Understanding inherited neutropenias
Maturation arrest of granulopoiesis in patients with severe congenital neutropenia
In patients with severe congenital neutropenia, the maturation of granulocytic precursor cells arrests at the stage of promyelocytes and myelocytes, which accumulate in the bone marrow. The morphology of promyelocytes is also different: in patients with severe congenital neutropenia, promyelocytes have multiple vacuoles, a bulked nucleus, and are much bigger than promyelocytes of healthy individuals.
Leukemia: pathogenesis and drug targeting
Model of leukemogenesis in severe congenital neutropenia
The development of leukemia is a multi-step process characterized by a series of genetic changes that predispose hematopoietic cells with inherited severe congenital neutropenia-associated mutations to malignant transformation. Prolonged exposure of these cells to high dosages of G-CSF might result in the acquisition of CSF3R mutations that generate truncated G-CSFRs, thereby leading to hypersensitivity to G-CSF, defective G-CSFR signal transduction, and clonal proliferation. Subsequently, the acquisition of additional leukemia-associated mutations (for example in RUNX1) or chromosomal aberrations, such as trisomy 21 and monosomy 7, ultimately lead to leukemogenic transformation.
Mechanisms of G-CSF-triggered myeloid differentiation of HSPCs
Tyrosine-protein kinase JAK2 (JAK-2), which is hyperactivated in myeloid cells of patients with severe congenital neutropenia, leads to phosphorylation and activation of the signal transducer and activator of transcription (STAT) 3 and STAT5A, promoting the proliferation of hematopoietic stem cells over granulocytic differentiation; sustained activation of STAT5A has been shown in patients with severe congenital neutropenia. Tyrosine-protein phosphatase non-receptor type 11 (SHP-2) is another component of G-CSF signal transduction; SHP-2 induces dephosphorylation and, thereby, activation of tyrosine-protein kinase Lyn, which in turn in association with another tyrosine kinase Syk phosphorylates and activates hematopoietic lineage cell-specific protein (HCLS1), inducing myeloid differentiation of hematopoietic cells. Substantially increased levels of SHP-2 have been observed in neutrophils from patients with severe congenital neutropenia. In addition, lymphoid enhancer-binding factor 1 (LEF-1) a transcription factor member of the Wnt signaling pathway, is severely diminished in myeloid progenitors of patients with severe congenital neutropenia. LEF-1 activates the granulocyte-specific transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) and its target genes cyclin D1 and c-MYC, as well as anti-apoptotic factor baculoviral IAP repeat-containing protein 5 (survivin). Expression of all these proteins is severely diminished in myeloid cells of patients with severe congenital neutropenia, along with the reduced activity of phosphatidylinositol 3-kinases (PI3K), serine/threonine-protein kinases (Akt), HCLS1-associated protein X-1 (HAX-1), and hematopoietic lineage cell-specific protein (HCLS1). As a consequence, G-CSF therapy activates NAMPT and by this the compensatory emergency granulopoiesis pathway: NAMPT converts nicotinamide (NA) into nicotinamide adenine dinucleotide (NAD+), activating NAD+-dependent protein deacetylases, sirtuins (SIRTs), which in turn activate C/EBPβ.
Post-translational modification of proteins by de-/acetylation and its role in hematopoietic differentiation and leukemogenic transformation
Posttranslational modiﬁcation (PTM) is crucial for regulating the functions of many eukaryotic proteins. Among the prominent PTMs are phosphorylation; lysine acetylation, ubiquitination, and methylation. In the past decade, Lysine acetylation has been found in a variety of proteins but not studied as widespread as phosphorylation. Post-translational protein de-/acetylation lead to activation or deactivation of their functions. Besides, Lysine acetylation dependent on the context could interplay with other PTMs in an agonistic or antagonistic manner.
We recently identified nicotinamide phosphoribosyltransferase (NAMPT), as an essential enzyme mediating granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis. G-CSF treatment of both healthy individuals and congenital neutropenia patients increase Intracellular NAMPT as well as it’s plasma levels. NAMPT activates NAD(+)-dependent sirtuins which are responsible for the post-translational deacetylation of many transcription factors and adaptor proteins (Nature Medicine, 2009).
Genetic diagnostics of congenital neutropenias
Genes with germline mutations associated with severe congenital neutropenia
Data based on 650 patients with severe congenital neutropenia registered in the European and North-American Branches of the Severe Chronic Neutropenia International Registry. *Mutations in JAGN1, LAMTOR2, GFI1, LYST, USB1, or mitochondrial DNA.
Main cellular localization of proteins mutated in patients with congenital neutropenia
High frequency of cooperative acquired mutations in CSF3R and RUNX1 in severe congenital neutropenia patients who overt leukemia
Patterns of acquisition of leukemia-associated mutations were investigated using next-generation deep-sequencing in 31 CN patients who developed leukemia or MDS. Twenty (64.5%) of the 31 patients had mutations in RUNX1. A majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R mutations.
High throughput drug screening to identify new therapeutic agents for congenital neutropenias, leukemias, and solid tumors