Steroid resistance in Diamond Blackfan anemia associates with p57Kip2 dysregulation in erythroid progenitors

Abstract

Despite the effective clinical use of steroids for the treatment of Diamond Blackfan anemia (DBA), the mechanisms through which glucocorticoids regulate human erythropoiesis remain poorly understood. We report that the sensitivity of erythroid differentiation to dexamethasone is dependent on the developmental origin of human CD34+ progenitor cells, specifically increasing the expansion of CD34+ progenitors from peripheral blood (PB) but not cord blood (CB). Dexamethasone treatment of erythroid-differentiated PB, but not CB, CD34+ progenitors resulted in the expansion of a newly defined CD34+CD36+CD71hiCD105med immature colony-forming unit-erythroid (CFU-E) population. Furthermore, proteomics analyses revealed the induction of distinct proteins in dexamethasone-treated PB and CB erythroid progenitors. Dexamethasone treatment of PB progenitors resulted in the specific upregulation of p57Kip2, a Cip/Kip cyclin-dependent kinase inhibitor, and we identified this induction as critical; shRNA-mediated downregulation of p57Kip2, but not the related p27Kip1, significantly attenuated the impact of dexamethasone on erythroid differentiation and inhibited the expansion of the immature CFU-E subset. Notably, in the context of DBA, we found that steroid resistance was associated with dysregulated p57Kip2 expression. Altogether, these data identify a unique glucocorticoid-responsive human erythroid progenitor and provide new insights into glucocorticoid-based therapeutic strategies for the treatment of patients with DBA.

Keywords: Bone marrow differentiation; Cell cycle; Development; Hematology; Hematopoietic stem cells.

Figure 1. Dexamethasone enhances the proliferation of human CFU-Es derived from PB CD34⁺ cells.

(A) Fold change in total expansion of PB-derived (red) and CB-derived (black, n = 5) CD34⁺ cells upon erythroid differentiation in the presence or absence of dexamethasone for 14 days. Data represent the mean ± SEM. (B) Total numbers of BFU-Es and CFU-Es generated from PB and CB cultures in the absence (PB–, CB–) or presence (PB+, CB+) of dexamethasone. Data are representative of 1 of 3 independent experiments. (C) Fold change in purified CFU-Es derived from PB (n = 6) and CB (n = 4) in the presence of dexamethasone relative to untreated controls after 14 days of expansion. Data represent the mean ± SEM. (D) Average size (area) of colonies generated from purified CFU-Es derived from untreated PB and CB after 5 days of culture in the absence (open circles) or presence (solid circles) of dexamethasone (n = 3). Data represent the mean ± SEM. Dex, dexamethasone. (E) Percentages of reticulocytes in 3 patients with DBA following treatment with prednisone, beginning on day 0. *P < 0.05, by 2-tailed Student’s t test (A, C, and D).

Figure 2. Dexamethasone specifically targets a transitional subpopulation of human CFU-Es derived from PB CD34⁺ cells.

(A) PB-derived CD34⁺ cells differentiated in the presence or absence of dexamethasone were evaluated as a function of their CD36 and CD34 expression profiles. Representative plots on day 4 of differentiation are presented. Data shown are representative of 1 of 3 independent experiments. (B) Gating strategy to define mature and immature transitional CFU-E progenitor populations based on the CD71/CD105 profiles of the CD34⁺CD36⁺ subset. Data are representative of 1 of 3 independent experiments. (C) Representative images of colonies formed by immature and mature CFU-Es as defined in B, in the presence of Epo alone or Epo, SCF, IL-3, IL-6, G-CSF, and GM-CSF (n = 3). Scale bars: 1 mm. (D) Colony size (area) generated by immature (red) and mature (black) CFU-Es, defined as in B and formed in the presence of Epo alone, in the absence (open circles) or presence (solid circles) of dexamethasone (n = 3). Data represent the mean ± SEM. (E) Representative histograms show CD71 and CD105 expression in PB-derived CD34⁺ cells differentiated in the absence (black) or presence (red) of dexamethasone (unsorted, day 4). Percentages of CD71^med and CD105^med are shown in the upper left quadrant of each plot. Data are representative of 1 of 3 independent experiments. (F) Quantification of CD105^med and CD105^hi cells following differentiation of PB-derived CD34⁺ cells in the absence (control, open circles) or presence (solid circles) of dexamethasone (day 4, n = 5). Data represent the mean ± SEM. *P < 0.05, by 2-tailed Student’s t test (D and F).

Figure 3. Proteomics studies highlight NR4A1 as a dexamethasone target in erythroid-differentiated PB progenitors.

(A) Ranked average log fold-change plots of differences in protein expression induced by dexamethasone in erythroid-differentiated PB progenitors. (B) Ranked average log fold-change plots of differences in protein expression induced by dexamethasone in erythroid-differentiated CB progenitors. (C) Top-20 proteins upregulated by dexamethasone in erythroid-differentiated PB progenitors based on the log fold change (log FC). (D) Top-20 proteins upregulated by dexamethasone in erythroid-differentiated CB progenitors based on the log fold change. (E) NR4A1 expression levels in purified PB- and CB-derived unsorted progenitors were evaluated by Western blotting on day 4 of expansion. Expression of NR4A1 relative to GAPDH is quantified below each lane. Data are representative of 1 of 3 independent experiments. (F) ssGSEA of proteins differentially regulated by dexamethasone. The top-10 upregulated and downregulated pathways between all samples are listed, and redundant pathways were eliminated.

