Brian Higgins

Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF -mutant melanoma

B-RAF is the most frequently mutated protein kinase in human cancers. The finding that oncogenic mutations in BRAF are common in melanoma, followed by the demonstration that these tumours are dependent on the RAF/MEK/ERK pathway, offered hope that inhibition of B-RAF kinase activity could benefit melanoma patients. Herein, we describe the structure-guided discovery of PLX4032 (RG7204), a potent inhibitor of oncogenic B-RAF kinase activity. Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF/MEK/ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts. Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase 1 clinical trials using a crystalline formulation of PLX4032 (ref. 5). In a subset of melanoma patients, pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment. This analysis revealed substantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumour regressions. At higher drug exposures afforded by a new amorphous drug formulation, greater than 80% inhibition of ERK phosphorylation in the tumours of patients correlated with clinical response. Indeed, the Phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily. These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity.

Antitumor activity of BRAF inhibitor vemurafenib in preclinical models of BRAF-mutant colorectal cancer.

The protein kinase BRAF is a key component of the RAS-RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAF(V600E) in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAF(V600)-expressing cell lines and inhibiting tumor growth in BRAF(V600E) bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAF(V600E)-mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206). The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAF(V600E) mutation.

In vitro and in vivo activity of R547: a potent and selective cyclin-dependent kinase inhibitor currently in phase I clinical trials.

The cyclin-dependent protein kinases are key regulators of cell cycle progression. Aberrant expression or altered activity of distinct cyclin-dependent kinase (CDK) complexes results in escape of cells from cell cycle control, leading to unrestricted cell proliferation. CDK inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells, and identifying small-molecule CDK inhibitors has been a major focus in cancer research. Several CDK inhibitors are entering the clinic, the most recent being selective CDK2 and CDK4 inhibitors. We have identified a diaminopyrimidine compound, R547, which is a potent and selective ATP-competitive CDK inhibitor. In cell-free assays, R547 effectively inhibited CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 (Ki = 1–3 nmol/L) and was inactive (Ki > 5,000 nmol/L) against a panel of >120 unrelated kinases. In vitro, R547 effectively inhibited the proliferation of tumor cell lines independent of multidrug resistant status, histologic type, retinoblastoma protein, or p53 status, with IC50s ≤ 0.60 μmol/L. The growth-inhibitory activity is characterized by a cell cycle block at G1 and G2 phases and induction of apoptosis. R547 reduced phosphorylation of the cellular retinoblastoma protein at specific CDK phosphorylation sites at the same concentrations that induced cell cycle arrest, suggesting a potential pharmacodynamic marker for clinical use. In vivo, R547 showed antitumor activity in all of the models tested to date, including six human tumor xenografts and an orthotopic syngeneic rat model. R547 was efficacious with daily oral dosing as well as with once weekly i.v. dosing in established human tumor models and at the targeted efficacious exposures inhibited phosphorylation of the retinoblastoma protein in the tumors. The selective kinase inhibition profile and the preclinical antitumor activity of R547 suggest that it may be promising for development for use in the treatment of solid tumors. R547 is currently being evaluated in phase I clinical trials. [Mol Cancer Ther 2006;5(11):2644–58]

Preclinical evaluation of tumor microvascular response to a novel antiangiogenic/antitumor agent RO0281501 by dynamic contrast-enhanced MRI at 1.5 T

Inhibition of tumor angiogenesis is a promising approach in cancer treatment. The purpose of this study was to evaluate the vascular response of human lung tumor xenografts in vivo to RO0281501, an inhibitor of tyrosine kinase receptors, including vascular endothelial growth factor receptor 2, fibroblast growth factor receptor, and platelet-derived growth factor receptor, using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Human non–small cell lung carcinoma (H460a) xenografts grown s.c. in athymic nu/nu mice were treated p.o. with the antiangiogenic agent RO0281501. Treatment-induced changes in tumor volume, epiphyseal growth plate thickness, and microvessel density assessed by CD31 immunohistochemistry were analyzed. Tumor vascular permeability and perfusion were measured in tumors using DCE-MRI with gadopentetate dimeglumine on a 1.5 T clinical scanner to assess vascular function. Treatment with RO0281501 resulted in significant growth retardation of H460a tumors. RO0281501-treated tumors showed histologic evidence of growth plate thickening and relatively lower microvessel density compared with the controls. Regarding DCE-MRI variables, the initial slope of contrast uptake and Akep were significantly decreased on day 7 of treatment. RO0281501 is a novel antiangiogenic/antitumor agent, which is active in the H460a xenograft model. Its effects on tumor vasculature can be monitored and assessed by DCE-MRI on a 1.5 T human MR scanner with clinically available gadopentetate dimeglumine contrast, which will facilitate clinical trials with this or similar agents. [Mol Cancer Ther 2006;5(8):1950–7]

