PMC

FOXP1 rescues growth defects of PUM1 and PUM2 KD cells. (A-D) Cord blood CD34⁺ HSPCs were transduced first with pLenti-PGK-USIL DEST lentiviral vectors that encode (+) or not (−) FOXP1, and then with shCtrl (−), shPUM1 or shPUM2 vectors, and sorted at day 3 posttransduction. (A) Immunoblot analysis of the indicated proteins at day 3 posttransduction. Actin was used as loading control. (B) Relative cell expansion at day 10 posttransduction (n = 3). (C) Annexin V labeling at day 7 (shPUM1) and at day 10 (shPUM2) posttransduction. (D) CFC assay at day 7 posttransduction (n = 3). (E-G) MOLM-14 cells were transduced first with pINDUCER21 inducible lentiviral vectors that encode (+) or not (−) FOXP1, then with shCtrl (−), shPUM1, or shPUM2 vectors. The day after, FOXP1 expression was induced with doxycycline (0.05 µg/mL). (E) Immunoblot analysis of the indicated proteins at day 4 posttransduction. Actin was used as loading control. (F) Relative cell expansion of double-transduced cells at day 8 posttransduction (n = 4). (G) Annexin V labeling at day 6 posttransduction (n = 4). Data are expressed as mean ± SEM. Ns, not significant. *P < .05; **P < .01 (Student t test).

FOXP1 contributes to human CD34⁺HSPC and leukemic cell growth. (A-E) Human cord blood CD34⁺ cells were transduced or not (nt) with lentiviral vectors encoding shRNAs targeting FOXP1 (shFOXP1#a, #b, #c; sequences detailed in supplemental Table 8) or luciferase as control (shCtrl). (A) Immunoblot analysis of the indicated proteins at day 3 posttransduction. Actin was used as loading control. (B) Cell expansion analysis along time posttransduction (n = 4). (C) Cell cycle analysis upon propidium iodide labeling at day 3 posttransduction (n = 3). (D) Annexin V labeling assessed at day 4 posttransduction (n = 4). (E) CFC assays at day 7 posttransduction (n = 3). (F-I) MOLM-14 leukemia cells were transduced or not (nt) with the indicated shRNA-encoding vectors. (F) Immunoblot analysis of the indicated proteins at day 3 posttransduction. Actin was used as loading control. (G) Cell expansion analysis along time posttransduction (n = 3). (H) Cell cycle analysis upon propidium iodide labeling at day 2 posttransduction (n = 3). (I) Annexin V labeling at day 3 posttransduction (n = 3). (J) Immunoblot analysis of the indicated proteins at day 3 (CD34⁺) and day 2 (MOLM-14) posttransduction. Actin was used as loading control. Data are expressed as mean ± SEM. *P < .05; **P < .01; ***P < .001 (Student t test).

PUM1 and PUM2 regulate FOXP1 expression in CD34⁺HSPCs and leukemic cells. (A) Immunoblot analysis of the indicated proteins in CD34⁺ HSPCs transduced or not (nt) with the indicated shRNA-encoding vectors at day 4 post-transduction. Actin was used as loading control. (B) RT-qPCR analysis of hFOXP1 mRNA in shRNA-transduced CD34⁺ cells at day 4 post-transduction. Results are normalized to GAPDH mRNA expression, and expressed relative to shCtrl-expressing cells (n = 3). (C) RT-qPCR analysis of hPUM1, hPUM2 and hFOXP1 transcripts in the indicated human stem/progenitor subpopulations (see ). Results are normalized to GAPDH expression, and expressed relative to expression in HSC (n = 3). (D) Immunoblot analysis of the indicated proteins in shRNA-transduced MOLM-14 cells at day 3 post-transduction. Actin was used as loading control. (E) RT-qPCR analysis of hFOXP1 mRNA in shRNA-transduced MOLM-14 cells at day 3 post-transduction. Results are normalized to GAPDH expression and expressed relative to shCtrl-expressing cells (n = 3). (F) RT-qPCR analysis of FOXP1 expression in AML patient samples grouped according to FAB classification, and in CD34⁺ cells from healthy donors. Each symbol represents data from a single patient. CD34⁺: 11 samples; AML₀: 4 samples; AML₁: 9 samples; AML₂: 17 samples; AML₃: 6 samples; AML₄: 6 samples; AML₅: 4 samples. Results are normalized to GAPDH, HPRT and Cyclophilin A mRNA levels. (G) Correlation between PUM1 or PUM2 and FOXP1 mRNA expressions in AML samples. r = Pearson correlation’s coefficient. Data are expressed as mean ± SEM. ns: not significant, * P < .05; **P < .01; ***P < .001 [Mann-Whitney test in (F); Student t test in (B, C, E)].

