPDCNs have complete loss of ZRSR2 protein. Activation marker (CD80) upregulation and type 1 interferon secretion after Toll-like receptor (TLR) stimulation with lipopolysaccharide (LPS) or R848 were reduced in ZRSR2-deficient cells. We found similar defective cytokine production in stimulated primary BPDCN cells compared to normal pDCs. After activation, normal pDCs undergo apoptosis in a negative feedback process. In contrast, ZRSR2-knockout, but not control cells, were protected from TLR activation-induced apoptosis. Reexpression of wild-type ZRSR2 in knockout cells restored activation-induced apoptosis (Figure 1B). These data suggested that ZRSR2-mutant BPDCNs have defects downstream of TLR stimulation. By RNA-sequencing, we found that IRF7 mRNA was mis-spliced in all ZRSR2- (2/2), SRSF2- (4/4), and SF3B1- (1/1) mutant BPDCNs compared to those with no mutated splicing gene (4/4). IRF7 (interferon regulatory factor 7) is a transcription factor activated by TLR signaling that is important for pDC activation and apoptosis. The IRF7 mRNA transcript contains a "weak intron" (intron 4) that is subject to intron retention, which leads to NMD and reduced IRF7 protein level in stimulated dendritic cells (Luke, Mol Cell 2019). IRF7 intron 4 was mis-spliced in ZRSR2-, SRSF2-, and SF3B1-mutant BPDCNs. ZRSR2-knockout CAL1 cells had severely impaired ability to upregulate IRF7 after LPS stimulation, which was partially rescued by reepxression of wild-type ZRSR2 (Figure 1C). Expression of constitutively activated IRF7 inhibited growth of both ZRSR2-knockout and control cells, confirming that the inability to activate IRF7 is important for the effect of ZRSR2 loss on TLR agonist-induced growth inhibition. In conclusion, male-biased ZRSR2 mutations are frequent in BPDCN and impair pDC activation and apoptosis, at least in part via TLR-IRF7. These data may explain why BPDCNs have an impaired activation state (Bierd, BCJ 2019). They also suggest that splicing factor mutations affect cell type-specific pathways to promote transformation, underscoring the importance of studying cancer genes in relevant contexts. Figure Disclosures Griffin: Moderna Therapeutics: Consultancy. Ghandi:Monte Rosa Therapeutics: Consultancy; Cambridge Data Science LLC: Current Employment, Current equity holder in private company. Seiler:Remix Therapeutics: Current Employment. Konopleva:Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Eli Lilly: Research Funding; Genentech: Consultancy, Research Funding; Agios: Research Funding; Rafael Pharmaceutical: Research Funding; Sanofi: Research Funding; AbbVie: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding; Calithera: Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; Cellectis: Research Funding; Ablynx: Research Funding; Kisoji: Consultancy; Stemline Therapeutics: Consultancy, Research Funding. Pemmaraju:Cellectis: Research Funding; Daiichi Sankyo: Research Funding; DAVA Oncology: Honoraria; Plexxikon: Research Funding; Blueprint Medicines: Honoraria; Incyte Corporation: Honoraria; SagerStrong Foundation: Other: Grant Support; Celgene: Honoraria; Pacylex Pharmaceuticals: Consultancy; Affymetrix: Other: Grant Support, Research Funding; MustangBio: Honoraria; Roche Diagnostics: Honoraria; Novartis: Honoraria, Research Funding; LFB Biotechnologies: Honoraria; Stemline Therapeutics: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Samus Therapeutics: Research Funding. Abdel-Wahab:H3 Biomedicine Inc.: Consultancy, Research Funding; Merck: Consultancy; Janssen: Consultancy; Envisagenics Inc.: Current equity holder in private company. Lane:Qiagen: Consultancy; Abbvie: Research Funding; Stemline Therapeutics: Research Funding

Sex-Biased ZRSR2 Mutations in Myeloid Malignancies Impair Plasmacytoid Dendritic Cell Activation and Apoptosis

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive leukemia of plasmacytoid dendritic cells (pDC). BPDCN occurs at least three times more frequently in men than in women, but the reasons for this sex bias are unknown. Here, studying genomics of primary BPDCN and modeling disease-associated mutations, we link acquired alterations in RNA splicing to abnormal pDC development and inflammatory response through Toll-like receptors. Loss-of-function mutations in ZRSR2, an X chromosome gene encoding a splicing factor, are enriched in BPDCN, and nearly all mutations occur in males. ZRSR2 mutation impairs pDC activation and apoptosis after inflammatory stimuli, associated with intron retention and inability to upregulate the transcription factor IRF7. In vivo, BPDCN-associated mutations promote pDC expansion and signatures of decreased activation. These data support a model in which male-biased mutations in hematopoietic progenitors alter pDC function and confer protection from apoptosis, which may impair immunity and predispose to leukemic transformation. SIGNIFICANCE: Sex bias in cancer is well recognized, but the underlying mechanisms are incompletely defined. We connect X chromosome mutations in ZRSR2 to an extremely male-predominant leukemia. Aberrant RNA splicing induced by ZRSR2 mutation impairs dendritic cell inflammatory signaling, interferon production, and apoptosis, revealing a sex- and lineage-related tumor suppressor pathway.This article is highlighted in the In This Issue feature, p. 275

How should we diagnose and treat blastic plasmacytoid dendritic cell neoplasm patients?

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia for which we developed a nationwide network to collect data from new cases diagnosed in France. In a retrospective, observational study of 86 patients (2000-2013), we described clinical and biological data focusing on morphologies and immunophenotype. We found expression of markers associated with plasmacytoid dendritic cell origin (HLA-DRhigh, CD3031, CD3041, and cTCL11) plus CD4 and CD56 and frequent expression of isolated markers from the myeloid, B-, and T-lymphoid lineages, whereas specific markers (myeloperoxidase, CD14, cCD3, CD19, and cCD22) were not expressed. Fifty-one percent of cytogenetic abnormalities impact chromosomes 13, 12, 9, and 15. Myelemia was associated with an adverse prognosis. We categorized chemotherapeutic regimens into 5 groups: acute myeloid leukemia (AML)-like, acute lymphoid leukemia (ALL)-like, lymphoma (cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP])-like, high-dose methotrexate with asparaginase (Aspa-MTX) chemotherapies, and not otherwise specified (NOS) treatments. Thirty patients received allogeneic hematopoietic cell transplantation (allo-HCT), and 4 patients received autologous hematopoietic cell transplantation. There was no difference in survival between patients receiving AML-like, ALL-like, or Aspa-MTX regimens; survival was longer in patients who received AML-like, ALL-like, or Aspa-MTX regimens than in those who received CHOP-like regimens or NOS. Eleven patients are in persistent complete remission after allo-HCT with a median survival of 49 months vs 8 for other patients. Our series confirms a high response rate with a lower toxicity profile with the Aspa-MTX regimen, offering the best chance of access to hematopoietic cell transplantation and a possible cure