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.
488 Background: BRAF mutations occur in about 10% of colorectal cancer (CRC). Most BRAF mutations involve the V600E amino acid substitution, resulting in constitutive activation of the MAPK signaling pathway. Vemurafenib (RG7204, PLX4032) is a first-in-class, BRAF V600E -specific small molecule inhibitor that dose-dependently inhibits tumor growth in V600E CRC xenografts. However, unlike responses observed in melanoma, single agent vemurafenib in a Phase I extension trial of 20 patients with previously treated metastatic CRC, resulted in only 5% response rate. The goal here was to explore in vitro and in vivo if addition of a MEK inhibitor (MEKi) could increase effects of vemurafenib on anti-proliferation and anti-tumor activity in BRAF V600E CRC cell lines. Methods: Combo of vemurafenib with a MEKi was tested in MTT assay for antiproliferative effect and combination effect was determined by combination index (CI) calculated by the CalcySyn software. Western analysis and Annexin V staining were utilized to evaluate combo effects on pathway inhibition and apoptosis induction. Optimal doses of both vemurafenib and MEKi were tested as single agent and in combination in the Colo205 and LS411N CRC xenograft models in nude mice. Results: Synergistic anti-proliferative effect was observed with the combo of vemurafenib and a MEKi in the V600E positive CRC cell lines tested. More effective pathway inhibition and apoptosis induction were observed with combo than either agent alone. Combination of vemurafenib and a MEKi delivered greater anti-tumor activity and increased life span of animals in the Colo205 and LS411N CRC xenograft models. Conclusions: These in vitro and in vivo data suggest that combined pharmacologic blockade within the RAS/RAF/MEK/ERK pathway is more effective than either agent alone and may be a way to exploit greater antitumor activity CRC patients in clinic.
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.
Abstract The BRAFV600E mutation is frequently found in human cancers (∼ 8%), with the highest incidence in melanoma (60–70%). Small molecules targeting BRAFV600E or MEK have shown significant clinical activity in melanoma patients carrying the BRAFV600E mutation. However, rapid acquisition of drug resistance is an emerging problem that limits patient survival benefits from these agents. MEK inhibition abrogates the intrinsic negative feedback on both RAS/RAF/MEK and PI3K/AKT pathways leading to induction of MEK and AKT phosphorylation. Induction of MEK and AKT phosphorylation may confer resistance and limit the clinical activity of MEK inhibitors. In this study, we investigated the molecular mechanisms of crosstalk between the two pathways. In NIH3T3 cells, engineered expression of BRAFV600E activates the MEK/ERK pathway leading to a significant growth advantage compared to control cells both in vitro and in vivo. MEK inhibition in NIH3T3 control cells strongly induces AKT phosphorylation (Thr308 and Ser473) and downstream signalling, whereas in NIH3T3 cells expressing BRAFV600E the AKT phosphorylation is significantly reduced. Knockdown of BRAFV600E restores MEK inhibition-induced elevation of AKT phosphorylation (pAKT). In the melanoma cell line with wild-type BRAF, MEK inhibition also induces pAKT (Thr308 and Ser473), whereas in the melanoma cell lines harbouring BRAFV600E, MEK inhibition-induced pAKT is not seen. Knockdown of BRAFV600E in those melanoma cell lines significantly elevates basal pAKT levels. These results suggest that BRAFV600E negatively regulates AKT pathway activation. In BRAFV600E melanoma cell lines, knockdown of MEK1/2 or ERK1/2 and inhibition of BRAF by RAF inhibitor have minimal effects on pAKT, suggesting that BRAFV600E suppresses AKT activation independent of the MEK/ERK downstream signals and its kinase activity. Furthermore, the elevation of pAKT(Thr308 and Ser473) caused by BRAFV600E knockdown can be abolished by Rictor, but not Raptor, knockdown, suggesting that the pAKT induction is Rictor dependent. Lack of pAKT induction in cells carrying BRAFV600E is associated with co-purification of Rictor-mTOR and Raptor-mTOR complexes, which is rarely seen in cells with wild type BRAF. Taken together, our data demonstrate that in BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT phosphorylation in a Rictor-dependent, MEK/ERK and kinase-independent manner, possibly via altering Rictor-mTOR complex formation. This study reveals a novel molecular mechanism of crosstalk between the RAF/MEK and the PI3K/AKT pathways, suggesting an underlying mechanism whereby a subset of BRAFV600E melanoma cells are exquisitely sensitive to MEK inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B130.
