A benzoxazole compound as a novel MEK inhibitor for the treatment of RAS/RAF mutant cancer

Mutations in RAS/RAF occur in large portion of malignancies and are associated with aggressive clinical behaviors and poor prognosis. Therefore, we developed a novel benzoxazole compound (KZ-001) as a highly potent and selective MEK 1/2 inhibitor. Our efforts were focused on enhancing the activity of the known MEK inhibitor AZD6244 and overcoming the shortcomings existing in current MEK inhibitors. Here we show that compound KZ-001 exhibits approximately 30-fold greater inhibition against BRAF- and KRAS-mutant tumor cells than that of AZD6244. These results were also demonstrated using in vivo xenograft models. Furthermore, pharmacokinetics (PK) analysis was performed for KZ-001, and this compound showed good orally bioavailability (28%) and exposure (AUC_0–∞ = 337 ± 169 ng h/mL). To determine its potential clinical application, the synergistic effect of KZ-001 with other agents was investigated both in vitro and in vivo (xenograft models). KZ-001 exhibited synergistic anti-cancer effect in combination with BRAF inhibitor vemurafenib and a microtubule-stabilizing chemotherapeutic agent docetaxel. In addition, KZ-001 inhibited the MAPK pathway like known MEK inhibitors. In summary, KZ-001, a structurally novel benzoxazole compound, was developed as a MEK inhibitor that has potential for cancer treatment.     

Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032

Purpose

To assess the relative radiosensitivities of a large collection of melanoma cell lines and to determine whether pharmacologic inhibition of mutant B-RAF with PLX-4032 can radiosensitize B-Raf+ melanoma cells.

Materials and methods

A large collection of melanoma cell lines (n = 37) were treated with 0-8 Gy IR and clonogenic survival assays used to generate survival curves to rank relative radiosensitivities among the cell lines. The ability of a B-RAF inhibitor, PLX-4032, to radiosensitize highly radioresistant B-Raf+ cells was also assessed by clonogenic cell survival and spheroid invasion assays and the effects of treatment on the cell cycle assessed by FACS.

Results

Melanoma cell lines displayed a very large, heterogeneous range of SF2 values (1.002-0.053) with a mean of 0.51. Cell lines with surviving fractions of 0.29 or less at SF2 and SF4 were observed at a high frequency of 18.9% and 70.2%, respectively. Treatment of B-Raf+ cells with the B-RAF inhibitor PLX-4032 in combination with radiation provided enhanced inhibition of both colony formation and invasion, and radiosensitized cells through an increase in G₁ arrest.

Conclusions

Our data suggest that melanomas are not uniformly radioresistant with a significant subset displaying inherent radiosensitivity. Pharmacologic inhibition of B-RAF with PLX-4032 effectively radiosensitized B-Raf+ melanoma cells suggesting that this combination approach could provide improved radiotherapeutic response in B-Raf+ melanoma patients.     

Synergistic effect of the inhibitors of RAF/MEK and AXL on KRAS‐mutated ovarian cancer cells with high AXL expression

KRAS mutation is frequently seen in a subtype of ovarian cancer categorized as type 1. The KRAS‐MAPK pathway, which is closely involved in type 1 cancer progression, is under the regulation of receptor tyrosine kinases (RTKs). AXL, one of the RTKs, has been reported to be overexpressed in ovarian cancer and contributes to the poor prognosis. However, there is no useful target‐based agent against such gene profiles. We examined the combined effect of the dual RAF/MEK inhibitor CH5126766 and AXL inhibitor R428 on the growth of ovarian cancer HEY‐T30 and OVCAR‐5 cell lines, both of which bear KRAS mutation and express AXL at a high level, using the WST‐8 assay and the colony formation assay. The synergistic effect of the combination was evaluated by the combination index. The apoptotic cells were analyzed by flow cytometry. The expression of apoptotic proteins and the phosphorylation of MAPK and AKT pathway proteins were investigated by western blotting. We found that CH5126766 and R428 suppressed the phosphorylation of ERK and AKT, respectively, and their combination synergistically inhibited the growth of both cell lines with enhancement of apoptosis accompanied by the Bim upregulation. Combined treatment with CH5126766 and R428 is expected as the novel therapeutic option for KRAS‐mutated ovarian cancer with high expression of AXL.     

