Research ArticleHematology Open Access | 10.1172/jci.insight.173665

Saravanan Ganesan,1 Sarah Awan-Toor,1 Fabien Guidez,1,2 Nabih Maslah,1,3 Rifkath Rahimy,4 Céline Aoun,1 Panhong Gou,1 Chloé Guiguen,1 Juliette Soret,5 Odonchimeg Ravdan,3 Valeria Bisio,6 Nicolas Dulphy,6,7 Camille Lobry,8 Marie-Hélène Schlageter,3 Michèle Souyri,1 Stéphane Giraudier,1,3 Jean-Jacques Kiladjian,1,5 Christine Chomienne,1 and Bruno Cassinat1,3

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Ganesan, S. in: JCI | PubMed | Google Scholar |

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Awan-Toor, S. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Guidez, F. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Maslah, N. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Rahimy, R. in: JCI | PubMed | Google Scholar |

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Aoun, C. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Gou, P. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Guiguen, C. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Soret, J. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Ravdan, O. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Bisio, V. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Dulphy, N. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Lobry, C. in: JCI | PubMed | Google Scholar |

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Schlageter, M. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Souyri, M. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Giraudier, S. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Kiladjian, J. in: JCI | PubMed | Google Scholar |

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Chomienne, C. in: JCI | PubMed | Google Scholar

1INSERM UMRS 1131, Institut de Recherche Saint-Louis, Université Paris Cité, Paris, France.

2INSERM U1232/LNC, Team Epi2THM, Université Bourgogne Franche-Comté, Dijon, France.

3Service de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France.

4Laboratoire de recherche en génétique et hématologie translationnelle, Institut Gonçalo Moniz, Salvador, Bahia, Brazil.

5INSERM CIC 1427, Université Paris Cité, Centre d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France.

6INSERM UMRS 1160, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

7Laboratoire d’Immunologie et d’Histocompatibilite, Hôpital Saint-Louis, AP-HP, Paris, France.

8INSERM U944, CNRS UMR7212, Institut de Recherche Saint-Louis, Université Paris-Cité, Paris, France.

Address correspondence to: Bruno Cassinat, Laboratoire de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France. Phone: 33.1.42.49.42.03; Email: bruno.cassinat@aphp.fr.

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Find articles by Cassinat, B. in: JCI | PubMed | Google Scholar

Authorship note: S Ganesan and SAT contributed equally to this work. CC and BC contributed equally to this work and are co–senior authors.

Published October 29, 2024 - More info

Published in Volume 9, Issue 23 on December 6, 2024
JCI Insight. 2024;9(23):e173665. https://doi.org/10.1172/jci.insight.173665.
© 2024 Ganesan et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published October 29, 2024 - Version history
Received: July 12, 2023; Accepted: October 22, 2024 View PDF Abstract

Despite the advances in the understanding and treatment of myeloproliferative neoplasm (MPN), the disease remains incurable with the risk of evolution to acute myeloid leukemia or myelofibrosis (MF). Unfortunately, the evolution of the disease to MF remains poorly understood, impeding preventive and therapeutic options. Recent studies in solid tumor microenvironment and organ fibrosis have shed instrumental insights on their respective pathogenesis and drug resistance, yet such precise data are lacking in MPN. In this study, through a patient sample–driven transcriptomic and epigenetic description of the MF microenvironment landscape and cell-based analyses, we identify homeobox B7 (HOXB7) overexpression and more precisely a potentially novel TGF-β/WNT/HOXB7 pathway as associated to a pro-fibrotic and pro-osteoblastic biased differentiation of mesenchymal stromal cells (MSCs). Using gene-based and chemical inhibition of this pathway, we reversed the abnormal phenotype of MSCs from patients with MF, providing the MPN field a potentially novel target to prevent and manage evolution to MF.

Graphical Abstract Introduction

Myelofibrosis (MF) is a life-threatening complication of BCR:ABL1-negative myeloproliferative neoplasms (MPNs) induced by the MPN cell clonal proliferation, leading to bone marrow fibrosis, extramedullary hematopoiesis, and acute leukemia transformation (1). For patients with MPN, this results in extremely poor quality of life and overall shortened survival (2). The progression of MPN to MF is reported with a frequency that could be as high as 10% at 10 years and 20% at 20 years (3) with an overall short median survival of 6 years when MF is established (2). So far, the knowledge gained on the MPN hematopoietic clone at the genetic and phenotypic level has provided instrumental descriptive information for MPN management but has not changed the prevention or treatment of MF. Retrospective MPN patient studies reveal that patients with polycythemia vera with homozygous or with a high allele burden for JAK2V617F mutations are more likely to progress to MF (4) while patients with post–essential thrombocythemia MF were more likely to have a high frequency of “nondriver” mutations affecting epigenetic regulation and the spliceosome machinery (ASXL1 and EZH2 mutations) (5). Recently, differential methylation of CpG sites of genes involved in cancer, embryonic development, inflammatory disease, or immunological diseases was identified in MF CD34+ cells (6). Unfortunately, no association between a single somatic gene mutation of the MPN clone and MPN evolution to MF was identified, spurring further studies to characterize alterations occurring in the microenvironment of the MPN clone.

