Fig 1: UHRF1 inhibitor UF146 suppresses AML cell survival by inhibiting proliferation and promoting apoptosis in vitro.a The scheme of the screening protocol for UHRF1 inhibitor. b The structural formula of UHRF1 inhibitor UF146. c The binding mode of UF146 to the G465, A463, G448 and V446-created 5mC cavity in SRA domain. d, e The SPR (d) and FRET (e) analysis were performed to examine the direct binding affinities of UF146 to the SRA domain of UHRF1 (n = 3). f The cellular viability of human CD34+ HSPCs, Kasumi-1 and THP-1 cells treated with UF146 or the vehicle was examined by MTT assay (n = 3). g The cellular viability of AML cells (Kasumi-1, SKNO-1, HL60, NB4, OCI-AML3, THP-1, NOMO-1, MOLM-13, MV4-11 and SKM-1 cells) treated with UF146 or the vehicle control for 48 h. h, i The representative flow cytometry analysis profiles (h) and quantification (i) analysis of the early apoptosis and late apoptosis in the primary AML patient cells 24 h after the treatment of UF146 or the vehicle control (n = 3). j The Wright’s staining of the primary AML cells treated with the vehicle or UF146. k The number of colonies generated from AE9a- or MLL-AF9-driven LICs treated with UF146 or the vehicle (n = 3). l The number of colonies generated from the primary AML patient cells treated with UF146 or the vehicle (n = 3). m The cluster dendrogram analysis of AML cells treated with UF146 and AML cells with knockdown of UHRF1. n The RNA-seq and GSEA analysis of the AML cells treated with UF146 or the vehicle. o, p The DNA methylation levels of MXD4 in UF146- or the vehicle-treated Kasumi-1 (o) and THP-1 (p) cells were analyzed by the bisulfite sequencing (n = 3). q Western blotting analysis of UHRF1/MXD4/SAP30 in AML cells 24 h after UF146 treatment. Statistical analyses were performed using Student’s unpaired t-test for i, k, l, o and p. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: The interaction of UHRF1 with SAP30 is critical for MXD4 repression and leukemogenesis.a The event free survival of AML patients was stratified by SAP30 expression into SAP30 high (survival days: 503 days, n = 211) and low (survival days: 646 days, n = 77) groups. b Correlation analysis of UHRF1/SAP30 expression in LSCs from AML patients was performed using the GSE76009 dataset. c The immunoprecipitation was performed using the anti-UHRF1 or anti-SAP30 antibody, and the anti-SAP30 and anti-UHRF1 antibodies were used for the Western blotting analysis in AML cells (n = 3). d The GST pull-down assay shows that UHRF1 interacts with SAP30 in vitro and the SRA domain of UHRF1 is required for the interaction with SAP30. e The schematic representation of the truncations of SRA domain. f The immunoprecipitation assay was performed to examine the interaction of HA-tagged mutant UHRF1 with Flag-SAP30 in 293T cells (n = 3). g The number of colonies generated from UHRF1-deficient AML cells transduced with WT or mutant UHRF1 (n = 3). h The survival of B-NDG recipient mice receiving Kasumi-1 (n = 6) or THP-1 (n = 5) cells with the restoration of UHRF1 and UHRF1-Mut2 after UHRF1 knockdown. i, j MXD4 expression was examined by q-PCR (i) and Western blotting (j) analysis in AML cells with SAP30 knockdown (n = 3). k The CUT&Tag analysis shows that SAP30 binds to the promoter of MXD4 in AML cells. l The ChIP-qPCR analysis of UHRF1 on the promoter of MXD4 in AML cells with the knockdown of SAP30 (n = 3). m MXD4 expression was examined by q-PCR analysis in UHRF1-deficient AML cells transduced with WT or mutant UHRF1 (n = 3). n, o The representative DNA methylation profiles (n) and quantification (o) analysis of MXD4 by the bisulfite sequencing in G572R and F573R mutant AML cells with UHRF1 knockdown. Data are all presented as means ± SD. Statistical analyses were performed using log-rank test for a, h, and Pearson’s correlation analysis for b. Student’s unpaired t-test was used for g, i, l, m, o. *P < 0.05, **P < 0.01, ***P < 0.001.
Supplier Page from Abcam for Anti-SAP30 antibody