TGF-β Inhibition Rescues Hematopoietic Stem Cell Defects and Bone Marrow Failure in Fanconi Anemia

Abstract

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by progressive bone marrow failure (BMF) from hematopoietic stem and progenitor cell (HSPC) attrition. A greater understanding of the pathogenesis of BMF could improve the therapeutic options for FA patients. Using a genome-wide shRNA screen in human FA fibroblasts, we identify transforming growth factor-β (TGF-β) pathway-mediated growth suppression as a cause of BMF in FA. Blocking the TGF-β pathway improves the survival of FA cells and rescues the proliferative and functional defects of HSPCs derived from FA mice and FA patients. Inhibition of TGF-β signaling in FA HSPCs results in elevated homologous recombination (HR) repair with a concomitant decrease in non-homologous end-joining (NHEJ), accounting for the improvement in cellular growth. Together, our results suggest that elevated TGF-β signaling contributes to BMF in FA by impairing HSPC function and may be a potential therapeutic target for the treatment of FA.

Figure 1. TGF-β Pathway Disruption Enhances Human FA Cellular Growth under Genotoxic Stress

(A) RIGER P value analysis to identify top candidate genes in genome-wide screen. Top candidates are highlighted with different colors. Genes of the TGF-β pathway (BMP2, SMAD3, PDCD4) are pointed out by arrow. Genes involved in MMC metabolism (NQO1 and NFE2L2) are pointed out by arrowhead. (B) Clonogenic survival of GM6914 (FA-A fibroblasts) and FANCA-corrected GM6914 cells after shRNA mediated knockdown of SMAD3. Cells were exposed MMC and cell survival was determined after 10–12 days in culture. The average of two independent experiments is presented. Immunoblot in the right panel shows SMAD3 knockdown efficiency in GM6914 cells. (C,D) MMC sensitivity of EUFA316 (FA-G lymphoblasts) and FANCG-corrected EUFA316 cells incubated with or without 10μM SMAD3 inhibitor SIS3 or 10μM TGF-β inhibitor SD208. Cells were cultured in triplicates in presence of MMC and DMS0 or inhibitor for 5–6 days and survival was determined. Data are representative of 3 independent experiments. (E) Acetaldehyde sensitivity of EUFA316 and FANCG-corrected EUFA316 cells incubated with or without 10μM SD208. Cells were exposed to acetaldehyde for 3 hours and survival was determined after culturing them for 5–6 days in presence of DMSO or SD208 in triplicates. The data are representative of 3 independent experiments. (F) Acetaldehyde sensitivity of EUFA316 cells after shRNA mediated knockdown SMAD3 and TP53. Right panel shows the immunoblots of the lysates from EUFA316 cells showing shRNA-mediated knockdown of SMAD3 or p53. Error bars represent mean ± s.e.m. *, p<0.05; **, p<0.01; ***, P<0.001. See also Figure S1.

Figure 2. Inhibition of the TGF-β Pathway Rescues the Functional Defects of HSPCs from FA Mice

