Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells
Ramesh C. Nayak, … , Carolyn Lutzko, Jose A. Cancelas
Ramesh C. Nayak, … , Carolyn Lutzko, Jose A. Cancelas
Published July 20, 2015
Citation Information: J Clin Invest. 2015;125(8):3103-3116. https://doi.org/10.1172/JCI80924.
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Research Article Hematology

Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells

Abstract

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE, which encodes neutrophil elastase (NE). However, a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end, we generated both normal and SCN patient–derived induced pluripotent stem cells (iPSCs), and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest, and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly, high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient–derived iPSCs through C/EBPβ-dependent emergency granulopoiesis. In contrast, sivelestat, an NE-specific small-molecule inhibitor, corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA, but not CEBPB; and promoting promyelocyte survival and differentiation. Together, these data suggest that SCN disease pathogenesis includes NE mislocalization, which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization.

Authors

Ramesh C. Nayak, Lisa R. Trump, Bruce J. Aronow, Kasiani Myers, Parinda Mehta, Theodosia Kalfa, Ashley M. Wellendorf, C. Alexander Valencia, Patrick J. Paddison, Marshall S. Horwitz, H. Leighton Grimes, Carolyn Lutzko, Jose A. Cancelas

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Figure 1

Impaired granulopoietic differentiation of hematopoietic progenitors derived from SCN patient iPSCs.

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Impaired granulopoietic differentiation of hemat...
(A) Colony forming cell assay of the hematopoietic progenitors derived from healthy donor (control 12 and control 13) and ELANE-mutant SCN iPSCs (SCN15 and SCN14). Cells were cultured in methyl cellulose semisolid medium with cytokines, and the myeloid (CFU-G, CFU-GM, CFU-M) and mix colonies were scored at day 14. (B) Schematic diagram of granulopoietic differentiation of iPSC-derived hematopoietic progenitors. (C) Top: Quantitative analyses of the granulopoietic differentiation of control iPSC–derived (control 12–derived) hematopoietic progenitors in liquid culture with myeloid differentiation cytokines. Bottom: Wright-Giemsa–stained cytospin at the end of the culture. (D) Top: Quantitative analyses of the granulopoietic differentiation of control iPSC–derived (control 13–derived) hematopoietic progenitors in liquid culture with myeloid differentiation cytokines. Bottom: Wright-Giemsa–stained cytospin at the end of the culture. (E) Top: Quantitative analyses of the granulopoietic differentiation of hematopoietic progenitors derived from SCN15 iPSCs in liquid culture condition with myeloid differentiation cytokines. Bottom: Wright-Giemsa–stained cytospin at the end of the culture. (F) Top: Quantitative analyses of the granulopoietic differentiation of hematopoietic progenitors derived from SCN14 iPSCs in liquid culture with myeloid differentiation cytokines. Bottom: Wright-Giemsa–stained cytospin at the end of the culture. (G) FACS-based ROS-generating-activity analyses of promyelocytes derived from control and SCN iPSCs. Scale bars: 10 μm. ProM, promyelocytes; My, myelocytes; MM, metamyelocytes; Neutr, neutrophils; Monos, monocytes. Data are presented as mean ± SD of individual values out of 4 or 5 independent experiments. Cont, control.