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SUMMARYGene editing theBCL11Aerythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of theBCL11A+58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of theBCL11A+58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.

Supplementary Figures 11-18

Chimeric antigen receptor (CAR) T-cell therapy has had limited success in early-phase clinical studies for solid tumors. Lack of efficacy is most likely multifactorial, including a limited array of targetable antigens. We reasoned that targeting the cancer-specific extra domain B (EDB) splice variant of fibronectin might overcome this limitation because it is abundantly secreted by cancer cells and adheres to their cell surface. In vitro, EDB-CAR T cells recognized and killed EDB-positive tumor cells. In vivo, 1 × 106 EDB-CAR T cells had potent antitumor activity in both subcutaneous and systemic tumor xenograft models, resulting in a significant survival advantage in comparison with control mice. EDB-CAR T cells also targeted the tumor vasculature, as judged by IHC and imaging, and their antivascular activity was dependent on the secretion of EDB by tumor cells. Thus, targeting tumor-specific splice variants such as EDB with CAR T cells is feasible and has the potential to improve the efficacy of CAR T-cell therapy.

Detailed methods, description of supplemental tables, and supplemental figures.

Supplementary Table S1

Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA-damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress and has been implicated as a potential biomarker to predict sensitivity to DNA-damaging agents (DDA). SLFN11 expression was quantified in 220 children with solid tumors using IHC. Sensitivity to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN) was assessed in sarcoma cell lines, including SLFN11 knock-out (KO) and overexpression models, and a patient-derived orthotopic xenograft model (PDOX). SLFN11 was expressed in 69% of pediatric sarcoma sampled, including 90% and 100% of Ewing sarcoma and desmoplastic small round-cell tumors, respectively, although the magnitude of expression varied widely. In sarcoma cell lines, protein expression strongly correlated with response to TAL and IRN, with SLFN11 KO resulting in significant loss of sensitivity in vitro and in vivo. Surprisingly, retrospective analysis of children with sarcoma found no association between SLFN11 levels and favorable outcome. Subsequently, high SLFN11 expression was confirmed in a PDOX model derived from a patient with recurrent Ewing sarcoma who failed to respond to treatment with TAL + IRN. Selective inhibition of BCL-xL increased sensitivity to TAL + IRN in SLFN11-positive resistant tumor cells. Although SLFN11 appears to drive sensitivity to replicative stress in pediatric sarcomas, its potential to act as a biomarker may be limited to certain tumor backgrounds or contexts. Impaired apoptotic response may be one mechanism of resistance to DDA-induced replicative stress.

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates β-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to β-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.

Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar β-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks. A custom adenine base editor can edit the variant of the β-globin gene that causes sickle cell disease into a non-pathogenic variant in human and mouse cells, and transplantation of the edited cells rescues sickle cell disease in mice.

Chimeric antigen receptor (CAR) T-cell therapy has had limited success in early-phase clinical studies for solid tumors. Lack of efficacy is most likely multifactorial, including a limited array of targetable antigens. We reasoned that targeting the cancer-specific extra domain B (EDB) splice variant of fibronectin might overcome this limitation because it is abundantly secreted by cancer cells and adheres to their cell surface. In vitro, EDB-CAR T cells recognized and killed EDB-positive tumor cells. In vivo, 1 × 106 EDB-CAR T cells had potent antitumor activity in both subcutaneous and systemic tumor xenograft models, resulting in a significant survival advantage in comparison with control mice. EDB-CAR T cells also targeted the tumor vasculature, as judged by IHC and imaging, and their antivascular activity was dependent on the secretion of EDB by tumor cells. Thus, targeting tumor-specific splice variants such as EDB with CAR T cells is feasible and has the potential to improve the efficacy of CAR T-cell therapy.

We previously reported a specific inverse agonist (SPA70) of the nuclear receptor pregnane X receptor (PXR). However, derivatization of SPA70 yielded only agonists and neutral antagonists, suggesting that inverse agonism of PXR is difficult to achieve. Therefore, we sought to design proteolysis targeting chimeras (PROTACs) aimed at inducing PXR degradation. Conjugation of a SPA70 derivative to ligands of the E3 substrate receptor cereblon (CRBN) resulted in one molecule, SJPYT-195, that reduced PXR protein level in an optimized degradation assay described here. Further analysis revealed that SJPYT-195 was a molecular glue degrader of the translation termination factor GSPT1 and that GSPT1 degradation resulted in subsequent reduction of PXR protein. GSPT1 has recently gained interest as an anticancer target, and our results give new insights into chemical determinants of drug-induced GSPT1 degradation. Additionally, we have developed assays and cell models for PXR degrader discovery that can be applied to additional protein targets.

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer–edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer– or control locus–targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.

Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9–mediated disruption of DNA regulatory elements that repress γ-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and β-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34(+) hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rγ(−/−)/Kit(W41/W41) immunodeficient mice. On-target editing did not impair CD34(+) cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.

