Bortezomib Induces G-CSF-Triggered Granulocytic Differentiation of CD34⁺ Cells From CN Patients by Rescue of STAT5-Dependent Degradation of LEF-1 Protein

Abstract 388

LEF-1 is a decisive transcription factor involved in G-CSFR triggered granulopoiesis and is downregulated in severe congenital neutropenia (CN). However the exact mechanism of LEF-1 downregulation is still unclear. CN patients show response to G-CSF therapy, however therapeutically high doses of G-CSF are required to increase the neutrophil counts. Also CN patients are found to be at increased risk of developing AML (cumulative incidence ∼ 20%). Since LEF-1 is normally expressed in monocytes and lymphocytes whose differentiation is not affected in CN, it must be a granulopoiesis-specific mechanism downstream of G-CSFR signalling leading to LEF-1 down-regulation. STAT5 is one of the factors activated by G-CSF signalling pathway and has also been found to be hyper-activated in AML blasts. Also STAT5 is known to suppress LEF-1 target gene C/EBPα. Therefore we investigated the effects of activated STAT5 on LEF-1 gene expression and functions. We performed chromatin immunoprecipitation (ChIP) assay from CD34⁺ cell nuclear lysates and found two putative STAT5 binding sites. Reporter gene assays performed using LEF-1 gene promoter, which contains these STAT5 binding sites co-transfected with LEF-1 and STAT5a cDNA showed significant down-regulation of LEF-1 dependent activation in presence of constitutive active STAT5a (caSTAT5a). However, when we introduced deletion mutations in both STAT5 binding regions and performed the reporter gene assays we found that STAT5 dependent down regulation was independent of its direct binding to the LEF-1 gene promoter. Therefore we further checked the effects of activated STAT5 on LEF-1 protein levels. We found that co-transfection of caSTAT5a cDNA with LEF-1 significantly reduces the LEF-1 protein levels. This reduction in LEF-1 protein levels could be normalised by treating the cells with proteosome inhibitors, bortezomib and MG132. Moreover addition of bortezomib or MG132 to the reporter assays could rescue the effect of STAT5 dependent downregulation on LEF-1 gene autoregulation. We indentified a novel endogenous interaction of STAT5 with LEF-1 protein in the co-immunoprecipites from jurkat cell lysates and this interaction was confirmed insitu using Duolink proximity ligation assay. Furthermore we analysed G-CSF dependent granulocytic differentiation of CD34⁺ from CN patients treated additionally with bortezomib. Granulocytic differentiation was assessed by FACS using myeloid-granulocytic surface markers CD15, CD16 and CD11b. Interestingly, we found that addition of bortezomib to G-CSF resulted in granulocytic differentiation of CD34⁺ cells of CN patients in vitro, which was abolished in CN cells treated with G-CSF alone. This granulocytic differentiation was accompanied by restoration of LEF-1 protein levels by bortezomib. Taken together we identified a novel mechanism of LEF-1 downregulation in CN patients, which is via elevated degradation of LEF-1 protein mediated by hyper-activated STAT5. This in turn leads to abrogated autoregulation of LEF-1 mRNA expression. Along with, this study opens gates to a new therapeutic approach using bortezomib in combination with G-CSF for CN patients who do not respond or need high doses of G-CSF.