PML Plays a Key Role in Severe Congenital Neutropenia with Mutant ELANE Causing Neutrophil Elastase Protein Misfolding

Introduction: Severe congenital neutropenia (SCN) is a genetically heterogeneous disease characterized by recurrent infections and a predisposition for malignant transformation. A wide variety of autosomal dominant or sporadic mutations in ELANE encoding neutrophil elastase (NE) are the most frequent cause of SCN, whereas recessive mutations in HAX1 are responsible for the autosomal recessive form of SCN known as Kostmann syndrome. How ELANE and HAX1 mutations cause SCN is still unclear. A prevailing hypothesis is that cellular stresses either caused by protein misfolding or malfunction in the case of ELANE-SCN, or by mitochondrial dysfunction in the case of HAX1-SCN, are drivers of the neutropenia. We focused on the role of the promyelocytic leukemia protein (PML) because PML is implicated in controlling cellular stress responses caused by reactive oxygen species (ROS) and protein misfolding and may exert both oncogenic and tumor-suppressive functions.

Aims: (1) To elucidate which cellular stress mechanisms are involved in different genetic subtypes of SCN. (2) To assess the role of PML in SCN with a predicted ELANE misfolding mutation.

Methods: We generated induced pluripotent stem cells (iPSCs) from healthy control and SCN patients with non-overlapping mutations: ELANE-I60F, ELANE-R103L and HAX1-W44X. CD34⁺CD45⁺ Hematopoietic Stem and Progenitor cells (HSPCs) were derived from iPSCs using the STEMdiff™ Hematopoietic Kit (STEMCELL Technologies). PML^-/- iPSCs were created by introducing a stop codon in exon 3, shared by all PML isoforms, using CRISPR/Cas9 mediated genome editing.

Results: HSPCs derived from the SCN-iPSCs showed increased ROS levels as measured with CellROX Deep Red. Consequently, nuclear translocation of the antioxidant regulatory factor NRF2 was significantly elevated in both ELANE- and HAX1-mutant SCN HSPCs relative to controls. Mutation prediction analysis (Venselaar, BMC Bioinformatics 2010) showed that ELANE-I60F likely causes NE protein misfolding, whereas the ELANE-R103L mutation predictably causes NE malfunction by disrupting interactions with other proteins. The mutation in HAX1 was predicted to result in nonsense-mediated mRNA decay. Transcriptome analysis using Gene Set Enrichment Analysis (GSEA) confirmed upregulation of the nonsense mediated decay pathway in HAX1 mutant HSPCs and in line with previous studies (Klein et al, 2008), FACS analysis using TMRM and Mitotracker Red showed that loss of HAX1 protein reduced mitochondrial membrane integrity. Surprisingly, and in apparent conflict with the mutation prediction analysis, GSEA on ELANE-I60F HSPCs did not show increased expression of the classical unfolded protein response (UPR) pathway. Because PML has been implicated as an alternative player involved in degrading misfolded proteins (Guo, Mol Cell 2014), we investigated a possible link between ELANE-I60F and PML. Immunofluorescent staining showed increased numbers of PML nuclear bodies (PML-NBs) in ELANE-I60F derived HPSCs, but not in ELANE-R103L or HAX1-W44X HSPCs. Furthermore, GSEA showed upregulation of transcripts associated with PML chromatin binding in ELANE-I60F, but not in ELANE-R103L or HAX1-W44X cells. Deletion of PML by CRISPR-Cas9 revealed that PML enhanced MYC and mTORC1-induced transcription and cell cycle signatures in HSPCs from ELANE-I60F, suggestive of an oncogenic role of PML by inducing proliferation and metabolism in ELANE-I60F. In contrast, PML inhibited these pathways in HSPCs derived from healthy control iPSCs, indicative of its tumor-suppressive function in normal HSPCs. Finally, and perhaps most intriguingly, transcriptome analysis revealed that ELANE-I60F HSPCs expressed 5-fold higher levels of (mutant) ELANE transcripts than control HSPCs, which were reduced to basal levels after deletion of PML.

Conclusion: HAX1 and ELANE mutations cause oxidative stress in SCN-HSPCs by distinct mechanisms. We provide evidence for a dual role of PML in the pathogenesis of SCN caused by an ELANE mutation (I60F) associated with NE misfolding: (1) NE misfolding and increased oxidative stress cause elevated formation of PML-NBs, leading to increased expression of proliferation, cell cycle and metabolism associated transcripts, (2) PML strongly enhances the levels of ELANE transcripts, thus driving the expression of the disease causative ELANE mutant through a feed-forward mechanism.