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Nuclear compartments are thought to play a role in 3-dimensional (3D) genome organization and gene expression. In mammalian brain, the architecture and dynamics of nuclear compartment-associated genome organization is not known. Here we develop Genome Organization using CUT and RUN Technology (GO-CaRT) to map genomic interactions with two nuclear compartments – the nuclear lamina and nuclear speckles – from different regions of the developing mouse, macaque and human brain. Lamina-associated domain (LAD) architecture in cells in vivo is distinct from that of cultured cells, including major differences in LADs previously considered cell-type invariant. In the mouse and human forebrain, dorsal and ventral neural precursor cells (NPCs) have differences in LAD architecture that correspond to their regional identity. LADs in the human and mouse cortex contain transcriptionally highly active sub-domains characterized by broad depletion of histone-3-lysine-9 di-methylation (H3K9me2). Evolutionarily conserved LADs in human, macaque and mouse brain are enriched for transcriptionally active neural genes associated with synapse function. By integrating GO-CaRT maps with genome-wide association study (GWAS) data, we find speckle-associated domains (SPADs) to be enriched for schizophrenia risk loci, indicating a physical relationship between these disease-associated genetic variants and a specific nuclear structure. Our work lays a framework for understanding the relationship between distinct nuclear compartments and genome function in brain development and disease.

Known fetal hemoglobin (HbF) silencers have potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here, through transcription factor (TF) CRISPR screening, we identify zinc-finger protein (ZNF) 410 as an HbF repressor. ZNF410 does not bind directly to the genes encoding γ-globins, but rather its chromatin occupancy is concentrated solely at CHD4, encoding the NuRD nucleosome remodeler, which is itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for the effects of ZNF410 on fetal globin repression. Knockout of ZNF410 or its mouse homolog Zfp410 reduces CHD4 levels by 60%, enough to substantially de-repress HbF while eluding cellular or organismal toxicity. These studies suggest a potential target for HbF induction for β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved TF with singular devotion to regulation of a chromatin subcomplex.

Human chromosomes are pervasively transcribed, but systematic understanding of coding and lncRNA genome function in cell differentiation is lacking. Using CRISPR interference (CRISPRi) in human induced pluripotent stem cells, we performed dual genome-wide screens – assessing 18,905 protein-coding and 10,678 lncRNA loci – and identified 419 coding and 201 lncRNA genes that regulate neural induction. Integrative analyses revealed distinct properties of coding and lncRNA genome function, including a 10-fold enrichment of lncRNA genes for roles in differentiation compared to proliferation. Further, we applied Perturb-seq to obtain granular insights into neural induction phenotypes. While most coding hits stalled or aborted differentiation, lncRNA hits were enriched for the genesis of diverse cellular states, including those outside the neural lineage. In addition to providing a rich resource (danlimlab.shinyapps.io/dualgenomewide) for understanding coding and lncRNA gene function in development, these results indicate that the lncRNA genome regulates lineage commitment in a manner fundamentally distinct from coding genes.

Author(s): Crawford, Emily D; Acosta, Irene; Ahyong, Vida; Anderson, Erika C; Arevalo, Shaun; Asarnow, Daniel; Axelrod, Shannon; Ayscue, Patrick; Azimi, Camillia S; Azumaya, Caleigh M; Bachl, Stefanie; Bachmutsky, Iris; Bhaduri, Aparna; Brown, Jeremy Bancroft; Batson, Joshua; Behnert, Astrid; Boileau, Ryan M; Bollam, Saumya R; Bonny, Alain R; Booth, David; Borja, Michael Jerico B; Brown, David; Buie, Bryan; Burnett, Cassandra E; Byrnes, Lauren E; Cabral, Katelyn A; Cabrera, Joana P; Caldera, Saharai; Canales, Gabriela; Castaneda, Gloria R; Chan, Agnes Protacio; Chang, Christopher R; Charles-Orszag, Arthur; Cheung, Carly; Chio, Unseng; Chow, Eric D; Citron, Y Rose; Cohen, Allison; Cohn, Lillian B; Chiu, Charles; Cole, Mitchel A; Conrad, Daniel N; Constantino, Angela; Cote, Andrew; Crayton-Hall, Tre'Jon; Darmanis, Spyros; Detweiler, Angela M; Dial, Rebekah L; Dong, Shen; Duarte, Elias M; Dynerman, David; Egger, Rebecca; Fanton, Alison; Frumm, Stacey M; Fu, Becky Xu Hua; Garcia, Valentina E; Garcia, Julie; Gladkova, Christina; Goldman, Miriam; Gomez-Sjoberg, Rafael; Gordon, M Grace; Grove, James CR; Gupta, Shweta; Haddjeri-Hopkins, Alexis; Hadley, Pierce; Haliburton, John; Hao, Samantha L; Hartoularos, George; Herrera, Nadia; Hilberg, Melissa; Ho, Kit Ying E; Hoppe, Nicholas; Hosseinzadeh, Shayan; Howard, Conor J; Hussmann, Jeffrey A; Hwang, Elizabeth; Ingebrigtsen, Danielle; Jackson, Julia R; Jowhar, Ziad M; Kain, Danielle; Kim, James YS; Kistler, Amy; Kreutzfeld, Oriana; Kulsuptrakul, Jessie; Kung, Andrew F

