No abstract

Mutations in , the gene for neutrophil elastase (NE), a protease expressed early in neutrophil development, are the most frequent cause of cyclic (CyN) and severe congenital neutropenia (SCN). We hypothesized that inhibitors of NE, acting either by directly inhibiting enzymatic activity or as chaperones for the mutant protein, might be effective as therapy for CyN and SCN. We investigated β-lactam-based inhibitors of human NE (Merck Research Laboratories, Kenilworth, NJ, USA), focusing on 1 inhibitor called MK0339, a potent, orally absorbed agent that had been tested in clinical trials and shown to have a favorable safety profile. Because fresh, primary bone marrow cells are rarely available in sufficient quantities for research studies, we used 3 cellular models: patient-derived, induced pluripotent stem cells (iPSCs); HL60 cells transiently expressing mutant NE; and HL60 cells with regulated expression of the mutant enzyme. In all 3 models, the cells expressing the mutant enzyme had reduced survival as measured with annexin V and FACS. Coincubation with the inhibitors, particularly MK0339, promoted cell survival and increased formation of mature neutrophils. These studies suggest that cell-permeable inhibitors of neutrophil elastase show promise as novel therapies for-associated neutropenia.

Heterozygous mutations in ELANE , the gene for neutrophil elastase, cause cyclic and congenital neutropenia through the programed cell death of neutrophil progenitors in the bone marrow. Granulocyte colony-stimulating factor is an effective therapy for these diseases, but alternative therapies are needed, especially for patients who do not respond well or are at high risk of developing myeloid malignancies. We developed an HL60 cell model for ELANE neutropenia and previously demonstrated that transient and regulated expression of mutant ELANE causes cell death by accelerated apoptosis. Knocking down the mutant gene or exposure to a potent inhibitor of neutrophil elastase rescued neutrophil development. Because of the great diversity in causative ELANE mutations, we generated stable HL60 clones expressing mutant P139L, C151Y and G214R and compared the effects of elastase inhibitor exposure to an ELANE knock-out line on cell development and function. ATRA induced differentiation demonstrated comparably impaired myeloid cell development for all three lines with upregulated expression of GRP78/BIP, an abnormality corrected by exposure of these cells to the elastase inhibitor MK-0339. The inhibitor and KO of mutant ELANE led to formation of neutrophils with comparable chemotactic and bactericidal capacities. We concluded that both strategies have great potential for the treatment of cyclic and congenital neutropenia. However, an orally absorbed, cell permeable inhibitor of neutrophil elastase, if proven safe and effective in a clinical trial, might be the better alternative to G-CSF or gene editing to treat ELANE neutropenia.

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. Disclosures Dale: Athelas, Inc.: Equity Ownership; Amgen: Consultancy, Research Funding; Sanofi-Aventi: Consultancy, Honoraria; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer-Ingelheim: Consultancy; Coherus: Consultancy.

Background : Mutations in ELANE, the gene for neutrophil elastase (NE), are the most frequent cause of both cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). G-CSF and HSCT are currently the only effective treatment options. We have examined the possibility that inhibitors of NE might also be treatment options. Methods: Cells: HL60 human promyelocytic cells, a commercially available cell line, and induced pluripotent stem cells (iPSc) derived from patient dermal fibroblasts or bone marrow stromal cells, reprogrammed using episomal vectors. NE inhibitors: Two cell permeable inhibitors were used: MK0339 provided by Merck and sivelestat from Sigma. Results: Expression of mutant NE in HL60 Cells Transient Expression:Transient transfection of HL60 cells with 4 different ELANE mutants (P139L, C151Y, V174_C181del, G214R) cloned in pcDNA 3.1 vector showed increased apoptosis after 48h of culture in 10% FBS RPMI and expression of mutant constructs. Addition of Merck NE inhibitor MK0339 with 1uM final concentration in culture significantly improved survival of cells expressing each of the mutations tested detected by Annexin V staining using flow cytometry. (p<0.0001) The effect of the sivelestat (100nM) was significantly effective on only G214R mutant. (p= 0.035) Regulated Expression:We tested the effects of Merck inhibitor MK0339 on tet-inducible HL60 cells with regulated expression of mutant NE (V174_C181del), a mutation found in a patient with severe congenital neutropenia who died of AML. We examined 3 characteristics of these cells: cell survival, myeloid differentiation, and proliferation. The cell lines, expressing wild type or mutant NE under a tetracycline regulated promoter, were treated with MK0339 and cultured for 72 hours in the presence or absence of doxycycline. MK0339 significantly reduced mutant NE triggered apoptosis and increased granulocytic differentiation induced by addition of all-trans retinoic acid (ATRA) in 5-day cultures. Expression of mutant NE also significantly reduced the growth rate of the HL60 cells, but addition of MK0339 promoted growth of these HL60 cells. Induced Pluripotent Stem Cells (iPSC) We have established iPS cell lines from three neutropenic patients (two brothers with P139L and another patient with G214R) and two healthy volunteers. Cell lines from all 3 patients consistently grew much slower and formed much smaller colonies than the controls using both mTeSR1/matrigel and DEF-CS feeder free culture medium systems. We observed more than 3-fold growth reduction of patient derived lines compared to healthy volunteers. We therefore tested the effects of MK-0339 and sivelestat under the same culture conditions. Growth of patient cell lines was enhanced and the cells appeared more robust, especially after addition of 1uM MK0339. The latter normalized patient derived iPSc proliferation. We also investigated survival of the iPS cells during growth in feeder free settings and the effect of the NE inhibitors. After 4 days of culture in DEF-CS system, the patient derived cell lines showed increased apoptosis measured by Annexin V. We observed more than 9-fold difference in increased apoptosis of patient derived cells compared to healthy volunteers. Addition of MK-0339 and sivelestat reduced the apoptosis. MK0339 significantly reduced the apoptosis in both mutant cell lines, while sivelestat significantly reduced the apoptosis in only P139L mutant cell lines. Again, sivelestat appeared to be less effective. Myeloid differentiation of iPSc was also impaired and inefficient in patient derived cell lines detected with granulocytic differentiation markers using flow cytometry. Introduction of MK0339 in this system normalizes the impaired myeloid differentiation capacities of patient derived iPSc lines. Summary/Conclusion: These studies clearly show effects of these selected inhibitors to enhance neutrophil formation in reproducible cellular models that mimics features of SCN and CyN. We believe this work will lead to novel therapies for ELANE associated neutropenia and open the door to other new therapies for neutropenia. Disclosures Dale: Amgen: Consultancy, Honoraria, Research Funding.