PUM1 and PUM2 contribute to human leukemic cell growth. (A) RT-qPCR analysis of hPUM1 and hPUM2 mRNA expression in AML patient samples grouped according to the French-American-British classification, and in CD34⁺ cells from healthy donors (peripheral blood or cord blood or BM). Each symbol represents data from a single patient. CD34⁺: 11 samples; AML₀: 5 samples; AML₁: 16 samples; AML₂: 26 samples; AML₃: 7 samples; AML₄: 6 samples; AML₅: 4 samples. Results are normalized to GAPDH, HPRT, and Cyclophilin A transcript levels. (B-E) MOLM-14 leukemia cells were transduced or not (nt) with the indicated shRNA-encoding vectors. (B) Immunoblot analysis of the indicated proteins at day 3 posttransduction. Actin was used as loading control. (C) Cell expansion analysis along time of culture posttransduction (n = 3). (D) Cell cycle analysis upon propidium iodide labeling at day 3 posttransduction (n = 3). (E) Annexin V labeling at day 4 posttransduction (n = 3). (F) Primary AML patient samples were transduced with shCtrl-, shPUM1-, or shPUM2-encoding vectors, and cultured onto MS5 stromal cells in CD34⁺ growth medium. At day 10, the cultures were disrupted by vigorous pipetting and scraping, and nucleated cells were counted by FACS. Results are expressed relative to shCtrl-transduced cell numbering (n = 6). Each symbol represents data from a single patient. Data are expressed as mean ± SEM. Ns, not significant. *P < .05; **P < .01; ***P < .001 (Mann-Whitney test in panel A; Student t test in panels C-F).

PUM1 and PUM2 positively regulate FOXP1 mRNA expression via two 3′UTR located PBE. (A) Schematic representation of human FOXP1 mRNA with the 2 PUM-binding elements (PBE), PBE1 and PBE2, boxed (red), and regions A, B, C delineated. The numbers indicate positions in 3′UTR, starting from translation stop codon. (B) RNA pull-down assay. MOLM-14 cell lysates were incubated with WT₁ or MUT₁ biotin-labeled FOXP1-PBE1 probes, and none or ×5 unlabeled PBE1 (WT₁ or MUT₁) or PBE2 (WT₂ or MUT₂) competitor riboprobes, as indicated. Pulled‐down PUM proteins were analyzed by immunoblotting. (C) RNA immunoprecipitation assays. Lysates from shRNA-transduced MOLM-14 cells were immunoprecipitated (IP) using control (IgG), PUM1 or PUM2 antibody, as indicated. Upper panel: Immunoblot analysis of the indicated proteins. Lower panel: RT-qPCR analysis of FOXP1 mRNA present in the IPs. The results are expressed as percent of input (n = 3). (D) Luciferase assay. MOLM-14 cells were transfected with control (siCtrl), PUM1 (siPUM1), or PUM2 (siPUM2) siRNA, and with the indicated luciferase reporter construct harboring the FL FOXP1-3′UTR. Relative luciferase activity was monitored 24 hours later and expressed as the ratio between Renilla luciferase (Rluc) activity and Firefly luciferase (Fluc) activity, used as transfection control. Data are expressed relative to expression in FOXP1-3′UTR FL and siCtrl transfected cells (n = 4). (E) Luciferase assays were performed as in panel D, with luciferase constructs harboring the indicated regions of the FOXP1-3′UTR (A-C) with WT, or doubly MUT1/2 PBEs. Data are expressed relative to expression in WT-FOXP1-3′UTR-C and siCtrl transfected cells (n = 4). (F) Luciferase assay. MOLM-14 cells were transfected with FOXP1-3′UTR-C luciferase reporter constructs harboring WT PBE1 and PBE2, mutated PBE1 (MUT1), mutated PBE2 (MUT2), or doubly mutated PBE1 and PBE2 (MUT1/2). Relative luciferase activity was monitored 24 hours later, expressed as the ratio Rluc/Fluc, and compared relative to expression in WT-FOXP1-3′UTR-C transfected cells (n = 4). Data are expressed as mean ± SEM. *P < .05; **P < .01; ***P < .001 (Student t test).

