Treatment of CN CD34⁺ progenitors with bortezomib or transduction of cells with LEF-1 lv promotes in vitro granulocytic differentiation. (A) CD34⁺ cells from CN patients (n = 2) were transduced with LEF-1 lv or ctrl lv, and granulocytic differentiation was induced as described in Materials and Methods; untransduced cells (controls) were cultured under the same conditions. Expression of LEF-1 mRNA and granulocytic (CD15) and myeloid (CD11b) surface markers as assessed on day 7 of culture. Cell surface marker expression was measured by FACS; representative histograms are presented. mRNA expression were assessed by qRT-PCR. LEF-1 mRNA levels were normalized to those of β-actin and are presented as AUs. Data represent means ± SD and are derived from 3 independent experiments, each in triplicate (**P < .01). (B) In vitro G-CSF–triggered granulocytic differentiation of CD34⁺ cells from 2 CN patients and 2 healthy individuals was performed in the presence or absence of bortezomib (10 nM), as described in Materials and Methods. Some cells from CN patients were transduced with lentivirus-based LEF-1 shRNA constructs. Surface expression of the granulocyte-specific markers, CD11b, CD15, and CD16, were assessed using FACS. Data represent means ± SD and are derived from 2 independent experiments, each in duplicate (*P < .05). (C) Schematic representation of the caSTAT5a-dependent degradation of LEF-1 protein in myeloid cells of CN patients. In CN patients, daily treatment with high pharmacological doses with G-CSF results in hyperactivation of STAT5a by phosphorylation owing to elevated JAK2 and diminished SOCS3 expression levels. Hyperactivated STAT5 activates NLK, promoting NLK–NARF interactions, and leading to recruitment of the NARF-NLK complex to LEF-1 protein and subsequent LEF-1 ubiquitination and degradation.