Carolyn A. Buser

MK-1775, a novel wee1 kinase inhibitor, radiosensitizes p53-defective human tumor cells

Purpose: Radiotherapy is commonly used to treat a variety of solid tumors. However, improvements in the therapeutic ratio for several disease sites are sorely needed, leading us to assess molecularly targeted therapeutics as radiosensitizers. The aim of this study was to assess the wee1 kinase inhibitor, MK-1775, for its ability to radiosensitize human tumor cells. Experimental Design: Human tumor cells derived from lung, breast, and prostate cancers were tested for radiosensitization by MK-1775 using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of MK-1775 to abrogate the radiation-induced G2 block, thereby allowing cells harboring DNA lesions to prematurely progress into mitosis, was determined using flow cytometry and detection of γ-H2AX foci. The in vivo efficacy of the combination of MK-1775 and radiation was assessed by tumor growth delay experiments using a human lung cancer cell line growing as a xenograft tumor in nude mice. Results: Clonogenic survival analyses indicated that nanomolar concentrations of MK-1775 radiosensitized p53-defective human lung, breast, and prostate cancer cells but not similar lines with wild-type p53. Consistent with its ability to radiosensitize, MK-1775 abrogated the radiation-induced G2 block in p53-defective cells but not in p53 wild-type lines. MK-1775 also significantly enhanced the antitumor efficacy of radiation in vivo as shown in tumor growth delay studies, again for p53-defective tumors. Conclusions: These results indicate that p53-defective human tumor cells are significantly radiosensitized by the potent and selective wee1 kinase inhibitor, MK-1775, in both the in vitro and in vivo settings. Taken together, our findings strongly support the clinical evaluation of MK-1775 in combination with radiation. Clin Cancer Res; 17(17); 5638–48. ©2011 AACR.

Downregulation of Notch Pathway by a γ-Secretase Inhibitor Attenuates AKT/Mammalian Target of Rapamycin Signaling and Glucose Uptake in an ERBB2 Transgenic Breast Cancer Model

ERBB2/neu and Notch signaling are known to be deregulated in many human cancers. However, pathway cross-talk and dependencies are not well understood. In this study, we use an ERBB2-transgenic mouse model of breast cancer (neuT) to show that Notch signaling plays a critical role in tumor maintenance. Inhibition of the Notch pathway with a gamma-secretase inhibitor (GSI) decreased both the Notch and the mammalian target of rapamycin/AKT pathways. Antitumor activity resulting from GSI treatment was associated with decreased cell proliferation as measured by Ki67 and decreased expression of glucose transporter Glut1. Positron emission tomography (PET) imaging showed that the functional consequences of decreased Glut1 translated to reduced glucose uptake and correlated with antitumor effects as measured by micro-computed tomography imaging. The decrease of Glut1 in neuT tumors was also observed in several human breast cancer cell lines following GSI treatment. We provide evidence that approximately 27% of ERBB2-positive human breast cancer specimens display high expression of HES1, phospho-S6RP, and GLUT1. Together, these results suggest that pathways downstream of Notch signaling are, at least in part, responsible for promoting tumor growth in neuT and also active in both neuT and a subset of human breast cancers. These findings suggest that GSI may provide therapeutic benefit to a subset of ERBB2-positive breast cancers and that [(18)F]FDG-PET imaging may be useful in monitoring clinical response.

RNA Interference-Mediated Silencing of Mitotic Kinesin KIF14 Disrupts Cell Cycle Progression and Induces Cytokinesis Failure

ABSTRACT KIF14 is a microtubule motor protein whose elevated expression is associated with poor-prognosis breast cancer. Here we demonstrate KIF14 accumulation in mitotic cells, where it associated with developing spindle poles and spindle microtubules. Cells at later stages of mitosis were characterized by the concentration of KIF14 at the midbody. Time-lapse microscopy revealed that strong RNA interference (RNAi)-mediated silencing of KIF14 induced cytokinesis failure, causing several rounds of endoreduplication and resulting in multinucleated cells. Additionally, less efficacious KIF14-specific short interfering RNAs (siRNAs) induced multiple phenotypes, all of which resulted in acute apoptosis. Our data demonstrate the ability of siRNA-mediated silencing to generate epiallelic hypomorphs associated with KIF14 depletion. Furthermore, the link we observed between siRNA efficacy and phenotypic outcome indicates that distinct stages during cell cycle progression are disrupted by the differential modulation of KIF14 expression.

