Samantha Goodstal

Inhibiting Aurora kinases reduces tumor growth and suppresses tumor recurrence after chemotherapy in patient-derived triple-negative breast cancer xenografts

Triple-negative breast cancers (TNBC) have an aggressive phenotype with a relatively high rate of recurrence and poor overall survival. To date, there is no approved targeted therapy for TNBCs. Aurora kinases act as regulators of mammalian cell division. They are important for cell-cycle progression and are frequently overexpressed or mutated in human tumors, including breast cancer. In this study, we investigated the therapeutic potential of targeting Aurora kinases in preclinical models of human breast cancers using a pan-inhibitor of Aurora kinases, AS703569. In vitro, AS703569 was tested in 15 human breast cancer cell lines. TNBC cell lines were more sensitive to AS703569 than were other types of breast cancer cells. Inhibition of proliferation was associated with cell-cycle arrest, aneuploidy, and apoptosis. In vivo, AS703569 administered alone significantly inhibited tumor growth in seven of 11 patient-derived breast cancer xenografts. Treatment with AS703569 was associated with a decrease of phospho-histone H3 expression. Finally, AS703569 combined to doxorubicin–cyclophosphamide significantly inhibited in vivo tumor recurrence, suggesting that Aurora kinase inhibitors could be used both in monotherapy and in combination settings. In conclusion, these data indicate that targeting Aurora kinases could represent a new effective approach for TNBC treatment. Mol Cancer Ther; 11(12); 2693–703. ©2012 AACR.

The MEK1/2 Inhibitor Pimasertib Enhances Gemcitabine Efficacy in Pancreatic Cancer Models by Altering Ribonucleotide Reductase Subunit-1 (RRM1)

Purpose: Gemcitabine, a nucleoside analogue, is an important treatment for locally advanced and metastatic pancreatic ductal adenocarcinoma (PDAC) but provides only modest survival benefit. Targeting downstream effectors of the RAS/ERK signaling pathway by direct inhibition of MEK1/2 proteins is a promising therapeutic strategy, as aberrant activation of this pathway occurs frequently in PDAC. In this study, the ability of pimasertib, a selective allosteric MEK1/2 inhibitor, to enhance gemcitabine efficacy was tested and the molecular mechanism of their interaction was investigated. Experimental Design: Cell survival and apoptosis were assessed by MTT and Caspase 3/7 Glo assays in human pancreatic cancer cell lines. Protein expression was detected by immunoblotting. The in vivo sensitivity of gemcitabine with pimasertib was evaluated in an orthotopic model of pancreatic tumor. Results: Synergistic activity was observed when gemcitabine was combined sequentially with pimasertib, in human pancreatic cancer cells. In particular, pimasertib reduced ribonucleotide reductase subunit 1 (RRM1) protein, and this was associated with sensitivity to gemcitabine. Pretreatment with MG132 impaired reduction of RRM1 protein induced by pimasertib, suggesting that RRM1 is degraded posttranslationally. Immunoprecipitation indicated enhanced MDM2-mediated polyubiquitination of RRM1 through Lys-48–mediated linkage following pimasertib treatment, an effect mediated, in part, by AKT. Finally, the combination treatment with pimasertib and gemcitabine caused significant tumor growth delays in an orthotopic pancreatic cancer model, with RRM1 downregulation in pimasertib-treated mice. Conclusions: These results confirm an important role of RRM1 in gemcitabine response and indicate MEK as a potential target to sensitize gemcitabine therapy for PDAC. Clin Cancer Res; 21(24); 5563–77. ©2015 AACR.

SAR and evaluation of novel 5H-benzo[c][1,8]naphthyridin-6-one analogs as Aurora kinase inhibitors

Several potent Aurora kinase inhibitors derived from 5H-benzo[c][1,8]naphthyridin-6-one scaffold were identified. A crystal structure of Aurora kinase A in complex with an initial hit revealed a binding mode of the inhibitor within the ATP binding site and provided insight for structure-guided compound optimization. Subsequent SAR campaign provided a potent and selective pan Aurora inhibitor, which demonstrated potent target modulation and antiproliferative effects in the pancreatic cell line, MIAPaCa-2. Furthermore, this compound inhibited phosphorylation of histone H3 (pHH3) in mouse bone morrow upon oral administration, which is consistent with inhibition of Aurora kinase B activity.