MLL rearrangements are translocation mutations that cause both acute lymphoblastic leukemia and acute myeloid leukemia (AML). These translocations can occur as sole clonal driver mutations in infant leukemias, suggesting that fetal or neonatal hematopoietic progenitors may be exquisitely sensitive to transformation by MLL fusion proteins. To test this possibility, we used transgenic mice to induce one translocation product, MLL-ENL, during fetal, neonatal, juvenile and adult stages of life. When MLL-ENL was induced in fetal or neonatal mice, almost all died of AML. In contrast, when MLL-ENL was induced in adult mice, most survived for >1 year despite sustained transgene expression. AML initiation was most efficient when MLL-ENL was induced in neonates, and even transient suppression of MLL-ENL in neonates could prevent AML in most mice. MLL-ENL target genes were induced more efficiently in neonatal progenitors than in adult progenitors, consistent with the distinct AML initiation efficiencies. Interestingly, transplantation stress mitigated the developmental barrier to leukemogenesis. Since fetal/neonatal progenitors were highly competent to initiate MLL-ENL–driven AML, we tested whether Lin28b, a fetal master regulator, could accelerate leukemogenesis. Surprisingly, Lin28b suppressed AML initiation rather than accelerating it. This may explain why MLL rearrangements often occur before birth in human infant leukemia patients, but transformation usually does not occur until after birth, when Lin28b levels decline. Our findings show that the efficiency of MLL-ENL–driven AML initiation changes through the course of pre- and postnatal development, and developmental programs can be manipulated to impede transformation.

Genome engineering using programmable nucleases is a rapidly evolving technique that enables precise genetic manipulations within complex genomes. Although this technology first surfaced with the creation of meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases, CRISPR-Cas9 has been the most widely adopted platform because of its ease of use. This comprehensive review presents a basic overview of genome engineering and discusses the major technological advances in the field. In addition to nucleases, we discuss CRISPR-derived base editors and epigenetic modifiers. We also delve into practical applications of these tools, including creating custom-edited cell and animal models as well as performing genetic screens. Finally, we discuss the potential for therapeutic applications and ethical considerations related to employing this technology in humans. © 2019 American Physiological Society. Compr Physiol 9:665-714, 2019.

Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is characterized by clonal expansion of natural killer (NK) cells where the underlying genetic mechanisms are incompletely understood. In the present study, we report somatic mutations in the chemokine gene CCL22 as the hallmark of a distinct subset of CLPD-NK. CCL22 mutations were enriched at highly conserved residues, mutually exclusive of STAT3 mutations and associated with gene expression programs that resembled normal CD16

Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA-damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress and has been implicated as a potential biomarker to predict sensitivity to DNA-damaging agents (DDA). SLFN11 expression was quantified in 220 children with solid tumors using IHC. Sensitivity to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN) was assessed in sarcoma cell lines, including SLFN11 knock-out (KO) and overexpression models, and a patient-derived orthotopic xenograft model (PDOX). SLFN11 was expressed in 69% of pediatric sarcoma sampled, including 90% and 100% of Ewing sarcoma and desmoplastic small round-cell tumors, respectively, although the magnitude of expression varied widely. In sarcoma cell lines, protein expression strongly correlated with response to TAL and IRN, with SLFN11 KO resulting in significant loss of sensitivity in vitro and in vivo. Surprisingly, retrospective analysis of children with sarcoma found no association between SLFN11 levels and favorable outcome. Subsequently, high SLFN11 expression was confirmed in a PDOX model derived from a patient with recurrent Ewing sarcoma who failed to respond to treatment with TAL + IRN. Selective inhibition of BCL-xL increased sensitivity to TAL + IRN in SLFN11-positive resistant tumor cells. Although SLFN11 appears to drive sensitivity to replicative stress in pediatric sarcomas, its potential to act as a biomarker may be limited to certain tumor backgrounds or contexts. Impaired apoptotic response may be one mechanism of resistance to DDA-induced replicative stress.

Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress, and SLFN11 gene silencing has been implicated as a common mechanism of drug resistance in tumors in adults. We found SLFN11 to be widely expressed in our cohort of pediatric sarcomas. In sarcoma cell lines, protein expression strongly correlated with response to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN), with SLFN11 knockout resulting in significant loss of sensitivity in vitro and in vivo. However, SLFN11 expression was not associated with favorable outcomes in a retrospective analysis of our patient cohort; instead, the protein was retained and promoted tumor growth and evasion. Furthermore, we show that pediatric sarcomas develop resistance to TAL and IRN through impaired intrinsic apoptosis, and that resistance can be reversed by selective inhibition of BCL-XL.Statement of SignificanceThe role of SLFN11 in pediatric sarcomas has not been thoroughly explored. In contrast to its activity in adult tumors, SLFN11 did not predict favorable outcomes in pediatric patients, was not silenced, and promoted tumor growth. Resistance to replicative stress in SLFN11-expressing sarcomas was reversed by selective inhibition of BCL-XL.

USP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development. In T-ALL cell lines, we showed the physical interaction of USP7 with E-proteins and TAL1 by mass spectrometry and ChIP-seq. Haploinsufficient but not complete CRISPR knock-out of USP7 showed accelerated cell growth and validated transcriptional down-regulation of E-protein targets. Our study unveiled the synergistic effect of USP7 haploinsufficiency with aberrant TAL1 activation on T-ALL, implicating USP7 as a haploinsufficient tumor suppressor in T-ALL. Our findings caution against a universal oncogene designation for USP7 while emphasizing the dosage-dependent consequences of USP7 inhibitors currently under development as potential cancer therapeutics.

