Page 172 - 2019_06-Haematologica-web

A.C. Glembotsky et al.
delayed MK maturation, and low ploidy levels.2 On this basis, it seems reasonable to consider the possibility that, in vivo, FPD/AML MK may not be able to fully compensate for the decrease in α2 and, in this scenario, α2 downregu- lation could possibly contribute to impaired platelet pro- duction in patients in addition to the above-mentioned MK abnormalities.
Considering that FPD/AML is a heterogeneous condition, study of additional pedigrees would be useful to determine whether patients harboring RUNX1 mutations different from those included in this study display similar defects.
In conclusion, gene expression analysis of RUNX1 knock-down or mutated MK proved to be a suitable approach to identify novel RUNX1 targets involved in platelet biology. TREML1 and ITGA2 may now be added to the growing list of genes regulated by RUNX1 in the megakaryocytic lineage. Down-regulated expression of these genes in patients contributes to the platelet defects induced by RUNX1 mutations. These findings highlight the key role of RUNX1 as a master regulator of the MK lineage, where it modulates the expression of a diverse array of genes crucial to platelet production and function. They also help further unravel the molecular mechanisms underlying the FPD/AML phenotype.
Funding
This study was supported by a French grant from the Ligue National Contre le Cancer (LNCC, équipe labellisée to HR, 2013 and 2016), the European grant ERA-NET (to C. Balduini, 2013), the Argentinian grant from the National Agency for Scientific and Technological Research (to PH, 2012), a grant from the Fondation Nelia et Amadeo Barletta (to PH, 2017) and the cooperation pro- gram between France and Argentina, Ecos-Sud-Ministerio de Ciencia, Tecnología e Innovación Productiva (MINCyT) (to HR and PH, 2016). DS was supported by a postdoctoral fellowship from LNCC. The microarray was funded by the apprenticeship tax from Gustave Roussy, Villejuif, France.
Acknowledgments
We thank the patients and their families for participating in this study. We thank P. Rameau for flow cell sorting and flow cytometry analysis (PFIC, Gustave Roussy, Villejuif, France), G. Meurice for transcriptome analysis (UMS AMMICA, INSERM US23/CNRS UMS S3665, Gustave Roussy, Villejuif, France), Gabriel Correa (Instituto Lanari, University of Buenos Aires, Argentina) for coagulation studies, Mirta Schattner (IMEX- CONICET, Buenos Aires, Argentina) for help with the flow chamber assay and Daniela Ayala (UE IDIM-CONICET) for help with Western blot experiments.
References
1. Tijssen MR, Cvejic A, Joshi A, et al. Genome-wide analysis of simultaneous GATA1/2, RUNX1, FLI1, and SCL binding in megakaryocytes identifies hematopoiet- ic regulators. Dev Cell. 2011;20(5):597-609.
2. Bluteau D, Glembotsky AC, Raimbault A, et al. Dysmegakaryopoiesis of FPD/AML pedigrees with constitutional RUNX1 mutations is linked to myosin II deregulat- ed expression. Blood. 2012;120(13):2708- 2718.
3. Zang C, Luyten A, Chen J, Liu XS, Shivdasani RA. NF-E2, FLI1 and RUNX1 collaborate at areas of dynamic chromatin to activate transcription in mature mouse megakaryocytes. Sci Rep. 2016;6:30255.
4. Ichikawa M, Asai T, Saito T, et al. AML-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis. Nat Med. 2004;10(3):299-304.
5. Schlegelberger B, Heller PG. RUNX1 defi- ciency (familial platelet disorder with pre- disposition to myeloid leukemia, FPDMM). Semin Hematol. 2017;54(2):75- 80.
6. Michaud J, Wu F, Osato M, et al. In vitro analyses of known and novel RUNX1/AML1 mutations in dominant familial platelet disorder with predisposi- tion to acute myelogenous leukemia: impli- cations for mechanisms of pathogenesis. Blood. 2002;99(4):1364-1372.
