A Novel PU.1 - Caspase 8/cFLIP Axis in Neutrophil and Macrophage Differentiation of AML Cells

The transcription factor PU.1 (SPI-1) plays an important role in numerous cellular processes of myeloid cells, such as cell survival, proliferation and differentiation. PU.1 is expressed at intermediate levels in hematopoietic progenitor cells, whereas progenitors expressing low amounts of PU.1 differentiate towards the lymphoid lineage, while increased amounts of PU.1 levels promoted macrophage or granulocyte differentiation. Additionally, low PU.1 expression levels contribute to the immature myeloid phenotype in acute myeloid leukemia (AML).

We recently linked PU.1 expression to TRAIL and chemotherapy sensitivity in AML cells. To further molecularly dissect the functions of PU.1 in myeloid cells, we focused on putative PU.1 targets associated with cell death responses emerging from a gene expression profiling experiment using Pu.1-null and Pu.1-restored 503 murine myeloid cells. We identified Caspase 8 (Casp8) and its paralogous gene c-Flip (aka FADD-like apoptosis regulator; Cflar) as PU.1-regulated genes, and showed putative PU.1-binding sites in their proximal promoter regions.

CASP8 and c-FLIP are known for their function downstream of death-receptor mediated apoptosis. Yet, it has been reported that CASP8 has non-apoptotic functions involving cell proliferation, differentiation, and inflammation. C-FLIP is the enzymatically inactive homolog of CASP8 and exists in three isoforms: a long isoform (c-FLIP_L), that partially inhibits CASP8 activity, and two short forms (c-FLIP_R and c-FLIP_S) that are anti-apoptotic.

To assess if CASP8 and/or c-FLIP are involved in PU.1-regulated cellular processes, we took advantage of the NB4 APL cell line model. These cells can be differentiated towards granulocytes with all-trans retinoic acid (ATRA) in a PU.1-dependent manner. We found an 8-10-fold induction of CASP8 and c-FLIP mRNA expression upon granulocytic differentiation of NB4 cells. Underlining a possible function of these two genes in granulocyte differentiation, we detected a markedly increased mRNA expression of both genes in human CD34⁺ hematopoietic cells differentiated towards neutrophils using G-CSF. Furthermore, knocking down PU.1 in NB4 cells significantly impaired CASP8 and cFLIP mRNA upregulation. Importantly, the anti-apoptotic cFLIP_s isoform was exclusively induced after prolonged ATRA-treatment in PU.1 knockdown cells, whereas cFLIP_L and CASP8 mRNA levels were reduced. The binding of PU.1 to the CFLAR promoter region together with altered cFLIP isoform ratio upon PU.1 expression indicates direct transcriptional activation of cFLIP and possibly an involvement of PU.1 in alternative splicing of cFLIP.

Based on previous reports linking non-apoptotic CASP8 functions to macrophage differentiation and our findings of PU.1-dependent CASP8 regulation, we next studied the role of CASP8 in more detail during monocyte and macrophage differentiation by knocking down CASP8 expression in HL60 cells. HL60 CASP8 knockdown cells, generated using two independent shRNAs, were treated with vitamin D₃ (VitD₃) or PMA to induce monocyte or macrophage differentiation, respectively. Surprisingly, knocking down CASP8 led to increased CD11b expression together with increased pseudopodia formation. Furthermore, analysis of secreted cytokines in HL60 CASP8 knockdown cells suggests activation of macrophages towards an M2 phenotype.

Our findings extend the role of PU.1 function to cell survival during granulocytic differentiation of APL cells. This occurs via distinct regulation of the pro-apoptotic CASP8, and anti-apoptotic cFLIP gene programs, respectively. Our findings suggest that increased expression of the anti-apoptotic, shorter cFLIP isoforms later in neutrophil differentiation may support short-term neutrophil cell survival. Lastly, our results implicate a novel PU.1-CASP8 pathway that may be necessary for alternative activation of M2 macrophages.