Z-DNA-binding protein 1 (ZBP1) is an innate immune sensor of nucleic acids that regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating receptor-interacting Ser/Thr kinase 3 (RIPK3), caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate immune sensors because of its N-terminal Zα1 and Zα2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Zα domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we generated Zbp1(ΔZα2/ΔZα2) mice that express ZBP1 lacking the Zα2 domain and demonstrate that this domain is critical for influenza A virus–induced PANoptosis and underlies perinatal lethality in mice in which the RIP homotypic interaction motif domain of RIPK1 has been mutated (Ripk1(mRHIM/mRHIM)). Deletion of the Zα2 domain in ZBP1 abolished influenza A virus–induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Zα2 domain of ZBP1 was sufficient to rescue Ripk1(mRHIM/mRHIM) mice from perinatal lethality caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Zα2 domain of ZBP1 in several physiological functions and establish a link between Z-RNA sensing via the Zα2 domain and promotion of influenza-induced PANoptosis and perinatal lethality.

Z-DNA-binding protein 1 (ZBP1) is an innate nucleic acid sensor which regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating RIPK3, caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate sensors because of its N-terminal Zα1 and Zα2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Zα domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we have generated Zbp1ΔZα2/ΔZα2 mice that lack the Zα2 domain of ZBP1 and demonstrate that this domain is critical for influenza A virus (IAV)-induced PANoptosis and perinatal lethality in RIPK1-RHIM mutated (Ripk1RHIM/RHIM) mice. Deletion of the Zα2 domain in ZBP1 abolished IAV-induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Zα2 domain of ZBP1 was sufficient to rescue Ripk1RHIM/RHIM mice from the perinatal lethality which is caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Zα2 domain of ZBP1 in physiological functions and establish a link between sensing of Z-RNAs via the Zα2 domain and the promotion of influenza-induced PANoptosis and perinatal lethality.

SUMMARYStress granules (SG) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SG act as a protective mechanism to cope with acute stress, but persistent SG have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Importantly, knockout of the MAGE-B2 mouse ortholog confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermo-sensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress through fine-tuning phase separation and could aid in the development of male fertility therapies.

Summary Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

Targeting the BCL11A erythroid enhancer by gene editing is a promising approach to fetal hemoglobin induction for beta-hemoglobinopathies. HbF levels vary widely among individuals, suggesting potential heterogeneity in HbF responses after therapeutic intervention. We hypothesize that maximizing both gene edit frequency and HbF induction potential could promote consistently favorable clinical outcomes. Here we compared CRISPR-Cas9 endonuclease editing of the BCL11A +58 enhancer with alternative gene modification approaches, including +55 erythroid enhancer editing alone or in combination with the +58 enhancer, as well as editing targeting the HBG1/2 promoter -115 BCL11A binding site and transduction by an shRNA knocking down the BCL11A transcript in erythroid precursors. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in the most potent HbF induction (52.4%±6.3%) of tested approaches (BCL11A +58 editing alone, 29.1%±3.9%; BCL11A +55 editing alone, 34.8±5.1%; HBG1/2 promoter editing, 34.1% ±5.4%; shmiR-BCL11A, 32.2%±4.4%; mock, 7.6%±3.4%). Based on assays in bulk and single cell derived erythroid cultures and xenografted immunodeficient mice, we found that disruption of core half E-box/GATA motifs at both the +58 and +55 enhancers was associated with greatest HbF induction, whether by small indels, interstitial 3.1 kb deletion, or 3.1 kb inversion. Rare gene edited clones with alleles that only partially disrupted these motifs were associated with intermediate HbF induction phenotypes. Combined editing of BCL11A +58 and +55 enhancers was compatible with HSC self-renewal in primary and secondary xenotransplant, with intact lymphoid, myeloid and erythroid repopulation. We conducted gene-edited cell product manufacturing process development and developed conditions using a MaxCyte electroporation instrument achieving mean 97.3±1.8% gene edits and 88.9%±6.4% viability 24 hours after electroporation in 3 engineering runs at clinical scale. We obtained similar results at small-scale with plerixafor-mobilized HSPCs from sickle cell disease (SCD) donors or G-CSF mobilized PBMCs from transfusion-dependent beta-thalassemia (TDT) donors, including 94.2%±4.4%, 99.5%±0.3% and 91.8%±6.3% of gene edits in engrafting cells from NBSGW 16 week mouse bone marrow of healthy, SCD and TDT donors respectively. Off-target analyses by pooled amplicon sequencing of 601 candidate off-target sites for the +58 and +55 targeting sgRNAs, nominated by a range of computational (CRISPRme) and experimental (GUIDE-seq and ONE-seq) methods, did not identify reference genome off-target edits at a sensitivity of 0.1% allele frequency. We evaluated +58/+55 enhancer combined targeting in nonhuman primates by performing ribonucleoprotein (RNP) electroporation in rhesus macaque mobilized peripheral blood CD34+ HSPCs with autologous re-infusion following busulfan myeloablation. We observed highly efficient gene edit frequency (85.2%, 88.8% and 84.9%) and durable HbF induction (26.4%, 57.5%, and 45.9% F-cells and 12.7%, 41.9%, and 28% gamma-globin) in the peripheral blood in 3 animals at most recent recorded time point post infusion (127, 78, and 54 weeks respectively). Single colony analyses and bulk ddPCR and unidirectional sequencing demonstrated that the long-term engrafting cells displayed a similar distribution of indels, 3.1 kb deletions, and 3.1 kb inversions as the input cell products. Erythroid stress due to hydroxyurea treatment, with or without phlebotomy, was associated with substantially augmented HbF responses (to 75.9%, 88.2%, and 57.8% F-cells and 47.9%, 68%, and 35.7% gamma-globin). No hematologic or other toxicities attributable to gene editing were observed. Together these results suggest that combined BCL11A +58 and +55 erythroid enhancer editing produces highly efficient on-target allelic disruption, erythroid-specific BCL11A downregulation, heightened HbF induction capacity compared to alternative approaches, preserved long-term multilineage engraftment potential by both human xenotransplant and rhesus autotransplant assays, and absence of evident genotoxicity, under clinically relevant SpCas9 RNP electroporation conditions. Disclosures No relevant conflicts of interest to declare.

