Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells

Abstract

Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.

Keywords: Shwachman-Diamond syndrome; ataluren; inherited bone marrow failure syndromes; myelodysplastic syndromes; translational readthrough-inducing drugs.

Figure 1.. Ataluren induces translational readthrough transfected HEK293T cells and in LCL carrying c.183-184TA>CT mutated SBDS, reducing p53 levels.

A, HEK293T cells transfected with the pcDNA3.1(+)-C-eGFP plasmid carrying the SBDS wild-type cDNA (wt) and SBDS carrying the c.183-184TA>CT stop mutation (magnification 10×, images were acquired by Olympus IX51 Inverted fluorescent microscope equipped with Olympus U-TV0.5XC-3 CCD Camera). The wt SBDS plasmid has been used as positive control. The green signal appears after transcription of the chimeric wtSBDS-P2A-eGFP construct and the subsequent translation and cleavage of the two separated SBDS and eGFP proteins (supplementary Figure 1). The SBDS c.183-184TA>CT mutated plasmid produces the green signal only in case of readthrough of the PTC, induced by ataluren. B, Representative experiment conducted on LCL generated from B cells of UPN58. Cells were incubated with ataluren 2.5-5 μM or with DMSO (1:2000) as control, for 24 h. SBDS protein levels were detected by western blot analysis. C, Scatter dot plots indicating the effect of ataluren on SBDS re-synthesis in LCL obtained from UPN24, UPN26, UPN58, UPN75, UPN82 and UPN106 after 24 h of treatment. Data are the mean ± SEM of 6 independent experiments. Normal distribution was tested by the Shapiro-Wilk test before running a two-tailed Student’s t-test for paired data (* p<0.05; ** p<0.01). D, Representative experiment conducted on LCL from UPN6, UPN43, UPN58, UPN82, and healthy donors (CTL1 and CTL2). Cells were grown in RPMI 1640 medium supplemented with 10% FBS for 24 h. Then, p53 protein levels were detected by western blot analysis (D) and densitometry analysis was carried out (E). Scatter dot plots represent the mean ± SEM of seven (n=7) experiments conducted on LCL derived from UPN6, UPN26, UPN43, UPN58, UPN82, UPN106. F, LCL obtained from UPN26 were treated with ataluren 2.5-5 μM for 24 h (representative experiment). Protein levels of p53 and SBDS were detected by western blot analysis, and densitometry analysis was performed (G). Scatter dot plots represent the mean ± SEM of five independent experiments (n=5) conducted in LCL from UPN26, UPN58, UPN82 as described in panel F. During western blot analyses, β-actin was quantified as loading control. Normal distribution was tested by the Shapiro-Wilk test before running the two-tailed Student’s t-test for paired data (*p<0.05).

Figure 2.. Ataluren improves 80S ribosome assembly and whole protein synthesis in SDS LCL.

A-C, Representative polysome profiles of LCL obtained from healthy donor (CTL8, A), and from UPN58 (B) and UPN24 (C) were evaluated in the absence (DMSO, 1:2000) or presence of 5 μM ataluren for 24h, by sucrose density gradient separation. D, The ratio of peak areas:total areas of fractions 40S, 60S, and 80S was calculated in six independent experiments (UPN58 n=2, UPN24 n=4) as performed in panels B-C. E-F, puromycin incorporation during protein synthesis was evaluated by SUnSET assay in LCL from UPN58 and UPN24 (representative experiments). G, Scatter dot plots represent the mean ± SEM of six independent experiments conducted in LCL from UPN58 (n=4) and UPN24 (n=2). Ponceau staining was used to evaluate the loading control. Normal distribution was tested by the Shapiro-Wilk test before running the two-tailed Student’s t-test for paired data (*p<0.05; **p<0.01, ns=non-significant).

Figure 3.. Ataluren improves myelopoiesis ex vivo in BM-MNC isolated from SDS patients.

