Successful treatment with MEK-inhibitor in a patient with NRAS-related cutaneous skeletal hypophosphatemia syndrome.

Cutaneous skeletal hypophosphatemia syndrome (CSHS) is caused by somatic mosaic NRAS variants and characterized by melanocytic/sebaceous naevi, eye, and brain malformations, and FGF23-mediated hypophosphatemic rickets. The MEK inhibitor Trametinib, acting on the RAS/MAPK pathway, is a candidate for CSHS therapy. A 4-year-old boy with seborrheic nevus, eye choristoma, multiple hamartomas, brain malformation, pleural lymphangioma and chylothorax developed severe hypophosphatemic rickets unresponsive to phosphate supplementation. The c.182A > G;p.(Gln61Arg) somatic NRAS variant found in DNA from nevus biopsy allowed diagnosing CSHS. We administered Trametinib for 15 months investigating the transcriptional effects at different time points by whole blood RNA-seq. Treatment resulted in prompt normalization of phosphatemia and phosphaturia, catch-up growth, chylothorax regression, improvement of bone mineral density, reduction of epidermal nevus and hamartomas. Global RNA sequencing on peripheral blood mononucleate cells showed transcriptional changes under MEK inhibition consisting in a strong sustained downregulation of signatures related to RAS/MAPK, PI3 kinase, WNT and YAP/TAZ pathways, reverting previously defined transcriptomic signatures. CSHS was effectively treated with a MEK inhibitor with almost complete recovery of rickets and partial regression of the phenotype. We identified "core" genes modulated by MEK inhibition potentially serving as surrogate markers of Trametinib action.

An integrative gene expression signature analysis identifies CMS4 KRAS-mutated colorectal cancers sensitive to combined MEK and SRC targeted therapy.

BACKGROUND: Over half of colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors, has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms that are not well understood. This study aims to investigate MEK inhibitor-associated resistance signaling and identify subpopulation(s) of CRC patients who may be sensitive to biomarker-driven drug combination(s). METHODS: We classified 2250 primary and metastatic human CRC tumors by consensus molecular subtypes (CMS). For each tumor, we generated multiple gene expression signature scores measuring MEK pathway activation, MEKi "bypass" resistance, SRC activation, dasatinib sensitivity, EMT, PC1, Hu-Lgr5-ISC, Hu-EphB2-ISC, Hu-Late TA, Hu-Proliferation, and WNT activity. We carried out correlation, survival and other bioinformatic analyses. Validation analyses were performed in two independent publicly available CRC tumor datasets (n = 585 and n = 677) and a CRC cell line dataset (n = 154). RESULTS: Here we report a central role of SRC in mediating "bypass"-resistance to MEK inhibition (MEKi), primarily in cancer stem cells (CSCs). Our integrated and comprehensive gene expression signature analyses in 2250 CRC tumors reveal that MEKi-resistance is strikingly-correlated with SRC activation (Spearman P < 10-320), which is similarly associated with EMT (epithelial to mesenchymal transition), regional metastasis and disease recurrence with poor prognosis. Deeper analysis shows that both MEKi-resistance and SRC activation are preferentially associated with a mesenchymal CSC phenotype. This association is validated in additional independent CRC tumor and cell lines datasets. The CMS classification analysis demonstrates the strikingly-distinct associations of CMS1-4 subtypes with the MEKi-resistance and SRC activation. Importantly, MEKi + SRCi sensitivities are predicted to occur predominantly in the KRAS mutant, mesenchymal CSC-like CMS4 CRCs. CONCLUSIONS: Large human tumor gene expression datasets representing CRC heterogeneity can provide deep biological insights heretofore not possible with cell line models, suggesting novel repurposed drug combinations. We identified SRC as a common targetable node--an Achilles' heel--in MEKi-targeted therapy-associated resistance in mesenchymal stem-like CRCs, which may help development of a biomarker-driven drug combination (MEKi + SRCi) to treat problematic subpopulations of CRC.

