The online version of this article has a Supplementary Appendix.The identification as cooperating targets of Proviral Integrations of Moloney virus in murine lymphomas suggested early on that PIM serine/threonine kinases play an important role in cancer biology. Whereas elevated levels of PIM1 and PIM2 were mostly found in hematologic malignancies and prostate cancer, increased PIM3 expression was observed in different solid tumors. PIM kinases are constitutively active and their activity supports in vitro and in vivo tumor cell growth and survival through modification of an increasing number of common as well as isoform-specific substrates including several cell cycle regulators and apoptosis mediators. PIM1 but not PIM2 seems also to mediate homing and migration of normal and malignant hematopoietic cells by regulating chemokine receptor surface expression. Knockdown experiments by RNA interference or dominant-negative acting mutants suggested that PIM kinases are important for maintenance of a transformed phenotype and therefore potential therapeutic targets. Determination of the protein structure facilitated identification of an increasing number of potent small molecule PIM kinase inhibitors with in vitro and in vivo anticancer activity. Ongoing efforts aim to identify isoform-specific PIM inhibitors that would not only help to dissect the kinase function but hopefully also provide targeted therapeutics. Here, we summarize the current knowledge about the role of PIM serine/threonine kinases for the pathogenesis and therapy of hematologic malignancies and solid cancers, and we highlight structural principles and recent progress on small molecule PIM kinase inhibitors that are on their way into first clinical trials. Haematologica 2010;95:1004-1015. doi:10.3324/haematol.2009 This is an open-access paper. ABSTRACTin blast crisis. 6 Abundant levels of PIM1 were found in hematopoietic cells. Moreover, sustained PIM1 expression was induced by cytokines that signal through structurally related receptors such as IL-3, GM-CSF, G-CSF or Subsequently, several studies have documented that PIM1 is a major downstream target of the signal transducer and activator of transcription (STATs) induced by a large variety of additional receptors such as IL-2, IL-7, IL-9, IFNγ, EPO, FLT3 or TPO.7 PIM1 expression is not only regulated at the transcriptional, but also at the posttranscriptional, translational and posttranslational levels ( Figure 1). Other studies have shown that PIM1 kinase is significantly protected from proteasomal degradation by heat shock proteins (Hsp70, Hsp90). 8,9 Moreover, it has been proposed that micro-RNAs, miR-1 and miR-210, might be implicated in regulation of PIM1 expression. 10,11 Germline inactivation of the PIM1 gene was associated with a mild phenotype as PIM1 deficient mice are ostensibly normal, healthy and fertile. However, subtle functional defects of the hematopoietic system have been identified: PIM1 -/-mice showed erythrocytic microcytosis and PIM1-/-B cells and bone marrow-derived mast c...

Much attention has recently been focused on PIM kinases as potential targets for the treatment of hematopoietic malignancies and some solid cancers. Using protein stability shift assays, we identified a family of imidazo [1,2-b]pyridazines to specifically interact with and inhibit PIM kinases with low nanomolar potency. The high-resolution crystal structure of a PIM1 inhibitor complex revealed that imidazo[1,2-b]pyrridazines surprisingly interact with the NH 2 -terminal lobe helix AC rather than with the kinase hinge region. Thus, the identified inhibitors are ATP competitive but not ATP mimetic compounds, explaining their enhanced selectivity with respect to conventional type I kinase inhibitors. One of the identified imidazo[1,2-b]pyridazines (K00135) was further tested in several hematopoietic cellular systems. First, K00135 dosedependently impaired survival of murine Ba/F3 cells that have been rendered cytokine independent by overexpression of human PIMs. Second, K00135 impaired survival and clonogenic growth of a panel of human acute leukemia cells. Third, exposure of K00135 significantly suppressed in vitro growth of leukemic blasts from five acute myelogenous leukemia patients but not of normal umbilical cord blood mononuclear cells. In vitro kinase assays and immunoblotting using lysates from human MV4;11 leukemic cells showed inhibition of phosphorylation of known PIM downstream targets, such as BAD and eukaryotic translation initiation factor 4E-binding protein 1, by K00135. Taken together, we report a family of small molecules that selectively interact and block PIM kinases and could serve as a lead to develop new targeted antileukemic therapeutics. [Cancer Res 2007;67(14):6916-24]

FLT3-ITD–mediated leukemogenesis is associated with increased expression of oncogenic PIM serine/threonine kinases. To dissect their role in FLT3-ITD–mediated transformation, we performed bone marrow reconstitution assays. Unexpectedly, FLT3-ITD cells deficient for PIM1 failed to reconstitute lethally irradiated recipients, whereas lack of PIM2 induction did not interfere with FLT3-ITD–induced disease. PIM1-deficient bone marrow showed defects in homing and migration and displayed decreased surface CXCR4 expression and impaired CXCL12–CXCR4 signaling. Through small interfering RNA–mediated knockdown, chemical inhibition, expression of a dominant-negative mutant, and/or reexpression in knockout cells, we found PIM1 activity to be essential for proper CXCR4 surface expression and migration of cells toward a CXCL12 gradient. Purified PIM1 led to the phosphorylation of serine 339 in the CXCR4 intracellular domain in vitro, a site known to be essential for normal receptor recycling. In primary leukemic blasts, high levels of surface CXCR4 were associated with increased PIM1 expression, and this could be significantly reduced by a small molecule PIM inhibitor in some patients. Our data suggest that PIM1 activity is important for homing and migration of hematopoietic cells through modification of CXCR4. Because CXCR4 also regulates homing and maintenance of cancer stem cells, PIM1 inhibitors may exert their antitumor effects in part by interfering with interactions with the microenvironment.

Development of both potent and selective kinase inhibitors is a challenging task in modern drug discovery. The innate promiscuity of kinase inhibitors largely results from ATP-mimetic binding to the kinase hinge region. We present a novel class of substituted 7,8-dichloro-1-oxo-β-carbolines based on the distinct structural features of the alkaloid bauerine C whose kinase inhibitory activity does not rely on canonical ATP-mimetic hinge interactions. Intriguingly, cocrystal structures revealed an unexpected inverted binding mode and the presence of halogen bonds with kinase backbone residues. The compounds exhibit excellent selectivity over a comprehensive panel of human protein kinases while inhibiting selected kinases such as the oncogenic PIM1 at low nanomolar concentrations. Together, our biochemical and structural data suggest that this scaffold may serve as a valuable template for the design and development of specific inhibitors of various kinases including the PIM family of kinases, CLKs, DAPK3 (ZIPK), BMP2K (BIKE), and others.

CORRESPONDENCE