Major effectors of adult-stage fetal globin silencing include the transcription factors (TFs) BCL11A and ZBTB7A/LRF and the NuRD chromatin complex, although each has potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here through CRISPR screening we discover ZNF410 to be a novel fetal hemoglobin (HbF) repressing TF. ZNF410 does not bind directly to the γ-globin genes but rather its chromatin occupancy is solely concentrated at CHD4, encoding the NuRD nucleosome remodeler, itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for ZNF410’s effects on γ-globin repression. Knockout of ZNF410 reduces CHD4 by 60%, enough to substantially de-repress HbF while avoiding the cellular toxicity of complete CHD4 loss. Mice with constitutive deficiency of the homolog Zfp410 are born at expected Mendelian ratios with unremarkable hematology. ZNF410 is dispensable for human hematopoietic engraftment potential and erythroid maturation unlike known HbF repressors. These studies identify a new rational target for HbF induction for the β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved transcription factor with singular devotion to regulation of a chromatin subcomplex.

Genome editing technologies are rapidly evolving, and analysis of deep sequencing data from target or off-target regions is necessary for measuring editing efficiency and evaluating safety. However, no software exists to analyze base editors, perform allele-specific quantification or that incorporates biologically-informed and scalable alignment approaches. Here, we present CRISPResso2 to fill this gap and illustrate its functionality by experimentally measuring and analyzing the editing properties of six genome editing agents.

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants from genome-wide association studies largely cluster in regulatory DNA. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating mutagenesis libraries with single or combinatorial nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, a locus associated with red blood cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false positive regions, which emphasizes the importance of off-target analysis in design of saturating mutagenesis experiments. Taken together, these data establish a widely applicable high-throughput and high-resolution methodology to reliably identify minimal functional sequences within large regions of disease- and trait-associated DNA.