Cotreatment with Vorinostat Enhances Activity of MK-0457 (VX-680) against Acute and Chronic Myelogenous Leukemia Cells

Purpose: We determined the effects of vorinostat (suberoylanalide hydroxamic acid) and/or MK-0457 (VX-680), an Aurora kinase inhibitor on the cultured human (HL-60, OCI-AML3, and K562) and primary acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML), as well as on the murine pro-B BaF3 cells with ectopic expression of the unmutated and mutant forms of Bcr-Abl. Experimental Design: Following exposure to MK-0457 and/or vorinostat, apoptosis, loss of viability, as well as activity and levels of Aurora kinase and Bcr-Abl proteins were determined. Results: Treatment with MK-0457 decreased the phosphorylation of Aurora kinase substrates including serine (S)10 on histone H3 and survivin, and led to aberrant mitosis, DNA endoreduplication as well as apoptosis of the cultured human acute leukemia HL-60, OCI-AML3, and K562 cells. Combined treatment with vorinostat and MK-0457 resulted in greater attenuation of Aurora and Bcr-Abl (in K562) kinase activity and levels as well as synergistically induced apoptosis of OCI-AML3, HL-60, and K562 cells. MK-0457 plus vorinostat also induced synergistic apoptosis of BaF3 cells with ectopic overexpression of wild-type or mutant Bcr-Abl. Finally, cotreatment with MK-0457 and vorinostat induced more loss of viability of primary AML and imatinib-refractory CML than treatment with either agent alone, but exhibited minimal toxicity to normal CD34+ progenitor cells. Conclusions: Combined in vitro treatment with MK-0457 and vorinostat is highly active against cultured and primary leukemia cells. These findings merit in vivo testing of the combination against human AML and CML cells, especially against imatinib mesylate–resistant Bcr-AblT315I–expressing CML Cells.

Development of thioquinazolinones, allosteric Chk1 kinase inhibitors.

A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K(m) for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site approximately 13A from the ATP binding site. Preliminary data is presented for several of these compounds.

In vitro anti-myeloma activity of the Aurora kinase inhibitor VE-465

This study characterized the preclinical anti‐myeloma activity of VE465, a low molecular weight pan‐Aurora kinase inhibitor. After 96‐h drug exposure, several multiple myeloma (MM) cell lines were more sensitive to VE465 compared to non‐malignant cells. The anti‐MM activity of VE465 was maintained in the presence of interleukin‐6 and, interestingly, enhanced by co‐culture with stromal cells. However, primary MM cells were less responsive than cell lines. Combinations with dexamethasone (Dex), doxorubicin (Doxo) and bortezomib showed no antagonism. Our study highlights the potential role of the tumour microenvironment in modulating the activity of this drug class.

Gene Signatures Derived from a c-MET-Driven Liver Cancer Mouse Model Predict Survival of Patients with Hepatocellular Carcinoma

Biomarkers derived from gene expression profiling data may have a high false-positive rate and must be rigorously validated using independent clinical data sets, which are not always available. Although animal model systems could provide alternative data sets to formulate hypotheses and limit the number of signatures to be tested in clinical samples, the predictive power of such an approach is not yet proven. The present study aims to analyze the molecular signatures of liver cancer in a c-MET-transgenic mouse model and investigate its prognostic relevance to human hepatocellular carcinoma (HCC). Tissue samples were obtained from tumor (TU), adjacent non-tumor (AN) and distant normal (DN) liver in Tet-operator regulated (TRE) human c-MET transgenic mice (nu200a=u200a21) as well as from a Chinese cohort of 272 HBV- and 9 HCV-associated HCC patients. Whole genome microarray expression profiling was conducted in Affymetrix gene expression chips, and prognostic significances of gene expression signatures were evaluated across the two species. Our data revealed parallels between mouse and human liver tumors, including down-regulation of metabolic pathways and up-regulation of cell cycle processes. The mouse tumors were most similar to a subset of patient samples characterized by activation of the Wnt pathway, but distinctive in the p53 pathway signals. Of potential clinical utility, we identified a set of genes that were down regulated in both mouse tumors and human HCC having significant predictive power on overall and disease-free survival, which were highly enriched for metabolic functions. In conclusions, this study provides evidence that a disease model can serve as a possible platform for generating hypotheses to be tested in human tissues and highlights an efficient method for generating biomarker signatures before extensive clinical trials have been initiated.