(A) qRT-PCR analysis showing the mRNA expression levels of Tgfb1, Smad3, Cdkn1a, Cdkn1c, Foxp3 and Atg5 in LSK populations from bone marrow of WT and Fancd2^−/− mice. (B) Quantification of the percentage of transduced (GFP⁺) and non-transduced (GFP⁻) LSK cells after 5 days in vitro culture. Bone marrow Lin⁻ cells from WT and Fancd2^−/− mice were transduced with lentiviruses encoding shSmad3-GFP or shControl-GFP and cultured in triplicates for 5 days. GFP+ and GFP- LSK cells were analyzed by flow cytometry. Data shown are the average of three independent experiments. (C) Depletion of Smad3 promotes the engraftment ability of Fancd2^−/− cells. Equal numbers of transduced Lin⁻GFP⁺ cells (20,000, CD45.2 cells) were transplanted into lethally irradiated recipients (CD45.1) along with 1×10⁵ helper cells (CD45.1). Percentages of donor-derived cells (GFP⁺CD45.2⁺) in peripheral blood of recipients were analyzed at 4 and 16 weeks post bone marrow transplantation (n=5 recipient mice per group). (D) Overexpression of FANCD2 in 293T cells suppresses TGF-β pathway activity as measured by TGF-β luciferase reporter activity. (E) Binding of FANCD2 to SMAD1 promoter upon DNA damage in 293T cells as detected by ChIP assay. FANCD2 biding was observed in the predicted binding region −2108 to −1950 bp of SMAD1 promoter. 293T cells were treated with MMC (1μM) for 8 hrs, and used in ChIP assays with an anti-FANCD2 antibody or IgG control antibody, followed by real-time PCR with indicated primer sets. ChIP data are presented as enrichment fold of FANCD2 binding to the specific regions after normalization with IgG. (F) Binding of FANCD2 to SMAD1 promoter upon DNA damage in corrected EUFA316 (+FAG) but not EUFA316 (+Vector) cells. ChIP assays with an anti-FANCD2 antibody or IgG control antibody were performed as described in (E) using a primer set for SMAD1 promoter region −2108 to −1950 bp. Error bars represent mean ± s.e.m. See also Figure S2.

Figure 3. TGF-β Pathway Inhibition Rescues Acetaldehyde-induced Genotoxicity in HSPCs from FA Mice

(A) Colony forming assay showing acetaldehyde resistance of Fancd2^−/− HSCs (CD48⁻CD150⁺LSK cells) with 1D11 treatment. HSCs from WT and Fancd2^−/− mice were exposed 2 mM acetaldehyde for 4h and then cultured in methycellulose medium containing 10 μg/mL 1D11 or its isotype control antibodies for 10 days. Survival of the hematopoietic progenitors was determined by colony quantification. (B) Inhibition of TGF-β pathway enhances acetaldehyde resistance of HSCs. Lin⁻ cells from bone marrow (BM) of WT and Fancd2^−/− mice were pretreated with 1D11 or isotype control antibody for 30min, followed by exposure to 2mM acetaldehyde for 4h. After 24h in culture with 1D11 or isotype control antibody, equal numbers of cells were transplanted into lethally irradiated recipients (CD45.1) along with 1×10⁵ helper cells (CD45.1). Donor-derived cells (CD45.2) in peripheral blood were analyzed by flow cytometric analysis at 4 and 16 weeks post transplantation (n=4~5 recipient mice per group). (C, D, E, F) 1D11 rescues acetaldehyde-induced DNA damage in HSCs and Lin⁻ cells. Representative images (C) and quantification (D) of γH2AX foci in HSCs from WT and Fancd2^−/− mice are shown. HSCs were pretreated with 1D11 or isotype control antibody followed by exposure to acetaldehyde for 4h, and harvested for immunofluorescence at the indicated time points. Hundred to 200 cells with more than 5 foci were counted for each sample. (Scale bar: 20μm). (E) Representative images of alkaline comets of bone marrow Lineage-negative cells from WT and Fancd2^−/− mice. Comet tails of Fancd2^−/− cells in the isotype plus acetaldehyde treatment group are highlighted by a star. (Scale bar: 50μm). (F) Olive tail moment demonstrating that 1D11 significantly prevents acetaldehyde-induced DNA damage in Fancd2^−/− bone marrow in vitro. Ninety-eight to 275 cells from each group were scored. Error bars represent s.e.m. See also Figure S3.

