HS1 (HCLS1) Protein, a Binding Partner of HAX1, Induces G-CSF-Triggered Granulopoiesis Via LEF-1 Transcription Factor.

Abstract 548

Mutations in HS1 (HCLS1) associated protein × 1 (HAX1) have been described in patients with severe congenital neutropenia (CN). HAX1 is a ubiquitously expressed and until now it was unclear how HAX1 mutations cause isolated neutropenia in CN patients. So we were interested in investigating G-CSF-dependent HS1 (HCLS1) protein expression and phosphorylation in CN patients compared to healthy individuals, since HS1 (HCLS1) is a hematopoietic-specific protein and may link HAX1 to the G-CSFR signaling via the tyrosine kinases lyn and syk. Indeed, we found that treatment of CD34⁺ hematopoietic cells with G-CSF leads to phosphorylation of HS1 (HCLS1) protein and recruitment of receptor thyrosine kinases lyn and syk. In CN patients harboring HAX1 mutations, G-CSF failed to upregulate expression and phosphorylation of HS1 (HCLS1), suggesting that for HS1 phosphorylation functional HAX1 protein is required. Indeed, inhibition of HAX1 in CD34⁺ hematopoietic cells from healthy individuals significantly abrogated G-CSF-triggered HS1 (HCLS1) phosphorylation. Previously, we found that LEF-1 is a master regulator of granulopoiesis and is severely diminished in myeloid cells of CN patients with HAX1 mutations. Moreover, these cells also exhibited severe defects in F-actin assembly. Using immunoprecipitation assay of endogenous proteins, we found that upon G-CSF stimulation of CD34⁺ hematopoietic cells, HS1 (HCLS1) protein additional to HAX1, also binds to LEF-1 protein modulating its expression, transcriptional activity and subsequently granulopoietic differentiation. Using CFU, qRT-PCR and reporter gene assays we demonstrated that inhibition of HS1 (HCLS1) severely disturbed G-CSF-triggered granulocytic differentiation in line with downregulation of LEF-1 expression and functions, as well as with pathological redistribution of F-actin.

Taken together, in this study we demonstrated for the first time the connection between G-CSF receptor, HAX1 mutations, downregulation of HS1 (HCLS1) and LEF-1, abnormal F-actin assembly and the defective granulocytic differentiation in CN patients. Moreover, we demonstrated for the first time an ultimate direct connection between Wnt signaling and G-CSF cytokine cascade in granulopoiesis.