Comprehensive Two- and Three-Dimensional RNAi Screening Identifies PI3K Inhibition as a Complement to MEK Inhibitor AS703026 for Combination Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a major cause of death among breast cancer patients that results from intrinsic and acquired resistance to systemic chemotherapies. To identify novel targets for effective treatment of TNBC through combination strategies with MEK inhibitor (AS703026), we used a novel method of combining high-throughput two- and three-dimensional (2D and 3D) RNAi screening. TNBC cells were transfected with a kinome siRNA library comprising siRNA targeting 790 kinases under both 2D and 3D culture conditions with or without AS703026. Molecule activity predictor analysis revealed the PI3K pathway as the major target pathway in our RNAi combination studies in TNBC. We found that PI3K inhibitor SAR245409 (also called XL765) combined with AS703026 synergistically inhibited proliferation compared with either drug alone (P < 0.001). Reduced in vitro colony formation (P < 0.001) and migration and invasion ability were also observed with the combination treatment (P<0.01). Our data suggest that SAR245409 combined with AS703026 may be effective in patients with TNBC. We conclude that a novel powerful high-throughput RNAi assays were able to identify anti-cancer drugs as single or combinational agents. Integrated and multi-system RNAi screening methods can complement difference between in vitro and in vivo culture conditions, and enriches targets that are close to the in vivo condition.

Combination of the MEK inhibitor pimasertib with BTK or PI3K-delta inhibitors is active in preclinical models of aggressive lymphomas

BACKGROUND Lymphomas are among the most common human cancers and represent the cause of death for still too many patients. The B-cell receptor with its downstream signaling pathways represents an important therapeutic target for B-cell lymphomas. Here, we evaluated the activity of the MEK1/2 inhibitor pimasertib as single agent and in combination with other targeted drugs in lymphoma preclinical models. MATERIALS AND METHODS Cell lines derived mature B-cell lymphomas were exposed to increasing doses of pimasertib alone. Immunoblotting and gene expression profiling were performed. Combination of pimasertib with idelalisib or ibrutinib was assessed. RESULTS Pimasertib as single agent exerted a dose-dependent antitumor activity across a panel of 23 lymphoma cell lines, although at concentrations higher than reported for solid tumors. Strong synergism was observed with pimasertib combined with the PI3K inhibitor idelalisib and the BTK inhibitor ibrutinib in cell lines derived from diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma. The data were confirmed in an in vivo experiment treating DLBCL xenografts with pimasertib and ibrutinib. CONCLUSION The data presented here provide the basis for further investigation of regimens including pimasertib in relapsed and refractory lymphomas.

Inhibition of aurora kinase B is important for biologic activity of the dual inhibitors of BCR-ABL and aurora kinases R763/ AS703569 and PHA-739358 in BCR-ABL transformed cells

ABL tyrosine kinase inhibitors (TKI) like Imatinib, Dasatinib and Nilotinib are the gold standard in conventional treatment of CML. However, the emergence of resistance remains a major problem. Alternative therapeutic strategies of ABL TKI-resistant CML are urgently needed. We asked whether dual inhibition of BCR-ABL and Aurora kinases A-C could overcome resistance mediated by ABL kinase mutations. We therefore tested the dual ABL and Aurora kinase inhibitors PHA-739358 and R763/AS703569 in Ba/F3- cells ectopically expressing wild type (wt) or TKI-resistant BCR-ABL mutants. We show that both compounds exhibited strong anti-proliferative and pro-apoptotic activity in ABL TKI resistant cell lines including cells expressing the strongly resistant T315I mutation. Cell cycle analysis indicated polyploidisation, a consequence of continued cell cycle progression in the absence of cell division by Aurora kinase inhibition. Experiments using drug resistant variants of Aurora B indicated that PHA-739358 acts on both, BCR-ABL and Aurora Kinase B, whereas Aurora kinase B inhibition might be sufficient for the anti-proliferative activity observed with R763/AS703569. Taken together, our data demonstrate that dual ABL and Aurora kinase inhibition might be used to overcome ABL TKI resistant CML.

Abstract LB-456: Evaluation of brain pharmacokinetics as a potential differentiation factor for the MEK inhibitors, MSC2015103 and pimasertib

