Jayasree S. Nair

Aurora B Kinase Regulates the Postmitotic Endoreduplication Checkpoint via Phosphorylation of the Retinoblastoma Protein at Serine 780

The phenotypic change characteristic of Aurora B inhibition is the induction of polyploidy. Utilizing specific siRNA duplexes and a selective small molecule inhibitor (AZD1152) to inhibit Aurora B activity in tumor cells, we sought to elucidate the mechanism by which Aurora B inhibition results in polyploidy. Cells treated with AZD1152 progressed through mitosis with misaligned chromosomes and exited without cytokinesis and subsequently underwent endoreduplication of DNA despite activation of a p53-dependent pseudo G1 checkpoint. Concomitant with polyploid cell formation, we observed the appearance of Rb hypophosphorylation, an event that occurred independently of cyclin-dependent kinase inhibition. We went on to discover that Aurora B directly phosphorylates Rb at serine 780 both in vitro and in vivo. This novel interaction plays a critical role in regulating the postmitotic checkpoint to prevent endoreduplication after an aberrant mitosis. Thus, we propose for the first time that Aurora B determines cellular fate after an aberrant mitosis by directly regulating the Rb tumor suppressor protein.

Impact of combined mTOR and MEK inhibition in uveal melanoma is driven by tumor genotype.

Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.

Drug Synergy Screen and Network Modeling in Dedifferentiated Liposarcoma Identifies CDK4 and IGF1R as Synergistic Drug Targets

Drug screening and computational modeling of oncogenic signaling pathways identifies synergistic drug pairs for liposarcoma. Predicting Synergistic Therapies Identifying oncogenic targets has improved therapeutic outcomes for cancer patients, but cancers notoriously show primary or acquired resistance to single-agent therapies. Using computational modeling derived from cell viability and high-throughput proteomics data, Miller et al. identified several synergistic pairs of targets in dedifferentiated liposarcoma (DDLS). In two patient-derived DDLS cell lines, combined inhibition of CDK4 (cyclin-dependent kinase 4) and IGF1R (insulin-like growth factor 1 receptor) induced a synergistic decrease in cell proliferation by repressing two pathways: that of retinoblastoma by CDK4 inhibitors and that of AKT and mTOR (mammalian target of rapamycin) by IGF1R inhibitors. The findings suggest that dual inhibition of CDK4 and IGF1R may be a treatment strategy for DDLS and that computational modeling may be applied to various cancers to predict improved combination therapies. Dedifferentiated liposarcoma (DDLS) is a rare but aggressive cancer with high recurrence and low response rates to targeted therapies. Increasing treatment efficacy may require combinations of targeted agents that counteract the effects of multiple abnormalities. To identify a possible multicomponent therapy, we performed a combinatorial drug screen in a DDLS-derived cell line and identified cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor 1 receptor (IGF1R) as synergistic drug targets. We measured the phosphorylation of multiple proteins and cell viability in response to systematic drug combinations and derived computational models of the signaling network. These models predict that the observed synergy in reducing cell viability with CDK4 and IGF1R inhibitors depends on the activity of the AKT pathway. Experiments confirmed that combined inhibition of CDK4 and IGF1R cooperatively suppresses the activation of proteins within the AKT pathway. Consistent with these findings, synergistic reductions in cell viability were also found when combining CDK4 inhibition with inhibition of either AKT or epidermal growth factor receptor (EGFR), another receptor similar to IGF1R that activates AKT. Thus, network models derived from context-specific proteomic measurements of systematically perturbed cancer cells may reveal cancer-specific signaling mechanisms and aid in the design of effective combination therapies.

