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Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.

Reconstitution of donor hematopoiesis after allogeneic hematopoietic stem cell transplantation (HSCT) forms the basis for effective graft-versus-leukemia responses, but mixed chimerism is not an infrequent outcome. How the donor and host hematopoietic system interact under conditions of mixed chimerism remains incompletely understood. Multi-modal single cell sequencing platforms are increasingly available and provide information regarding cell identities and interactions at high resolution. However, the analysis of post-transplant immune reconstitution requires consistent distinguishing of donor- and recipient-derived cells, which for sparse single cell sequencing data until now has remained a challenge. Recently, mitochondrial DNA (mtDNA) mutations have been recognized for their potential as personal genetic barcodes that can be detected with mitochondrial single cell assay for transposase-accessible chromatin with sequencing (mtscATAC-seq). We hypothesized that individual-specific mtDNA mutations could provide a sensitive and robust approach for distinguishing donor- from recipient-derived cells, and therefore tested this approach on bone marrow (BM) samples from patients with relapsed acute myeloid leukemia (AML) post-HSCT. We employed ATAC with select cell surface antigen profiling by sequencing (ASAP-seq), which enables the detection of mtDNA mutations within distinct surface marker-defined cell populations alongside chromatin accessibility. We selected serial samples collected from the ETCTN 10026 study, which tested combined decitabine (days 1-5, every 4 weeks, start cycle 0) and ipilimumab (day 1, every 4 weeks, start cycle 1) in relapsed AML post-HSCT. We focused on 13 samples (study entry, on treatment and disease progression) from 3 patients: AML1012 (HSCT from a matched related donor [MRD]), and AML1010 and AML1026 (matched unrelated donor [MUD]-HSCT). In total, we obtained 33,943 ASAP-seq profiles, including 3,283 single T cells. While clustering using single cell chromatin profiles alone only allowed identification of either CD4 + or CD8 + T cells, integration with surface marker expression enabled more detailed annotations of 8 T cell subpopulations and NK cells. Further, phenotypically distinct subpopulations such as CD57 + CD4 + and CD8 + T cells shared highly similar chromatin profiles, and 11.1% of CD4 + and 33.7% of CD8 + T cells would have been mislabeled based on clustering of chromatin profiles alone. Thus, ASAP-seq identified T cell subsets with markedly improved accuracy and resolution than scATAC-seq alone. Upon evaluation of mtDNA mutations to discriminate donor- and recipient-derived single T cells, we found that this was unreliable for MRD-HSCT (AML1012), but highly robust in the setting of MUD-HSCT (AML1010, AML1026), consistent with maternal inheritance of mitochondrial genomes. For the latter two patients, we identified 48 donor- and 26 recipient-specific mtDNA mutations, all with high heteroplasmy (range 82 - 99%). Presence of donor- and recipient-derived mtDNA mutations was mutually exclusive, and recipient-specific mtDNA mutations were also detectable in AML cells. Clinical bulk and mtDNA mutation-based single T cell chimerisms were highly correlated (r = 0.97). AML1010 had sustained complete T cell chimerism (>97%) during study treatment. In AML1026, the mtDNA mutation-based T cell chimerism rose from 55% to 71% after 1 cycle of decitabine and then remained stable until disease progression 3 months later. This was associated with increased percentage of donor-derived CD4 + T cells (45% [study entry] vs. 71% [after 1 cycle of decitabine], p < 0.01), while donor-derived CD8 + T cells remained unchanged at 76%. Across all studied timepoints in AML1026, donor versus recipient skewing was also highest in CD4 + T cell subsets, with fewer naïve (20% vs. 31%, p < 0.01) but more donor-derived CD57 + CD4 + T cells (13% vs. 3%, p < 0.01). We demonstrate that mtDNA mutations can discriminate between donor- and recipient-derived single cells, enabling detection and in-depth characterization of chimeric immune cell dynamics after MUD HSCT. This approach will allow to systematically dissect conditions of mixed chimerism in the post-transplant setting with larger studies. Disclosures DeAngelo: Abbvie: Research Funding; Takeda: Consultancy; Servier: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Incyte: Consultancy; Forty-Seven: Consultancy; Autolus: Consultancy; Amgen: Consultancy; Agios: Consultancy; Blueprint: Research Funding; Glycomimetrics: Research Funding. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Sankaran: Cellarity: Consultancy; Forma: Consultancy; Novartis: Consultancy; Branch Biosciences: Consultancy; Ensoma: Consultancy. Soiffer: Jasper: Consultancy; Jazz Pharmaceuticals, USA: Consultancy; Precision Biosciences, USA: Consultancy; Juno Therapeutics, USA: Other: Data Safety Monitoring Board; Kiadis, Netherlands: Membership on an entity's Board of Directors or advisory committees; Rheos Therapeutics, USA: Consultancy; Gilead, USA: Other: Career Development Award Committee; NMPD - Be the Match, USA: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy. Garcia: Genentech: Research Funding; Prelude: Research Funding; Pfizer: Research Funding; AstraZeneca: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company. OffLabel Disclosure: ipilimumab to modulate anti-leukemia immunity in the post-transplant and transplant-naive context

