Targeting Host PIM Protein Kinases Reduces Mayaro Virus Replication

Mayaro virus (MAYV) manipulates cell machinery to successfully replicate. Thus, identifying host proteins implicated in MAYV replication, represents an opportunity to discover potential antiviral targets. PIM kinases are enzymes that regulate essential cell functions and also appear to be critical factors in the replication of certain viruses. In this study we explored the consequences of PIM kinase inhibition in the replication of MAYV and other arboviruses. Cytopathic effects or viral titers in samples from MAYV-, Chikungunya-, Una- or Zika-infected cells treated with PIM kinase inhibitors were evaluated using an inverted microscope or plaque-forming assays. The expression of viral proteins E1 and nsP1 in MAYV-infected cells was assessed using an immunofluorescence confocal microscope or Western blot. Our results revealed that PIM kinase inhibition partially prevented MAYV-induced cell damage and also promoted a decrease in viral titers for MAYV, UNAV and ZIKV. The inhibitory effect of PIM kinase blocking was observed for each of the MAYV strains tested and also occurred as late as 8 hpi. Finally, PIM kinase inhibition suppressed the expression of MAYV E1 and nsP1 proteins. Taken together, these findings suggest that PIM kinases could represent an antiviral target for MAYV and other arboviruses.

Feedback Loop Regulation Between Pim Kinases and Tax Keeps HTLV-I Viral Replication in Check.

The Pim family of serine/threonine kinases promote tumorigenesis by enhancing cell survival and inhibiting apoptosis. Three isoforms exist, Pim-1, -2, and -3 that are highly expressed in hematological cancers, including Pim-1 in Adult T-cell leukemia (ATL). Human T-cell leukemia virus type-1 (HTLV-I) is the etiological agent of ATL, a dismal lymphoproliferative disease known as adult T-cell leukemia. The HTLV-I virally encoded oncogene Tax promotes CD4+ T-cell transformation through disruption of DNA repair pathways and activation of survival and cellular proliferation pathways. In this study, we found Tax increases the expression of Pim-1 and Pim-3, while decreasing Pim-2 expression. Furthermore, we discovered that Pim-1, -2, and -3 bind Tax protein to reduce its expression thereby creating a feedback regulatory loop between these two oncogenes. The loss of Tax expression triggered by Pim kinases led to loss in Tax-mediated transactivation of the HTLV-I LTR and reductions in HTLV-I virus replication. Since Tax is also the immunodominant cytotoxic T cell lymphocytes (CTL) target, our data suggest that Pim kinases may play an important role in immune escape of HTLV-1-infected cells. IMPORTANCE The Pim family of protein kinases have established pro-oncogenic functions. They are often up regulated in cancer; especially leukemias and lymphomas. In addition, a role for Pim kinases in control of virus expression and viral latency is important for KSHV and HIV-1. Our data demonstrate that HTLV-I encodes viral genes that promote and maintain Pim kinase activation, which in turn may stimulate T-cell transformation and maintain ATL leukemic cell growth. HTLV-I Tax increases expression of Pim-1 and Pim-3, while decreasing expression of Pim-2. In ATL cells, Pim expression is maintained through extended protein half-life and heat shock protection. In addition, we found that Pim kinases have a new role during HTLV-I infection. Pim-1, -2, and -3 can subvert Tax expression and HTLV-I virus production. This may lead to partial suppression of the host immunogenic responses to Tax and favor immune escape of HTLV-1-infected cells. Therefore, Pim kinases have not only pro-oncogenic roles but also favor persistence of the virus-infected cell.

Dibenzofuran Derivatives Inspired from Cercosporamide as Dual Inhibitors of Pim and CLK1 Kinases

Pim kinases (proviral integration site for Moloney murine leukemia virus kinases) are overexpressed in various types of hematological malignancies and solid carcinomas, and promote cell proliferation and survival. Thus, Pim kinases are validated as targets for antitumor therapy. In this context, our combined efforts in natural product-inspired library generation and screening furnished very promising dibenzo[b,d]furan derivatives derived from cercosporamide. Among them, lead compound 44 was highlighted as a potent Pim-1/2 kinases inhibitor with an additional nanomolar IC50 value against CLK1 (cdc2-like kinases 1) and displayed a low micromolar anticancer potency towards the MV4-11 (AML) cell line, expressing high endogenous levels of Pim-1/2 kinases. The design, synthesis, structure–activity relationship, and docking studies are reported herein and supported by enzyme, cellular assays, and Galleria mellonella larvae testing for acute toxicity.

PIM Kinases in Multiple Myeloma

Multiple myeloma (MM) remains an incurable disease and novel therapeutic agents/approaches are urgently needed. The PIM (Proviral insertion in murine malignancies) serine/threonine kinases have 3 isoforms: PIM1, PIM2, and PIM3. PIM kinases are engaged with an expansive scope of biological activities including cell growth, apoptosis, drug resistance, and immune response. An assortment of molecules and pathways that are critical to myeloma tumorigenesis has been recognized as the downstream targets of PIM kinases. The inhibition of PIM kinases has become an emerging scientific interest for the treatment of multiple myeloma and several PIM kinase inhibitors, such as SGI-1776, AZD1208, and PIM447 (formerly LGH447), have been developed and are under different phases of clinical trials. Current research has been focused on the development of a new generation of potent PIM kinase inhibitors with appropriate pharmacological profiles reasonable for human malignancy treatment. Combination therapy of PIM kinase inhibitors with chemotherapeutic appears to create an additive cytotoxic impact in cancer cells. Notwithstanding, the mechanisms by which PIM kinases modulate the immune microenvironment and synergize with the immunomodulatory agents such as lenalidomide have not been deliberately depicted. This review provides a comprehensive overview of the PIM kinase pathways and the current research status of the development of PIM kinase inhibitors for the treatment of MM. Additionally, the combinatorial effects of the PIM kinase inhibitors with other targeted agents and the promising strategies to exploit PIM as a therapeutic target in malignancy are highlighted.

Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family

We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.

IL-15 and PIM kinases direct the metabolic programming of intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IEL) are an abundant population of tissue-resident T cells that protect and maintain the intestinal barrier. IEL respond to epithelial cell-derived IL-15, which is complexed to the IL-15 receptor α chain (IL-15/Rα). IL-15 is essential both for maintaining IEL homeostasis and inducing IEL responses to epithelial stress, which has been associated with Coeliac disease. Here, we apply quantitative mass spectrometry to IL-15/Rα-stimulated IEL to investigate how IL-15 directly regulates inflammatory functions of IEL. IL-15/Rα drives IEL activation through cell cycle regulation, upregulation of metabolic machinery and expression of a select repertoire of cell surface receptors. IL-15/Rα selectively upregulates the Ser/Thr kinases PIM1 and PIM2, which are essential for IEL to proliferate, grow and upregulate granzyme B in response to inflammatory IL-15. Notably, IEL from patients with Coeliac disease have high PIM expression. Together, these data indicate PIM kinases as important effectors of IEL responses to inflammatory IL-15.

PIM kinases inhibit AMPK activation and promote tumorigenicity by phosphorylating LKB1

Background The oncogenic PIM kinases and the tumor-suppressive LKB1 kinase have both been implicated in the regulation of cell growth and metabolism, albeit in opposite directions. Here we investigated whether these kinases interact with each other to influence AMPK activation and tumorigenic growth of prostate and breast cancer cells. Methods We first determined how PIM and LKB1 kinases affect AMPK phosphorylation levels. We then used in vitro kinase assays to demonstrate that LKB1 is phosphorylated by PIM kinases, and site-directed mutagenesis to identify the PIM target sites in LKB1. The cellular functions of PIM and LKB1 kinases were evaluated using either pan-PIM inhibitors or CRISPR/Cas9 genomic editing, with which all three PIM family members and/or LKB1 were knocked out from PC3 prostate and MCF7 breast cancer cell lines. In addition to cell proliferation assays, we examined the effects of PIM and/or LKB1 loss on tumor growth using the chick embryo chorioallantoic membrane (CAM) xenograft model. Results We provide both genetic and pharmacological evidence to demonstrate that inhibition of PIM expression or activity increases phosphorylation of AMPK at Thr172 in both PC3 and MCF7 cells, but not in their derivatives lacking LKB1. This is explained by our observation that all three PIM family kinases can phosphorylate LKB1 at Ser334. Wild-type LKB1, but not its phosphodeficient derivative, can restore PIM inhibitor-induced AMPK phosphorylation in LKB1 knock-out cells. In the CAM model, loss of LKB1 enhances tumorigenicity of PC3 xenografts, while cells lacking both LKB1 and PIMs exhibit slower proliferation rates and form smaller tumors. Conclusion PIM kinases are novel negative regulators of LKB1 that affect AMPK activity in an LKB1-dependent fashion. The impairment of cell proliferation and tumor growth in cells lacking both LKB1 and PIMs indicates that these kinases possess a shared signaling role in the context of cancer. These data also suggest that PIM inhibitors may be a rational therapeutic option for LKB1-deficient tumors.

Novel Pan-Pim Kinase Inhibitors With Imidazopyridazine and Thiazolidinedione Structure Exert Potent Antitumor Activities

Pim kinases are overexpressed in various types of hematological malignancies and solid carcinomas, and promote cell proliferation and survival. Here in this study, we investigated the preclinical profile of novel pan-Pim kinase inhibitors with imidazopyridazine and thiazolidinedione structure. Imidazopyridazine-thiazolidinediones inhibited activities of Pim kinases with IC50 values of tens to hundreds nanomolar. With YPC-21440 and/or YPC-21817, which exhibited especially high inhibitory activities against Pim kinases, we investigated in vitro and in vivo activities of imidazopyridazine-thiazolidinediones. In silico analysis of binding mode of YPC-21440 and Pim kinases revealed that it directly bound to ATP-binding pockets of Pim kinases. In the kinase panel tested, YPC-21440 and YPC-21817 were highly specific to Pim kinases. These compounds exerted antiproliferative activities against various cancer cell lines derived from hematological malignancies and solid carcinomas. Furthermore, they suppressed phosphorylation of Pim kinase substrates, arrested cell cycle at the G1 phase, and induced apoptosis in cultured cancer cells. In tumor xenograft models, YPC-21440 methanesulfonate and YPC-21817 methanesulfonate exerted antitumor activities. Furthermore, pharmacodynamic analysis with a xenograft model suggested that YPC-21817 methanesulfonate inhibited Pim kinases in tumors. In conclusion, our data revealed that imidazopyridazine-thiazolidinediones are novel Pim kinases inhibitors, effective on various types of cancer cell lines both in vitro and in vivo.