The Checkpoint Kinase Inhibitor AZD7762 Potentiates Chemotherapy-Induced Apoptosis of p53-Mutated Multiple Myeloma Cells

DNA cross-linking agents are frequently used in the treatment of multiple myeloma–generating lesions, which activate checkpoint kinase 1 (Chk1), a critical transducer of the DNA damage response. Chk1 activation promotes cell survival by regulating cell-cycle arrest and DNA repair following genotoxic stress. The ability of AZD7762, an ATP-competitive Chk1/2 inhibitor to increase the efficacy of the DNA-damaging agents bendamustine, melphalan, and doxorubicin was examined using four human myeloma cell lines, KMS-12-BM, KMS-12-PE, RPMI-8226, and U266B1. The in vitro activity of AZD7762 as monotherapy and combined with alkylating agents and the “novel” drug bortezomib was evaluated by studying its effects on cytotoxicity, signaling, and apoptotic pathways. The Chk1/2 inhibitor AZD7762 potentiated the antiproliferative effects of bendamustine, melphalan, and doxorubicin but not bortezomib in multiple myeloma cell lines that were p53-deficient. Increased γH2AX staining in cells treated with bendamustine or melphalan plus AZD7762 indicates a greater degree of DNA damage with combined therapy. Abrogation of the G2–M checkpoint by AZD7762 resulted in mitotic catastrophe with ensuing apoptosis evidenced by PARP and caspase-3 cleavage. In summary, the cytotoxic effects of bendamustine, melphalan and doxorubicin on p53-deficient multiple myeloma cell lines were enhanced by the coadministration of AZD7762. These data provide a rationale for testing these combinations in patients with relapsed and/or refractory multiple myeloma. Mol Cancer Ther; 11(8); 1781–8. ©2012 AACR.

The Topoisomerase I Poison CPT-11 Enhances the Effect of the Aurora B Kinase Inhibitor AZD1152 both In vitro and In vivo

Purpose: AZD1152 is an Aurora B kinase inhibitor currently in clinical trials. As the topoisomerase I poison CPT-11 induces a G2 arrest, a mechanistic understanding of the cell cycle interactions between these agents may prove critical for combination therapy. Methods: AZD1152 was tested in vitro and in vivo with SN-38 and CPT-11 against HCT-116 cells. Inhibition of clonogenicity, induction of apoptosis, effects on polyploidy, and tumor growth were examined. Results: AZD1152 alone induced polyploidy of HCT-116 cells at low nanomolar concentrations. The induction of apoptosis required prolonged exposure (48 hours) and higher concentrations of drug. When SN-38 was given before or concomitantly with AZD1152, SN-38 blocked the AZD1152 effect by arresting cells in G2 and inhibiting cells from undergoing polyploidy. With the reverse combination (AZD1152 followed by SN-38), there was a significant induction of polyploidy and apoptosis, even with shorter exposure (24 hours) of AZD1152. In vivo, AZD1152 alone suppressed HCT-116 xenograft tumor growth in a dose-dependent manner with target inhibition of phosphoH3, induction of multinucleated giant cells, but without induction of apoptosis. In combination, both sequences in vivo (CPT->AZD, AZD->CPT, P = 0.008, AUC/d) proved superior to either single agent therapy. However, AZD->CPT still showed a greater increase in apoptosis and greater suppression of tumor regrowth than CPT->AZD (P = 0.02, AUC/d). Conclusions: The results from these studies indicate a promising therapeutic strategy for combining AZD1152 with CPT-11, and suggest that the sequence of drug administration is pivotal when an Aurora B kinase inhibitor is administered with a topoisomerase I poison.

The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53-/- cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53- dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A.

Imaging Colon Cancer Response Following Treatment with AZD1152: A Preclinical Analysis of [18F]Fluoro-2-deoxyglucose and 3′-deoxy-3′-[18F]Fluorothymidine Imaging