Extensive studies in MF point to MPN mesenchymal stromal cells (MSCs) as key players. TGF-β has been shown for decades to have a direct role in fibrosis whether in MF or in other fibrotic diseases (1), and murine MF models show efficient targeting of the TGF-β receptor I (7) or TGF-β results in the reduction or inhibition of fibrosis (8). In patients with MPN, other cytokines such as IL-1β, IL-8, IL-2R, IL-6, TNF-α (9), CCL2 (10), lipocalin-2 (11), oncostatin-M (12), PDGF (13), FGF, and VEGF (14); inhibitors of metalloproteinase (15); and more recently IL-13 (16) have also been associated with the establishment of MF. An overall remodeling of the local hematopoietic niche by cytokine storms such as induced by IL-1β may also contribute to fibrosis (17). Recently, pro-fibrotic MSC subpopulations with increased expression of alarmins have been identified in patients with MPN who progress to fibrosis (18). Thus, clear evidence exists that during MPN evolution, both the MPN clone(s) and the MPN MSCs are subject to numerous external and internal altered signals. How these signals activate which pathways leading to fibrosis needs to be further unraveled.

To contribute to the ongoing research, we designed a study to comprehensively analyze the MSCs of patients with MF MPN. Leaning on a well-characterized MPN patient cohort both at the pathological and genomic level, we identify the TGF-β/WNT/homeobox B7 (HOXB7) axis in MF patients’ MSCs that is efficiently targeted in vitro.

Results

MSCs from patients with fibrotic MPN have distinct signatures and are biased toward osteoblast differentiation. While expanded MSCs from MPN patients meeting criteria of fibrosis (19), which will be referred to as fibrotic MPN MSCs (F-MPNs, n = 12) (Figure 1A and Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.173665DS1; and described in Methods), presented as stellar shaped cells in a clustered growth pattern, MSCs from age-matched controls (n = 17) showed a normal spindle shape (Figure 1B). The expansion and growth appeared slower in F-MPNs than in control MSCs albeit at a patient-dependent level (data not shown). F-MPNs expressed well-known cell surface MSC markers (CD105, CD90, CD73). However, their level of expression differed from control MSCs with an increased CD105 expression and a decreased CD90 and CD73 expression (Figure 1C). As reported previously (20), these F-MPNs also expressed high levels of the cell surface fibrosis marker LEPR (CD295), but a lower-than-reported expression of PDGFR-α (CD140a) (Figure 1C), which may indicate hyperstimulation of the PDGFR-α by PDGF. Indeed, analysis of the cytokine profile secreted from these expanded MSCs highlighted a pro-inflammatory profile for the F-MPNs as seen through an increased secretion of pro-inflammatory cytokines, such as IL-1α, IL-1β, IL-8, IL-6, CCL2, and PDGF. As expected, these F-MPNs secreted high levels of TGF-β1, TGF-β2, and TGF-β3, of which TGF-β1 is a known driver of fibrosis in MPN (1) (Figure 1D). None of the expanded F-MPNs (n = 12) expressed MPN driver mutations (Supplemental Table 1). RNA-Seq analysis of these F-MPNs (n = 7) equally underscored their distinct features compared with control MSCs (n = 4) at the transcriptional level (Figure 1E), with more than 1,600 differentially regulated genes (Figure 1F). F-MPNs’ gene expression profiles were enriched for fibrosis-associated genes (Figure 1, F and G), further validating the clinical and pathology selection criteria used for the MPN samples of this analysis (Supplemental Table 1). GSEA identified a dysregulation of fibrotic tissue pathways, such as the TGF-β pathway, cytoskeleton regulation, and NOTCH signaling (Figure 1G). Interestingly, we discovered a significant enrichment of WNT signaling and a disrupted MSC differentiation pattern, with an increase of osteoblast differentiation–associated genes and a decrease of adipocyte-associated genes, suggesting a differentiation bias of F-MPNs (Figure 1, H and I).