MSC2015103 is an orally bio-available, selective, and highly potent small molecule inhibitor of MEK1/2. As a follower to the front-runner MEK inhibitor, pimasertib (MSC1936369/AS703026), a prime objective for the program is to differentiate the two compounds. Results from previous pharmacokinetic (PK) studies of MSC2015103 and pimasertib in mice had shown that both compounds could effectively cross the blood-brain barrier, but that MSC2015103 was retained in the brain longer than pimasertib. This was further confirmed in a study examining the whole-body distribution of radio-labeled compounds in mice. It is unclear whether the differential brain PK characteristics of the two agents will be clinically significant. To begin to address this, a series of pre-clinical studies were performed. Exposure levels and concurrent target modulation in normal murine brain tissue were examined over time following administration of multiple doses of MSC2015103 and pimasertib. Notable differences in the magnitude and temporal dynamics of exposure were observed; while concentrations of pimasertib in the brain peaked ∼1 hour post-administration (with a correlative decrease in phospho-ERK of ∼90%) with a relatively rapid clearance out of the tissue, concentrations of MSC2015103 increased in the brain over time and remained relatively elevated until 24 hours post-administration (the last time point) with only marginal target modulation observed. As more dramatic inhibition of pERK has previously been observed in subcutaneous tumors in mice in the presence of the same exposure of MSC2015103, murine brain orthotopic models of glioblastoma were employed to compare the relative target modulation of MSC2015103 and pimasertib in tumor tissue within the brain compartment, as well as their potential anti-tumor effects. Data from these orthotopic studies demonstrated similar high levels of phospho-ERK inhibition for both MSC2015103 and pimasertib in glioblastoma tumor tissue, which correlated with similar anti-tumor activity in the U87 model. The PK of MSC2015103 varied considerably from pimasertib, with a higher exposure (AUC1-24h) ratio of brain tumor-to-normal brain with MSC2015103 as compared with pimasertib. In line with the PK findings, MSC2015103 inhibited phospho-ERK in brain tumors but with relatively less target modulation in normal brain tissue, whereas pimasertib showed similar inhibition of phospho-ERK in both tissues. Collectively, these findings suggest a potential improvement in the safety/therapeutic index in the brain for MSC2015103. This may translate into other tissues, such as the eye, which has a similar blood barrier, and may be important in light of ocular toxicities observed in clinical trials with other MEK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-456. doi:1538-7445.AM2012-LB-456

Abstract 2676: The MEK-inhibitor pimasertib is synergistic with PI3K-delta and BTK inhibitors in lymphoma models

Pimasertib (AS-703026) is a potent and highly selective ATP noncompetitive MEK1/2 inhibitor that has shown anti-tumor activity in different pre-clinical models. We have previously reported pimasertib activity as single agent in lymphoma models and preliminary combinations results (Gaudio et al, AACR 2014). Here, we report detailed data on combinations of pimasertib with the PI3K-delta inhibitor idelalisib and with the BTK-inhibitor ibrutinib. Methods. Cell lines derived from activated B-cell like (ABC) diffuse large B-cell lymphoma (DLBCL) (OCI-Ly10, TMD8), germinal center B-cell like (GCB) DLBCL (DOHH2, RCK8) and from mantle cell lymphoma (REC1, JEKO1) were exposed to increasing doses of pimasertib alone and in combination with idelalisib and ibrutinib. Synergy was assessed by Chou-Talalay combination index (CI). ERK phosphorylation level and PARP cleavage were detected by western blotting in cell treated with single agents or combination of pimasertib with idelalisib or ibrutinib. NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice were subcutaneously inoculated with OCI-Ly-10 (10 x106) DLBCL cell line. Mice developed palpable tumors (100 mm3) and were randomized to receive pimasertib, orally once per day (30 mg/kg), ibrutinib (5 mg/Kg), the combination of the two, or control vehicle alone. Tumor size was measured two times per week using a digital caliper [tumor volume (mm3) = (I x W x W)/2]. Results. Strong synergism was observed with pimasertib combined with the PI3K inhibitor idelalisib in the ABC-DLBCL OCI-Ly10 (median CI = 0.026) and TMD8 (CI = 0.25), whereas synergistic/additive effects were detected in GCB-DLBCL (DOHH2, RCK8) and MCL (REC1, JEKO1). Synergism was observed with the BTK-inhibitor ibrutinib in all the cell lines: OCI-Ly10 (CI = 0.32), TMD8 (CI = 0.63), DOHH2 (CI = 0.66), RCK8 (CI = 0.87), REC1 and JEKO1 (CI = 0.2). Thirty minutes of exposure time with pimasertib were sufficient to knock-down phospho-ERK1/2 proteins in the mentioned ABC-DLBCL and MCL cell lines stimulated with anti-IgM (20μg/mL) for 20 minutes. Stronger down-regulation of phospho-ERK1/2 was seen in ABC-DLBCL and MCL cell lines treated with combination of pimasertib with idelalisib or pimasertib with ibrutinib rather than single agent treatment conditions. Notably, apoptosis and PARP cleavage was observed in cell lines treated with pimasertib in combination with ibrutinib or idelalisib. OCI-Ly10 xenograft tumors that received a combination of pimasertib (30mg/Kg) and ibrutinib (5mg/Kg) for 14 days, showed a five-fold reduction of both tumor volume and weight as compared to the control and single compound groups. Conclusions. Pimasertib-containing combinations with PI3K-delta and BTK inhibitors show very promising activity in preclinical models of mature lymphomas. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Chiara Barassi, Elena Bernasconi, Ivo Kwee, Luciano Cascione, Maurilio Ponzoni, Andrea Rinaldi, Anastasios Stathis, Samantha Goodstal, Emanuele Zucca, Francesco Bertoni. The MEK-inhibitor pimasertib is synergistic with PI3K-delta and BTK inhibitors in lymphoma models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2676. doi:10.1158/1538-7445.AM2015-2676

