Conditional Expression of Mutant ELANE Produces Unfolded Protein Response but Fails to Promote Cell Death or Differentiation Block: What Is the Mechanism for Severe Congenital Neutropenia?

Introduction: Germline mutations in the neutrophil elastase gene ELANE cause ~60% of severe congenital neutropenia (SCN). SCN frequently transforms to acute myeloid leukemia concomitant with acquisition of a mutant, truncated CSF3R, e.g. CSF3R d715.Two hypotheses have been advanced to explain the neutropenia: mislocalization of ELANE or unfolded protein response (UPR) induced by mutant ELANE. We present here data that demonstrates UPR induction by mutant ELANE but fails to results in either enhanced cell death or block in neutrophilic differentiation.

Methods: We expressed mutant ELANE G185R found in patients with SCN. We used the IL-3-dependent murine myeloid 32D cell line that can terminally differentiate into neutrophils. Using the 32D cell, we developed 32D cell lines that express a chimeric rabbit growth hormone receptor (rGHR)/CSF3R receptor to prevent crosstalk with endogenous murine CSF3R. We used 32D cells that express either the wt or mutant d715 chimeric receptors: rGHR/wtCSF3R or rGHR/d715 respectively. The goal of our study was to identify how mutant ELANE expression is tolerated in the presence of wt CSF3R or CSF3R d715 signaling. We wanted to determine the effect of ELANE expression on cell survival, proliferation, differentiation, and UPR in context of wt vs mutant CSF3R signaling. To stimulate cytoplasmic CSF3R signaling the cells are treated with human growth hormone (hGH). We generated: rGHR/wtCSF3R-wtELANE, rGHR/wtCSF3R-G185R, rGHR/d175-wtELANE, and rGHR/d715-G185R. Cell proliferation, viability and differentiation assays were performed ± hGH.

Results: No differences were observed in viability or differentiation. Interestingly cells expressing the mutant ELANE G185R showed enhanced proliferation vs wt hELANE, observed only in the context of rGHR/wtCSF3R and not with rGHR/d715. Morphological and flow cytometry based differentiation assays identified cell lines expressing GHR/wtCSF3R showed terminal differentiation. However, as expected rGHR/d715 expressing cell lines did not differentiate. We next determined if UPR is observed in these cells. Quantitative reverse transcribed PCR (qPCR) and western blot analysis identified increased expression of BiP at mRNA level and CHOP at both mRNA and protein levels in cells expressing the mutant ELANE G185R. CHOP has been identified to be the pro-apoptotic component of the UPR. Thus we observed enhanced CHOP expression, but we observed a paradoxical increase in proliferation for cells expressing ELANE G185R. We next identified that only rGHR/wtCSF3R-G185R cells were sensitive to either tunicamycin or bortezomib treatment, potent inducers of UPR. Because these cell lines were stable transfectants and might have adapted to mutant ELANE expression, we next put wt or mutant ELANE in a doxycycline-inducible expression system and transduced parental 32D cells. Induction of ELANE was dose and time dependent. Similar to the stable expression system, phenotypic differences between ± doxycycline treatments were not observed even though an increase in mRNA of UPR mediators was detected: CHOP, BiP, GADD34, PERK ATF4, and ATF6. An interesting observation was that, although there was a pan increase in UPR protein mRNA, there was a reduction in XBP1 splicing. IRE-1 mRNA levels were also low or undetectable in our cell lines. This might explain reduced XBP1 splicing. However, when 32D parent cells were treated with thapsigargin, a potent inducer of UPR and cell death, an increase in XBP1 splicing was observed.

Conclusions: Our observations suggest that UPR induction by mutant ELANE is not strong enough to promote cell death and that mutant ELANE causes SCN through an alternative mechanism.

This work was supported by NIH R01R01HL128173.