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CONGENITAL AMEGAKARYOCYTIC THROMBOCYTOPENIA: FUNCTIONAL RESCUE OF A NOVEL MPL MUTANT IN PRIMARY HEMATOPOIETIC CELLS USING CRISPR-CAS9
Author(s):
Cédric Cleyrat
,
Cédric Cleyrat
Affiliations:
Pathology,University of New Mexico Cancer Center,Albuquerque,United States
Romain Girard
,
Romain Girard
Affiliations:
Pathology,University of New Mexico Cancer Center,Albuquerque,United States
Eun Ho Choi
,
Eun Ho Choi
Affiliations:
Pathology,University of New Mexico Cancer Center,Albuquerque,United States
Éric Jeziorski
,
Éric Jeziorski
Affiliations:
Service de Pédiatrie III,Hôpital Arnaud de Villeneuve,Montpellier,France
Thierry Lavabre-Bertrand
,
Thierry Lavabre-Bertrand
Affiliations:
Laboratory of Clinical Cytology and Cytogenetics,University Hospital of Nîmes,Nîmes,France
Sylvie Hermouet
,
Sylvie Hermouet
Affiliations:
Inserm UMR892/CNRS UMR6299,Centre de Recherche en Cancérologie Nantes-Angers (CRCNA) and Institut de Recherche en Santé - 2,Nantes,France
Serge Carillo
,
Serge Carillo
Affiliations:
Laboratory of Clinical Cytology and Cytogenetics,University Hospital of Nîmes,Nîmes,France
Bridget S. Wilson
Bridget S. Wilson
Affiliations:
Pathology,University of New Mexico Cancer Center,Albuquerque,United States
(Abstract release date: 05/18/17) EHA Library. Cleyrat C. 06/24/17; 181786; S499
Cédric Cleyrat
Cédric Cleyrat
Contributions
PDF Abstract
Abstract

Abstract: S499

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 16:45 - 17:00

Location: Room N104

Background
Thrombopoietin (Tpo) and its receptor, Mpl, are the principal regulators of early/late thrombopoiesis and hematopoietic stem cells maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is a rare inherited syndrome characterized by thrombocytopenia at birth, progressing to bone marrow failure and pancytopenia. The functional impact of CAMT mutations on Mpl is yet to be determined. Here we report unique familial cases of CAMT presenting with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T (K39N or Baltimore) mutation.

Aims
This study focuses on the functional characterization of this novel Mpl mutant and the use of genome editing as a novel therapeutic option for CAMT.

Methods
Human megakaryoblastic UT-7 and murine Ba/F3 cells stably expressing human wild-type (WT) Mpl or mutant Mpl fused to mNeonGreen were used as models. Confocal microscopy, proliferation and surface biotinylation assays, as well as co-immunoprecipitation and western blotting analysis, were used to elucidate the function and trafficking of Mpl mutants. Multiplex, flow-based, CRISPR-Cas9 gene editing was used to repair mutant MPL and rescue its function. Cord blood from the younger male sibling was used as a source of primary homozygous Mpl K39N/W272R CD34+ cells. CD34+ cells were edited using ribonucleoproteins electroporation followed by sequencing and functional assays such as flow cytometry and single colony assays.

Results
Consanguineous parents and their eldest daughter, all heterozygous for Mpl K39N/W272R, do not present any signs of disease. Their monozygotic twin daughters presented at birth with severe thrombocytopenia leading to a diagnosis of CAMT type I. Whole blood sequencing revealed the presence of a homozygous double Mpl K39N/W272R mutation, as their younger male sibling. One of the twins died after bone marrow transplant.

Confocal microscopy shows that a significant fraction of chimeric WT Mpl protein reaches the cell surface. Significant surface expression is also noted for Mpl K39N. In contrast, the chimeric Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, showed no detectable surface expression of the Tpo receptor while being strongly co-localized with ER marker calreticulin. Both WT and K39N-mutated Mpl were found signaling competent, while single or double mutants bearing W272R were unresponsive to Tpo. Tpo-induced signaling was partially restored via GRASP55 over-expression (forcing ER-trapped Mpl to traffic to the cell surface). Genome editing performed on cells carrying the W272R mutation restored the WT sequence and the response to Tpo, with similar cell proliferation as WT Mpl cells. Finally, when applied to primary Mpl K39N/W272R CD34+ cells, CRISPR-based gene editing rescued surface expression of Mpl and response to Tpo, as assessed by flow cytometry. Furthermore, edited CD34+ cells were able to generate a similar number of megakaryocytic colonies as control CD34+ cells in a single colony assay. Non-edited cells failed to do so.