MEK inhibition leads to BRCA2 downregulation and sensitization to DNA damaging agents in pancreas and ovarian cancer models

Targeting the DNA damage response (DDR) in tumors with defective DNA repair is a clinically successful strategy. The RAS/RAF/MEK/ERK signalling pathway is frequently deregulated in human cancers. In this study, we explored the effects of MEK inhibition on the homologous recombination pathway and explored the potential for combination therapy of MEK inhibitors with DDR inhibitors and a hypoxia-activated prodrug. We studied effects of combining pimasertib, a selective allosteric inhibitor of MEK1/2, with olaparib, a small molecule inhibitor of poly (adenosine diphosphate [ADP]-ribose) polymerases (PARP), and with the hypoxia-activated prodrug evofosfamide in ovarian and pancreatic cancer cell lines. Apoptosis was assessed by Caspase 3/7 assay and protein expression was detected by immunoblotting. DNA damage response was monitored with γH2AX and RAD51 immunofluorescence staining. In vivo antitumor activity of pimasertib with evofosfamide were assessed in pancreatic cancer xenografts. We found that BRCA2 protein expression was downregulated following pimasertib treatment under hypoxic conditions. This translated into reduced homologous recombination repair demonstrated by levels of RAD51 foci. MEK inhibition was sufficient to induce formation of γH2AX foci, suggesting that inhibition of this pathway would impair DNA repair. When combined with olaparib or evofosfamide, pimasertib treatment enhanced DNA damage and increased apoptosis. The combination of pimasertib with evofosfamide demonstrated increased anti-tumor activity in BRCA wild-type Mia-PaCa-2 xenograft model, but not in the BRCA mutated BxPC3 model. Our data suggest that targeted MEK inhibition leads to impaired homologous recombination DNA damage repair and increased PARP inhibition sensitivity in BRCA-2 proficient cancers.

Abstract 2603: Combination activity of the MEK inhibitor Pimasertib and the hypoxia-activated prodrug TH-302 in pancreatic and biliary tract tumor xenograft models

The efficacy and tolerability of the MEK inhibitor Pimasertib and the hypoxia-activated prodrug TH-302 combination were explored in pancreatic and biliary tract preclinical tumor xenograft models. These studies were based on the hypotheses that combination efficacy may occur by several possible mechanisms including inhibition of DNA repair or increase of hypoxia by Pimasertib, or by differential targeting of the oxic and hypoxic tumor compartments by Pimasertib and TH-302 respectively. Since Pimasertib and TH-302 had each been tested in separate clinical trials in pancreatic cancer with gemcitabine (NCT01016483 and NCT01144455 respectively), these data could provide the rationale for clinical testing of this novel combination in pancreatic cancer. The combination was also tested in biliary tract cancer models (cholangiocarcinoma and gall bladder) based on the reported sensitivity of these tumor types to MEK inhibitors, their hypoxic nature, and the prevalence and high unmet need of these indications, particularly in Asia. The results showed that in the MIA PaCa-2 human pancreatic xenograft model, combination efficacy was observed and found to be schedule dependent. The greatest combination efficacy occurred when Pimasertib was administered first, 2 hours prior to TH-302, suggesting a unique combination mechanism in cells when the MAPK signaling pathway is suppressed first. The combination was subsequently explored in 2 additional pancreatic models and a panel of biliary tract patient-derived xenograft (PDX) models. In 10 cholangiocarcinoma PDX models tested, 3 models were responders to Pimasertib monotherapy and 4 models converted from being a non-responder with either monotherapy to a responder with the Pimasertib TH-302 combination using the%TV (tumor volume) criteria, which is related to a clinical PR RECIST response. Combination effects were not observed in 2 additional pancreatic or 6 gall bladder models tested. Some models were highly sensitive to Pimasertib or TH-302 monotherapy, making combination effects difficult to observe. Biomarker analyses are ongoing to identify markers that could predict which tumors are the most sensitive to this combination therapy. Citation Format: Jianguo Ma, Sakeena Syed, Lindsey Crowley, Jamie Shaw, Janet Ogden, Brian Elenbaas, Samantha Goodstal. Combination activity of the MEK inhibitor Pimasertib and the hypoxia-activated prodrug TH-302 in pancreatic and biliary tract tumor xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2603. doi:10.1158/1538-7445.AM2015-2603