piperidines and Rhabdoid-Tumor

Rhabdoid tumors (RTs) are rare, highly aggressive pediatric malignancies with poor prognosis and with no standard or effective treatment strategies. RTs are characterized by biallelic inactivation of the INI1 tumor suppressor gene. INI1 directly represses CCND1 and activates cyclin-dependent kinase (cdk) inhibitors p16(Ink4a) and p21(CIP). RTs are exquisitely dependent on cyclin D1 for genesis and survival. To facilitate translation of unique therapeutic strategies, we have used genetically engineered, Ini1(+/-) mice for therapeutic testing. We found that PET can be used to noninvasively and accurately detect primary tumors in Ini1(+/-) mice. In a PET-guided longitudinal study, we found that treating Ini1(+/-) mice bearing primary tumors with the pan-cdk inhibitor flavopiridol resulted in complete and stable regression of some tumors. Other tumors showed resistance to flavopiridol, and one of the resistant tumors overexpressed cyclin D1, more than flavopiridol-sensitive cells. The concentration of flavopiridol used was not sufficient to down-modulate the high level of cyclin D1 and failed to induce cell death in the resistant cells. Furthermore, FISH and PCR analyses indicated that there is aneuploidy and increased CCND1 copy number in resistant cells. These studies indicate that resistance to flavopiridol may be correlated to elevated cyclin D1 levels. Our studies also indicate that Ini1(+/-) mice are valuable tools for testing unique therapeutic strategies and for understanding mechanisms of drug resistance in tumors that arise owing to loss of Ini1, which is essential for developing effective treatment strategies against these aggressive tumors.

Topics: Animals; Cell Line, Tumor; Chromosomal Proteins, Non-Histone; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; DNA Primers; Drug Resistance, Neoplasm; Flavonoids; Gene Expression Regulation, Neoplastic; Gene Silencing; Histological Techniques; Immunoblotting; Immunohistochemistry; In Situ Hybridization, Fluorescence; Longitudinal Studies; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron, Transmission; Piperidines; Polymerase Chain Reaction; Positron-Emission Tomography; Rhabdoid Tumor; SMARCB1 Protein

Rhabdoid Tumors (RTs) are highly aggressive pediatric malignancies with poor prognosis. There are currently no standard or effective treatments for RTs in part because treatments are not designed to specifically target these tumors. Our previous studies indicated that targeting the cyclin/cdk pathway is a novel therapeutic strategy for RTs and that a pan-cdk inhibitor, flavopiridol, inhibits RT growth. Since the toxicities and narrow window of activity associated with flavopiridol may limit its clinical use, we tested the effect of combining flavopiridol with 4-hydroxy-Tamoxifen (4OH-Tam) in order to reduce the concentration of flavopiridol needed for inhibition of RTs.. The effects of flavopiridol, 4OH-Tam, and their combination on RT cell cycle regulation and apoptosis were assessed by: i) cell survival assays, ii) FACS analysis, iii) caspase activity assays, and iv) immunoblot analysis. Furthermore, the role of p53 in flavopiridol- and 4OH-Tam-mediated induction of cell cycle arrest and apoptosis was characterized using RNA interference (siRNA) analysis. The effect of p53 on flavopiridol-mediated induction of caspases 2, 3, 8 and 9 was also determined.. We found that the combination of flavopiridol and 4OH-Tam potently inhibited the growth of RT cells. Low nanomolar concentrations of flavopiridol induced G₂ arrest, which was correlated to down-modulation of cyclin B1 and up-regulation of p53. Addition of 4OH-Tam did not affect flavopiridol-mediated G₂ arrest, but enhanced caspase 3,7-mediated apoptosis induced by the drug. Abrogation of p53 by siRNA abolished flavopiridol-induced G₂ arrest, but enhanced flavopiridol- (but not 4OH-Tam-) mediated apoptosis, by enhancing caspase 2 and 3 activities.. Combining flavopiridol with 4OH-Tam potently inhibited the growth of RT cells by increasing the ability of either drug alone to induce caspases 2 and 3 thereby causing apoptosis. The potency of flavopiridol was enhanced by abrogation of p53. Our results warrant further studies investigating the combinatorial effects of flavopiridol and 4OH-Tam as a novel therapeutic strategy for RTs and other tumors that have been shown to respond to flavopiridol.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspases; Cell Cycle; Cell Survival; Drug Screening Assays, Antitumor; Flavonoids; Flow Cytometry; G2 Phase; Humans; Piperidines; Rhabdoid Tumor; RNA Interference; Tamoxifen; Tumor Suppressor Protein p53

Rhabdoid tumors are aggressive and incurable pediatric malignancies. INI1/hSNF5, a tumor suppressor biallelically deleted/inactivated in rhabdoid tumors, directly represses cyclin D1. Rhabdoid tumors and cells are exquisitely dependent on cyclin D1 for genesis and survival, suggesting that targeting the cyclin/cyclin-dependent kinase (cdk) axis may be an effective therapeutic strategy for these tumors. Because cdk inhibitors have not been used for preclinical or clinical testing on rhabdoid tumors, we investigated the effect of flavopiridol, a pan-cdk inhibitor with promising clinical activity, on rhabdoid tumors.. The effect of flavopiridol on rhabdoid cells was tested in vitro using survival, cell cycle, and apoptosis assays. Its effect was assessed in vivo using xenografted rhabdoid tumor models. Immunoblot and immunohistochemical analysis was used to assess the effect of flavopiridol on cyclin D1 and p21 expression in vitro and in vivo, respectively.. Nanomolar concentrations of flavopiridol inhibited rhabdoid cell growth (IC(50) approximately 200 nmol/L), induced G(1) and G(2) arrest, and apoptosis in vitro in a concentration-dependent manner. These effects were correlated with the down-modulation of cyclin D1, up-regulation of p21, and induction of caspase 3/7 activities. Flavopiridol (at 7.5 mg/kg) significantly inhibited the growth of xenografted rhabdoid tumors, and its effect was correlated with the induction of p21 and down-modulation of cyclin D1.. Flavopiridol is effective in inducing cell cycle arrest and cytotoxicity in rhabdoid tumors. Its effects are correlated with the down-regulation of cyclin D1 and the up-regulation of p21. Flavopiridol is potentially a novel chemotherapeutic agent for rhabdoid tumors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Drug Screening Assays, Antitumor; Flavonoids; Humans; Mice; Mice, SCID; Neoplasm Transplantation; Piperidines; Protein Kinase Inhibitors; Rhabdoid Tumor; Xenograft Model Antitumor Assays