A Carboxy-Terminally Truncated G-CSF Receptor Abolishes Apoptosis Induction By Neutrophil Elastase G185R Mutant In Myeloid Cells,

Mutations in ELANE encoding neutrophil elastase (NE) have been identified in the majority of patients with severe congenital neutropenia (SCN). SCN patients are predisposed to acute myeloid leukemia (AML), and progression from SCN to AML is accompanied by acquisition of mutations in CSF3R encoding the G-CSFR receptor (G-CSFR) in approximately 80% of the patients. The mutations, which cause receptor’s carboxy-terminal truncation, are highly specific for SCN/AML, but the reason for the prevalence of the CSF3R mutations in patients with SCN/AML is unknown. We show here that NE expression was induced by G-CSF in myeloid 32D cells expressing the wild type (WT) G-CSFR (32D/GR), but not in cells expressing an SCN-derived truncated G-CSFR, d715 (32D/d715). Unlike the WT G-CSFR, the d715 receptor was unable to activate a 1.8-kb promoter fragment of mouse Elane. The NE mutants associated with SCN have been shown to cause premature apoptosis of differentiating granulocytic precursors. To address whether the d715 receptor might abolish apoptosis induction by the NE mutants in part by suppressing their expression, we placed the cDNA encoding the SCN-derived NE G185R mutant under the control of the Elane promoter and the resultant expression construct was transfected into 32D/GR and 32D/d715 cells. G-CSF treatment resulted in increased expression of G185R mutant and premature apoptosis in 32D/GR cells, but had no effect on G185R expression and cell survival in 32D/d715 cells. We further addressed whether the d715 receptor was capable of directly suppressing apoptosis induced by the G185R mutant. As expected, forced expression of the G185R mutant in 32D/GR cells led to accelerated apoptosis in response to G-CSF, but did not negatively influence the viability of 32D/d715 cells cultured in G-CSF. To evaluate whether the d715 receptor inhibited apoptosis induction by the G185R mutant in other hematopoietic cells, we expressed the G185R mutant in murine multipotent FDCP-mix A4 cells transfected with WT G-CSFR (FDCP/GR) or d715 receptor (FDCP/d715). Although FDCP/GR cells expressing G185R mutant showed no significant increase in apoptotic cell death upon treatment with G-CSF, they exhibited increased sensitivity to tunicamycin-induced endoplasmic reticulum (ER) stress. In contrast, the G185R mutant failed to sensitize FDCP/d715 cells to apoptosis induction by tunicamycin. Thus, it appears that the d715 receptor may abolish G185R mutant-induced apoptosis by two distinct mechanisms, i.e., suppression of its expression and direct inhibition of its pro-apoptotic action. Our data may have implications for understanding the prevalence of CSF3R mutations in patients with SCN/AML. We propose that the CSF3R mutations might represent a mechanism by which myeloid cells carrying the ELANE mutations evade the pro-apoptotic effect of the NE mutants in SCN patients.