AAV-mediated genome editing is influenced by the formation of R-loops.

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

Phase 1b trial of tagraxofusp in combination with azacitidine with or without venetoclax in acute myeloid leukemia.

ABSTRACT: CD123, a subunit of the interleukin-3 receptor, is expressed on ∼80% of acute myeloid leukemias (AMLs). Tagraxofusp (TAG), recombinant interleukin-3 fused to a truncated diphtheria toxin payload, is a first-in-class drug targeting CD123 approved for treatment of blastic plasmacytoid dendritic cell neoplasm. We previously found that AMLs with acquired resistance to TAG were re-sensitized by the DNA hypomethylating agent azacitidine (AZA) and that TAG-exposed cells became more dependent on the antiapoptotic molecule BCL-2. Here, we report a phase 1b study in 56 adults with CD123-positive AML or high-risk myelodysplastic syndrome (MDS), first combining TAG with AZA in AML/MDS, and subsequently TAG, AZA, and the BCL-2 inhibitor venetoclax (VEN) in AML. Adverse events with 3-day TAG dosing were as expected, without indication of increased toxicity of TAG or AZA+/-VEN in combination. The recommended phase 2 dose of TAG was 12 µg/kg/day for 3 days, with 7-day AZA +/- 21-day VEN. In an expansion cohort of 26 patients (median age 71) with previously untreated European LeukemiaNet adverse-risk AML (50% TP53 mutated), triplet TAG-AZA-VEN induced response in 69% (n=18/26; 39% complete remission [CR], 19% complete remission with incomplete count recovery [CRi], 12% morphologic leukemia-free state [MLFS]). Among 13 patients with TP53 mutations, 7/13 (54%) achieved CR/CRi/MLFS (CR = 4, CRi = 2, MLFS = 1). Twelve of 17 (71%) tested responders had no flow measurable residual disease. Median overall survival and progression-free survival were 14 months (95% CI, 9.5-NA) and 8.5 months (95% CI, 5.1-NA), respectively. In summary, TAG-AZA-VEN shows encouraging safety and activity in high-risk AML, including TP53-mutated disease, supporting further clinical development of TAG combinations. The study was registered on ClinicalTrials.gov as #NCT03113643.

Cyclin-Dependent Kinase-Mediated Phosphorylation of FANCD2 Promotes Mitotic Fidelity.

Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs upon exposure to DNA-damaging agents and during the S phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a cyclin-dependent kinase (CDK) regulatory phosphosite (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by immunoblotting with an S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during the S phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity.

FANCD2 Binding to H4K20me2 via a Methyl-Binding Domain Is Essential for Efficient DNA Cross-Link Repair.

Fanconi anemia (FA) is an inherited disease characterized by bone marrow failure and increased cancer risk. FA is caused by mutation of any 1 of 22 genes, and the FA proteins function cooperatively to repair DNA interstrand cross-links (ICLs). A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs within chromatin. How FANCD2 and FANCI are anchored to chromatin remains unknown. In this study, we identify and characterize a FANCD2 histone-binding domain (HBD) and embedded methyl-lysine-binding domain (MBD) and demonstrate binding specificity for H4K20me2. Disruption of the HBD/MBD compromises FANCD2 chromatin binding and nuclear focus formation and its ability to promote error-free DNA interstrand cross-link repair, leading to increased error-prone repair and genome instability. Our study functionally describes the first FA protein chromatin reader domain and establishes an important link between this human genetic disease and chromatin plasticity.

Modulation of the Fanconi anemia pathway via chemically induced changes in chromatin structure.

Fanconi anemia (FA) is a rare disease characterized by congenital defects, bone marrow failure, and atypically early-onset cancers. The FA proteins function cooperatively to repair DNA interstrand crosslinks. A major step in the activation of the pathway is the monoubiquitination of the FANCD2 and FANCI proteins, and their recruitment to chromatin-associated nuclear foci. The regulation and function of FANCD2 and FANCI, however, is poorly understood. In addition, how chromatin state impacts pathway activation is also unknown. In this study, we have examined the influence of chromatin state on the activation of the FA pathway. We describe potent activation of FANCD2 and FANCI monoubiquitination and nuclear foci formation following treatment of cells with the histone methyltransferase inhibitor BRD4770. BRD4770-induced activation of the pathway does not occur via the direct induction of DNA damage or via the inhibition of the G9a histone methyltransferase, a mechanism previously proposed for this molecule. Instead, we show that BRD4770-inducible FANCD2 and FANCI monoubiquitination and nuclear foci formation may be a consequence of inhibition of the PRC2/EZH2 chromatin-modifying complex. In addition, we show that inhibition of the class I and II histone deacetylases leads to attenuated FANCD2 and FANCI monoubiquitination and nuclear foci formation. Our studies establish that chromatin state is a major determinant of the activation of the FA pathway and suggest an important role for the PRC2/EZH2 complex in the regulation of this critical tumor suppressor pathway.