A-B, BM-MNCs isolated from bone marrow aspirates of SDS patients and healthy controls (as specified in Supplementary Table 2) were seeded at a density of 1×10⁵ cells/ml in 3 cm Petri dishes containing StemMACS Lite medium supplemented with 20 ng/ml G-CSF and incubated in the absence (DMSO, 1:4000) or in the presence of 2.5 μM ataluren for 21 days at 37°C. CFU-GM (A) and BFU-E (B) were counted every 7 days. Histograms represent the mean ± SEM of 32 bone marrow biopsies (n=32) isolated from 21 SDS patients. Normal distribution was tested by the Shapiro-Wilk test before running the two-tailed Student’s t-test for paired data (**p<0.01; ***p<0.001; ****p<0.0001). C, Representative western blot analysis of SBDS carried out in myeloid colonies obtained from HD13 and HD14 after 21 days of incubation in the absence (DMSO, 1:4000) or in the presence of 2.5 μM ataluren as indicated in panels A-B. Protein extracts obtained from CTL5 LCL were used as internal control of SBDS protein levels. D, Representative western blot analysis of SBDS carried out in myeloid colonies obtained from UPN74 after 21 days of incubation in the absence (DMSO, 1:4000) or in the presence of 2.5 μM ataluren as indicated in panels A-B. E, Freshly isolated bone marrow sample from UPN37 was depleted by red blood cells using osmotic lysis, and then incubated with or without (DMSO, 1:2000) 5 μM ataluren for 24 h in IMDM supplemented with 10% FBS and 20 ng/ml G-CSF. Levels of CD11b, CD16, CD13, and CD45 were evaluated by flow cytometry (representative experiment). Flow cytometry analysis using the quadruple immunostaining for the granulocytic differentiation based on CD45, CD13, CD16 and CD11b was then performed. The analysis of the combination of CD16 expression with the granulocytic-monocytic lineage marker CD13 was used as a useful indicator of granulocytic differentiation. The percentage of each gated population is indicated within the plots.

Figure 4.. Chemotaxis-on-Chip assay reveals that ataluren improves chemotaxis in SDS BM-MNC differentiated into neutrophils.

BM-MNCs were isolated from bone marrow aspirates of 4 SDS patients (UPN26, UPN75, UPN80, and UPN147) and incubated in IMDM supplemented with 20 ng/ml G-CSF in the absence (DMSO, 1:2000) or in the presence of 5 μM ataluren for 24 h to stimulate neutrophil differentiation. Neutrophil maturation marker CD66b was evaluated by flow cytometry before testing chemotaxis. Single cell analysis of chemotaxis was performed on AVI video generated by capturing one image every minute for 60 mins in time-lapse through a CCD camera connected to the microscope. Untreated BM-MNC-derived neutrophils were exposed to IL-8 and fMLP chemical gradient (positive control) or tested in the absence of chemotactic stimuli (negative control) to normalize random movements of the cells, regardless of chemotaxis. A, Representative analysis of manual cell tracking conducted with ImageJ software in BM-MNC isolated from SDS patients (UPN80, UPN147) and healthy donor HD15 and differentiated into neutrophils as described above, in the absence (DMSO, 1:2000) or in the presence of 5 μM ataluren treatment. Each dot and line represent a single cell analysis of cell movement under chemical gradient sustained by IL-8 and fMLP. B, Forward migration index as calculated for each bone marrow sample tested. C, Scatter dot plots are mean ± SEM independent experiments as depicted in panel B. Normal distribution was tested by the Shapiro-Wilk test before running a two-tailed Student’s t-test (**** p<0.0001). E, Centre of mass has been calculated for each bone marrow sample tested under the same conditions indicated in panel A.

Figure 5.. Ataluren restores SBDS protein synthesis in primary osteoblasts isolated from SDS patients.

Primary OBs were isolated from bone biopsies obtained from 4 SDS patients (UPN24, UPN26, UPN72 and UPN106) and Healthy donors (HD1, HD2 and HD3). Cells (p1) were incubated in the absence (DMSO, 1:2000) or in the presence of 5-10 μM ataluren for 24-96 hr. Proteins were extracted in RIPA lysis buffer and western blot were performed to evaluate SBDS and β-actin levels. A, Representative experiment conducted on OBs isolated from UPN106 at 24 hr. B, Representative experiment conducted on OBs isolated from UPN26 at 24 hr. C, Scatter dot plots indicating the maximal efficacy of ataluren treatment on SBDS re-synthesis in OBs within the 96 h of treatment. Data are the mean ± SEM of 3-5 independent experiments. Normal distribution was tested by the Shapiro-Wilk test before running a two-tailed Student’s t-test (* p<0.05; ** p<0.01).