Figure 4. Dexamethasone increases p57^Kip2 expression in Epo-induced PB CD34⁺ cells.

(A) Expression of p57^Kip2 and p27^Kip1 was evaluated by Western blotting in CD34⁺ progenitors during Epo-induced erythroid differentiation. Erythroid differentiation was controlled by evaluating α-globin expression. Data are representative of 5 independent experiments. (B) Quantification of the differences in p57Kip2 protein levels in PB and CB cultures between days 4 and 7 of erythroid differentiation (n = 5; values for day 4 were arbitrarily set at 1.). (C) Expression of p57^Kip2, p27^Kip1, and GAPDH in PB-derived and CB-derived progenitors was evaluated in sorted CFU-Es following 4 days of Epo-induced differentiation in the absence (–) or presence (+) of dexamethasone. Representative Western blots from 1 of 3 independent experiments are shown. (D) Quantification of the fold changes in p57^Kip2 and p27^Kip1 expression in purified PB-derived (n = 7, red circles) and CB-derived (n = 6, black circles) CFU-Es in the presence of dexamethasone relative to control conditions (arbitrarily set at 1). Data represent the mean ± SEM. (E) Quantification of the percentages of PB-derived BFU-Es, immature CFU-Es, and mature CFU-Es that were in S phase in the absence (open circles) and presence (solid circles) of dexamethasone. S phase was quantified by Hoechst 33342 staining (n = 5). Data represent the mean ± SEM. *P < 0.05, by 2-tailed Student’s t test (D and E).

Figure 5. Aberrant steroid-mediated induction of p57^Kip2 in erythroid progenitors from transfusion-dependent patients with DBA.

(A) The expansion of CD34⁺ cells derived from healthy controls (Ctrl) (black, n = 8), transfusion-dependent (TD) patients with DBA (blue, n = 5), and steroid-responsive (SR) patients with DBA (red, n = 3) is presented following a 7-day stimulation. Data represent the mean ± SEM. (B) Expression of p57^Kip2 and p27^Kip1 in erythroid progenitors from healthy controls and patients with DBA, either transfusion dependent or steroid responsive, was evaluated by Western blotting on day 7 of differentiation. Expression of p57^Kip2 relative to GAPDH is quantified below each lane, with control levels in the healthy donor arbitrarily set at 1. (C) Quantification of the fold change in dexamethasone-induced p57^Kip2 expression following expansion of CD34⁺ cells from healthy controls (n = 8) as compared with transfusion-dependent patients with DBA (n = 5) and steroid-responsive patients with DBA (n = 3). Data represent the mean ± SEM. *P < 0.05, by Kruskal-Wallis test with Dunn’s post hoc analysis with corrections for multiple comparisons (A and C).

Figure 6. Downregulation of CDKN1C (p57^Kip2) in CD34⁺ progenitors attenuates the impact of dexamethasone, accelerating erythroid differentiation, whereas olomoucine, a CKI, mimics the effect of dexamethasone.

(A) CD34⁺ progenitors were transduced with a lentiviral vector harboring an shRNA targeting luciferase (shLuc) or p57^Kip2 (shp57). Expression of p57^Kip2 was evaluated by Western blotting relative to GAPDH levels on day 7 after transduction. Data are representative of 1 of 3 independent experiments. (B) Expansion of control (shLuc) and p57^Kip2-downregulated (shp57) PB CD34⁺ cells was evaluated in the absence (open circles) or presence (solid circles) of dexamethasone after 14 days of culture (n = 5). Data represent the mean ± SEM. *P < 0.05, by 2-tailed Student’s t test. (C) Representative histograms of GPA expression in control (shLuc) and p57^Kip2-downregulated (shp57) progenitors on day 14 of differentiation. MFIs are indicated. Data are representative of 1 of 3 independent experiments. (D) Quantification of the ratio of immature CFU-Es to mature CFU-Es following expansion of PB-derived CD34⁺ cells in the absence (black circles) or presence of 100 nM dexamethasone (red circles) or 1 μM olomoucine (blue circles) on day 4 of culture. n = 5. Data represent the mean ± SEM. *P < 0.05, by Kruskal-Wallis test with Dunn’s post hoc analysis with corrections for multiple comparisons.

Figure 7. Dexamethasone increases p57^Kip2 levels in Epo-induced, BM-derived progenitors, expanding the immature CD105^med CFU-E subset.

(A) BM CD34⁺ progenitors were differentiated in the absence (open circles) or presence (solid circles) of dexamethasone, and total cell numbers on day 14 are presented (n = 5 independent experiments). Data represent the mean ± SEM. *P < 0.05, by 2-tailed Student’s t test. (B) Representative histograms of CD105 expression in BM-derived CD34⁺ cells differentiated in the absence (black) or presence (red) of dexamethasone on day 4 are shown, and the percentages of CD105^med cells are indicated. (C) Expression of p57^Kip2 and GAPDH in purified BM-derived and CB-derived mature CFU-Es, generated in the absence or presence of dexamethasone, was evaluated by Western blotting (blots from 1 of 3 independent experiments are shown).