Several major effectors of adult-stage fetal globin silencing have been identified, including the transcription factors (TFs) BCL11A and ZBTB7A/LRF and the NuRD chromatin complex, although each has potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here through CRISPR screening of 1591 transcription factors, we discover ZNF410 to be a novel fetal hemoglobin (HbF) repressing TF. ZNF410 does not bind directly to the γ-globin genes but rather its chromatin occupancy is solely concentrated at CHD4, encoding the NuRD nucleosome remodeler, itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements, one at its promoter and the other at an enhancer 6 kb upstream, with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. Peaks at these clusters display up to 57- and 77-fold enrichment respectively for ZNF410 binding compared to controls. Footprint analysis shows that the ZNF410 motifs are protected from nuclease cleavage. No other bona fide peaks could be detected across the genome in HUDEP-2 cells or primary CD34+ HSPC-derived erythroid precursors. We demonstrate that the ZNF410-bound elements at CHD4 completely account for ZNF410's effects on γ-globin repression. Knockout of ZNF410 reduces CHD4 by 60%, enough to substantially de-repress HbF while avoiding the cellular toxicity of complete CHD4 loss. Mice with constitutive deficiency of the homolog Zfp410 are born at expected Mendelian ratios (6 homozygous Zfp410Gt/Gt gene-trap allele mice out of 20 live births) with unremarkable hematology. ZNF410 is dispensable for human hematopoietic engraftment potential and erythroid maturation unlike known HbF repressors. Engrafting human erythrocytes induce HbF from ~2.5% in controls to ~17% in ZNF410 edited recipients, by hemoglobin HPLC. These studies identify a new rational target for HbF induction for the β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved transcription factor with singular devotion to regulation of a chromatin subcomplex. FIGURE LEGEND: ZNF410 chromatin occupancy is restricted to CHD4. (A) Dense mutagenesis of ZNF410 shows its five C2H2 zinc-finger domains (red rectangles) are especially critical for HbF repression. (B) Genome-wide ZNF410 binding was identified by CUT&RUN in HUDEP-2 cells. Enrichment for ZNF410 binding was concentrated at just two sites in the genome - at the promoter and at an enhancer ~6 kb upstream of CHD4. (C) Genome-wide ZNF410 motif occurrences (JASPAR motif shown in center) were mapped using a 3 kb sliding window. Clusters of ZNF410 motifs were found only at three locations in the genome, including the CHD4 promoter and CHD4 -6 kb enhancer. (D) CHD4 locus showing ZNF410 binding (red peaks) at the CHD4 promoter and CHD4 -6 kb enhancer regions with anti-HA antibody recognizing epitope-tagged ZNF410 and anti-ZNF410 antibody recognizing endogenous ZNF410 in both HUDEP-2 cells and CD34+ HSPC derived erythroid precursors. CUT&RUN footprints demonstrate ZNF410 motif protection, ATAC-seq chromatin accessibility and phyloP DNA sequence conservation. Disclosures Bauer: Pfizer: Consultancy; Syros Pharmaceuticals: Consultancy; Fulcrum Therapeutics: Consultancy; Sanofi: Research Funding.

Genome editing technologies are rapidly evolving, and analysis of deep sequencing data from target or off-target regions is necessary for measuring editing efficiency and evaluating safety. However, no software exists to analyze base editors, perform allele-specific quantification or that incorporates biologically-informed and scalable alignment approaches. Here, we present CRISPResso2 to fill this gap and illustrate its functionality by experimentally measuring and analyzing the editing properties of six genome editing agents.

CRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets we have developed CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genome, transcript, and protein coordinates and structure. No available tool has similar functionality. The ensuing genotype-phenotype linear and 3D maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro.

To identify novel targets for acute myeloid leukemia (AML) therapy, we performed genome-wide CRISPR-Cas9 screening using AML cell lines, followed by a second screen in vivo. Here, we show that the mRNA decapping enzyme scavenger (DCPS) gene is essential for AML cell survival. The DCPS enzyme interacted with components of pre-mRNA metabolic pathways, including spliceosomes, as revealed by mass spectrometry. RG3039, a DCPS inhibitor originally developed to treat spinal muscular atrophy, exhibited anti-leukemic activity via inducing pre-mRNA mis-splicing. Humans harboring germline biallelic DCPS loss-of-function mutations do not exhibit aberrant hematologic phenotypes, indicating that DCPS is dispensable for human hematopoiesis. Our findings shed light on a pre-mRNA metabolic pathway and identify DCPS as a target for AML therapy. Yamauchi et al. perform in vitro and in vivo CRISPR-Cas9 genetic screening of p53 WT AML to identify potential therapeutic targets. They find that AML relies on the DCPS decapping enzyme, and a DCPS inhibitor shows anti-leukemia activity in tumor models without impacting normal hematopoiesis.

Tiling screens using CRISPR-Cas technologies provide a powerful approach to map regulatory elements to phenotypes of interest, but computational methods that effectively model these experimental approaches for different CRISPR technologies are not readily available. Here we present CRISPR-SURF, a deconvolution framework to identify functional regulatory regions in the genome from data generated by CRISPR-Cas nuclease, CRISPR interference (CRISPRi), or CRISPR activation (CRISPRa) tiling screens. We validated CRISPR-SURF on previously published and new data, identifying both experimentally validated and new potential regulatory elements. With CRISPR tiling screens now being increasingly used to elucidate the regulatory architecture of the non-coding genome, CRISPRSURF provides a generalizable and accessible solution for the discovery of regulatory elements.

Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for β-hemoglobinopathy therapeutic intervention. The NuRD chromatin complex participates in γ-globin repression. Here we use pooled CRISPR screening to comprehensively disrupt NuRD protein coding sequences in human adult erythroid precursors. We find essential for fetal hemoglobin (HbF) control a nonredundant subcomplex of NuRD protein family paralogs, whose composition we corroborate by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identifies key protein interfaces where in-frame alleles result in loss-of-function due to destabilization or altered function of subunits. We ascertain mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrate that sequestering CHD4 from NuRD phenocopies these mutations. This work indicates a generalizable approach to discover protein complex features amenable to rational biochemical targeting., Editorial summary: Comprehensive CRISPR mutagenesis targeting all members of the NuRD complex identifies a specific sub-complex required for fetal globin silencing and informs a rational targeting strategy for elevating globin levels while avoiding cytotoxicity.

Although therapeutic genome editing of autologous hematopoietic stem cells (HSCs) in principle could cure β-hemoglobinopathies, CRISPR-Cas9 mediated gene modification has demonstrated variable efficiency, specificity, and persistence in HSCs. Here we demonstrate selection-free on-target editing of the BCL11A erythroid enhancer by Cas9:sgRNA ribonucleoprotein in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. First we screened a set of 20 guide RNAs targeting the functional core of the +58 BCL11A enhancer for maximal HbF induction by RNP delivery. We used SpCas9 protein with additional NLS sequences, synthetic modified sgRNA, and optimized electroporation buffer to produce >95% on-target indels disrupting a critical GATA1 binding site within the +58 BCL11A enhancer in CD34+ HSPCs. Clonal analysis showed that even 1 bp indels around the cleavage site were sufficient for HbF reactivation. Specificity was evaluated by CIRCLE-seq, a method to define genome-wide target sequences susceptible to RNP cleavage in vitro. Amplicon deep sequencing of 24 possible off-target sites from edited CD34+ cells did not reveal any off-target editing with limit of detection 0.1% allele frequency. Despite transient induction of a p53 transcriptional response peaking at 4-8 hours after RNP electroporation, we found no evidence of selection for TP53 or 94 other hematologic malignancy associated mutations by targeted deep sequencing. Edited CD34+ HSPCs from healthy donors contributed to multilineage engraftment of primary and secondary immunodeficient mouse recipients at similar levels as unedited control cells. Likewise we found that edited CD34+ HSPCs from a plerixafor-mobilized SCD donor contributed to marrow engraftment and multilineage hematopoiesis in immunodeficient NBSGW mice after 16 weeks at similar levels as unedited cells, with ~95% indel frequency for engrafting healthy and SCD donor cells. Edited engrafting SCD cells were similarly competent for secondary transplantation as unedited controls. Erythroid progeny of edited engrafting sickle cell disease HSCs expressed therapeutic levels of fetal hemoglobin (HbF) and resisted sickling. Erythroid progeny of edited CD34+ HSPCs from 7 transfusion-dependent ß-thalassemia donors showed restored globin chain balance and amelioration of microcytosis and poikilocytosis. We found that compared to the bulk CD34+ HSPC pool, HSCs preferentially underwent nonhomologous as compared to microhomology mediated end-joining repair based on three assays: sorting of CD34+ CD38- CD90+ CD45RA- cells; isolation of cells in G0, G1, S, and G2/M cell cycle phases; and evaluation of long-term engrafting cells in immunodeficient mice. Together these data show that NHEJ-based BCL11A enhancer editing approaching complete allelic disruption is a practicable therapeutic strategy to produce durable HbF induction in SCD and ß-thalassemia. Disclosures Esrick: Bluebird Bio: Honoraria. Williams:Bluebird Bio: Research Funding.