Hematopoiesis is a well-characterized paradigm of cellular differentiation that is highly regulated to ensure balanced proportions of mature blood cells. However, many aspects of this process remain poorly understood in humans. For example, there is extensive variation in commonly measured blood cell traits, which can manifest as diseases at extreme ends of the spectrum, yet the vast majority of genetic loci responsible for driving these differences are currently unknown. Here, we integrate fine-mapped population genetics with high-resolution chromatin landscapes to gain novel insights into regulatory mechanisms critical for human blood cell production and disease. First, we conducted a genome-wide association study in 115,000 individuals from the UK Biobank, measuring the effects of genetic variation on 16 blood traits spanning 7 hematopoietic lineages (erythroid, platelet, lymphocyte, monocyte, neutrophil, eosinophil, basophil). Within each region of association (n = 2,056), we performed Bayesian fine-mapping on all common variants to resolve the most likely causal hits. Going further, we were interested in whether genetic variants predominantly act in terminal cell states or less differentiated progenitors. To this end, we overlapped fine-mapped variants with chromatin accessibility profiles (ATAC-seq) of 18 primary hematopoietic populations sorted from healthy donors. Across all lineages, 21% of regulatory variants were restricted to accessible chromatin (AC) peaks in terminal progenitors. Interestingly, 59% of variants fell in AC regions of one or more upstream progenitor states, suggesting that a significant amount of variation in blood traits stems from regulatory signaling in earlier stages of hematopoiesis. Motivated by this finding, we hypothesized that different branches of hematopoiesis (e.g., monocyte and red blood cell count) could be co-regulated by pleiotropic variants acting in common progenitor populations. Therefore, we investigated variants associated with 2 or more of the 7 blood cell types for which phenotypes were available. Remarkably, across 172 such variants, there was an average of 60% more open chromatin in progenitors than terminal cell types (mean 4.01 vs. 2.44 counts per million; p = 0.025). Examining the directional effects of these variants on distinct lineages, we discovered that 91% of pleiotropic variants exhibited a tune mechanism by changing the levels of different blood cells in the same direction. One such example was rs17758695 located in intron 1 of BCL2, an anti-apoptotic protein known to regulate cell death similarly across multiple hematopoietic cell types. In contrast, the remaining 9% of pleiotropic variants favored one lineage at the expense of others (switch mechanism), including novel variants near key myeloid-determining transcription factors CEBPA and MYC (rs78744187 and rs562240450). Together, these results suggest that pleiotropic variants 1) preferentially act in common progenitor rather than terminal cell types, and 2) predominantly tune multiple traits in the same direction, but may favor one at the expense of others when influencing lineage commitment. Finally, given the enrichment of fine-mapped variants in common progenitor states, we set out to determine whether classically defined hematopoietic populations could be divided into lineage-biased subpopulations based on differential genetic regulation of blood traits. To do so, we measured the enrichment of fine-mapped variants in the chromatin landscapes of 2,034 single cells isolated from 8 hematopoietic progenitor populations. Strikingly, we discovered significant heterogeneity within the common myeloid progenitor (CMP) population, in which one subset of cells exhibited greater open-chromatin enrichment for myeloid trait variants and relevant transcription factor (TF) binding (CEBPA, IRF8), whereas the other subset showed enrichment for erythroid trait variants and TFs (GATA1, KLF1). By integrating genetic fine-mapping with chromatin data, we identified hundreds of causal variants regulating 16 blood traits, characterized novel mechanisms of pleiotropic effects, and discovered cell states enriched for blood trait regulation. These findings provide new insights into the importance of genetic regulation in progenitor cell states and will contribute to knowledge of how these processes go awry in diseases of blood cell production. Disclosures No relevant conflicts of interest to declare.

