A phase I study of the HDM2 antagonist SAR405838 combined with the MEK inhibitor pimasertib in patients with advanced solid tumours.

BACKGROUND: This phase I, open-label, dose-escalation study evaluated the safety, pharmacokinetics and pharmacodynamics of combination therapy with the HDM2 inhibitor SAR405838 and the MEK1/2 inhibitor pimasertib administered orally once daily (QD) or twice daily (BID) in locally advanced or metastatic solid tumours (NCT01985191). METHODS: Patients with locally advanced or metastatic solid tumours with documented wild-type TP53 and RAS or RAF mutations were enroled. A 3 + 3 dose-escalation design was employed. The primary objective was to assess maximum tolerated dose (MTD). RESULTS: Twenty-six patients were treated with SAR405838 200 or 300 mg QD plus pimasertib 60 mg QD or 45 mg BID. The MTD was SAR405838 200 mg QD plus pimasertib 45 mg BID. The most common dose-limiting toxicity was thrombocytopenia. The most frequently occurring treatment-related adverse events were diarrhoea (81%), increased blood creatine phosphokinase (77%), nausea (62%) and vomiting (62%). No significant drug-drug interactions were observed. The biomarkers MIC-1 and pERK were, respectively, upregulated and downregulated in response to study treatment. In 24 efficacy-evaluable patients, one patient (4%) had a partial response and 63% had stable disease. CONCLUSIONS: The safety profile of SAR405838 and pimasertib combined was consistent with the safety profiles of both drugs. Preliminary antitumour activity was observed.

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.

Preclinical evidence for the effective use of TL-895, a highly selective and potent second-generation BTK inhibitor, for the treatment of B-cell malignancies.

TL-895 (formerly known as M7583) is a potent, highly selective, adenosine triphosphate (ATP)-competitive, second-generation, irreversible inhibitor of Bruton's tyrosine kinase (BTK). We characterized its biochemical and cellular effects in in vitro and in vivo models. TL-895 was evaluated preclinically for potency against BTK using IC50 concentration-response curves; selectivity using a 270-kinase panel; BTK phosphorylation in Ramos Burkitt's lymphoma cells by ProteinSimple Wes analysis of one study; anti-proliferative effects in primary chronic lymphocytic leukemia (CLL) blasts; cell viability effects in diffuse large B-cell lymphoma (DLBCL) and mantle-cell lymphoma (MCL) cell lines; effects on antibody-dependent cell-mediated cytotoxicity (ADCC) from Daudi cells and chromium-51 release from human tumor cell lines; and efficacy in vivo using four MCL xenograft model and 21 DLBCL patient-derived xenograft (PDX) models (subtypes: 9 ABC, 11 GCB, 1 Unclassified). TL-895 was active against recombinant BTK (average IC50 1.5 nM) and inhibited only three additional kinases with IC50 within tenfold of BTK activity. TL-895 inhibited BTK auto-phosphorylation at the Y223 phosphorylation site (IC50 1-10 nM). TL-895 inhibited the proliferation of primary CLL blasts in vitro and inhibited growth in a subset of activated DLBCL and MCL cell lines. TL-895 inhibited the ADCC mechanism of therapeutic antibodies only at supra-clinical exposure levels. TL-895 significantly inhibited tumor growth in the Mino MCL xenograft model and in 5/21 DLBCL PDX models relative to vehicle controls. These findings demonstrate the potency of TL-895 for BTK and its efficacy in models of B-cell lymphoma despite its refined selectivity.

Selective Oral MEK1/2 Inhibitor Pimasertib in Metastatic Melanoma: Antitumor Activity in a Phase I, Dose-Escalation Trial.

BACKGROUND: Pimasertib is a selective, potent mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor. OBJECTIVES: The aim of this study was to describe the efficacy, safety, and pharmacodynamics of pimasertib at pharmacologically active doses in a cohort of patients with locally advanced/metastatic melanoma from a first-in-human study of pimasertib. METHODS: This was a phase I, open-label, two-part, dose-escalation study. Part 1 was conducted in patients with solid tumors and identified the maximum tolerated dose, while Part 2 was restricted to patients with advanced/metastatic melanoma. Endpoints included safety, pharmacodynamics, and antitumor activity. We present data for patients with melanoma only from both parts of the study. RESULTS: In total, 93 patients with melanoma received pimasertib, 89 of whom received pharmacologically active doses (28-255 mg/day) across four dose regimens in the two parts of the study. The objective response rate was 12.4% (11/89): complete response (n = 1) and partial response (PR; n = 10). Six patients responded for > 24 weeks. Nine of the 11 responders had tumors with B-Raf Proto-Oncogene, Serine/Threonine Kinase (BRAF; n = 6) and/or NRAS Proto-Oncogene, GTPase (NRAS; n = 3) mutations. Forty-six patients had stable disease (SD). In patients with ocular melanoma (n = 13), best overall response was PR (n = 1), SD (n = 11), and disease progression (n = 1). Phosphorylated extracellular signal-regulated kinase (pERK) levels were substantially reduced within 2 h of treatment and inhibition was sustained with continuous twice-daily dosing. Treatment-related, recurrent, grade 3 or higher adverse events were reported in eight patients, including diarrhea, and skin and ocular events. CONCLUSION: Results from this phase I study indicate that pimasertib has clinical activity in patients with locally advanced/metastatic melanoma, particularly BRAF- and NRAS-mutated tumors, at clinically relevant doses associated with pERK inhibition in peripheral blood mononuclear cells. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00982865.

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.

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.

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.

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.

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.