Sickle cell disease (SCD) is a chronic, life-altering multisystem disorder that affects millions of individuals worldwide. Strategies for genetic therapy of autologous SCD hematopoietic stem cells (HSCs) include lentiviral vector (LV) delivery of an anti-sickling β-like globin gene and genome editing, either to revert the SCD mutation by homology-directed repair (HDR) or to induce the expression of fetal hemoglobin (HbF, α2γ2) in red blood cell (RBC) by non-homologous end-joining repair (NHEJ). Base editing is a newer technology that offers the potential for facile generation of more precise genetic alterations with improved safety features. Adenosine base editors (ABEs) convert A-T base pairs to G-C pairs at loci targeted by a single guide (sg) RNA and Cas9 nickase. In contrast to LV vectors and standard Cas9 genome editing, ABEs function through mechanisms that are independent of double-stranded DNA breaks (DSBs), which can cause large deletions, structural DNA rearrangements and TP53-mediated DNA damage responses leading to cell death or malignant transformation. Base editors cannot generate the T-to-A transversion required to revert the mutant SCD codon (Val, GTG) to wild-type (Glu, GAG). However, ABEs can convert the Val codon to Ala (GCG) to generate the naturally occurring, non-sickling variant hemoglobin "Makassar" (HbG). Hemoglobin Makassar heterozygotes and 1 reported homozygote exhibit normal RBC indices, indicating that the variant is benign. We used protein directed evolution to generate a new ABE (ABE8e-NRCH) that accesses a nearby CACC PAM to convert HbS alleles to HbG efficiently in heterologous cells. To demonstrate therapeutic proof of concept, we electroporated ABE8e-NRCH mRNA and targeting sgRNA or ABE8e-NRCH/sgRNA ribonucleoprotein (RNP) complex into 3 different SCD donor CD34+ hematopoietic stem and progenitor cells (HSPCs). After 48 hours, conversion of the HbS allele to HbG was 58±5% with ABE8e-NRCH mRNA/sgRNA and 34±5% with RNP (n=3). On target editing was maximal at 144 hours: 80±2% with ABE8e-NRCH mRNA/sgRNA and 44±7% with ABE8e-NRCH protein (n=3). The indel rate resulting from inadvertent DSBs was To assess base editing in repopulating HSCs, we transplanted ABE8e-NRCH-edited SCD CD34+ cells into NBSGW mice. The editing frequency before transplantation was ~64% for ABE8e-NRCH mRNA/sgRNA and 40% for RNP at 48 hours after electroporation. Mice were euthanized after 16 weeks and base editing was analyzed in human donor-derived cells. Overall editing rates were preserved in repopulating cells: 68%±3% for ABE8e-NRCH mRNA/sgRNA and 36%±3% for RNP (n=4). The editing frequencies were similar in donor cell-derived myeloid, erythroid, and B cell-lineages, indicating that base editing did not alter hematopoietic development. Bone marrow erythroblasts derived from base-edited and control CD34+ HSPCs exhibited similar maturation profiles and enucleation. Erythroblasts generated in vivo from HSPCs from SCD patient exhibited potentially therapeutic levels of HbG protein: 58±3% with ABE8e-NRCH mRNA/sgRNA and 27%±3% with RNP (n=4). Adenine base editor conversion of the HbS allele to the Makassar variant in autologous HSCs represents a new therapeutic approach for SCD. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Henderson:Trilink Biotech: Current Employment. Mccaffrey:Trilink Biotech: Current Employment. Liu:Pairwise Plants: Consultancy, Patents & Royalties; Prime Medicine: Consultancy, Patents & Royalties; Editas Medicine: Consultancy, Patents & Royalties; Beam Therapeutics: Consultancy, Patents & Royalties. Weiss:Rubius Inc.: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Beam Therapeuticcs: Consultancy, Current equity holder in private company.