Sickle cell disease and β-thalassemia are major hemoglobin disorders for which induction of fetal hemoglobin (HbF) can mitigate disease severity. However, the molecular mechanisms underlying the developmental repression of HbF remain incompletely understood. The nucleosome remodeling and deacetylase (NuRD) complex is a major negative regulator of HbF level. In this study, we sought to identify possible rational therapeutic strategies targeting critical NuRD determinants. We employed comprehensive dense mutagenesis using pooled CRISPR screening in HUDEP-2 human erythroid precursors to disrupt protein coding sequences of all 13 genes of the NuRD complex, including CHD, MTA, GATAD2, HDAC, MBD, and RBBP family members. The custom sgRNA library included 5,038 sgRNAs. We found that only 5 genes, CHD4, MTA2, GATAD2A, HDAC2, and MBD2, were required for HbF repression, suggesting that a non-redundant NuRD sub-complex contributes to HbF silencing. We validated the existence of this NuRD sub-complex by mass spectrometry analysis after immunoprecipitation of CHD4 and MTA2 as well as MTA2-BioID2 mediated proximity labeling. Remarkably, 5 of the 6 NuRD subunit proteins commonly detected by these three methods were identified as functional by CRISPR screening (MTA2, RBBP4, CHD4, GATAD2A, HDAC2). Disruption of CHD4 resulted in the highest HbF induction of any of the NuRD subunits. However, unlike the other NuRD genes, CHD4 disruption also led to cellular toxicity. We observed a small group of sgRNAs within the CHDCT2 domain of CHD4 associated with high HbF induction yet relatively modest negative fitness. We validated by electroporation of Cas9:sgRNA to CD34+ HSPC primary erythroid precursors that in-frame mutations of CHD4 CHDCT2 escape cellular toxicity while inducing HbF. Similarly, we targeted homologous amino acid residues within mouse Chd4 CHDCT2 domain by Cas9 mutagenesis in mouse oocytes. While loss of Chd4 is lethal at the blastocyst stage, homozygous in-frame deletions within the Chd4 CHDCT2 domain are tolerated in mouse embryos and result in increased γ-globin expression in mid-gestation embryos bearing transgenic human β-globin gene clusters. To investigate the mechanism whereby in-frame deletions at CHD4 CHDCT2 impact NuRD, we performed glycerol gradient density sedimentation, which revealed that these in-frame mutations impair the recruitment of CHD4 to the NuRD complex. A recent study demonstrated that the previously poorly characterized CHD4 CHDCT2 domain directly binds to GATAD2 factors (Torrado et al, FEBS J, 2017). We observed a cluster of sgRNAs associated with heightened HbF enrichment scores at the C-terminal region of GATAD2A encompassing a C2C2-type GATA zinc finger. We hypothesized that ectopic expression of this GATAD2A zinc finger might competitively bind to CHD4 and displace CHD4 from NuRD. Overexpression of the GATAD2A zinc finger in both HUDEP-2 and CD34+ HSPC derived primary erythroid precursors led to robust induction of HbF without negatively impacting cellular fitness. Immunoprecipitation of the GATAD2A zinc finger enriched CHD4 but not other endogenous NuRD components, such as GATAD2A or MBD2. Moreover, glycerol gradient density sedimentation showed that the GATAD2A zinc finger co-sedimented with sub-NuRD fractions of CHD4. Together these data suggest that expression of the GATAD2A zinc finger sequesters CHD4 from NuRD, yet spares cytotoxicity. In summary, we show that biochemical disruption of the CHD4-GATAD2A interaction could serve as a rational therapeutic strategy to potently induce HbF for the β-hemoglobin disorders while preventing cellular toxicity associated with complete CHD4 inhibition. Disclosures No relevant conflicts of interest to declare.

Discovery of molecular mechanisms responsible for trait associations as discovered by genome-wide association studies (GWAS) is hampered by difficulty in identifying causal genetic variants due to linkage disequilibrium. Typical assays of genetic function are low throughput or evaluate sequences in heterologous ectopic settings. Genome editing enables perturbation of trait-associated genetic sequences within relevant genomic, chromatin and cellular context. Here we perform comprehensive analysis of genetic variants associated with red blood cell traits by pooled CRISPR screening. We performed a genome-wide Cas9 gene knockout screen in immortalized erythroid precursors (HUDEP-2 cells) during erythroid maturation to define functional erythroid genes required for cell growth or differentiation. We evaluated 952 loci associated with nine red blood cell traits (Astle et al, Cell 2016) comprising 24,843 SNPs. We linked 7,187 (28.9%) of these SNPs to genes by at least one of four routes: sharing topological associated domain, physical proximity ( Disclosures No relevant conflicts of interest to declare.