Upstream mitogen-activated protein kinase (MAPK) pathway inhibition: MEK inhibitor followed by a BRAF inhibitor in advanced melanoma patients

BRAF-mutant melanoma can be successfully treated by BRAF kinase inhibitors (BRAFi) and MEK kinase inhibitors (MEKi). However, the administration of BRAFi followed by MEKi did not generate promising response rate (RR). The purpose of this investigation was to evaluate the time to progression (TTP) with a mitogen-activated protein kinase (MAPK) pathway upstream inhibition strategy in BRAF mutated melanoma patients.BRAF mutation positive metastatic melanoma patients were identified within the Dermatology Cooperative Oncology Group (DeCOG) network and were treated first with a MEKi and upon progression with a selective BRAFi.A total of 23 melanoma patients (six females, 17 males, aged 47–80 years) were retrospectively analysed for TTP. The total median TTP was 8.9 months. The median TTP for MEKi was 4.8 (1.2–23.2) and subsequent for BRAFi 4.5 (1.2–15.7) months, respectively. A higher RR for MEKi (39%, nine partial responses and 0 complete responses) than previously reported was observed.Our analysis suggests that the reversed inhibition of the MAPK pathway is feasible in BRAF mutated melanoma. The median TTP (8.9 months) is close to the promising BRAF- and MEKi combination therapy (median progression-free survival (PFS) 9.4 months). The total treatment duration of the MAPK inhibition when a MEKi is administered first is similar compared to the reversed sequence, but TTP shifts in favour to the MEKi. This approach is feasible with reasonable tolerability. This clinical investigation encourages further studies in prospective clinical trials to define the optimal treatment schedule for the MAPK pathway inhibition and should be accompanied by molecular monitoring using repeated biopsies.     

MEK inhibitor-associated retinopathy (MEKAR) in metastatic melanoma: Long-term ophthalmic effects

BackgroundMitogen-activated protein kinase kinase (MEK) inhibitors have aroused considerable interest in oncology. Activity has been demonstrated in various types of cancer, especially melanoma. MEK inhibitors induce a transient retinopathy, considered to be a class effect. At present, only sparse data are available on retinal effects with long-term MEK inhibition.Patients and methodsIn this prospective, observational study, patients with advanced melanoma participating in different phase 1/2 or phase 3 clinical trials were treated with the MEK inhibitor binimetinib, with a v-Raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor, or with combination therapy. They underwent regular ophthalmological examinations including determination of visual function, biomicroscopy, dilated fundoscopy and optical coherence tomography (OCT) for a period of up to 2 years. Retinopathy was diagnosed on defined OCT criteria.ResultsSixty-two patients were investigated between 1st October 2011 and 31st July 2015: 13 were treated with the MEK inhibitor binimetinib alone, 10 with a selective BRAF inhibitor, and 39 with combination therapy. In 92% of patients on monotherapy and 100% of those on combination treatment, binimetinib caused dose-related lesions with serous neuroretinal detachments and oedema, strongly dependent on the time after medication. With continued treatment, retinal volume and thickness decreased to levels below baseline, without any apparent functional deficits or changes in structural integrity.ConclusionsBinimetinib induces a specific retinopathy with daily fluctuations depending on the time interval after medication. The retinopathy partially recovers, but can still be detected many months later. Retinal thinning, possible first signs of retinal atrophy have been observed after long-term treatment, but, so far, without functional relevance.     