Rare variants in the γ-globin (HBG2 and HBG1) promoters cause sustained postnatal expression of fetal hemoglobin (HbF, α2γ2) in red blood cells (RBCs). This benign condition is termed hereditary persistence of fetal hemoglobin (HPFH). Individuals with HPFH variants are protected from β-hemoglobinopathies including sickle cell disease and β-thalassemia. Our group and others have used CRIPSR/Cas9-mediated non-homologous end joining to generate HPFH-like insertion-deletion (indel) mutations in the γ-globin promoter. However, simultaneous double-stranded breaks (DSBs) in the tandem duplicated γ-globin genes can result in loss or inversion of the intervening genetic material and/or chromosomal rearrangements. More generally, Cas9-associated DSBs can elicit a cytotoxic DNA repair response leading to cell death or evoke p53 loss with malignant transformation. Base editor (BE) proteins represent a promising approach to install precise nucleotide substitutions without DSBs. Adenosine base editors (ABEs), consisting of catalytically impaired Cas9 fused to a modified adenosine deaminase, create targeted A:T-to-G:C mutations. Here we describe the use of ABEs to recapitulate naturally occurring HPFH variants in hematopoietic stem cells (HSCs). We electroporated ABE7.10-single guide (sg) RNA ribonucleoprotein (RNP) complex into mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) to recreate 3 different HPFH variants in the HBG1/2 promoters (-198 T>C, -175 T>C and -113 A>G). Measured editing frequency was maximal on day 10 after electroporation and transferred to erythroid differentiation media. 20% editing efficiency was observed for the -198 site, 58% for -175 and 50% for -113. Indel frequencies were C), 60±10% (-175 T>C), and 42±7% (-113 A>G) versus14±2% in unedited control cells. Base editing at the -175 site in sickle cell disease (SCD) donor CD34+ HSPCs resulted in the induction of HbF to 55% in erythroid progeny compared to 6% in controls. After exposure to hypoxia (2% oxygen), reticulocytes generated from -175 T>C-edited CD34+ HSPCs exhibited sickling rates of 24%, compared to 52% in controls. Thus, creation of this variant, which generates a de novo binding site for the transcriptional activator TAL1, reactivates erythroid cell HbF to levels that inhibit sickle hemoglobin polymerization and cell sickling. To assess base editing in HSCs, we used ABE RNP to modify the -175 site in SCD donor CD34+ HSPCs, followed by transplantation into NBSGW mice. The editing frequency in CD34+ HSPCs before transplantation was ~30% and declined to approximately 20% in bone marrow-repopulating donor cells at 16 weeks post-transplantation. Editing frequencies were similar in CD34+ donor cell-derived myeloid, erythroid, and B cells, indicating that hematopoietic differentiation was not altered. Bone marrow erythroblasts derived from base-edited and control CD34+ HSPCs exhibited similar maturation profiles and enucleation. Erythroblasts generated in vivo from SCD patient HSPCs exhibited 32±2% HbF compared to unedited controls (4±1%) (n=4, P>0.0001). Our studies provide proof of concept that adenosine base editors can be used therapeutically for β-hemoglobinopathies. Specifically, generation of the -175 T>C HPFH mutation in patient HSCs followed by autologous transplantation represents a new therapeutic approach for SCD and β-thalassemia. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Sharma:Spotlight Therapeutics: Consultancy; Magenta Therapeutics: Other: Research Collaboration; CRISPR Therapeutics, Vertex Pharmaceuticals, Novartis: Other: Clinical Trial PI. Liu:Pairwise Plants: Consultancy, Patents & Royalties; Editas Medicine: Consultancy, Patents & Royalties; Beam Therapeutics: Consultancy, Patents & Royalties; Prime Medicine: Consultancy, Patents & Royalties. Weiss:Beam Therapeuticcs: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Rubius Inc.: Consultancy, Current equity holder in private company.

Summary Alternative polyadenylation (APA) contributes to transcriptome complexity by generating mRNA isoforms with varying 3′ UTR lengths. APA leading to 3′ UTR shortening (3′ US) is a common feature of most cancer cells; however, the molecular mechanisms are not understood. Here, we describe a widespread mechanism promoting 3′ US in cancer through ubiquitination of the mRNA 3′ end processing complex protein, PCF11, by the cancer-specific MAGE-A11–HUWE1 ubiquitin ligase. MAGE-A11 is normally expressed only in the male germline but is frequently re-activated in cancers. MAGE-A11 is necessary for cancer cell viability and is sufficient to drive tumorigenesis. Screening for targets of MAGE-A11 revealed that it ubiquitinates PCF11, resulting in loss of CFIm25 from the mRNA 3′ end processing complex. This leads to APA of many transcripts affecting core oncogenic and tumor suppressors, including cyclin D2 and PTEN. These findings provide insights into the molecular mechanisms driving APA in cancer and suggest therapeutic strategies.

With advancements in gene editing technologies, our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate, paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases. CRISPR-Cas9, short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9, is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells. This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic. The β-hemoglobinopathies are a group of monogenic diseases, which despite their high prevalence and chronic debilitating nature, continue to have few therapeutic options available. In this review, we will discuss our existing comprehension of the genetics and current state of treatment for β-hemoglobinopathies, consider potential genome editing therapeutic strategies, and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.

Summary Fetal and adult hematopoietic stem cells (HSCs) have distinct proliferation rates, lineage biases, gene expression profiles, and gene dependencies. Although these differences are widely recognized, it is not clear how the transition from fetal to adult identity is coordinated. Here we show that murine HSCs and committed hematopoietic progenitor cells (HPCs) undergo a gradual, rather than precipitous, transition from fetal to adult transcriptional states. The transition begins prior to birth and is punctuated by a late prenatal spike in type I interferon signaling that promotes perinatal HPC expansion and sensitizes progenitors to the leukemogenic FLT3ITD mutation. Most other changes in gene expression and enhancer activation are imprecisely timed and poorly coordinated. Thus, heterochronic enhancer elements, and their associated transcripts, are activated independently of one another rather than as part of a robust network. This simplifies the regulatory programs that guide neonatal HSC/HPC ontogeny, but it creates heterogeneity within these populations.