High tumor levels of IL6 and IL8 abrogate preclinical efficacy of the γ-secretase inhibitor, RO4929097

Interest continues to build around the early application of patient selection markers to prospectively identify patients likely to show clinical benefit from cancer therapies. Hypothesis generation and clinical strategies often begin at the preclinical stage where responder and nonresponder tumor cell lines are first identified and characterized. In the present study, we investigate the drivers of in vivo resistance to the γ‐secretase inhibitor RO4929097. Beginning at the tissue culture level, we identified apparent IL6 and IL8 expression differences that characterized tumor cell line response to RO4929097. We validated this molecular signature at the preclinical efficacy level identifying additional xenograft models resistant to the in vivo effects of RO4929097. Our data suggest that for IL6 and IL8 overexpressing tumors, RO4929097 no longer impacts angiogenesis or the infiltration of tumor associated fibroblasts. These preclinical data provide a rationale for preselecting patients possessing low levels of IL6 and IL8 prior to RO4929097 dosing. Extending this hypothesis into the clinic, we monitored patient IL6 and IL8 serum levels prior to dosing with RO4929097 during Phase I. Interestingly, the small group of patients deriving some type of clinical benefit from RO4929097 presented with low baseline levels of IL6 and IL8. Our data support the continued investigation of this patient selection marker for RO4929097 and other types of Notch inhibitors undergoing early clinical evaluation.

In vivo activity of novel capecitabine regimens alone and with bevacizumab and oxaliplatin in colorectal cancer xenograft models

Modifying the capecitabine dosing schedule from 14 days on, 7 days off (14/7) to 7 days on, 7 days off (7/7) may enable higher doses and improved antitumor efficacy in colorectal cancer xenografts. Capecitabine 14/7 (267 or 400 mg/kg) and 7/7 (467 or 700 mg/kg) schedules in doublet and triplet combinations with optimally dosed bevacizumab (5 mg/kg) and oxaliplatin (6.7 mg/kg) were studied in female athymic nude mice bearing HT29 colorectal xenografts. Additional studies of suboptimally dosed bevacizumab (2.5 mg/kg) and capecitabine 7/7 (360 mg/kg) were done in a similar Colo205 tumor xenograft model. Monotherapy and combination regimens were administered to groups of 10 animals and compared with vehicle controls. In the HT29 model, tumor growth inhibition and increase in life span (ILS) were significantly greater with capecitabine 7/7 than with 14/7 (P < 0.05). The additional benefit of capecitabine 7/7 versus 14/7 was biologically significant according to National Cancer Institute criteria (>25% ILS). Adding bevacizumab to capecitabine 7/7 resulted in significantly greater survival relative to either agent alone (P < 0.0001). When oxaliplatin was added, efficacy was significantly better with the triplet combination including capecitabine 7/7 (tumor growth inhibition >100% and ILS 234%) compared with 14/7 (95% and 81%, respectively). In the Colo205 model, combination therapy with capecitabine 7/7 plus bevacizumab resulted in significantly greater survival relative to either agent alone (P < 0.0001). In conclusion, in athymic nude mice bearing moderately thymidine phosphorylase–expressing HT29 or Colo205 colorectal xenografts, a capecitabine 7/7 schedule permits increased drug delivery compared with traditional 14/7 regimens, greatly improving monotherapy activity without major toxicity. [Mol Cancer Ther 2009;8(1):75–82]

Discovery of potent and selective spiroindolinone MDM2 inhibitor, RO8994, for cancer therapy

The field of small-molecule inhibitors of protein-protein interactions is rapidly advancing and the specific area of inhibitors of the p53/MDM2 interaction is a prime example. Several groups have published on this topic and multiple compounds are in various stages of clinical development. Building on the strength of the discovery of RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound, we have developed additional scaffolds that provide opportunities for future development. Here, we report the discovery and optimization of a highly potent and selective series of spiroindolinone small-molecule MDM2 inhibitors, culminating in RO8994.

