Kenneth Kolinsky

RG7204 (PLX4032), a Selective BRAFV600E Inhibitor, Displays Potent Antitumor Activity in Preclinical Melanoma Models

The BRAF(V600E) mutation is common in several human cancers, especially melanoma. RG7204 (PLX4032) is a small-molecule inhibitor of BRAF(V600E) kinase activity that is in phase II and phase III clinical testing. Here, we report a preclinical characterization of the antitumor activity of RG7204 using established in vitro and in vivo models of malignant melanoma. RG7204 potently inhibited proliferation and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and ERK phosphorylation in a panel of tumor cell lines, including melanoma cell lines expressing BRAF(V600E) or other mutant BRAF proteins altered at codon 600. In contrast, RG7204 lacked activity in cell lines that express wild-type BRAF or non-V600 mutations. In several tumor xenograft models of BRAF(V600E)-expressing melanoma, we found that RG7204 treatment caused partial or complete tumor regressions and improved animal survival, in a dose-dependent manner. There was no toxicity observed in any dose group in any of the in vivo models tested. Our findings offer evidence of the potent antitumor activity of RG7204 against melanomas harboring the mutant BRAF(V600E) gene.

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.

MDM2 Small-Molecule Antagonist RG7112 Activates p53 Signaling and Regresses Human Tumors in Preclinical Cancer Models

MDM2 negatively regulates p53 stability and many human tumors overproduce MDM2 as a mechanism to restrict p53 function. Thus, inhibitors of p53-MDM2 binding that can reactivate p53 in cancer cells may offer an effective approach for cancer therapy. RG7112 is a potent and selective member of the nutlin family of MDM2 antagonists currently in phase I clinical studies. RG7112 binds MDM2 with high affinity (K(D) ~ 11 nmol/L), blocking its interactions with p53 in vitro. A crystal structure of the RG7112-MDM2 complex revealed that the small molecule binds in the p53 pocket of MDM2, mimicking the interactions of critical p53 amino acid residues. Treatment of cancer cells expressing wild-type p53 with RG7112 activated the p53 pathway, leading to cell-cycle arrest and apoptosis. RG7112 showed potent antitumor activity against a panel of solid tumor cell lines. However, its apoptotic activity varied widely with the best response observed in osteosarcoma cells with MDM2 gene amplification. Interestingly, inhibition of caspase activity did not change the kinetics of p53-induced cell death. Oral administration of RG7112 to human xenograft-bearing mice at nontoxic concentrations caused dose-dependent changes in proliferation/apoptosis biomarkers as well as tumor inhibition and regression. Notably, RG7112 was highly synergistic with androgen deprivation in LNCaP xenograft tumors. Our findings offer a preclinical proof-of-concept that RG7112 is effective in treatment of solid tumors expressing wild-type p53.

Interleukin-12 Antagonist Activity of Mouse Interleukin-12 p40 Homodimer in Vitro and in Vivo

Mo(p40)2 is a potent IL-12 antagonist that interacts strongly with the beta 1 subunit of the IL-12R to block binding of moIL-12 to the high-affinity mouse IL-12R. Mo(p40)2, alone or in synergy with the 2B5 mAb specific for the moIL-12 heterodimer, blocked IL-12-induced responses in vitro, Mo(p40)2 was thus used alone or with 2B5 mAb to examine the role of IL-12 in vivo, Mo(p40)2 caused a dose-dependent inhibition of both the rise in serum IFN-gamma levels in mice injected with endotoxin and the Th1-like response to immunization with KLH. Treatment with mo(p40)2 plus 2B5 anti-moIL-12 mAb also suppressed DTH responses to methylated bovine serum albumin but not specific allogeneic CTL responses in vivo. In each of these models, responses seen in mice treated with mo(p40)2 +/- 2B5 anti-moIL-12 mAb were similar to those observed in IL-12 knockout mice. Thus, mo(p40)2 can act as a potent IL-12 antagonist in vivo, as well as in vitro, and is currently being used to investigate the role of IL-12 in the pathogenesis of some Th1-associated autoimmune disorders in mice.

Resistance to Selective BRAF Inhibition Can Be Mediated by Modest Upstream Pathway Activation

A high percentage of patients with BRAF(V600E) mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF(V600E) melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF(V600E) inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance.

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]

Preclinical Optimization of MDM2 Antagonist Scheduling for Cancer Treatment by Using a Model-Based Approach

Purpose: Antitumor clinical activity has been demonstrated for the MDM2 antagonist RG7112, but patient tolerability for the necessary daily dosing was poor. Here, utilizing RG7388, a second-generation nutlin with superior selectivity and potency, we determine the feasibility of intermittent dosing to guide the selection of initial phase I scheduling regimens. Experimental Design: A pharmacokinetic–pharmacodynamic (PKPD) model was developed on the basis of preclinical data to determine alternative dosing schedule requirements for optimal RG7388-induced antitumor activity. This PKPD model was used to investigate the pharmacokinetics of RG7388 linked to the time-course of the antitumor effect in an osteosarcoma xenograft model in mice. These data were used to prospectively predict intermittent and continuous dosing regimens, resulting in tumor stasis in the same model system. Results: RG7388-induced apoptosis was delayed relative to drug exposure with continuous treatment not required. In initial efficacy testing, daily dosing at 30 mg/kg and twice a week dosing at 50 mg/kg of RG7388 were statistically equivalent in our tumor model. In addition, weekly dosing of 50 mg/kg was equivalent to 10 mg/kg given daily. The implementation of modeling and simulation on these data suggested several possible intermittent clinical dosing schedules. Further preclinical analyses confirmed these schedules as viable options. Conclusion: Besides chronic administration, antitumor activity can be achieved with intermittent schedules of RG7388, as predicted through modeling and simulation. These alternative regimens may potentially ameliorate tolerability issues seen with chronic administration of RG7112, while providing clinical benefit. Thus, both weekly (qw) and daily for five days (5 d on/23 off, qd) schedules were selected for RG7388 clinical testing. Clin Cancer Res; 20(14); 3742–52. ©2014 AACR.