Pluripotency markers are differentially induced by MEK inhibition in thyroid and melanoma BRAFV600E cell lines

Oncogenic mutations in BRAF are common in melanoma and thyroid carcinoma and drive constitutive activation of the MAPK pathway. Molecularly targeted therapies of this pathway improves survival compared to chemotherapy; however, responses tend to be short-lived as resistance invariably occursCell line models of melanoma and thyroid carcinoma, +/? BRAF^V600E activating mutation, were treated with the MEK inhibitor PD0325901. Treated and naive samples were assayed for expression of key members of the MAPK pathway. Global microRNA expression profiling of naive and resistant cells was performed via next generation sequencingand indicated pluripotency pathways in resistance. Parental cell lines were progressed to holoclones to confirm the miRNA stemness profileMembers of the MIR302/373/374/520 family of embryonic stem cell specific cell cycle regulating (ESCC) microRNAs were identified as differentially expressed between resistant BRAF^V600E melanoma and thyroid cell lines. Upregulated expression of gene and protein stemness markers, upregulated expression of MAPK pathway genes and downregulation of the ESCC MIR302 cluster in BRAF^V600E melanoma indicated an increased stem-like phenotype in resistant BRAF^V600E melanoma. Conversely, downregulated expression of gene and protein stemness markers, downregulated expression of MAPK pathway genes, upregulation of the ESCC MIR520 cluster, reeexpression of cell surface receptors, and induced differentiation-associated morphology in resistant BRAF^V600E indicate a differentiated phenotype associated with MEK inhibitor resistance in BRAF^V600E thyroid cellsThe differential patterns of resistance observed between BRAF^V600E melanoma and thyroid cell lines may reflect tissue type or de novo differentiation, but could have significant impact on the response of primary and metastatic cells to MEK inhibitor treatment. This study provides a basis for the investigation of the cellular differentiation/self-renewal access and its role in resistance to MEK inhibition.     

Radiosensitization and downregulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) upon inhibition of mitogen/extracellular signal-regulated kinase (MEK) in malignant melanoma cells

Background

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an important cofactor in the p53-mediated DNA damage response pathway upon ionizing radiation (IR) and exerts anti-apoptotic effects also independent of p53 pathway activation. Furthermore, hnRNP K is overexpressed in various neoplasms including malignant melanoma (MM). Here, we investigate the role of hnRNP K in the radioresistance of MM cells.

Methods and results

Our results show cytoplasmic expression of hnRNP K in human MM surgical specimens, but not in benign nevi, and a quick dose- and time-dependent upregulation in response to IR accompanied by cytoplasmic redistribution of the protein in the IPC-298 cellular tumor model carrying an activating NRAS mutation (p.Q61L). SiRNA-based knockdown of hnRNP K induced a delayed decline in γH2AX/53BP1-positive DNA repair foci upon IR. Pharmacological interference with MAPK signaling abrogated ERK phosphorylation, diminished cellular hnRNP K levels, impaired γH2AX/53BP1-foci repair and proliferative capability and increased apoptosis comparable to the observed hnRNP K knockdown phenotype in IPC-298 cells.

Conclusion

Our results indicate that pharmacological interference with MAPK signaling increases vulnerability of NRAS-mutant malignant melanoma cells to ionizing radiation along with downregulation of endogenous hnRNP K and point towards a possible use for combined MEK inhibition and localized radiation therapy of MM in the NRAS-mutant setting where BRAF inhibitors offer no clinical benefit.     

MEK inhibition in non-small cell lung cancer

KRAS mutations are the most common mutations in non-small cell lung cancer (NSCLC) with adenocarcinoma histology. KRAS mutations result in the activation of the RAF–MEK–ERK pathway, and agents that target RAF–MEK–ERK pathways have been investigated in KRAS mutant NSCLC. The two agents furthest in development are selumetinib and trametinib. Trametinib has greater binding for the MEK1/2 allosteric site, and generally has superior pharmacokinetics. A randomized phase II trial of docetaxel with and without selumetinib revealed that the combination resulted numerically superior overall survival, and a statistically significant improvement in progression-free survival and objective response rate. However, a concerning rate of hospital admission, grade 3 or 4 neutropenia, and febrile neutropenia was observed with the combination. Trials have investigated MEK inhibitors as single agents and in combination with erlotinib, and the data do not support the further development. The activity of MEK inhibitors appears to be similar in patients with KRAS mutant and wild-type NSCLC suggesting KRAS mutation status is not a reliable biomarker for efficacy. It is possible that mutations of genes in addition to KRAS mutations impact the activity of MEK inhibitors, or specific subsets of KRAS mutations may be resistant or susceptible to MEK inhibition. Other potential explanations are gene amplifications, alternative RNA splicing of genes resulting in activation of their protein products, and deregulation of noncoding RNAs and consequent altered protein expression.     