MDM2 antagonists boost antitumor effect of androgen withdrawal: implications for therapy of prostate cancer

BackgroundHormone therapy is the standard of care for newly diagnosed or recurrent prostate cancers. It uses anti-androgen agents, castration, or both to eliminate cancer promoting effect of testicular androgen. The p53 tumor suppressor controls a major pathway that can block cell proliferation or induce apoptosis in response to diverse forms of oncogenic stress. Activation of the p53 pathway in cancer cells expressing wild-type p53 has been proposed as a novel therapeutic strategy and recently developed MDM2 antagonists, the nutlins, have validated this in preclinical models of cancer. The crosstalk between p53 and androgen receptor (AR) signaling suggest that p53 activation could augment antitumor outcome of androgen ablation in prostate cancer. Here, we test this hypothesis in vitro and in vivo using the MDM2 antagonist, nutlin-3 and the p53 wild-type prostate cancer cell line, LNCaP.ResultsUsing charcoal-stripped serum as a cellular model of androgen deprivation, we show an increased apoptotic effect of p53 activation by nutlin-3a in the androgen-dependent LNCaP cells and to a lesser extent in androgen-independent but responsive 22Rv1 cell line. This effect is due, at least in part, to an enhanced downregulation of AR expression by activated p53. In vivo, androgen deprivation followed by two weeks of nutlin administration in LNCaP-bearing nude mice led to a greater tumor regression and dramatically increased survival.ConclusionsSince majority of prostate tumors express wild-type p53, its activation by MDM2 antagonists in combination with androgen depletion may offer an efficacious new approach to prostate cancer therapy.

Pyrazolobenzodiazepines: Part I. Synthesis and SAR of a potent class of kinase inhibitors

A novel series of pyrazolobenzodiazepines 3 has been identified as potent inhibitors of cyclin-dependent kinase 2 (CDK2). Their synthesis and structure-activity relationships (SAR) are described. Representative compounds from this class reversibly inhibit CDK2 activity in vitro, and block cell cycle progression in human tumor cell lines. Further exploration has revealed that this class of compounds inhibits several kinases that play critical roles in cancer cell growth and division as well as tumor angiogenesis. Together, these properties suggest a compelling basis for their use as antitumor agents.

Abstract 1370: RO5323441, a humanized monoclonal antibody against the placenta growth factor, blocks PlGF-induced VEGFR-1 phosphorylation in vitro and tumor growth in vivo

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Solid tumor growth requires new blood vessel formation or angiogenesis. Inhibition of angiogenesis as a therapeutic strategy in oncology has been validated by treatments that block vascular endothelial growth factor (VEGF) or its receptors, such as bevacizumab (Avastin®), which is an anti-VEGF antibody that prolongs survival in colorectal, lung and other cancer patients in combination with chemotherapy. However, since not all tumors are sensitive to VEGF blockade and resistance mechanisms to VEGF therapies can develop, inhibition of additional targets may be necessary in order to control tumor growth and achieve better clinical effects. PlGF is a member of the VEGF family that is found only in very low levels under normal physiological conditions, but is up-regulated in almost all major malignant diseases. PlGF expression has shown to correlate with tumor stages and patient survival in breast cancer, CRC and gastric cancer [1-3]. Pre-clinical data support a role for PlGF in tumor angiogenesis, and demonstrate that blocking PlGF can inhibit tumor growth [4]. RO5323441, a humanized IgG1 monoclonal antibody directed against PlGF, has demonstrated anti-tumor activity in human tumor xenograft models in mice, has a benign preclinical toxicology profile and is being developed for the treatment of multiple advanced cancer indications. RO5323441 binds to both PlGF-1 and PlGF-2 in a dose dependent manner, and is not cross-reactive with murine PlGF or human VEGF. RO5323441 inhibits the binding of human PlGF-1 or PlGF-2 to VEGFR −1 with IC50 values of 0.1 and 0.2 nM, respectively. RO5323441 blocks PlGF-induced VEGFR-1 phosphorylation in Flt-1-transfected HEK293 cells. Antitumor activity has been demonstrated in mutliple tumor models including ACHN and Caki-1 renal cell carcinoma and Huh-7, hepatocellular carcinoma xenografts. Inhibition of established tumors ranged from 43-97% with twice weekly dosing. A refractory model of non-small cell lung cancer has also been identified. Studies to understand the mechanism of action and to identify pharmacodynamic markers and have been carried out in ACHN-tumor bearing mice. References 1. Wei SC, Tsao PN, Yu SC, et al. Placenta growth factor expression is correlated with survival of patients with colorectal cancer. Gut. 2005 May; 54(5):666-72. 2. Parr C, Watkins G, Boulton M et al. Placenta growth factor is over-expressed and has prognostic value in human breast cancer. Eur J Cancer. 2005 Dec;41(18):2819-27. 3. Chen CN, Hsieh FJ, Cheng YM, et al. The significance of placenta growth factor in angiogenesis and clinical outcome of human gastric cancer. Cancer Lett. 2004 Sep 15;213(1):73-82. 4. Fischer C, Jonckx B, Mazzone M, et al. Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell. 2007. 131 : 463-75. 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 1370.