Preclinical activity of cobimetinib alone or in combination with chemotherapy and targeted therapies in renal cell carcinoma.

Background: The poor outcome of advanced renal cell carcinoma (RCC) necessitates new treatments. Cobimetinib is a MEK inhibitor and approved for the treatment of melanoma. This work investigated the efficacy of cobimetinib alone and in combination with anti-RCC drugs. Methods: Proliferation and apoptosis assays were performed, and combination index was analyzed on RCC cell lines (CaKi-2, 786-O, A-704, ACHN and A489) and xenograft models. Immunoblotting analysis was conducted to investigate the MAPK pathway. Results: Cobimetinib was active against RCC cells, with IC50 at 0.006-0.8µM, and acted synergistically with standard-of-care therapy. Cobimetinib at nontoxic doses prevented tumor formation, inhibited tumor growth and enhanced efficacy of 5-fluorouracil, sorafenib and sunitinib via suppressing Raf/MEK/ERK, leading to MAPK pathway inhibition. Conclusion: Our findings demonstrate the potent anti-RCC activity of cobimetinib and its synergism with RCC standard-of-care drugs, and confirm the underlying mechanism of the action of cobimetinib.

An integrative pharmacogenomics analysis identifies therapeutic targets in KRAS-mutant lung cancer.

BACKGROUND: KRAS mutations are the most frequent oncogenic aberration in lung adenocarcinoma. KRAS mutant isoforms differentially shape tumour biology and influence drug responses. This heterogeneity challenges the development of effective therapies for patients with KRAS-driven non-small cell lung cancer (NSCLC). METHODS: We developed an integrative pharmacogenomics analysis to identify potential drug targets to overcome MEK/ERK inhibitor resistance in lung cancer cell lines with KRAS(G12C) mutation (n = 12). We validated our predictive in silico results with in vitro models using gene knockdown, pharmacological target inhibition and reporter assays. FINDINGS: Our computational analysis identifies casein kinase 2A1 (CSNK2A1) as a mediator of MEK/ERK inhibitor resistance in KRAS(G12C) mutant lung cancer cells. CSNK2A1 knockdown reduces cell proliferation, inhibits Wnt/ß-catenin signalling and increases the anti-proliferative effect of MEK inhibition selectively in KRAS(G12C) mutant lung cancer cells. The specific CK2-inhibitor silmitasertib phenocopies the CSNK2A1 knockdown effect and sensitizes KRAS(G12C) mutant cells to MEK inhibition. INTERPRETATION: Our study supports the importance of accurate patient stratification and rational drug combinations to gain benefit from MEK inhibition in patients with KRAS mutant NSCLC. We develop a genotype-based strategy that identifies CK2 as a promising co-target in KRAS(G12C) mutant NSCLC by using available pharmacogenomics gene expression datasets. This approach is applicable to other oncogene driven cancers. FUND: This work was supported by grants from the National Natural Science Foundation of China, the National Key Research and Development Program of China, the Lung Cancer Research Foundation and a Mildred-Scheel postdoctoral fellowship from the German Cancer Aid Foundation.

Investigating the potential clinical benefit of Selumetinib in resensitising advanced iodine refractory differentiated thyroid cancer to radioiodine therapy (SEL-I-METRY): protocol for a multicentre UK single arm phase II trial.

