Overexpression of the CXCR4 receptor is a hallmark of chronic lymphocytic leukemia (CLL) and is important for CLL cell survival, migration, and interaction with their protective microenvironment. In acute myelogenous leukemia (AML), PIM1 was shown to regulate the surface expression of the CXCR4 receptor. Here, we show that PIM (proviral integration site for Moloney murine leukemia virus) kinases 1-3 are overexpressed and that the CXCR4 receptor is hyperphosphorylated on Ser339 in CLL compared with normal lymphocytes. Furthermore, CXCR4 phosphorylation correlates with PIM1 protein expression and PIM1 transcript levels in CLL. PIM kinase inhibition with three different PIM kinase inhibitors induced apoptosis in CLL cells independent of the presence of protective stromal cells. In addition, PIM inhibition caused dephosphorylation of the CXCR4 receptor on Ser339, resulting in enhanced ligand-dependent CXCR4 internalization and reduced re-externalization after withdrawal of CXCL12. Furthermore, PIM inhibition in CLL cells blocked CXCR4 functions, such as migration toward CXCL12-or CXCL12-induced extracellular signal-regulated kinase (ERK) phosphorylation. In concordance, pretreatment of CLL cells with PIM kinase inhibitors strongly reduced homing of CLL cells toward the bone marrow and the spleen of Rag2

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LETTERS TO THE EDITOR © F e r r a t a S t o r t i F o u n d a t i o n

FLT3 kinase has become an attractive drug target in AML with up to 30% of cases harboring internal-tandem-duplication (ITD) mutations. For these, conferring a worse prognosis and decreased overall survival, several FLT3 tyrosine kinase inhibitors (TKIs) are currently being tested in clinical trials. However, when using these drugs as monotherapy, the problem of short duration of remissions and high incidence of TKI resistance has emerged. Here, we investigated two members of a novel class of tyrosine kinase inhibitors, 3,4-diarylmaleimides, for their efficacy on mutated FLT3 kinase. These compounds inhibit FLT3 kinase in an ATP-competitive manner and effectively inhibit phosphorylation of downstream targets. 3,4-Diarylmaleimides (DHF125 and 150) induce apoptosis in FLT3-ITD-dependent cells lines and patient blasts at low micromolar concentrations. They are retained in the cytoplasm of exposed cells for more than 24 h and synergize with chemotherapy and midostaurin. Both 3,4-diarylmaleimides show inhbition of FLT3-ITD-related kinase autophosphorylation at distinct tyrosine residues when compared to midostaurin. In conclusion, this novel group of compounds shows differential inhibition patterns with regard to FLT3 kinase and displays a promising profile for further clinical development. Currently, experiments evaluating toxicity in murine models and unraveling the exact binding mechanism are under way to facilitate a potential clinical application.

<div>Abstract<p>Overexpression of the CXCR4 receptor is a hallmark of chronic lymphocytic leukemia (CLL) and is important for CLL cell survival, migration, and interaction with their protective microenvironment. In acute myelogenous leukemia (AML), PIM1 was shown to regulate the surface expression of the CXCR4 receptor. Here, we show that PIM (proviral integration site for Moloney murine leukemia virus) kinases 1–3 are overexpressed and that the CXCR4 receptor is hyperphosphorylated on Ser339 in CLL compared with normal lymphocytes. Furthermore, CXCR4 phosphorylation correlates with PIM1 protein expression and <i>PIM1</i> transcript levels in CLL. PIM kinase inhibition with three different PIM kinase inhibitors induced apoptosis in CLL cells independent of the presence of protective stromal cells. In addition, PIM inhibition caused dephosphorylation of the CXCR4 receptor on Ser339, resulting in enhanced ligand-dependent CXCR4 internalization and reduced re-externalization after withdrawal of CXCL12. Furthermore, PIM inhibition in CLL cells blocked CXCR4 functions, such as migration toward CXCL12- or CXCL12-induced extracellular signal–regulated kinase (ERK) phosphorylation. In concordance, pretreatment of CLL cells with PIM kinase inhibitors strongly reduced homing of CLL cells toward the bone marrow and the spleen of Rag2^−/−γc^−/− mice <i>in vivo</i>. Interestingly, the knockdown of PIM kinases in CLL cells demonstrated diverging functions, with PIM1 regulating CXCR4 surface expression and PIM2 and PIM3 as important for the survival of CLL cells. Our results show that PIM kinase inhibitors are an effective therapeutic option for CLL, not only by impairing PIM2/3-mediated CLL cell survival, but also by blocking the PIM1/CXCR4-mediated interaction of CLL cells with their protective microenvironment. <i>Mol Cancer Ther; 13(5); 1231–45. ©2014 AACR</i>.</p></div>