Somatic genetic alterations of IKZF1, which encodes the lymphoid transcription factor IKAROS, are common in high-risk B-progenitor acute lymphoblastic leukemia (ALL) and are associated with poor prognosis. Such alterations result in the acquisition of stem cell-like features, overexpression of adhesion molecules causing aberrant cell-cell and cell-stroma interaction, and decreased sensitivity to tyrosine kinase inhibitors. Here we report coding germline IKZF1 variation in familial childhood ALL and 0.9% of presumed sporadic B-ALL, identifying 28 unique variants in 45 children. The majority of variants adversely affected IKZF1 function and drug responsiveness of leukemic cells. These results identify IKZF1 as a leukemia predisposition gene, and emphasize the importance of germline genetic variation in the development of both familial and sporadic ALL.

Acute myeloid leukemia (AML) cells often co-opt normal hematopoietic stem cell (HSC) programs to drive neoplastic proliferation, and HSC-related gene expression signatures have been identified as biomarkers for poor prognosis in AML patients. We sought to identify new regulators of HSCs and AML cells from previously published HSC and leukemia stem cell (LSC) gene expression signatures. We identified PRKCH (protein kinase C eta) as a gene that is highly expressed in both mouse and human HSCs, as well as in LSCs from independent cohorts of AML patients. Prkch deletion in mice resulted in impaired HSC function. PRKCH was most highly expressed in undifferentiated (FAB M0) subtype AML, and high expression correlated with TP53 and RUNX1 mutations, high-risk cytogenetic features, and poor overall survival. Prkch deletion in an Flt3-ITD/Runx1 mutant mouse AML model did not extend survival. Thus, PRKCH is necessary for normal HSC function; its expression predicts poor survival in AML patients, but it is not required for AML to develop.

Induction of fetal hemoglobin (HbF, α2γ2) via genome editing-mediated disruption of DNA regulatory elements that repress expression of γ-globin genes (HBG1 and HBG2) is a promising therapeutic strategy for b-hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia. Optimal technical approaches and safety profiles are yet to be fully defined. We used CRISPR/Cas9 to target a DNA repressor element near the distal CCAAT box of the HBG1/HBG2 promoters. This region contains a "TGACC" motif recognized by BCL11A, a transcriptional repressor protein that regulates γ-to-β globin switch after birth. Rare germline variants at or near this motif are associated with hereditary persistence of fetal hemoglobin, a benign genetic condition that alleviates the clinical manifestations of co-inherited b-hemoglobinopathies. Previously, we showed that transduction of human CD34+ cells with lentiviral vector encoding Cas9 and guideRNA (gRNA) targeting the HBG1/HBG2 promoter caused induction of HbF in red blood cell (RBC) progeny generated in vitro (Traxler et. al, Nature Medicine v22,2016). Here we present a clinically tractable approach for disrupting the HBG1/HBG2 BCL11A binding site in human hematopoietic stem cells (HSCs). Electroporation of Cas9:gRNA ribonucleoprotein (RNP) complex into healthy or SCD donor CD34+ cells resulted in up to 80% on-target insertion-deletion (indel) mutations and 35% HbF in erythroid progeny generated in vitro. Sixteen to 17 weeks after transplantation of gene edited CD34+ cells into immunodeficient NBSGW mice, up to 75% of donor CD34+ cells in recipient bone marrow contained on-target indels, demonstrating efficient modification of repopulating human HSCs. No differences in CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages were observed between edited and control cells. Moreover, up to 78% of gene edited erythroid cells stained with anti-HbF antibody ("F-cells") compared to 15% in control erythroid cells, suggesting a "pan-cellular" pattern of HbF expression after editing. Strikingly, human donor-derived erythroid cells in recipient bone marrow expressed up to 40% HbF compared to 3% HbF in controls. Although the editing frequencies of HBG1 and HBG2 promoters varied between different donor CD34+ cells, an engineered variant of Cas9 containing 3 nuclear localization sequences (Wu et. al,Nature Medicine v25, 2019) edited repopulating HSCs more efficiently and consistently than conventional Cas9 with two nuclear localization signals. Simultaneous on-target RNP-induced DSBs at both HBG1 and HBG2 can result in the deletion of the intervening 4.9-kb region, leaving a single hybrid gene with HBG2 promoter sequences fused to the downstream HBG1 gene. We detected this deletion in approximately 30% of edited cells, with no associated decline in HbF expression determined by clonal analysis of erythroid colonies. No off-target mutations were detected by targeted sequencing of the 26 top candidate sites identified by CIRCLE-seq, an in vitro genome-scale method for detecting Cas9 activity. Analysis of gene edited human donor cells purified from mouse bone marrow showed no chromosomal rearrangements by G-banding (n=20) or fluorescence in situ hybridization with a probe located distal to the HBG1/HBG2 loci (n=225). Taken together, our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a CRISPR-Cas9 genome editing approach to induce HbF for treating hemoglobinopathies. Figure. Gene editing of the HBG1/HBG2 promoters in HSCs and HbF induction of erythroid progeny in vivo. Plerixafor-mobilized CD34+ cells from an individual with SCD were edited with RNP and transplanted into NBSGW mice, which were analyzed 16-17 weeks later. A. On-target indel frequency before (Pre) and after bone marrow transplantation (BM). The black bars represent a 13-nucleotide deletion associated with human hereditary persistence of fetal hemoglobin. B. Human erythroblasts and reticulocytes derived from RNP-edited and non-edited Control CD34+ donor cells. Scale bar = 10 mm. C. HbF immunostaining control and RNP edited erythroid cells in recipient bone marrow assessed by flow cytometry. D. %HbF protein in hemolysates of control (C) and RNP edited erythroid cells assessed by ion-exchange HPLC. n= 3 biological replicates. **** P < 0.0001. Figure Disclosures Metais: MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Sharma:Doris Duke Foundation: Research Funding; Vertex Pharmaceuticals: Other: Study PI. Weiss:Beam Therapeutics: Consultancy; Rubius INC: Consultancy; GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy.