Figure 4. Inhibition of TGF-β Pathway Rescues Physiological Stress-induced Bone Marrow Failure in FA Mice

(A) TGF-β pathway inhibition by 1D11 prevents pI:pC induced DNA damage in HSCs in vivo. WT and Fancd2^−/− mice were injected intraperitoneally with pI:pC (5mg/kg) and 1D11 or isotype control antibody (10mg/kg). Forty-eight hours after the treatments, HSCs were sorted for DNA damage analysis by immunofluorescence staining or by the single cell comet tail assay. (B) Percentages of HSCs with γH2AX foci) and (C) 53BP1 foci are shown. Hundred to 150 cells were counted for each sample. (D) Olive tail moment in a comet assay demonstrating that 1D11 significantly reduces pI:pC-induced DNA damage in Fancd2^−/− HSCs. Ninety-two to 196 HSCs from each group were scored. (E) Schematic of pI:pC-induced bone marrow failure mouse model. (F) Peripheral blood analysis of pI:pC plus isotype or 1D11-treated WT and Fancd2^−/− mice as shown in (E). Red blood cell (RBC) counts, white blood cell counts (WBCs) and hemoglobin levels are shown (n=4–5 mice per group). (G) CFU-S content in the bone marrow of WT or Fancd2^−/− mice after four weeks of pI:pC plus isotype or 1D11 treatment as shown in (E) (H) Inhibition of TGF-β pathway rescues pI;pC-mediated functional defects of HSPCs. WT and Fancd2^−/− mice were exposed to pI:pC plus isotype or 1D11 for four weeks as shown in (E) and bone marrow cells were transplanted into lethally irradiated recipients (CD45.1) along with 1×10⁵ helper cells (CD45.1). Donor-derived cells (CD45.2) in peripheral blood were analyzed at 4 weeks post transplantation (n=4~5 recipient mice per group). (I, J) 1D11 rescues pI:pC-induced DNA damage in HSPCs. (I) Representative images and (J) quantification of γH2AX foci in HSPCs from WT and Fancd2^−/− mice after four weeks treatment with pI:pC plus isotype or 1D11 as shown in (E). (Scale bar: 20μm). Error bars represent s.e.m. See also Figure S4.

Figure 5. TGF-β Pathway Blockade Rescues the Function of Primary HSPCs from Patients with FA

(A) Hierarchial clustering and heat-map of the expression of TGF-β pathway genes in human FA and non-FA bone marrow samples using gene set enrichment analysis. (B–F) Colony forming assays using primary CD34⁺ bone marrow cells from five FA patients. Cells were transduced with lentiviruses encoding shControl, shTP53 or shSMAD3 followed by selection for puromycin resistance and then plated in methycellulose cultures or the cells were directly plated in methycellulose cultures containing GC1008 for colony growth. The hematopoietic colonies were counted after 10 days in culture. (G–I) Colony forming assays using FA-like CD34⁺ cells from human cord blood. As shown in the schematic (G), FA-like CD34⁺ cells were generated by transducing human cord blood CD34⁺ cells with lentivirus encoding shFANCD2. After selection for puromycin resistance, cells were transduced with lentivirus encoding shSMAD3-GFP and GFP⁺ cells were sorted by FACS and subjected to clonogenic assay in triplicates. qRT-PCR analysis (H) shows significant reduction of FANCD2 and SMAD3 expression in cells. Hematopoietic colonies were counted after 10–14 days in culture (I). (J) Colony forming assay of FA-like CD34⁺ cells with GC1008 treatment. Hematopoietic colonies were counted after 10–14 days in culture. (K) In vivo xenograftment assay. Transduced human cord blood CD34⁺ cells with shFANCD2 or shControl were selected with puromycin and transplanted into sub-lethally irradiated NSG mice. Recipient mice were treated with GC1008 at 3 doses per week for 2 weeks. Human cells were analyzed in the peripheral blood at 2 weeks post transplantation. Data shown are combined from two independent experiments (n= 4–5 recipient mice). (L,M) GC1008 rescues MMC-induced DNA damage in primary FA-like HSPCs. Representative images (L) and quantification (M) of γH2AX foci in cord blood CD34⁺ cells transduced with lentivirus encoding shFANCD2 or shControl. Puromycin resistant cord blood CD34⁺ cells transduced with lentivirus were exposed to MMC (100 ng/ml) for 2 hrs and allowed to recover for 24 hrs in presence of GC1008. Cells were then analyzed for γH2AX foci by immunofluorescence. Thirty to hundred cells with more than 5 foci were counted for each sample. (Scale bar: 20μm). Error bars represent mean ± s.e.m. See also Figure S5.