Figure 1

Expanded bone marrow MSCs from patients with MPN MF show an inflammatory profile and a biased osteoblast differentiation. (A) Experimental workflow of this study (illustration created using BioRender). ATAC-Seq, assay for transposase-accessible chromatin using sequencing. (B) Representative phase contrast images of expanded bone marrow MSCs from either patients with MPN MF patients or healthy age-matched controls. The spindle shape morphology of control MSCs (left) shifts to a stellar morphology in F-MPNs (right). Pictures were taken on passage 2. Scale bar = 20× original magnification. (C) Immunophenotypic characterization of expanded F-MPNs and control MSCs. All markers were present but differentially expressed in F-MPNs compared with controls. P values (*<0.05, **<0.01, ***<0.001) were calculated using unpaired t test. (D) Cytokine secretion profile of expanded MSCs. Day 3 supernatants at passage 2 were analyzed using a multiplexed Luminex assay. F-MPNs show a significant increase in inflammatory cytokine levels. P values (*<0.05, **<0.01, ***<0.001) were calculated using unpaired t test. (E) Heatmap and unsupervised hierarchical clustering by sample and gene were performed using the 300 genes (RNA-Seq data) that had the largest coefficients of variation based on DESeq2 analysis. The data are based on samples from the F-MPNs (n = 7) and control (n = 4) MSC group. (F) Volcano plot showing the relationship between the P values and the log2 fold-change in normalized expression (DESeq2) between F-MPNs (n = 7) and control MSCs (n = 4). Genes found to be the most differentially expressed are shown in the plot by P value. (G) Gene set enrichment analysis (GSEA) of RNA-Seq data between F-MPNs (n = 7) and control MSCs (n = 4) demonstrates an enrichment of gene sets of the fibrosis pathway. (H) Bar graph showing the pathways differentially expressed in the RNA-Seq analysis from control MSCs versus F-MPNs identified by DAVID pathway analysis. (I) Bar graph showing upregulation of osteoblast-associated genes and downregulation of adipocyte-associated genes in RNA-Seq data of F-MPNs.

To further verify and decipher the dysregulated gene expression observed in F-MPNs, we first looked to chromatin accessibility in samples of the RNA-Seq cohort (3 each for F-MPNs and control MSCs). Based on differential peak calling (DESeq2 analysis), the F-MPNs showed a distinct chromatin accessibility profile from control age-matched MSCs (Figure 2A). A further analysis provided insights into the accessible transcription factor (TF) binding sites. The motif analysis (HOMER-based motif analysis) pointed to an enrichment of binding sites for TFs involved in fibrosis (Figure 2E). In line with the upregulation of the TGF-β pathway genes observed in these F-MPN samples, we noted an enrichment of SMAD3, RUNX2, and SP5 TF motifs (Figure 2E). Interestingly, these TFs are associated with osteoblast differentiation (21, 22). We also noted a significantly distinct enrichment of other osteoblast lineage TF motifs (23–26), such as FRA-1, ATF-3, FRA-2, AP-1, and TEAD4, in F-MPNs (Supplemental Figure 1). Integrating RNA-Seq data and ATAC-Seq data of genes involved in osteoblast lineage differentiation clearly identified an increased accessibility of the promoters of ACTA2 (coding for α–smooth muscle actin, α-SMA) and PLZF1 genes (Figure 2B). However, other osteoblast lineage differentiation genes, such as those highly expressed in the RNA-Seq study (Figure 1I) or reported in the literature, did not come out as significant at the time of analysis (27–30). More significantly, the C/EBPα TF motif involved in adipocyte differentiation (31) was reduced, corroborating the RNA-Seq results (Supplemental Figures 2 and 3) and in line with the significantly reduced chromatin accessibility for a C/EBPα target gene, PPARγ, a master gene of adipocyte differentiation (32). These omics data of the F-MPNs’ differentiation capacity were validated by quantitative reverse transcription PCR (qRT-PCR) on a larger cohort of F-MPN and control samples including samples used for RNA-Seq and ATAC-Seq (n = 10 and 12 F-MPN and control MSC samples), which showed an increased expression of osteoblast genes (ACTA2, PLZF1) and decreased expression of adipocyte gene (PPARγ) and chondrocyte gene (SOX9; Figure 2C). Finally, to verify these molecular findings, we performed a functional differentiation assay of F-MPNs (n = 12) and control MSCs (n = 10). After 21 days of culture and using lineage-specific staining (alizarin red and Oil Red O, respectively), F-MPNs showed an increased osteodifferentiation and reduced adipocyte differentiation (Figure 2D). Overall, the findings of this integrated transcriptome and epigenome analysis in patients with MF MPN point to an upregulation of osteoblast differentiation and fibrosis genes in the MSCs of MPN patients with fibrosis. These results are in line with previous studies (18, 33) and validate this cohort for the following studies of MF patient MSCs continued below.