Adaptive upregulation of FOXD3 and resistance to PLX4032/4720-induced cell death in mutant B-RAF melanoma cells

Melanoma cells driven by mutant v-raf murine sarcoma viral oncogene homolog B1 (B-RAF) are highly resistant to chemotherapeutic treatments. Recent phase 1 results with PLX4032/RG7204/vemurafenib, which selectively inhibits B-RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)1/2 signaling in mutant B-RAF cells, has given encouragement to this struggling field. Nearly all patients in the phase 1-3 studies saw at least some response and the overall response rates ranged from 48 and 81%. However, despite initial tumor shrinkage, most responders in the trial experienced tumor relapse over time. These findings indicate that both intrinsic and acquired resistance may affect the clinical efficacy of PLX4032. It is critical to optimize PLX4032 activity to improve response rates and understand why some patients with the B-RAF mutation do not respond. We have previously shown that the stemness factor, Forkhead box D3 (FOXD3), is upregulated following inhibition of B-RAF-MEK signaling in mutant B-RAF melanoma cells. Here, we show that upregulation of FOXD3 following treatment with PLX4032 and PLX4720 (the non-clinical tool compound for PLX4032) confers resistance to cell death. Small interfering RNA-mediated knockdown of FOXD3 significantly enhanced the cell death response after PLX4032/4720 treatment in mutant B-RAF melanoma cell lines. Additionally, upregulation of FOXD3 after PLX4720 treatment was attenuated in non-adherent conditions and correlated with enhanced cell death. Ectopic expression of FOXD3 in non-adherent cells significantly reduced cell death in response to PLX4720 treatment. Together, these data indicate that upregulation of FOXD3 is an adaptive response to RAF inhibitors that promotes a state of drug resistance.     

Therapeutic efficacy of combined BRAF and MEK inhibition in metastatic melanoma: a comprehensive network meta-analysis of randomized controlled trials

Background

Several recent randomized clinical trials have preliminarily demonstrated that initial targeted therapy with combined BRAF and MEK inhibition is more effective in metastatic melanoma (MM) than single agent. To guide therapeutic decisions, we did a comprehensive network meta-analysis to identify evidence to robustly support whether combined BRAF and MEK inhibition is the best initial targeted therapeutic strategy for patients with MM.

Methods

The databases of PubMed and trial registries were researched for randomized clinical trials of targeted therapy. Data of outcome were extracted on progression-free survival (PFS), objective response rate (ORR), and overall survival (OS). Network meta-analysis using a Bayesian statistical model was performed to evaluate relative hazard ratio (HR) for PFS and OS, odds ratio (OR) for ORR.

Results

Finally, 16 eligible trials comprising 5976 participants were included in this meta-analysis. PFS were significantly prolonged in patients who received combined BRAF-MEK inhibition compared with those who received BRAF inhibition (HR: 0.58, 95%CI: 0.51-0.67, P < 0.0001) or MEK inhibition alone (HR: 0.29, 95%CI: 0.22-0.37, P < 0.0001). Combined BRAF-MEK inhibition also improved the OS over BRAF inhibition (HR: 0.67, 95%CI: 0.56-0.81, P < 0.0001) or MEK inhibition alone (HR: 0.48, 95%CI: 0.36-0.65, P < 0.0001). The ORR was superior in combined BRAF and MEK inhibition comparing with BRAF inhibition (OR: 2.00, 95%CI: 1.66-2.44, P < 0.0001) or MEK inhibition alone (OR: 20.66, 95%CI: 12.22-35.47, P < 0.0001).

Conclusions

This study indicates that concurrent inhibition of BRAF and MEK improved the most effective therapeutic modality as compared as single BRAF or MEK inhibition for patients with MM.     

