Fig 2: NLK inhibition increases expansion of erythroid progenitors from human and murine models of DBA.a Human cord blood CD34+ progenitors were transduced with lentivirus co-expressing GFP with shRNA against luciferase (shLuc), RPS19 (shRPS19), or RPL11 (shRPL11). After 36 h GFP+ cells were differentiated in erythroid media in the presence or absence of 5 µM SD208 for 15 days. Cells were counted and assessed for cell surface expression of CD235. b Lin-Kit+ hematopoietic progenitors were obtained from three mouse embryos expressing tetracycline-inducible shRNA against RPS19 at day E14.5 or three untreated mice, and thee mature RPL11+/+ or three mature RPL11+/lox mice treated with tamoxifen for eight weeks. Cells were grown in the presence or absence of doxycycline and/or SD208 for 8 days prior to counting and assessing for Ter119 surface expression. Values were expressed as a percentage relative to untreated controls. c, d Intracellular phosphorylation of NLK at Thr298 was determined by capillary electrophoresis using the Peggy Sue™ Automated Western blotting platform. After lysis, 4 µg of protein from the CD235+ population was probed against pThr298-NLK and normalized to GAPDH. Detected NLK phosphorylation is plotted relative to untreated control. e CD34+ HSPCs were isolated from three healthy control and three DBA patient mononuclear bone marrow aspirates by magnetic bead sorting and differentiated in the presence or absence of SD208 for 14 days. After counting the total cell population, the ratio of CD235+ erythroblasts was determined by flow cytometry and number of CD235+ erythroblasts calculated. This was expressed as a percentage of the average number of erythroid cells present in untreated healthy controls. f NLK phosphorylation was assessed by capillary electrophoresis as above. Bars represent means ± SD with individual data points overlaid. Purple depicts untreated controls, yellow depicts controls treated with SD208, red depicts untreated DBA progenitors, while blue depicts DBA progenitors treated with SD208. n = 3 independent experiments performed in triplicate. Statistics: two-tailed Student’s t test, significant *p < 0.05. Also see Supplementary Fig. 8. Source data are provided as a Source Data file.

Fig 3: Deregulated SATB1 identified as master regulator of RPS19-sensitive early erythroid genes. (A) RNA transcripts differentially regulated by RPS19 insufficiency in differentiating fetal liver-derived hematopoietic stem and progenitor cells (HSPCs) were cross referenced with RNA transcripts differentially expressed between cord blood-derived CD34+ HSPCs and early burst-forming unit–erythroid (BFU-E) progenitors. Data sets are represented diagrammatically, with differentially regulated transcripts from one data set designated in blue, the other data set in green. Forty-two transcripts are found in both data sets, represented by the overlapping region, and are designated RPS19-sensitive early erythroid genes. (B) CD34+ HSPC cord blood or fetal liver were transduced with shRNA against luciferase and differentiated for 0 or 5 days in erythroid-promoting medium, and expression of RPS19-sensitive early erythroid genes was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Upregulation is denoted in green while downregulation is in red. CD34+ HSPCs transduced and comparatively designated as upregulated (green), further upregulated (dark green), downregulated (red), or further downregulated (dark red). No deregulation is depicted in yellow. (C) miRNA-seq was performed comparing fetal liver HSPCs transduced with control (shLuc) or shRNA against RPS19 (shRPS19). Upregulated miRNAs are represented in darker shades of orange/red. (D) The 3′-untranslated region (3′UTR) sequences of RPS19-senstive early erythroid genes were screened for consensus sequences for the two most highly RPS19-deregulated miRNAs, identifying six transcripts. Expression of (E) SATB1 mRNA and (F) protein was analyzed at days 0 and 5 in fetal liver-derived differentiating HSPCs by qRT-PCR (n = 5) (E) and Western blot analysis (n = 3, representative image shown) (F), respectively. Cells were left untransduced or were transduced with shRNA against luciferase or RPS19. Data are represented as means ± SD. (G) iPSCs clones were generated from mononuclear cells from bone marrow aspirates from a healthy control and RPS26 mutation-bearing DBA patient. iPSCs were differentiated to CD34+ iHSCs through wnt stimulation. Three iHSC clones (n = 3) for each group were transduced with tetracycline-inducible shRNA against RPS19 or cDNA for RPS26. After 5 days of differentiation, cultures were lysed and SATB1 mRNA was assessed by qRT-PCR. Data are displayed as means ± SD. *Two-tailed Student t test significant at p < 0.05. See also Supplementary Figure E1 (online only, available at www.exphem.org).

Fig 4: Induction of miR-34 in RPS19 insufficiency is p53 dependent. (A) CB CD34+ hematopoietic stem and progenitor cells (HSPCs) were transduced in erythropoiesis-promoting medium with shRNA against luciferase or RPS19 and nontargeting or p53 before miR-30, miR-34, and SATB1 analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) at day 5 (n = 3). (B) Lymphoblastic cell lines derived from DBA patients were assessed for miR-30 and miR-34 by qRT-PCR. These were compared with a lymphoblastic cell line derived from a healthy control, or the same control transduced with shRNA against RPS19. Significance is indicated (*) when expression is greater (p ≤ 0.05) than observed in the corresponding DR07 control (n = 7). (C) Diagrammatic representation of the proposed myeloid lineage-specific premature downregulation of SATB1. In lymphoblastic cells, RPS19 insufficiency induces miR-34 through p53 but this is insufficient to downregulate SATB1. In myeloid lineage cells, RPS19 insufficiency induces both miR-30 and miR-34, which synchronize to prematurely downregulate SATB1. Data are represented as means ± SD. *Two-tailed Student t test significant at p < 0.05. See also Supplementary Figure E4 (online only, available at www.exphem.org).

Fig 5: SATB1 re-expression modestly increases erythroid expansion but increases megakaryocyte and other myeloid lineages. CB CD34+ hematopoietic stem and progenitor cells (HSPCs) were transduced with shRNA against RPS19 in the presence or absence of SATB1 cDNA and plated in methylcellulose containing hematopoiesis-promoting medium (n = 5). Resulting burst-forming unit–erythroid (BFU-E) (A) and colony-forming unit–granulocyte/macrophage (CFU-GM) colonies (B) were scored. Representative images of plates are shown (C, left) along with representative BFU-E colonies (C, right). Bar = 200 μm. (D) Methylcellulose was dissolved in 4°C water, and cell number assessed by hemacytometer. The proportion of CD235+ cells was assessed, and the number of CD235+ cells was calculated by dividing the number of totals cells by the percentage of CD235+. Values are expressed as a fold of control to normalize for experimental variability. (E) CB CD34+ HSPCs (n = 3) were transduced with shRNA against RPS19 in the presence or absence of SATB1 cDNA and grown in liquid culture for 12 days before flow cytometry and calculation of CD235+, CD11b+, and CD41a+ populations. Data are represented as means ± SD. *Two-tailed Student t-test significant at p < 0.05. See also Supplementary Figure E2 (online only, available at www.exphem.org).