Gain-of-function mutations in Notch receptor genes occur in 10-15% of cases of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), and are associated with inferior clinical outcomes. Nearly all Notch mutations reported in B cell tumors lead to loss of the C-terminal negative regulatory PEST domain and result in stabilization of the activated form of Notch (intracellular Notch [ICN]), whereas mutations that lead to ligand-independent Notch activation (which are common in T cell acute lymphoblastic leukemia [T-ALL]) are rare. ICN can be detected in tumor cells within lymph nodes of >80% of patients with CLL, suggesting that Notch may have a broader oncogenic role than the incidence of Notch mutations would suggest. However, the downstream targets of Notch in B-cell tumors have not been identified. We used a gamma-secretase inhibitor (GSI) washout strategy to determine the immediate, direct effects of Notch activation in three MCL cell lines with Notch gain-of-function mutations, including two cell lines with unusual Notch gene rearrangements that lead to ligand-independent Notch activation, as well as a third line with a Notch PEST domain mutation in which signaling was activated with recombinant Notch ligand. Using these models, we identified likely direct target genes and their associated genomic Notch response elements using RNA-seq and ChIP-Seq in the Notch-on and Notch-off states. Most of these response elements corresponded to long-range enhancers that showed Notch-dependent changes in H3K27 acetylation, and were bound by components of the Notch transcription complex (NTC) in both cell lines. We confirmed these associations by performing ChIP-Seq on primary CLL and MCL biopsies, and by identifying specific looping interactions with Notch target gene promoters in public genome-wide proximity ligation datasets (RNA Pol2 ChIA-PET) from a lymphoblastoid cell line expressing the EBV-encoded Notch surrogate protein EBNA2. MYC was among the most strongly Notch-activated genes in Notch-dependent MCL cell lines and was associated with NTC binding at two B cell-specific 5' enhancers distinct from the Notch-dependent MYC enhancer previously identified in T-ALL. MCL cell line proliferation was blocked by Cas9 nuclease or epigenetic repressors targeting the 5' MYC enhancers, whereas cells were rescued from Notch inhibition by GSI via transduction with MYC. Gene set enrichment analysis of other direct Notch target genes identified in MCL models showed enrichment for regulators of B cell receptor (BCR) signaling, including the Src family kinase genes FYN, LYN, and BLK, and the signaling complex adaptor BLNK, as well as regulators of CD40 and cytokine signaling. RNA-seq analysis of primary CLL lymph node biopsies revealed significantly higher expression of many Notch target genes in biopsies with high levels of ICN. To functionally validate Notch target genes in primary tumors, we co-cultured CLL and MCL cells obtained from peripheral blood with Notch ligand-expressing stromal cells in the presence ("notch off") or absence ("notch on") of GSI, and demonstrated increased expression of Notch target genes, including MYC, in the "notch-on" cells. Furthermore, "notch-on" CLL cells showed increased phosphorylation of the BCR signaling intermediates SYK and PLCg2 upon BCR crosslinking compared to GSI-treated cells. Finally, we validated Notch-dependent regulation of target genes in vivo in a patient-derived xenograft model of NOTCH1-mutant MCL. Notch target gene expression was significantly higher in MCL cells within the spleen versus bone marrow or blood, but was markedly reduced in animals treated for five days with GSI. Additional xenograft studies are ongoing, and will be described at the meeting. Our data link active Notch signaling to two well-characterized oncogenic drivers in B cell lymphoma, MYC and BCR signaling, and may have important implications for the development of treatment strategies involving Notch antagonists and other targeted therapeutics, such as BCR targeting agents. Disclosures Weinstock: Novartis: Consultancy, Research Funding.