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Therapy of acute myeloid leukemia (AML) is unsatisfactory. Histone deacetylase inhibitors (HDACi) are active against leukemic cells in vitro and in vivo. Clinical data suggest further testing of such epigenetic drugs and to identify mechanisms and markers for their efficacy. Primary and permanent AML cells were screened for viability, replication stress/DNA damage, and regrowth capacities after single exposures to the clinically used pan-HDACi panobinostat (LBH589), the class I HDACi entinostat/romidepsin (MS-275/FK228), the HDAC3 inhibitor RGFP966, the HDAC6 inhibitor marbostat-100, the non-steroidal anti-inflammatory drug (NSAID) indomethacin, and the replication stress inducer hydroxyurea (HU). Immunoblotting was used to test if HDACi modulate the leukemia-associated transcription factors β-catenin, Wilms tumor (WT1), and myelocytomatosis oncogene (MYC). RNAi was used to delineate how these factors interact. We show that LBH589, MS-275, FK228, RGFP966, and HU induce apoptosis, replication stress/DNA damage, and apoptotic fragmentation of β-catenin. Indomethacin destabilizes β-catenin and potentiates anti-proliferative effects of HDACi. HDACi attenuate WT1 and MYC caspase-dependently and -independently. Genetic experiments reveal a cross-regulation between MYC and WT1 and a regulation of β-catenin by WT1. In conclusion, reduced levels of β-catenin, MYC, and WT1 are molecular markers for the efficacy of HDACi. HDAC3 inhibition induces apoptosis and disrupts tumor-associated protein expression.
Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia.
The taxanes are effective microtubule-stabilizing chemotherapy drugs that inhibit mitosis, induce apoptosis, and produce regression in a fraction of cancers that arise at many sites including the ovary. Novel therapeutic targets that augment taxane effects are needed to improve clinical chemotherapy response in CCNE1-amplified high grade serous ovarian cancer (HGSOC) cells. In this study, we conducted an siRNA-based kinome screen to identify modulators of mitotic progression in CCNE1-amplified HGSOC cells that may influence clinical paclitaxel response. PLK1 is overexpressed in many types of cancer, which correlates with poor prognosis. Here, we identified a novel synthetic lethal interaction of the clinical PLK1 inhibitor BI6727 and the microtubule-targeting drug paclitaxel in HGSOC cell lines with CCNE1-amplification and elucidated the underlying molecular mechanisms of this synergism. BI6727 synergistically induces apoptosis together with paclitaxel in different cell lines including a patient-derived primary ovarian cancer culture. Moreover, the inhibition of PLK1 reduced the paclitaxel-induced neurotoxicity in a neurite outgrowth assay. Mechanistically, the combinatorial treatment with BI6727/paclitaxel triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of anti-apoptotic BCL-2 family proteins, followed by significant loss of the mitochondrial membrane potential and activation of caspase-dependent effector pathways. This conclusion is supported by data showing that BI6727/paclitaxel-co-treatment stabilizes FBW7, a component of SCF-type ubiquitin ligases that bind and regulate key modulators of cell division and growth including MCL-1 and Cyclin E. This identification of a novel synthetic lethality of PLK1 inhibitors and a microtubule-stabilizing drug has important implications for developing PLK1 inhibitor-based combination treatments in CCNE1-amplified HGSOC cells.
High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions.
In eukaryotes, double-stranded (ds) RNA induces sequence-specific inhibition of gene expression referred to as RNA interference (RNAi). We exploited RNAi to define the role of HER2/neu in the neoplastic proliferation of human breast cancer cells. We transfected SK-BR-3, BT-474, MCF-7, and MDA-MB-468 breast cancer cells with short interfering RNA (siRNA) targeted against human HER2/neu and analyzed the specific inhibition of HER2/neu expression by Northern and Western blots. Transfection with HER2/neu-specific siRNA resulted in a sequence-specific decrease in HER2/neu mRNA and protein levels. Moreover, transfection with HER2/neu siRNA caused cell cycle arrest at G0/G1 in the breast cancer cell lines SKBR-3 and BT-474, consistent with a powerful RNA silencing effect. siRNA treatment resulted in an antiproliferative and apoptotic response in cells overexpressing HER2/neu, but had no influence in cells with almost no expression of HER2/neu proteins like MDA-MB-468 cells. These data indicate that HER2/neu function is essential for the proliferation of HER2/neuoverexpressing breast cancer cells. Our observations suggest that siRNA targeted against human HER2/neu may be valuable tools as anti proliferative agents that display activity against neoplastic cells at very low doses.
Background Cyclin B1, the regulatory subunit of cyclin-dependent kinase 1 (Cdk1), is essential for the transition from G2 phase to mitosis. Cyclin B1 is very often found to be overexpressed in primary breast and cervical cancer cells as well as in cancer cell lines. Its expression is correlated with the malignancy of gynecological cancers. Methods In order to explore cyclin B1 as a potential target for gynecological cancer therapy, we studied the effect of small interfering RNA (siRNA) on different gynecological cancer cell lines by monitoring their proliferation rate, cell cycle profile, protein expression and activity, apoptosis induction and colony formation. Tumor formation in vivo was examined using mouse xenograft models. Results Downregulation of cyclin B1 inhibited proliferation of several breast and cervical cancer cell lines including MCF-7, BT-474, SK-BR-3, MDA-MB-231 and HeLa. After combining cyclin B1 siRNA with taxol, we observed an increased apoptotic rate accompanied by an enhanced antiproliferative effect in breast cancer cells. Furthermore, control HeLa cells were progressively growing, whereas the tumor growth of HeLa cells pre-treated with cyclin B1 siRNA was strongly inhibited in nude mice, indicating that cyclin B1 is indispensable for tumor growth in vivo. Conclusion Our data support the notion of cyclin B1 being essential for survival and proliferation of gynecological cancer cells. Concordantly, knockdown of cyclin B1 inhibits proliferation in vitro as well as in vivo. Moreover, targeting cyclin B1 sensitizes breast cancer cells to taxol, suggesting that specific cyclin B1 targeting is an attractive strategy for the combination with conventionally used agents in gynecological cancer therapy.