Reticular Dysgenesis-associated Adenylate Kinase 2 deficiency causes failure of myelopoiesis through disordered purine metabolism

Abstract

Reticular Dysgenesis is a particularly grave from of severe combined immunodeficiency (SCID) that presents with severe congenital neutropenia and a maturation arrest of most cells of the lymphoid lineage. The disease is caused by biallelic loss of function mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) as substrate for adenosine triphosphate (ATP) synthesis in the mitochondria. Accordingly, it has long been hypothesized that a decline in OXPHOS metabolism is the driver of the disease. The mechanistic basis for Reticular Dysgenesis, however, remained incompletely understood, largely due to lack of appropriate model systems to phenocopy the human disease. We have used single cell RNA-sequencing of bone marrow cells from 2 reticular dysgenesis patients to gain insight into the disease pathology. Gene set enrichment for differentially expressed genes in different subsets of myeloid and lymphoid progenitor cells pointed to processes involving RNA and ribonucleoprotein assembly and catabolism as well as cell cycle defects. To investigate these findings and precisely mimic the failure of human myelopoiesis in culture, we developed a cell-tracible model of Reticular Dysgenesis based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. In this model, we have identified that AK2-deficienct myeloid progenitor cells exhibit NAD+ depletion and high levels of reductive stress accompanied by an accumulation of AMP and IMP while ADP and ATP are only mildly decreased. Our studies further show that AK2-deficienct cells have decreased de novo purine synthesis and increased purine breakdown, accompanied by decreased RNA and ribosome subunit cellular content. These data highlight the profound impact of mitochondrial dysfunction on the cellular redox state and nucleotide pool and identify the mechanistic basis of Reticular Dysgenesis as a defect in purine metabolism.

Single cell profiling of bone metastasis ecosystems from multiple cancer types reveals convergent and divergent mechanisms of bone colonization

“…We initially categorized the major cell groups (immune, epithelium, and stromal cells) based on the expression levels of conventional cell markers (immune: PTPRC/CD45 for pan-immune cells; CD3E/G for T cells; CD79A for B cells; CD68 for myeloid cells; EPCAM, KRT8/18/19 for epithelia cells; and PECAM, ACTA2, MYL9 and MYLK for stromal cells). For immune cell annotations, we employed machine learning support vector machines (SVM) 39,40 , we integrated, well-annotated single-cell datasets from various publications: (GSE179346 45 ; E-MTAB-8884, E-MTAB-9139 (https://www.ebi.ac.uk/arrayexpress/), GSE175604 46 , GSE120221 47 , GSE193138 48 , GSE159929 49 , GSE181989 50 , GSE159624 51 , GSE130430 52 , GSE139369 53 , GSE165645 54 , GSE128639 55 , GSE185381 56 , GSE166895 57 , GSE133181 58 , GSE135194 59 , resulting in a reference dataset comprising over 670,000 immune cells and encompassing more than 40 cell types and cellular states ( Table S2 ). Following cell type predictions in our dataset using the trained references, we conducted manual validation of classical cell marker expression 60 .…”