Purpose: To determine whether treatment response to the Aurora B kinase inhibitor, AZD1152, could be monitored early in the course of therapy by noninvasive [18F]-labeled fluoro-2-deoxyglucose, [18F]FDG, and/or 3′-deoxy-3′-[18F]fluorothymidine, [18F]FLT, PET imaging. Experimental design: AZD1152-treated and control HCT116 and SW620 xenograft-bearing animals were monitored for tumor size and by [18F]FDG, and [18F]FLT PET imaging. Additional studies assessed the endogenous and exogenous contributions of thymidine synthesis in the two cell lines. Results: Both xenografts showed a significant volume-reduction to AZD1152. In contrast, [18F]FDG uptake did not demonstrate a treatment response. [18F]FLT uptake decreased to less than 20% of control values in AZD1152-treated HCT116 xenografts, whereas [18F]FLT uptake was near background levels in both treated and untreated SW620 xenografts. The EC50 for AZD1152-HQPA was approximately 10 nmol/L in both SW620 and HCT116 cells; in contrast, SW620 cells were much more sensitive to methotrexate (MTX) and 5-Fluorouracil (5FU) than HCT116 cells. Immunoblot analysis demonstrated marginally lower expression of thymidine kinase in SW620 compared with HCT116 cells. The aforementioned results suggest that SW620 xenografts have a higher dependency on the de novo pathway of thymidine utilization than HCT116 xenografts. Conclusions: AZD1152 treatment showed antitumor efficacy in both colon cancer xenografts. Although [18F]FDG PET was inadequate in monitoring treatment response, [18F]FLT PET was very effective in monitoring response in HCT116 xenografts, but not in SW620 xenografts. These observations suggest that de novo thymidine synthesis could be a limitation and confounding factor for [18F]FLT PET imaging and quantification of tumor proliferation, and this may apply to some clinical studies as well. Clin Cancer Res; 17(5); 1099–110. ©2011 AACR.

MLN-8237: A dual inhibitor of aurora A and B in soft tissue sarcomas.

Aurora kinases have become an attractive target in cancer therapy due to their deregulated expression in human tumors. Liposarcoma, a type of soft tissue sarcoma in adults, account for approximately 20% of all adult soft tissue sarcomas. There are no effective chemotherapies for majority of these tumors. Efforts made to define the molecular basis of liposarcomas lead to the finding that besides the amplifications of CDK4 and MDM2, Aurora Kinase A, also was shown to be overexpressed. Based on these as well as mathematic modeling, we have carried out a successful preclinical study using CDK4 and IGF1R inhibitors in liposarcoma. MLN8237 has been shown to be a potent and selective inhibitor of Aurora A. MLN-8237, as per our results, induces a differential inhibition of Aurora A and B in a dose dependent manner. At a low nanomolar dose, cellular effects such as induction of phospho-Histone H3 (Ser10) mimicked as that of the inhibition of Aurora kinase A followed by apoptosis. However, micromolar dose of MLN-8237 induced polyploidy, a hallmark effect of Aurora B inhibition. The dose dependent selectivity of inhibition was further confirmed by using siRNA specific inhibition of Aurora A and B. This was further tested by time lapse microscopy of GFP-H2B labelled cells treated with MLN-8237. LS141 xenograft model at a dose of 30 mg/kg also showed efficient growth suppression by selective inhibition of Aurora Kinase A. Based on our data, a dose that can target only Aurora A will be more beneficial in tumor suppression.

Inhibition of polo like kinase 1 in sarcomas induces apoptosis that is dependent on Mcl-1 suppression

Sarcomas are rare cancers and the current treatments in inoperable or metastatic disease have not been shown to prolong survival. In order to develop novel targeted therapies, we tested the efficacy of polo-like kinase 1 (PLK-1) inhibitor (TAK-960) in sarcoma. All the sarcoma cell lines were sensitive to TAK-960 with IC50s in the low nanomolar range. We chose MPNST, CHP100 and LS141 for our studies and of which MPNST cells exclusively underwent polyploidy after a delay in mitosis for about 18 hours; CHP100 cells, after a 24h mitotic delay, died of apoptosis; LS141, after a delay in mitosis stayed at 4N with mild apoptosis. Apoptosis induced by TAK-960 in CHP100 was associated with down-regulation of Mcl-1 and the effect was recapitulated by down-regulating PLK1 by siRNA, confirming that the effect of TAK-960 on Mcl-1 expression is target specific. With suppression of Mcl-1 by siRNA, TAK-960 induced apoptosis in MPNST cells as well. These effects were confirmed in vivo, such that TAK-960 more effectively inhibited CHP100 than MPNST xenografts. In the setting of PLK-1 inhibition, Mcl-1 down regulation is shown to be an important determinant of apoptosis. Collectively, the net effect of this is to drive cells to apoptosis, resulting in a greater anti-tumor effect in vivo. Therefore, targeting PLK-1 should have a greater impact in treating sarcomas provided there is concomitant suppression of Mcl-1. These results further indicate that Mcl-1 could be an important biomarker to predict sensitivity to the induction of apoptosis by PLK-1 targeted therapy in sarcoma.