Abstract In acute myeloid leukemia (AML), TP53 mutations and dysregulation of wild-type p53 is common and supports an MDM2 antagonist as a therapy. RO6839921 is an inactive pegylated prodrug of the oral MDM2 antagonist idasanutlin (active principle [AP]) that allows for IV administration. This phase 1 monotherapy study evaluated the safety, pharmacokinetics, and pharmacodynamics of RO6839921 in patients with AML. Primary objectives identified dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD). Secondary objectives assessed pharmacokinetic, pharmacodynamic, and antileukemic activity. A total of 26 patients received 120–300 mg AP of idasanutlin. The MTD was 200 mg, with DLTs at 250 (2/8 patients) and 300 mg (2/5). Treatment–related adverse events in >20% of patients were diarrhea, nausea, vomiting, decreased appetite, and fatigue. Six deaths (23.1%) occurred, all unrelated to treatment. Pharmacokinetics showed rapid and near-complete conversion of the prodrug to AP and dose-proportional exposure across doses. Variability ranged from 30%–47% (22%–54% for idasanutlin). TP53 was 21 (87.5%) wild-type and 3 mutant (12.5%). The composite response rate (complete remission [CR], CR with incomplete hematologic recovery/morphological leukemia-free state [CRi/MLFS], or CR without platelet recovery [CRp]) was 7.7%. Antileukemic activity (CR, CRi/MLFS, partial response, hematologic improvement/stable disease) was observed in 11 patients (disease control rate, 42%): 10/11 were TP53 wild-type; 1 had no sample. p53 activation was demonstrated by MIC-1 induction and was associated with AP exposure. There was not sufficient differentiation or improvement in the biologic or safety profile compared with oral idasanutlin to support continued development of RO6839921. NCT02098967.
Idasanutlin, an MDM2 antagonist, showed clinical activity and a rapid reduction in JAK2 V617F allele burden in patients with polycythemia vera (PV) in a phase 1 study. This open-label phase 2 study evaluated idasanutlin in patients with hydroxyurea (HU)-resistant/-intolerant PV, per the European LeukemiaNet criteria, and phlebotomy dependence; prior ruxolitinib exposure was permitted. Idasanutlin was administered once daily on days 1 through 5 of each 28-day cycle. The primary end point was composite response (hematocrit control and spleen volume reduction > 35%) in patients with splenomegaly and hematocrit control in patients without splenomegaly at week 32. Key secondary end points included safety, complete hematologic response (CHR), patient-reported outcomes, and molecular responses. All patients (n = 27) received idasanutlin; 16 had response assessment (week 32). Among responders with baseline splenomegaly (n = 13), 9 (69%) attained any spleen volume reduction, and 1 achieved composite response. Nine patients (56%) achieved hematocrit control, and 8 patients (50%) achieved CHR. Overall, 43% of evaluable patients (6/14) showed a ≥50% reduction in the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (week 32). Nausea (93%), diarrhea (78%), and vomiting (41%) were the most common adverse events, with grade ≥ 3 nausea or vomiting experienced by 3 patients (11%) and 1 patient (4%), respectively. Reduced JAK2 V617F allele burden occurred early (after 3 cycles), with a median reduction of 76%, and was associated with achieving CHR and hematocrit control. Overall, the idasanutlin dosing regimen showed clinical activity and rapidly reduced JAK2 allele burden in patients with HU-resistant/- intolerant PV but was associated with low-grade gastrointestinal toxicity, leading to poor long-term tolerability. This trial was registered at www.clinincaltrials.gov as #NCT03287245.
Abstract Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53 , encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi’s ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.
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