BCR-ABL Tyrosine Kinase Inhibitors Interfere with the PI3K/Akt Signalling Pathway and Activate the Microphthalmia-Associated Transcription Factor (MITF) in Human Dendritic Cells

Introduction: Dendritic cells (DC) are the most powerful antigen-presenting cells (APC) and play a key role in balancing T cell responses, depending on their expression of costimulatory and/or coinhibitory molecules. Inhibitory factors such as the anti-inflammatory cytokine IL-10 suppress the activity of NF-κB in DC resulting in downregulation of costimulatory molecules and impaired immunostimulatory capacity. GPNMB (osteoactivin, DC-HIL), a type I transmembrane protein well known for its role in osteoblastogenesis, is also expressed on APC and acts as a coinhibitory molecule that strongly inhibits T cell responses upon binding to its counterpart syndecan (SD)-4. Interestingly, GPNMB expression on human monocyte-derived dendritic cells (moDC) is dramatically upregulated upon exposure to IL-10 but also to the BCR-ABL tyrosine kinase inhibitors (TKI) imatinib, nilotinib or dasatinib (Schwarzbich et al., Cancer Immunol Immunother 2012). Here we aimed to elucidate the molecular switch that facilitates transcriptional activation upon TKI-mediated inhibition of cellular signalling in moDC.

Methods: MoDC were generated in vitro by culturing CD14⁺ peripheral blood monocytes with GM-CSF and IL-4 in the presence or absence of TKI (imatinib, nilotinib) or specific inhibitors of signalling pathways (Akt inhibitor MK2206, Erk inhibitor FR180204, PI3K inhibitor LY294002, or c-Raf inhibitor 553003). After 7 days, moDc were harvested followed by immunophenotyping and analysis of signalling cascades by FACS, real-time RT-PCR and western-blotting. Functionality of moDC was analyzed by mixed lymphocyte reactions (MLR).

Results: We found that BCR-ABL TKI lead to dephosphorylation and thereby activation of the serine/threonine protein kinase GSK3ß via inhibition of PI3K/Akt signalling. This leads to phosphorylation of microphthalmia-associated transcription factor (MITF) and translocation of this transcription factor to the nucleus. Moreover, treatment with PI3K/Akt inhibitors resulted in profound upregulation of GPNMB in moDC, indicating that MITF activation and the resulting GPNMB expression are in fact facilitated by dephosphorylation of GSK3ß following inhibition of PI3K/Akt signalling. In line, both BCR-ABL TKI treatment and specific inhibition of PI3K/Akt resulted in reduced stimulatory capacity of moDC in MLR with allogenic T cells that could be restored by addition of the (recombinant) GPNMB T cell ligand SD-4.

Conclusions: BCR-ABL TKI exert immunosuppressive effects by activation of GSK3ß, which we thereby identify as molecular switch causing MITF-mediated transcriptional activity upon TKI-mediated inhibition of PI3K/Akt signalling. MITF activation upregulates GPNMB expression which impairs the immunostimulatory capacity of moDC. Our data extend the current understanding regarding the molecular mechanisms that balance activating and inhibitory signals in DC. Manipulation of the involved signalling cascades and in particular GPNMB expression/function may constitute a promising strategy in combinatory approaches using TKI and DC-based immunotherapy and may also allow for manipulation of T cell responses in graft-versus-host disease (GvHD).