Dual inhibition of (V600E)BRAF and the PI3K/AKT/mTOR pathway cooperates to induce apoptosis in melanoma cells through a MEK-independent mechanism

BRAF is a main oncogene in human melanomas. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with RAF inhibitor Sorafenib induces apoptosis in NPA melanoma cells expressing oncogenic ^V600EBRAF. This effect is mediated through a MEK/ERK-independent mechanism, since treatment with the MEK inhibitor U0126 does not exert any effect. Moreover, we demonstrate that inhibition of the PI3K/AKT/mTOR cascade alone does not increase apoptosis in these cells. However, the blockage of this pathway in cells lacking either BRAF expression or activity cooperates to induce higher levels of apoptosis than those achieved by inhibition of BRAF alone. Consistently, we demonstrate that abrogation of BRAF expression increases AKT and mTOR phosphorylation, suggesting the existence of a compensatory pro-survival mechanism after BRAF depletion. Together, our data provide a rationale for dual targeting of BRAF and PI3K/AKT/mTOR signalling to effectively control melanoma disease.     

KRAS mutational subtype and copy number predict in vitro response of human pancreatic cancer cell lines to MEK inhibition

Background:

To study the molecular mechanism regulating sensitivity to MEK inhibition in pancreatic cancer cell lines.

Methods:

A growth inhibition assay determined sensitivity to MEK162 in a panel of 29 pancreatic cancer cell lines. For the same panel, KRAS mutational status and copy-number variation (CNV) was determine using PCR, array CGH and FISH. Two sensitive and two resistant cell lines were further interrogated for difference in baseline and MEK162-induced gene expression, as well as signal transduction using microarray and western blotting. Cell cycle and apoptosis analysis was measured by flow cytometry.

Results:

We report a strong correlation between both specific KRAS mutational subtype and CNV, and sensitivity to MEK inhibition. Cell lines with a KRAS (V12) mutation and KRAS gains or loss (n=7) are ∼10 times more resistant than those having neither a KRAS (V12) mutation nor KRAS CNV (n=14). Significant differences in baseline and MEK162-induced gene expression exist between the sensitive and resistant lines, especially in genes involved in RAS, EGF receptor and PI3K pathways. This was further supported by difference in signal transduction. MEK 162 blocked ERK1/2, as well as inhibited PI3K and S6 and increased p27KIP1 levels in the sensitive lines.

Conclusions:

Given the potency of MEK162, it may be a promising new therapy for patients with pancreatic cancer and KRAS mutational subtypes, and CNV may serve as important biomarkers for selecting patients that benefit from MEK-targeting based on these preclinical data.     

MEK inhibition overcomes chemoimmunotherapy resistance by inducing CXCL10 in cancer cells

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Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells

Anticancer activities of cinnamic acid derivatives include induction of apoptosis by irreversible DNA damage leading to cell death. The present work aimed to compare the cytotoxic and genotoxic potential of cinnamic acid in human melanoma cell line (HT-144) and human melanocyte cell line derived from blue nevus (NGM). Viability assay showed that the IC₅₀ for HT-144 cells was 2.4 mM, while NGM cells were more resistant to the treatment. The growth inhibition was probably associated with DNA damage leading to DNA synthesis inhibition, as shown by BrdU incorporation assay, induction of nuclear aberrations and then apoptosis. The frequency of cell death caused by cinnamic acid was higher in HT-144 cells. Activated-caspase 3 staining showed apoptosis after 24 hours of treatment with cinnamic acid 3.2 mM in HT-144 cells, but not in NGM. We observed microtubules disorganization after cinnamic acid exposure, but this event and cell death seem to be independent according to M30 and tubulin labeling. The frequency of micronucleated HT-144 cells was higher after treatment with cinnamic acid (0.4 and 3.2 mM) when compared to the controls. Cinnamic acid 3.2 mM also increased the frequency of micronucleated NGM cells indicating genotoxic activity of the compound, but the effects were milder. Binucleation and multinucleation counting showed similar results. We conclude that cinnamic acid has effective antiproliferative activity against melanoma cells. However, the increased frequency of micronucleation in NGM cells warrants the possibility of genotoxicity and needs further investigation.