Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation
Dominique Bluteau, Alessandra Balduini, Nathalie Balayn, Manuela Currao, Paquita Nurden, Caroline Deswarte, Guy Leverger, Patrizia Noris, Silverio Perrotta, Eric Solary, William Vainchenker, Najet Debili, Remi Favier, Hana Raslova
Dominique Bluteau, Alessandra Balduini, Nathalie Balayn, Manuela Currao, Paquita Nurden, Caroline Deswarte, Guy Leverger, Patrizia Noris, Silverio Perrotta, Eric Solary, William Vainchenker, Najet Debili, Remi Favier, Hana Raslova
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Research Article Hematology

Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation

Abstract

Point mutations in the 5′ UTR of ankyrin repeat domain 26 (ANKRD26) are associated with familial thrombocytopenia 2 (THC2) and a predisposition to leukemia. Here, we identified underlying mechanisms of ANKRD26-associated thrombocytopenia. Using megakaryocytes (MK) isolated from THC2 patients and healthy subjects, we demonstrated that THC2-associated mutations in the 5′ UTR of ANKRD26 resulted in loss of runt-related transcription factor 1 (RUNX1) and friend leukemia integration 1 transcription factor (FLI1) binding. RUNX1 and FLI1 binding at the 5′ UTR from healthy subjects led to ANKRD26 silencing during the late stages of megakaryopoiesis and blood platelet development. We showed that persistent ANKRD26 expression in isolated MKs increased signaling via the thrombopoietin/myeloproliferative leukemia virus oncogene (MPL) pathway and impaired proplatelet formation by MKs. Importantly, we demonstrated that ERK inhibition completely rescued the in vitro proplatelet formation defect. Our data identify a mechanism for development of the familial thrombocytopenia THC2 that is related to abnormal MAPK signaling.

Authors

Dominique Bluteau, Alessandra Balduini, Nathalie Balayn, Manuela Currao, Paquita Nurden, Caroline Deswarte, Guy Leverger, Patrizia Noris, Silverio Perrotta, Eric Solary, William Vainchenker, Najet Debili, Remi Favier, Hana Raslova

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Figure 6

Decrease in MAPK signaling pathway is necessary for PPT formation by MKs.

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Decrease in MAPK signaling pathway is necessary for PPT formation by MKs...
MKs were differentiated from CD34+ cells in the presence of TPO. (A and B) Analysis of the MAPK pathway. Starved MKs were stimulated by TPO (100 ng/ml) for 5, 30, 60, and 120 minutes. (A) Western blot analysis of ERK and PERK performed at days 6, 10, and 14. (B) Flow cytometry analysis of PERK at days 7 and 10. The ratio of fluorescence intensity represents the ratio between the value obtained using PERK and control IgG isotype Abs for each time point of TPO stimulation. For A and B, experiments were performed 3 times with similar results. (C) Inhibition of MAPK pathway increases PPT formation. The CD41+ cells were sorted at day 6, the PD98059 inhibitor was added at day 8, and the percentage of PPT-bearing MKs was evaluated at days 11–13. One of 3 independent experiments with similar results is presented. Data were collected from triplicate wells and represent mean ± SD of triplicate. *P < 0.05, Student’s t test. (D) PPT-forming MKs cultured in presence or absence of PD98059 at day 13. (E and F) Inhibition or activation of MAPK pathway displayed an opposite effect on PPT formation. (E) CD34+ cells were transduced by retroviruses encoding for a dominant negative form of MEK1 and GFP (DNMEKGFP) or (F) for an active form of MEK1 (MEKa) and GFP. A retrovirus MIGR encoding for GFP was used as control. CD41+GFP+ cells were sorted at day 10 and the percentage of PPT-bearing MKs was evaluated at day 13 of culture. Data were collected from triplicate wells and represent mean ± SD.