The FLT3 Internal Tandem Duplication (FLT3ITD) mutation is common in adult acute myeloid leukemia (AML) but rare in early childhood AML. It is not clear why this difference occurs. Here we show that Flt3ITD and cooperating Flt3ITD/Runx1 mutations cause hematopoietic stem cell depletion and myeloid progenitor expansion during adult but not fetal stages of murine development. In adult progenitors, FLT3ITD simultaneously induces self-renewal and myeloid commitment programs via STAT5-dependent and STAT5-independent mechanisms, respectively. While FLT3ITD can activate STAT5 signal transduction prior to birth, this signaling does not alter gene expression until hematopoietic progenitors transition from fetal to adult transcriptional states. Cooperative interactions between Flt3ITD and Runx1 mutations are also blunted in fetal/neonatal progenitors. Fetal/neonatal progenitors may therefore be protected from leukemic transformation because they are not competent to express FLT3ITD target genes. Changes in the transcriptional states of developing hematopoietic progenitors may generally shape the mutation spectra of human leukemias.

Summary Pten negatively regulates the phosphatidylinositol 3-kinase (PI3K) pathway and is required to maintain quiescent adult hematopoietic stem cells (HSCs). Pten has been proposed to regulate HSCs cell autonomously and non-cell autonomously, but the relative importance of each mechanism has not been directly tested. Furthermore, the cytokines that activate the PI3K pathway upstream of Pten are not well defined. We sought to clarify whether Pten cell autonomously or non-cell autonomously regulates HSC mobilization. We also tested whether Pten deficiency affects the HSC response to granulocyte colony-stimulating factor (G-CSF) and interferon-α (IFNα) since these cytokines induce HSC mobilization or proliferation, respectively. We show that Pten regulates HSC mobilization and expansion in the spleen primarily via cell-autonomous mechanisms. Pten-deficient HSCs do not require G-CSF to mobilize, although they are hyper-sensitized to even low doses of exogenous G-CSF. Pten-deficient HSCs are similarly sensitized to IFNα. Pten therefore modulates the HSC response to inflammatory cytokines., Graphical Abstract, Highlights • Pten regulates HSC mobilization primarily via cell-autonomous mechanisms • Pten modulates the HSC response to G-CSF • Pten modulates the HSC response to interferon-α, Magee and colleagues show that Pten suppresses HSC mobilization and extramedullary expansion primarily through cell-autonomous mechanisms. The authors also show that Pten-deficient HSCs are hyper-sensitive to mobilizing effects of G-CSF and interferon-α, even at low-cytokine concentrations. These findings suggest that a key function of Pten in HSCs is to blunt signal transduction downstream of inflammatory cytokines.

KMT2C/MLL3 is one of the most commonly mutated tumor suppressor genes in human cancer. Haploinsufficiency at the KMT2C/MLL3 locus often occurs in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) as a result of mono-allelic 7q deletions. Mll3 haploinsufficiency has been shown to accelerate AML in mice, but it is not clear how it regulates normal hematopoietic progenitors or why it is required to suppress leukemogenesis. We used a novel loss-of-function mouse allele to characterize hematopoiesis in Mll3-deficient fetal livers and adult bone marrow. Mll3 deletion caused a modest increase in fetal liver hematopoietic stem cells (HSCs) and a modest decrease in lineage restricted progenitors (HPCs). In contrast, Mll3 heterozygosity caused significant expansion of the HSC population in adult bone marrow without affecting more committed progenitor populations. Mll3+/- and Mll3-/- HSCs were able to repopulate lethally irradiated mice, and repopulating activity was enhanced in secondary and tertiary transplants relative to wild type HSCs. Thus, Mll3 haploinsufficiency enhances HSC self-renewal capacity. We next tested whether Mll3 regulates the balance between self-renewal and commitment in non-transplanted HSCs. We used H2B-GFP pulse-chase assays to assess the division histories of HSCs over time. After a 6-week chase, wild type HSCs retained high levels of H2B-GFP expression, consistent with prior studies showing that adult HSCs divide infrequently. In contrast, almost all Mll3+/- HSCs had low to moderate H2B-GFP expression levels, indicative of an extensive division history. This difference was not associated with an increase in the percentage of cycling (S/G2/M) HSCs, and it was not associated with a loss of self-renewal capacity that occurs with cumulative divisions in wild type HSCs. Thus, Mll3 haploinsufficiency allows cumulative HSC divisions to accrue without compromising self-renewal. Mll3 encodes a histone methyltransferase that binds active enhancers and promotes transcription. This raised the question of whether Mll3 deletion results in a loss of commitment-related gene expression, increased self-renewal gene expression or altered epigenetic landscapes. To our surprise, gene expression was indistinguishable between wild type and Mll3+/- HSCs. However, ATAC-seq analysis revealed patterns of nucleosome depletion in Mll3+/- HSCs that closely resembled wild type HPCs. No differences were observed in the ATAC-seq profiles of wild type and Mll3+/- HPCs. These observations suggest that as Mll3+/- HSCs self-renew, they remodel their epigenetic landscapes to resemble committed progenitors, yet they do not activate commitment related genes, and they do exit the stem cell pool. The data suggest that stress conditions that drive HSCs into cycle, such as the chemotherapy regimens that often precede 7q- MDS, may select for Mll3 haploinsufficient HSC clones. Citation Format: Theresa Owuor, Shaina Porter, Cluster Andrew, Riddhi Patel, Jeffrey Magee. MLL3 haploinsufficiency preserves self-renewal capacity in HSCs with extensive cumulative division histories [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4996.