7. Antony-Debré I, Manchev VT, Balayn N, et al. Level of RUNX1 activity is critical for leukemic predisposition but not for throm- bocytopenia. Blood. 2015;125(6):930-940.
8. Glembotsky AC, Bluteau D, Espasandin YR, et al. Mechanisms underlying platelet function defect in a pedigree with familial
platelet disorder with a predisposition to acute myelogenous leukemia: potential role for candidate RUNX1 targets. J Thromb Haemost. 2014;12(5):761-772.
9. Latger-Cannard V, Philippe C, Bouquet A, et al. Haematological spectrum and geno- type-phenotype correlations in nine unre- lated families with RUNX1 mutations from the French network on inherited platelet disorders. Orphanet J Rare Dis. 2016;11:49.
10. Sun L, Gorospe JR, Hoffman EP, Rao AK. Decreased platelet expression of myosin regulatory light chain polypeptide (MYL9) and other genes with platelet dysfunction and CBFA2/RUNX1 mutation: insights from platelet expression profiling. J Thromb Haemost. 2007;5(1):146-154.
11. Heller PG, Glembotsky AC, Gandhi MJ, et al. Low Mpl receptor expression in a pedi- gree with familial platelet disorder with predisposition to acute myelogenous leukemia and a novel AML1 mutation. Blood. 2005;105(12):4664-4670.
12. Antony-Debré I, Bluteau D, Itzykson R, et al. MYH10 protein expression in platelets as a biomarker of RUNX1 and FLI1 alter- ations. Blood. 2012;120(13):2719-2722.
13. Gilles L, Guièze R, Bluteau D, et al. P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulat- ed by AML-1. Blood. 2008;111(8):4081- 4091.
14. Washington AV, Schubert RL, Quigley L, et al. A TREM family member, TLT-1, is found exclusively in the alpha-granules of megakaryocytes and platelets. Blood. 2004; 104(4):1042-1047.
15. Smith CW, Raslan Z, Parfitt L, et al. TREM- like transcript 1: a more sensitive marker of platelet activation than P-selectin in humans and mice. Blood Adv. 2018;2(16):2072-2078.
16. Gattis JL, Washington AV, Chisholm MM,
et al. The structure of the extracellular domain of triggering receptor expressed on myeloid cells like transcript-1 and evidence for a naturally occurring soluble fragment. J Biol Chem. 2006;281(19):13396-13403.
17. Washington AV, Gibot S, Acevedo I, et al. TREM-like transcript-1 protects against inflammation-associated hemorrhage by facilitating platelet aggregation in mice and humans. J Clin Invest. 2009;119(6):1489- 1501.
18. Barrow AD, Astoul E, Floto A, et al. Cutting edge: TREM-like transcript-1, a platelet immunoreceptor tyrosine-based inhibition motif encoding costimulatory immunoreceptor that enhances, rather than inhibits, calcium signaling via SHP-2. J Immunol. 2004;172(10):5838-5842.
19. Giomarelli B, Washington VA, Chisholm MM, et al. Inhibition of thrombin-induced platelet aggregation using human single- chain Fv antibodies specific for TREM-like transcript-1. Thromb Haemost. 2007; 97(6):955-963.
20. Morales-Ortíz J, Deal V, Reyes F, et al. TLT- 1 is a prognostic indicator in ALI/ARDS and prevents tissue damage in the lungs in a mouse model. Blood. 2018;132(23):2495- 2505.
21. Derive M, Bouazza Y, Sennoun N, et al. Soluble TREM-like transcript-1 regulates leukocyte activation and controls microbial sepsis. J Immunol. 2012;188(11):5585-5592.
22. Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor? Blood. 2003;102(2):449-461.
23. Elagib KE, Racke FK, Mogass M, Khetawat R, Delehanty LL, Goldfarb AN. RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation. Blood. 2003;101(11):4333-4341.
24. Aneja K, Jalagadugula G, Mao G, Singh A, Rao AK. Mechanism of platelet factor 4
1254
haematologica | 2019; 104(6)