Conclusion
We report a new double in cis mutation of Mpl (K39N/W272R) in the context of CAMT. Function of the deficient Mpl receptor could be rescued using two separate approaches: GRASP55 over-expression and CRISPR-Cas9 genome engineering. Successful editing of primary hematopoietic stem cells indicates direct therapeutic applications for gene editing in this disease.

Session topic: 11. Bone marrow failure syndromes incl. PNH - Biology

Keyword(s): Hematopoietic stem and progenitor cells, Gene therapy, C-mpl, Thrombocytopenia

Abstract: S499

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 16:45 - 17:00

Location: Room N104

Background
Thrombopoietin (Tpo) and its receptor, Mpl, are the principal regulators of early/late thrombopoiesis and hematopoietic stem cells maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is a rare inherited syndrome characterized by thrombocytopenia at birth, progressing to bone marrow failure and pancytopenia. The functional impact of CAMT mutations on Mpl is yet to be determined. Here we report unique familial cases of CAMT presenting with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T (K39N or Baltimore) mutation.

Aims
This study focuses on the functional characterization of this novel Mpl mutant and the use of genome editing as a novel therapeutic option for CAMT.

Methods
Human megakaryoblastic UT-7 and murine Ba/F3 cells stably expressing human wild-type (WT) Mpl or mutant Mpl fused to mNeonGreen were used as models. Confocal microscopy, proliferation and surface biotinylation assays, as well as co-immunoprecipitation and western blotting analysis, were used to elucidate the function and trafficking of Mpl mutants. Multiplex, flow-based, CRISPR-Cas9 gene editing was used to repair mutant MPL and rescue its function. Cord blood from the younger male sibling was used as a source of primary homozygous Mpl K39N/W272R CD34+ cells. CD34+ cells were edited using ribonucleoproteins electroporation followed by sequencing and functional assays such as flow cytometry and single colony assays.

Results
Consanguineous parents and their eldest daughter, all heterozygous for Mpl K39N/W272R, do not present any signs of disease. Their monozygotic twin daughters presented at birth with severe thrombocytopenia leading to a diagnosis of CAMT type I. Whole blood sequencing revealed the presence of a homozygous double Mpl K39N/W272R mutation, as their younger male sibling. One of the twins died after bone marrow transplant.

Confocal microscopy shows that a significant fraction of chimeric WT Mpl protein reaches the cell surface. Significant surface expression is also noted for Mpl K39N. In contrast, the chimeric Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, showed no detectable surface expression of the Tpo receptor while being strongly co-localized with ER marker calreticulin. Both WT and K39N-mutated Mpl were found signaling competent, while single or double mutants bearing W272R were unresponsive to Tpo. Tpo-induced signaling was partially restored via GRASP55 over-expression (forcing ER-trapped Mpl to traffic to the cell surface). Genome editing performed on cells carrying the W272R mutation restored the WT sequence and the response to Tpo, with similar cell proliferation as WT Mpl cells. Finally, when applied to primary Mpl K39N/W272R CD34+ cells, CRISPR-based gene editing rescued surface expression of Mpl and response to Tpo, as assessed by flow cytometry. Furthermore, edited CD34+ cells were able to generate a similar number of megakaryocytic colonies as control CD34+ cells in a single colony assay. Non-edited cells failed to do so.

Conclusion
We report a new double in cis mutation of Mpl (K39N/W272R) in the context of CAMT. Function of the deficient Mpl receptor could be rescued using two separate approaches: GRASP55 over-expression and CRISPR-Cas9 genome engineering. Successful editing of primary hematopoietic stem cells indicates direct therapeutic applications for gene editing in this disease.

Session topic: 11. Bone marrow failure syndromes incl. PNH - Biology

Keyword(s): Hematopoietic stem and progenitor cells, Gene therapy, C-mpl, Thrombocytopenia

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