Much progress has been made toward identifying the mutations that cause human acute myeloid leukemia (AML), and these studies have shown that pediatric and adult AML are often caused by different mutations. Genetic differences between pediatric and adult AML may underlie differences in outcomes and necessitate different treatment strategies, yet we have few insights into why these differences occur. One possibility is that the mechanisms that regulate normal hematopoiesis change with age, and mutations therefore have age-specific phenotypes in pre-leukemic progenitors. To fully understand how AML evolves in children and adults, and how targeting individual pathways might impact cell physiology at different ages, it is important to understand how somatic mutations interface with the normal, temporally dynamic programs that regulate hematopoiesis. The FLT3-Internal Tandem Duplication (FLT3-ITD) mutation is common in adult AML but rare in early childhood AML (30-40% of adult AML, 5-10% of AML in children We used FLT3-ITD knock-in mice to test whether FLT3-ITD has developmental context specific effects on hematopoiesis. In adult mice, FLT3-ITD depleted the HSC pool and expanded multipotent progenitor (MPP) and myeloid progenitor populations consistent with prior studies. In fetal mice, FLT3-ITD had no effect on HSC or MPP numbers, HSC function (as determined by limit dilution transplants) or myelopoiesis. FLT3-ITD did not affect hematopoiesis until shortly after birth. These temporal differences were evident even in the presence of cooperating Runx1 mutations. To understand why fetal and adult progenitors responded differently to FLT3-ITD, we characterized signal transduction and gene expression in fetal, neonatal and adult progenitors. We found that STAT5 was activated by FLT3-ITD at all stages of development, but MAPK was activated only in post-natal progenitors concordant with the onset of HSC and myeloid phenotypes. To our surprise, conditional Stat5a/b deletion exacerbated the HSC depletion and myeloid expansion phenotypes of adult FLT3-ITD mice rather than rescuing them. This suggests that STAT5 helps to maintain adult, FLT3-ITD mutant progenitors in an undifferentiated state even as other effectors promote myeloid differentiation. We next used microarrays to test whether FLT3-ITD has age-specific effects on gene expression in HSCs and MPPs, and to identify normal temporal changes in gene expression that may modulate the FLT3-ITD phenotypes. These studies made several key points: 1) In wild type HSCs, most fetal-specific genes were inactivated and most adult-specific genes were activated between birth and P14. This transition was earlier than prior studies have suggested, and it correlated with the age at which FLT3-ITD induced HSC depletion and myeloid expansion. 2) FLT3-ITD did not alter gene expression until after birth, coincident with onset of the HSC depletion and myeloid expansion phenotypes. 3) FLT3-ITD target genes were more differentially expressed in MPPs than in HSCs, consistent with recent data suggesting that MPPs are a cell of origin for FLT3-ITD driven AML. 4) Most, but not all, FLT3-ITD target genes were STAT5 dependent. Our analyses have identified novel, adult-specific candidate effectors of FLT3-ITD. Moreover, our findings raise the question of whether fetal genetic programs can suppress FLT3-ITD driven leukemogenesis, and we have begun to address this question with gain of function models. AML cells may exhibit "context addiction" (i.e.a sustained requirement for normal adult gene products and a toxic response to fetal gene products), that could be exploited therapeutically. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cell transplantation (HSCT) has been used to cure patients for over four decades, yet the kinetics and clonal dynamics of repopulation by multipotent HSCs remains unresolved. Two sequence-based methods have been used to measure HSC contribution in preclinical models, integration site (IS) analysis and DNA barcoding. Both methods are accomplished by retrovirus tagging of HSCs and each confers different methodological constraints. We hypothesized that IS analysis and DNA barcoding together would provide more robust HSCT reconstitution data if applied to the clinically relevant nonhuman primate autologous HSCT model. We developed a high complexity (~1.2 million), DNA-barcoded LV library encoding the chemotherapy resistance gene MGMT(P140K) and enhanced green fluorescent protein (GFP) to measure hematopoietic reconstitution following total body irradiation (1020 cGy) and autologous HSCT in two pigtailed macaques. In both animals, hematopoietic recovery from TBI was achieved within 1-2 months after HSCT. We observed ~1 and 12% GFP+ peripheral blood white blood cells (PB WBCs) following hematopoietic recovery in these animals, respectively. We performed barcode and IS retrieval from PB WBCs collected early (1 month) after HSCT and observed 1 in 10,000 infused CD34+ cells is capable of trilineage hematopoiesis. In sum, the combination of retrovirus integration site analysis with molecular barcode analysis provides synergistic methods in understanding the kinetics and clonal dynamics of hematopoietic reconstitution following autologous HSCT and revealed a successive pattern of multi-lineage clonal behavior. Disclosures No relevant conflicts of interest to declare.