A novel combination therapy with histone deacetylase inhibitor and Aurora kinase inhibitor for “triple negative” breast cancers.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 AbstractsnnAbstract #401 nnBackground: Aurora A and B are essential for normal mitosis. Aurora A (AA) is amplified and/or co-overexpressed with Aurora B (AB) in over 50% of breast cancers, including the basaloid variety, “Triple Negative” breast cancers. MK-0457 (M) is a pan-Aurora kinase inhibitor that inhibits in vitro and in vivo tumor growth, as well as induces apoptosis of cancer cells. Aurora kinases are chaperoned by hsp90, and their down regulation is known to sensitize transformed cells to paclitaxel. Vorinostat (V) is a pan-histone deacetylase inhibitor (HA-HDI) that induces in vitro growth arrest, differentiation and apoptosis of human breast cancer cells. By inhibiting HDAC6 and inducing hyper-acetylation and inhibition of hsp90, V depletes the levels of hsp90 chaperoned proteins, including AA and AB kinase. Methods: AA amplification in MB-231 (“Triple Negative”) and BT-474 (HER-2 amplified) cells were determined by FISH and array-CGH. The cells were treated with M (50 to 250 nM) and/or V (0.5 to 2.0 μM) for 24 to 48 hours. Cell cycle status, DNA endoreduplication (PI staining) and apoptosis (Annexin V/PI staining) were determined by flow cytometry. Intracellular protein levels of p-AA or p-AB, AA and AB, p-survivin, survivin, were determined by immunoblot analyses. Results: Treatment with M or V induces cell cycle G2/M phase accumulation and apoptosis of the breast cancer cells. Dose-dependently, treatment with M depleted phosphorylated (p)-AA, -survivin, and -Ser10 H3 levels. V induced hsp90 acetylation and depleted the levels of AA, AB and survivin. Both M and V induced formation of multi-polar aberrant mitotic spindles. Co-treatment with M and V mediated more depletion of p-survivin, AA and AB levels, as well as synergistically induced apoptosis of MB-231 and BT-474 cells. In the orthotopic MB-231 xenograft breast cancer model, co-treatment with M and V caused significantly more tumor growth delay, lung metastases and survival than treatment with either agent alone. Treatment with V induced in vivo hsp90 hyper-acetylation in the mouse xenografts, as well as in the accessible tumors from patients with breast cancer. This was determined by immunoblot analysis of post-treatment versus pre-treatment tumors with the anti-acetylated-K69-hsp90 antibody, which was developed for these studies. Hsp90 hyper-acetylation was associated with in vivo depletion of AA and AB levels in the mouse xenografts. Co-treatment with M and V, versus treatment with either M or V, also led to greater in vivo attenuation of AA, AB and p-survivin. Conclusion: These studies support the rationale to evaluate the safety and efficacy of the combination of M and V, without or with paclitaxel, against breast cancers with AA amplification and/or overexpression.nnCitation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 401.

Abstract 1759: MK-1775, a novel Wee1 kinase inhibitor, radiosensitizes p53-defective non-small cell lung cancer (NSCLC) cells

Wee1 is a tyrosine kinase involved in the S>G2 and G2>M cell cycle transitions. Following DNA damage, Wee1 is phosphorylated by upstream kinases and stabilized. Stabilized Wee1, in turn, phosphorylates and inhibits CDC2, preventing the cells from entering mitosis before repairing their damaged DNA. Abrogation of Wee1 activity, either by decreasing expression using siRNA, or by inhibiting protein activity with selective small molecule inhibitors, sensitizes tumor cells to DNA-damaging agents. This sensitization preferentially takes place in a p53 null or mutant context. We have investigated the ability of a novel, small molecule inhibitor of Wee1, MK-1775, to radiosensitize NSCLC cells treated in vitro. Four NSCLC cell lines were used in the study: A549 and H460 with wild-type p53 status and H1299 and Calu6 with null or mutant p53 status, respectively. A 24 hr pre-irradiation treatment with 200 nM MK-1775 suppressed phosphorylation of CDC2 in both A549 and H1299 cells, however, this treatment did not produce a radiosensitizing effect in either line. An 18 hr post-irradiation treatment slightly radiosensitized the H1299 line but not the A549 line. The optimal treatment of a 1 hr pre-irradiation followed by an 18 hr post-irradiation treatment substantially radiosensitized the H1299 and Calu6 lines, yielding dose-enhancement factors (DEFs) at 10% survival of 1.39 and 1.47 respectively, but did not sensitize the A549 or H460 cells, DEFs of 1.0. To determine the mechanism responsible for the radiosensitizing effect of MK-1775, cells were treated with nocodazole following a dose of 4 Gy and analyzed for phospho-histone H3 as an indication of mitotic index after 4 hrs. Unirradiated cells accumulated in mitosis during the 4 hrs in nocodazole and this accumulation was substantially enhanced by MK-1775 in both p53 wild type A549 and p53 null H1299 cells suggesting that Wee1 inhibition accelerates unirradiated cells into mitosis prematurely. Neither A549 nor H1299 cells, irradiated with 4 Gy, accumulated in mitosis during the 4 hr nocodazole treatment in the absence of MK-1775 indicating a robust G2 block for both lines. However, H1299 cells irradiated with 4 Gy and treated with MK-1775 accumulated in mitosis to a level higher than the unirradiated cells treated with MK-1775 alone. This enhanced entry of irradiated cells into mitosis was not evident to nearly the same degree in the A549 cells. Thus, we conclude that the Wee1 inhibitor, MK-1775, accelerates cells harboring DNA-damage prematurely into mitosis in a p53-dependent manner correlating with its radiosensitizing effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1759.