PUM1 and PUM2 contribute to human HSPC expansion. (A) RT-qPCR analysis of hPUM1 and hPUM2 transcripts in human stem/progenitor cell subpopulations: HSC (CD34⁺CD38^lowCD90⁺), MPP (CD34⁺CD38^lowCD90⁻), CMP (CD34⁺CD38⁺CD45RA⁻IL-3Rα^low), MEP (CD34⁺CD38⁺IL-3Rα⁻CD45RA⁺), and GMP (CD34⁺CD38⁺IL-3Rα⁺CD45RA⁺). Results are normalized to GAPDH expression and expressed relative to PUM expression in HSCs (n = 3). (B-F) Cord blood CD34⁺ cells were transduced or not (nt) with lentiviral vectors encoding shRNA targeting PUM1 (shPUM1-a, sequence #a in supplemental Table 8), PUM2 (shPUM2-a, sequence #a in supplemental Table 8), or luciferase as control (shCtrl), and were maintained in culture posttransduction. (B) Immunoblot analysis of the indicated proteins at day 4 posttransduction. Actin was used as loading control. (C) Cell expansion analysis along time of culture posttransduction (n = 4). (D) Cell cycle analysis upon propidium iodide labeling at day 3 posttransduction (n = 4). (E) Annexin V labeling at day 5 posttransduction (n = 4). (F) At day 7 posttransduction, shRNA/GFP⁺ output cells were seeded in methylcellulose medium and numbering of CFC was assessed after 2 weeks (n = 3); representative micrographs of colonies are presented on the right side (×20). BFU-E (burst forming unit-erythroid), GM-CFC (granulomacrophagic colony forming cell). (G) Cord blood CD34⁺ cells from the same batch were transduced with lentiviral vectors encoding shRNA targeting PUM1 (shPUM1-a), PUM2 (shPUM2-a), or luciferase as control (shCtrl). Three days posttransduction, sublethally irradiated NSG-immunodeficient mice received a 1:1 mixture of sorted shPUM/Tomato⁺ CD34⁺ cells and shCtrl/GFP⁺ CD34⁺ cells. Presence of GFP⁺ and Tom⁺ cells among human BM CD45⁺ cells of engrafted mice was scored 12 weeks later by FACS analysis. Each symbol represents data from a single chimeric mouse. Data are expressed as mean ± SEM. *P < .05; **P < .01; ***P < .001 (Student t test).

PUM1 and PUM2 contribute to murine HSPC expansion. (A) RT-qPCR analysis of mPum1 and mPum2 transcripts in mouse stem/progenitor subpopulations: LT-HSC (long-term HSC, Lin⁻Sca⁺cKit⁺CD34⁻CD150⁺), ST-HSC (short-term HSC, Lin⁻Sca⁺cKit⁺CD34⁺CD150⁺), MPP (Lin⁻Sca⁺cKit⁺CD34⁺CD150⁻), CMP (Lin⁻Sca⁻cKit⁺CD34⁺CD16⁻), MEP (Lin⁻Sca⁻cKit⁺CD34⁻CD16⁻), and GMP (Lin⁻Sca⁻cKit⁺CD34⁺CD16⁺). Results are normalized to Gapdh expression and expressed relative to Pum expression in LT-HSCs. (B-F) LSK cells were transduced with lentiviral vectors encoding the indicated shRNA targeting mPum1 (shmPum1) or mPum2 (shmPum2) or both (shmPum1a+2a) or luciferase as control (shCtrl) (sequences detailed in supplemental Table 8). shRNA/GFP⁺ LSK cells were sorted 2 days after transduction and maintained in liquid culture. (B) Immunoblot analysis of the indicated proteins at day 7 posttransduction. p85-PI3kinase was used as loading control. (C) Cell expansion analysis along time of culture posttransduction (n = 3). (D) Cell cycle analysis upon propidium iodide labeling at day 5 posttransduction (n = 4). (E) Apoptosis analysis at day 7 posttransduction through AnnexinV-PE/7AAD labeling (n = 4). (F) At day 7 posttransduction, shRNA/GFP⁺ output LSK-derived cells were seeded in methylcellulose medium and numbering of CFC was assessed after 7 days (n = 5); representative micrographs of colonies are presented on the right side (×20). (G-I) Murine LSKCD150⁺ cells were transduced with lentiviral vectors encoding shRNA targeting mPum1 (shmPum1-a) or mPum2 (shmPum2-a) or luciferase as control (shCtrl). shRNA/GFP⁺ cells were sorted 2 days after transduction and maintained in liquid culture. (G) Immunoblot analysis of the indicated proteins at day 7 posttransduction. β-Tubulin was used as loading control. (H) Cell expansion analysis along time of culture posttransduction (n = 3). (I) Three days posttransduction, lethally irradiated C57BL/6-Ly5.2 mice received 15 000 sorted shRNA/GFP⁺ LSKCD150⁺ cells from C57Bl/6 (Ly5.1) donors, in competition with the same amount of GFP⁻ LSKCD150⁺ cells from Ly5.1 mice, together with 1.5 × 10⁵ Ly5.2 BM cells. Presence of GFP⁺ cells in CD45.1⁺ BM cells of engrafted mice was assessed 4 months later by flow cytometry (at least 10⁵ events). Each symbol represents data from a single chimeric mouse. (Figure representative of 1 experiment out of 2). Data are expressed as mean ± SEM. *P < .05; **P < .01; ***P < .001 (Student t test).