TP53 suppression promotes erythropoiesis in del(5q) MDS, suggesting a targeted therapeutic strategy in lenalidomide-resistant patients

Abstract

Stabilization of p53 in erythroid precursors in response to nucleosomal stress underlies the hypoplastic anemia in myelodysplastic syndromes (MDS) with chromosome 5q deletion [del(5q)]. We investigated whether cenersen, a clinically active 20-mer antisense oligonucleotide complementary to TP53 exon10, could suppress p53 expression and restore erythropoiesis in del(5q) MDS. Cenersen treatment of ribosomal protein S-14-deficient erythroblasts significantly reduced cellular p53 and p53-up-regulated modulator of apoptosis expression compared with controls, accompanied by a significant reduction in apoptosis and increased cell proliferation. In a two-stage erythroid differentiation assay, cenersen significantly suppressed nuclear p53 in bone marrow CD34+ cells isolated from patients with del(5q) MDS, whereas erythroid burst recovery increased proportionally to the magnitude of p53 suppression without evidence of del(5q) clonal suppression (r = -0.6; P = 0.005). To explore the effect of p53 suppression on erythropoiesis in vivo, dexamethasone, a glucocorticoid receptor-dependent p53 antagonist, was added to lenalidomide treatment in eight lower-risk, transfusion-dependent, del(5q) MDS patients with acquired drug resistance. Transfusion independence was restored in five patients accompanied by expansion of erythroid precursors and decreased cellular p53 expression. We conclude that targeted suppression of p53 could support effective erythropoiesis in lenalidomide-resistant del(5q) MDS.

Conflict of interest statement

Conflict of interest statement: L.J.S. has equity ownership in Smith Holdings, which owns rights to the drug involved in this study. F.J.D. has employment, equity ownership, and membership on the board of directors or advisory committees in Eleos, Inc. R.A.W. is a consultant for Novartis, Celgene Corporation, Janssen, and Alexion. A.F.L. is a consultant for Celgene Corporation.

Figures

Fig. 1.

Cenersen-treated, RPS14-deficient erythroblasts show improved cell growth. (A) RPS14-deficient erythroblasts (sh41 and sh44) generated from human CD34+ bone marrow cells have a significantly diminished growth capability compared with the scramble control. Scramble, open circles; sh41, asterisks; sh44, open squares. (B–D) RPS14-deficient erythroblasts showed increased cell growth when treated with cenersen compared with treatment with control oligonucleotide. Cenersen, filled circles; control oligonucleotide, open circles. Viability was assessed by trypan blue exclusion. Results are means ± SEM (n = 7). P values were calculated by two-way ANOVA (A) or Wilcoxon signed-rank test (B–D). *P < 0.05; **P < 0.01.

Fig. 2.

Cenersen reduces p53 and PUMA expression in RPS14-deficient erythroblasts and decreases apoptosis. Cenersen treatment resulted in significant down-regulation of p53 (A) and its downstream target PUMA (B) in RPS14-deficient erythroblasts compared with control oligonucleotide-treated cells. Cenersen, black bars; control oligonucleotide, open bars. Expression levels are shown as means ± SEM of fluorescence intensity (n = 8). Representative photomicrographs are shown of p53 (C) and PUMA (D) immunofluorescence in RPS14-deficient erythroblasts that have undergone cenersen (Right) or control oligonucleotide treatment (Left), with p53 staining shown in green, PUMA staining in red, and DAPI in blue (magnification, ×630). (E) Percentage apoptosis as assessed by Annexin V-positive staining, shown as means ± SEM (n = 10). Cenersen, black bars; control oligonucleotide, open bars. (F) Representative flow cytometry contour plots showing a significant increase in the viable, nonapoptotic (Annexin V-negative, propidium iodide-negative) cell population in RPS14-deficient erythroblasts after cenersen treatment. P values were calculated by Wilcoxon signed-rank test: *P < 0.05; **P < 0.01.

Fig. 3.

Cenersen treatment decreased p53 expression in primary MDS erythroid progenitors. (A) Representative photomicrograph of p53 immunofluorescence in del(5q) MDS progenitors, with p53 staining shown in green and DAPI in blue (magnification: Upper, ×630; Lower, ×3,780). (B–D) Nuclear and cytoplasmic p53 FI levels in del(5q) MDS (n = 10; B), nondel(5q) MDS (n = 11; C), and normal controls (n = 6; D) with cenersen and control oligonucleotide treatment. Cenersen, black bars; control oligonucleotide, open bars. Values are mean FI ± SEM. P values were calculated by Wilcoxon signed-rank test: *P < 0.05; NS, not significant.

Fig. 4.

Cenersen treatment promotes erythroid colony-forming capacity. (A) BFU-E numbers were significantly increased in del(5q) MDS specimens (n = 10). This increase was not significant in nondel(5q) MDS (n = 11) or control (n = 6) specimens. (B) There is a significant correlation between the magnitude of BFU-E improvement and magnitude of nuclear p53 suppression in MDS erythroid progenitors treated with cenersen (Pearson correlation factor r = −0.6; P = 0.005). (C) Cenersen treatment increased the number of CFU-E in del(5q) (n = 9), nondel(5q) (n = 10), and control samples (n = 5). Results are means ± SEM. P values were calculated by Wilcoxon signed-rank test: **P < 0.01; NS, not significant.

Fig. 5.

Lenalidomide plus dexamethasone treatment suppresses p53 expression and promotes erythroid expansion. Bone marrow biopsies of treated patients were stained with p53 (A) and spectrin (B), with percentage of positive staining shown. Representative micrographs of a single responder are shown at both baseline (Left) and time of response (Right). (Upper) p53 staining was decreased between baseline and the time of response. (Lower) Spectrin staining increased from baseline to the time of response (magnification: ×600). Results are means ± SEM. P values were calculated by t-test: *P < 0.05; NS, not significant.