Genome-wide knockout screening employing CRISPR-Cas9 genome-editing is a powerful tool for functional genomics. However, identifying actionable targets for cancer therapy has been challenging due, in part, to the complex genetic background of cell lines used for screening. To overcome this obstacle, we generated two acute myeloid leukemia (AML) lines from mouse leukemia models based on activity of the leukemia oncogenes CALM/AF10 or MLL/AF9. Both lines exhibited a normal karyotype and intact Tp53 activity. Using these lines, we performed genome-wide CRISPR-Cas9 screening, followed by a second screen in vivo . We then selected genes meeting the following criteria: 1) they encoded a protein with an available inhibitor or 2) their germline mutation loss-of-function phenotype was relatively moderate based on the literature or the human exome-sequencing database. We excluded genes with a well-defined function in leukemogenesis as well as those encoding components of basal cellular machineries. Among genes significantly depleted in our primary screen was the mRNA decapping enzyme scavenger (Dcps), which encodes a mRNA 5' cap binding protein implicated in mRNA decay. Read counts for each Dcps-targeted single-guide RNA (sgRNA) significantly decreased in vitro (AML cell lines) and in vivo (mouse AML model). A negative selection CRISPR-Cas9 mutagenesis scan of all Dcps coding exons revealed that the C-terminal Dcps domain, namely aa 230-240, plays a critical role in AML survival. RG3039, an orally active quinazoline derivative, is a DCPS inhibitor that was originally developed to treat spinal muscular atrophy (SMA) and has been judged safe in a phase I trial in healthy volunteers (Van Meerbeke JP et al. Hum Mol Genet. 2013). We validated RG3039 binding to DCPS protein in AML cells via a cellular thermal shift assay (CETSA). We then assessed anti-leukemia effects of RG3039 by treating human AML lines with RG3039 and generating growth curves. That analysis showed that RG3039 had dose-dependent anti-proliferative effects. RG3039 treatment induced cell cycle arrest and apoptosis, revealed by EdU incorporation assay and Annexin V stain, respectively. Since DCPS protein was predominantly nuclear in human primary AML cells, we hypothesized that DCPS primarily functions in that compartment rather than in the cytoplasmic mRNA 3' end decay pathway. To search for nuclear proteins potentially interacting with DCPS, we undertook immunoprecipitation with an anti-DCPS antibody of lysates of AML cells followed by mass spectrometry analysis. Among highly significant interactors, we identified components of pre-mRNA processing machineries including spliceosomes. To assess whether DCPS inhibition would impair pre-mRNA splicing, we treated AML cells with RG3039 and performed RNA-sequencing to determine potential transcriptome-wide splicing changes. Alternative 5' splice site selection was most frequently observed in RG3039-treated cells, and most aberrant splicing events involved the first exon. Bioinformatic prediction analysis revealed that approximately 40% of the aberrantly-spliced genes were NMD (nonsense-mediated mRNA decay)-sensitive. Gene Set Enrichment Analysis identified gene signatures representing a type-I interferon response, reminiscent of that observed in RNAi-treated cells, in RG3039-treated AML cells. We next explored effects of DCPS deficiency on normal hematopoiesis in humans. To do so, we examined peripheral blood counts of three children harboring germline homozygous loss-of-function mutations as well as heterozygous relatives in a family reported previously (Ng et al. Hum Mol Genet 2015). They exhibited normal blood counts, indicating that DCPS is dispensable for steady-state hematopoiesis in humans. Finally, to investigate potential anti-leukemia effects of RG3039 in vivo, we tested RG3039 efficacy using patient-derived xenograft (PDX) AML models established from three human AML lines. RG3039 exhibited anti-leukemic activity, as evidenced by the lower leukemia burden in PB and BM of RG3039-treated mice. They survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of RG3039 monotherapy against AML in vivo. In summary, our findings shed a new light on a pre-mRNA metabolic pathway regulated by DCPS. They also identify DCPS as a novel target for AML therapy and suggest potential "repurposing" of RG3039 as an anti-leukemia drug. Disclosures Seiler: H3 Biomedicine, Inc.: Employment. Orkin: Epizyme Inc.: Consultancy; Bioverativ: Consultancy. Buonamici: H3 Biomedicine Inc.: Employment.