P751: UNRAVELING THE DIFFERENT PATHOMECHANISMS OF CONGENITAL AND CYCLIC NEUTROPENIAS: CYCLIC EXPRESSION OF HEMATOPOIETIC STEM-CELL-SPECIFIC TRANSCRIPTION FACTORS IN CYCLIC NEUTROPENIA
2023-08-01

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Severe congenital neutropenia (CN) and cyclic neutropenia (CyN) are disorders of hematopoiesis that differ markedly in disease severity. Mutations in the ELANE gene, encoding the neutrophil elastase (NE) protein, are responsible for most CyN and CN cases. These mutations lead to accelerated myeloid progenitor cell apoptosis due to the production of misfolded NE and subsequently enhanced unfolded protein response (UPR). Aims: Unraveling the different molecular pathomechanisms of congenital and cyclic neutropenias. Methods: 1) Identification of ELANE negative CD49f+ “escaper” hematopoietic stem cells by flow cytometry analysis; 2) qRT-PCR analysis of expression levels of the hematopoietic stem-cell-specific transcription factors CEBPA, MLL1, HOXA9, MEIS1, HLF; and 3) single-cell RNA-seq analysis. Results: We found that in CyN (n=9), independently of the cycle stage, a median of 2.8 % (range 1.2 – 9,6 %) CD34+ cells were CD49f+ early hematopoietic stem cells (eHSCs). Single-cell RNA expression analysis demonstrated that CD49f+ eHSCs did not express ELANE and thus escape from the UPR caused by mutated NE (escaper cells). The ELANE negative CD49f+ eHSCs were stimulated during the nadir and the ascending arm of the cycle by G-CSF that induced asymmetric division of eHSCs and production of equal percentages of CD49f+ eHSC and CD49f- progenitor cells. The resulting cell populations (CD49f+ and CD49f-) exhibited G-CSF-induced upregulation of the hematopoietic stem-cell-specific transcription factors, CEBPA, MLL1, HOXA9, and MEIS1 during the ascending arm of the cycle up to the peak. This G-CSF-induced upregulation showed an up to 8-fold increase in the mRNA level and resulted in the differentiation of myeloid progenitor cells to mature neutrophils at the CyN peak. Expression of hepatic leukemia factor (HLF), a biomarker for self-renewing early eHSCs, which was also 6-fold higher at the peak of the cycle than at the nadir, additionally supported the asymmetric division of CD49f+ cells. However, NE protein released by neutrophils at the cycle’s peak caused a negative feedback loop on granulopoiesis through the proteolytic digestion of G-CSF, limiting the biologically active G-CSF in the downward arm of the cycle and leading to neutropenia. CD34+ cells from six CN patients contained equal percentages of CD49f+ cells as CyN patients but failed to express mRNA levels of CEBPA, MLL1, HOXA9, and MEIS1, and therefore are not stimulated to proliferate and differentiate at the same level as CD49f+ cells from CyN patients. The lack of expression of these hematopoietic transcription factors in CN is caused by the lack of expression of Wnt-signaling peptides, especially of LEF1, as has been previously reported by us (Skokowa et al., Nat Med 2012). In CyN expression of LEF1 is normal. Summary/Conclusion: Summary/Conclusion: The differences between the pathomechanisms of congenital and cyclic neutropenia are the different expression of hematopoietic stem-cell-specific transcription factors in CD34+ cells, including CD49f+ eHSC in CyN, and the lack of expression of these transcription factors in CN. Keywords: Severe congenital neutropenia