Ataluren improves hematopoietic and pancreatic disorders in Shwachman-Diamond syndrome patients: a compassionate program case-series

Abstract

Shwachman-Diamond syndrome (SDS) is characterized by exocrine pancreatic insufficiency, neutropenia, and a high risk of myeloid malignancy. Most patients with SDS harbor nonsense mutations in Shwachman-Bodian-Diamond syndrome gene (SBDS), which encodes a ribosome assembly factor. We investigated the translational read-through effect of ataluren in three patients with SDS undergoing a compassionate use program for twelve months. The primary and secondary endpoints were restoring SBDS protein levels in hematopoietic cells and improving myelopoiesis, respectively. SBDS synthesis increased in hematopoietic cells, whereas the bone marrow showed improved cellularity with the maturation of myeloid progenitors. In parallel, absolute neutrophil count was improved in two out of three patients, whereas platelet count increased in all recruited patients. Ataluren treatment normalized mTOR phosphorylation in peripheral blood monocytes and lymphocytes, suggesting a reduction of ribosomal stress. The exocrine pancreatic function also improved. Although the reduced sample size may represent a major limitation of this work, our findings strongly encourages the further clinical development of ataluren to treat SDS.

Fig. 1. Ataluren-dependent restoration of SBDS protein synthesis was followed by improved bone marrow cellularity.

Bone marrow biopsies and aspirates were performed in three male SDS patients aged 13 to 20 (mean 16.3) before and after 6 months of therapy (6 m). Total proteins were isolated from BM-MNC and paraffin-embedded bone marrow biopsies were processed for immunohistochemical analysis. Colony assays were performed, seeding 1 × 10⁵ BM-MNC of each patient and healthy donor. a Western blot analyses (UPN26, representative) for SBDS before and after ataluren treatment with densitometry analysis. Other western blot analyses are included in Supplementary Fig. 1. b Immunohistochemical analysis of bone marrow biopsies collected from UPN26, UPN58, and UPN74 before (0) and after (6 m) therapy. c Representative colony assays demonstrating differences between patients with SDS and healthy donors (upper panel) and the effect of ataluren on SDS BM-MNC maturation after 6 months of treatment (lower panel). d Counts of myeloid (CFU-GM) colonies obtained from healthy donors (HD, white bars, n = 11, males 50%, mean age 38.3 years, range 15–52) and SDS patients at the baseline (NT, gray bars, n = 22, males 72%, mean age 18, range 7–44) and after 6 months of ataluren treatment (6 m, n = 3) after 14 days and 21 days of incubation. e Counts of erythroid (BFU-E) colonies from healthy donors (HD, white bars, n = 15) and SDS patients at the baseline (NT, gray bars, n = 11) and after 6 months of ataluren treatment (6 m, n = 3) after 14 days and 21 days of incubation. Data are mean ± SEM.

Fig. 2. Ataluren treatment improved myeloid maturation in bone marrow.

Immunophenotype of myeloid lineage was analyzed in whole bone marrow aspirates by flow cytometry (n = 3, mean age 16.3, range 13–20). Analysis of bone marrow aspirates obtained from UPN26 (a), UPN58 (b), UPN74 (c) before (0) and after 6 months of ataluren treatment (6 m). Granulocytes (PMN, white) were detected by morphological analyses using CD45 as leukocyte marker. PMN were gated, and promyelocytes (light blue), myelocytes (green), metamyelocytes (orange), and neutrophils (yellow) were detected, staining CD16 and CD11b.

Fig. 3. Ataluren treatment increased absolute neutrophil and platelet counts in peripheral blood of SDS patients.

a Comparison of absolute neutrophil count before (from −24 to 0 months) and during treatment (from 0.5 to 12 months) in UPN26 (red, solid dot), UPN58 (blue, hollow dot), and UPN74 (green, inverted solid triangle). Initiation of therapy is indicated by the red dotted line. b Comparison of peripheral neutrophil number before (pre) and after (post) ataluren treatment. Data are mean ± SEM (pre n = 3, post n = 9). Student’s t test has been indicated. c Comparison of platelet count before (from −24 to −12 months) and during treatment (from 0.5 to 12 months), as depicted in (a). d Comparison of peripheral platelet number before and after ataluren treatment. Data are mean ± SEM (pre n = 3, post n = 9). Mean age 16.3, range 13–20.

Fig. 4. Ataluren treatment improved the secretion of pancreatic enzymes including fecal elastase-1 and amylase.

a Fecal elastase-1 levels were measured by ELISA in stool samples before (0) and after 6 and 12 months of therapy in UPN26 (red, solid dot), UPN58 (blue, hollow dot), and UPN74 (green, inverted solid triangle) (n = 3, mean age 16.3, range 13–20). Data are represented as a gain of Fecal elastase-1 release relative to the baseline. b Amylase levels released into serum in each patient throughout 12 months of therapy (n = 3, mean age 16.3, range 13–20). Data are represented as a gain of serum amylase release relative to the baseline.

Fig. 5. Ataluren reduced the levels of phospho-mTOR (S2248) in leukocytes from patients with SDS.

Peripheral blood samples were collected from healthy donors (HD, white bars) and SDS patients (gray bars) before (0, dark gray), and after 3 and 6 months of therapy. Phospho-mTOR S2448 levels were analyzed by Phospho-Flow analysis in whole blood. Immunophenotypic markers were used to isolate different leukocyte populations. Data represent phospho-mTOR level in white blood cells (WBC) (a), lymphocytes (b), monocytes (c), and polymorphonuclear granulocytes (PMN) (d) of healthy donors (HD, n = 6) and SDS patients (n = 3, mean age 16.3, range 13–20) UPN26 (red lines, solid dot), UPN58 (blue lines, hollow dot), and UPN74 (green lines, inverted solid triangle). Data are mean ± SEM.