CXCR4 surface expression is strongly elevated on CLL cells and is important for CLL cell survival and homing to the bone marrow. PIM kinases are overexpressed and mutated in various hematologic malignancies including B-cell lymphomas. Recent findings in AML suggest a direct link in between PIM1 kinase expression and the surface expression level of the CXCR4 receptor and Ser339 phosphorylation in CXCR4. Therefore the aim of the presented study was to investigate the role of PIM kinases in regulating the CXCR4-CXCL12 pathway in CLL.
By using a phospho-CXCR4 Ser339 antibody for both, western blot analysis of CLL cells from the peripheral blood (n=11) and IHC staining of bone marrow from CLL patients (n=30), we can show that in contrast to normal B-cells the majority of CLL cells have a hyperphosphorylated CXCR4 receptor. Furthermore high phosphorylation levels of the CXCR4 receptor in the bone marrow correlated with poor prognosis in CLL. Besides increased phosphorylation of the CXCR4 receptor, also the 3 PIM kinases, especially PIM1 (P=.0060) and PIM2 (P=.0404) are strongly overexpressed in CLL compared to normal B-lymphocytes on mRNA- and protein level. Transcript levels of PIM1 (P=.0020), PIM2 (P=.0006) and PIM3 (P=.0206) positively correlated with the median CXCR4 surface expression. Furthermore PIM1 protein expression demonstrated a positive correlation with the CXCR4 phosphorylation on Ser339. PIM kinase inhibition with 3 different PIM kinase inhibitors (K00135, K00486 and CMV-05–144–01) resulted in a dose-dependent induction of apoptosis in primary human CLL cells independent of the presence of stromal cells, indicating that pathways activated by stromal cells are directly targeted by PIM kinase inhibitors. Additionally, inhibition of PIM with the inhibitor K00135 induced dephosphorylation of the CXCR4 receptor on Ser339 and resulted in enhanced ligand-dependent CXCR4 internalization and reduced reexternalization after withdrawal of CXCL12. Furthermore PIM inhibition induced degradation of internalized CXCR4 receptor and blocked CXCR4 function regarding survival, ERK-phosphorylation and migration towards a CXCL12 gradient. We could further demonstrate that pretreatment of primary CLL cells with PIM kinase inhibitors and resulting down regulation of the CXCR4 receptor strongly reduced homing of CLL cells towards the bone marrow (P=.0293) and the spleen (P=.0104) of Rag2−/−γc−/− mice in vivo.
Our results demonstrate, that PIM kinase inhibitors are an effective treatment option for CLL by directly inducing pro-apoptotic pathways in CLL cells, but also by blocking the CXCR4 pathway which is important for the interaction of CLL cells with their protective microenvironment.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.