Inhibition of PIM1 blocks the autophagic flux to sensitize glioblastoma cells to ABT-737-induced apoptosis.

Overcoming apoptosis resistance is one major issue in glioblastoma (GB) therapies. Accumulating evidence indicates that resistance to apoptosis in GB is mediated via upregulation of pro-survival BCL2-family members. The synthetic BH3-mimetic ABT-737 effectively targets BCL2, BCL2 like 1 and BCL2 like 2 but still barely affects cell survival which is presumably due to its inability to inhibit myeloid cell leukemia 1 (MCL1). The constitutively active serine/threonine kinase proviral integration site for moloney murine leukemia virus 1 (PIM1) was recently found to be overexpressed in GB patient samples and to maintain cell survival in these tumors. For different GB cell lines, Western Blot, mitochondrial fractionation, fluorescence microscopy, effector caspase assays, flow cytometry, and an adult organotypic brain slice transplantation model were used to investigate the putative PIM1/MCL1 signaling axis regarding potential synergistic effects with ABT-737. We demonstrate that combination of the PIM1 inhibitor SGI-1776 or the pan-PIM kinase inhibitor AZD1208 with ABT-737 strongly sensitizes GB cells to apoptosis. Unexpectedly, this effect was found to be MCL1-independent, but could be partially blocked by caspase 8 (CASP8) inhibition. Remarkably, the analysis of autophagy markers in combination with the observation of massive accumulation and hampered degradation of autophagosomes suggests a completely novel function of PIM1 as a late stage autophagy regulator, maintaining the autophagic flux at the level of autophagosome/lysosome fusion. Our data indicate that PIM1 inhibition and ABT-737 synergistically induce apoptosis in an MCL1-independent but CASP8-dependent manner in GB. They also identify PIM1 as a suitable target for overcoming apoptosis resistance in GB.

The proto-oncogene Pim-1 is a target of miR-33a.

The constitutively active serine/threonine kinase Pim-1 is upregulated in different cancer types, mainly based on the action of several interleukines and growth factors at the transcriptional level. So far, a regulation of oncogenic Pim-1 by microRNAs (miRNAs) has not been reported. Here, we newly establish miR-33a as a miRNA with potential tumor suppressor activity, acting through inhibition of Pim-1. A screen for miRNA expression in K562 lymphoma, LS174T colon carcinoma and several other cell lines revealed generally low endogenous miR-33a levels relative to other miRNAs. Transfection of K562 and LS174T cells with a miR-33a mimic reduced Pim-1 levels substantially. In contrast, the cell-cycle regulator cyclin-dependent kinase 6 predicted to be a conserved miR-33a target, was not downregulated by the miR-33a mimic. Seed mutagenesis of the Pim-1 3'-untranslated region in a luciferase reporter construct and in a Pim-1 cDNA expressed in Pim-1-deficient Skov-3 cells demonstrated specific and direct downregulation of Pim-1 by the miR-33a mimic. The persistence of this effect was comparable to that of a small interfering RNA-mediated knockdown of Pim-1, resulting in decelerated cell proliferation. In conclusion, we demonstrate the potential of miR-33a to act as a tumor suppressor miRNA, which suggests miR-33a replacement therapy through delivery of miR mimics as a novel therapeutic strategy.