Figure 6. TGF-β Pathway Inhibition Upregulates HR and Downregulates NHEJ in HSCs from Fancd2^−/− mice. FA

(A) TGF-β pathway inhibition induces expression of the majority genes involved in DNA damage repair in HSCs from WT mice. HSCs were sorted from WT mice after 48h treatment with 1D11 or isotype control antibody, and used for qRT-PCR analysis. (B) The expression levels of DNA damage repair genes in Fancd2^−/− and WT HSCs. Some genes involved in HR and NHEJ pathways were pointed out by arrow. (C) Blockade of TGF-β pathway induces HR gene expression and downregulates NHEJ gene expression in HSCs from Fancd2^−/− mice. HSCs were sorted from Fancd2^−/− mice after 48h treatment with 1D11 or isotype control antibody, and used for qRT-PCR analysis. Some genes involved in HR and NHEJ pathways were pointed out by arrow. (D, E) Gene expression of representative NHEJ (D) and HR (E) genes Lig4, Prkdc, Brca2, and Xrcc1 in HSCs. (F) The frequency of HSCs in Fancd2^−/− mice after 48h treatment with 1D11 or isotype control antibody (n=4 mice per group). (G) Schematic of acetaldehyde sensitivity assay in bone marrow HSPCs from WT or Fancd2^−/− mice. (H) 1D11 does not protect the Fancd2^−/− HSPCs from genotoxic stress when HR is inhibited. HSPCs from WT or Fancd2^−/− mice were exposed to 1D11 and RAD51 inhibitors (10 μM) for 30 min followed by exposure to acetaldehyde for 4 hrs. The cells were then washed and cultured in presence of 1D11 and RAD51 inhibitors for five days and survival was determined. Error bars represent mean ± s.e.m. *p < 0.05; **p < 0.01. See also Figure S6.

Figure 7. TGF-β Pathway Inhibition Increases HR and Decreases NHEJ activities in FA Cells

(A, B) TGF-β pathway inhibition affects the choice of HR versus NHEJ pathways in repairing individual DNA breakpoints in FA cells. GM6914 cells (FA-A cells) or FANCA corrected GM6914 cells with shControl or shSMAD3 were used to generate traffic light reporter system, and then were infected with GFP-donor template and I-SceI lentivirus to generate DNA breakpoints. Quantification analysis of HR and NHEJ repair events is shown. (B) The ratio of HR to NHEJ activity based on the data in (A). (C) SD208 mediated TGF-β pathway inhibition increases HR events and decreases NHEJ events. Quantification of HR and NHEJ repair events in GM6914 cells exposed to SD-208 for 72 hrs as detected by traffic light reporter assay described in (A). (D) SMAD3 knockdown significantly increases HR efficiency. HR assay was measured in U2OS cells with DR-GFP reporter after transduction with lentivirus encoding indicated shRNAs. The representative of three independent experiments is presented. (E) NHEJ reporter assay showing decreased NHEJ activity in U2OS cells after inhibition of the TGF-β pathway by small molecule inhibitors. (F, G) TGF-β pathway inhibition promotes HR activity in FA cells. Representative images (F) and quantification (G) of RAD51 foci in MMC treated GM6914 (FA-A) cells or FANCA-corrected GM6914 cells with shRNA-mediated knockdown of SMAD3. Cells were exposed to 1μM MMC for 6h, and allowed to recover for 24h and 48h. RAD51 foci were then identified. One hundred cells were scored for RAD51 foci. (Scale bar: 20μm) Error bars represent mean ± s.e.m. See also Figure S7.