CRISPR/Cas9 Knock-in HL60 Cells Closely Simulate Cellular and Functional Abnormalities of ELANE associated Neutropenia; Phenotype Rescue with MK-0339 Neutrophil Elastase Inhibitor

Background: Mutations in ELANE are the most common cause of both cyclic and severe congenital neutropenia. Congenital neutropenia is characterized by low neutrophil counts in peripheral blood and impaired survival and maturation of myeloid precursors in bone marrow. G-CSF and HSCT are currently the only effective treatment options. To date, more than 100 different ELANE mutations have been reported. We previously described that some mutations (e.g. G214R and C151Y) are associated with more severe outcomes, while others (e.g. P139L and R220Q) result in relatively mild clinical phenotypes. (Curr Op Hematol. 2015;22:3-11) To explain the biological effects of various ELANE mutants we have created CRISPR/Cas9 edited ELANE mutants using the commercially available human promyelocytic cell line HL60.

Methods: We used CRISPR/Cas9 editing technology to create engineered HL60 cell lines with ELANE P139L and C151Y single point heterozygous mutations. Both ELANE mutant cell lines as well as wild-type HL60 cells were cultured for 7 days in complete RPMI supplemented with 2uM all-trans retinoic acid (ATRA) to trigger myeloid differentiation. Survival of these cell lines was investigated using Annexin V-PE staining and flow cytometric analysis. Granulocytic differentiation was evaluated using CD11b surface marker staining and flow cytometry and by performing manual differential cell counts. Unfolded protein response (UPR) was measured by western blotting using UPR specific antibodies.

MK-0339 is a potent, cell permeable, orally absorbed inhibitor of neutrophil elastase (NE), previously investigated in preclinical and clinical studies by Merck/DuPont as a potential anti-inflammatory drug. We have recently reported that MK-0339 increases cell survival and myeloid differentiation in cellular models of ELANE associated neutropenia. (Makaryan, et al, J Leukoc Biol. 2017;102(4):1143-1151). We examined the effects of MK-0339 on these cell lines.

Results: Annexin V staining showed more than 2-fold increase in apoptotic cells in both mutant cell lines compared to wild-type.

Granulocytic differentiation measured by surface CD11b expression was significantly impaired in both mutant cell lines (p-values <0.0001). Cytospins stained with Diff-Quik showed a typical block of myeloid differentiation and a significant deficiency of mature myelocytes.

Western blot analysis using antibodies to GRP78/BiP and ATF6 showed a typical UPR signature in both ELANE mutant cell lines compared to wild type. It is important to note that the C151Y mutant, the mutant clinically associated with more severe disease, shows more severe impairment compared to P139L. Addition of 1uM MK-0339 to the culture completely restored normal survival and myeloid differentiation of both mutant cell lines.

Conclusions: We believe CRISPR/Cas9 engineered HL60 cell lines expressing mutant NE are a highly reproducible and reliable cellular model for investigating genetic neutropenias. These results suggest that a panel of different mutant ELANE HL60 cell lines will help to elucidate the molecular and biochemical origin of phenotypic variability in ELANE associated neutropenia.