BACKGROUND: Thyroid cancer is the most common endocrine malignancy. Some advanced disease is, or becomes, resistant to radioactive iodine therapy (refractory disease); this holds poor prognosis of 10% 10-year overall survival. Whilst Sorafenib and Lenvatinib are now licenced for the treatment of progressive iodine refractory thyroid cancer, these treatments require continuing treatment and can be associated with significant toxicity. Evidence from a pilot study has demonstrated feasibility of Selumetinib to allow the reintroduction of I-131 therapy; this larger, multicentre study is required to demonstrate the broader clinical impact of this approach before progression to a confirmatory trial. METHODS: SEL-I-METRY is a UK, single-arm, multi-centre, two-stage phase II trial. Participants with locally advanced or metastatic differentiated thyroid cancer with at least one measureable lesion and iodine refractory disease will be recruited from eight NHS Hospitals and treated with four-weeks of oral Selumetinib and assessed for sufficient I-123 uptake (defined as any uptake in a lesion with no previous uptake or 30% or greater increase in uptake). Those with sufficient uptake will be treated with I-131 and followed for clinical outcomes. Radiation absorbed doses will be predicted from I-123 SPECT/CT and verified from scans following the therapy. Sixty patients will be recruited to assess the primary objective of whether the treatment schedule leads to increased progression-free survival compared to historical control data. DISCUSSION: The SEL-I-METRY trial will investigate the effect of Selumetinib followed by I-131 therapy on progression-free survival in radioiodine refractory patients with differentiated thyroid cancer showing increased radioiodine uptake following initial treatment with Selumetinib. In addition, information on toxicity and dosimetry will be collected. This study presents an unprecedented opportunity to investigate the role of lesional dosimetry in molecular radiotherapy, leading to greater personalisation of therapy. To date this has been a neglected area of research. The findings of this trial will be useful to healthcare professionals and patients alike to determine whether further study of this agent is warranted. It is hoped that the development of the infrastructure to deliver a multicentre trial involving molecular radiotherapy dosimetry will lead to further trials in this field. TRIAL REGISTRATION: SEL-I-METRY is registered under ISRCTN17468602 , 02/12/2015.

Discovery of carbazole derivatives as novel allosteric MEK inhibitors by pharmacophore modeling and virtual screening.

We report in this work the discovery of novel allosteric MEK inhibitors by pharmacophore modeling and virtual screening. Two out of 13 virtual hit compounds were identified as MEK kinase inhibitors using a MEK1 binding assay. Structural derivations on the hit compound M100 (IC50 = 27.2 ±â€¯4.5 µM in RAF-MEK cascading assay) by substituent transformation and bioisosterism replacement have led to the synthesis of a small library of carbazoles. The enzymatic studies revealed the preliminary structure-activity relationships and the derivative 22k (IC50 = 12.8 ±â€¯0.5 µM) showed the most potent inhibitory effect against Raf-MEK cascading. Compound 7 was discovered as toxic as M100 to tumor cells whereas safer to HEK293 cells (IC50 > 100 µM) than M100 (IC50 = 8.9 ±â€¯2.0 µM). It suggests that carbazole is a good scaffold for the design of novel MEK inhibitors for therapeutic uses. More importantly, the developed pharmacophore model can serve as a reliable criterion in novel MEK inhibitor discovery.

Bevacizumab, irinotecan, temozolomide, tyrosine kinase inhibition, and MEK inhibition are effective against pleomorphic xanthoastrocytoma regardless of V600E status.

INTRODUCTION: Pleomorphic xanthoastrocytoma (PXA) is a rare Grade II and III glioma. Surgical resection is the mainstay of treatment, however, adjuvant therapy is sometimes necessary. Given the rarity of PXA, chemotherapeutic efficacy data is limited. The importance of the BRAF V600E mutation in the context of MAP kinase pathway inhibition is unknown. The purpose of this study was to perform an in vivo screen of a variety to agents to determine efficacy against both V600E mutant and non-mutant PXA. METHODS: The efficacy of bevacizumab, temozolomide, lomustine (CCNU), irinotecan (CPT 11), a tyrosine kinase inhibitor (sorafenib), a selective MEK1/2 inhibitor (cobimetinib), and a BRAF inhibitor (vemurafenib) were assessed in two subcutaneous xenografts: D645 PXA (V600E-mutant) and D2363 PXA (V600E-non-mutant) (n = 5-10 mice). Select agents were also assessed in an intracranial model of D2363 PXA (n = 6-9). Subcutaneous tumor growth and survival were the endpoints. RESULTS: Temozolomide, bevacizumab, CPT 11, and sorafenib significantly inhibited subcutaneous tumor growth in both V600E-mutant and V600E-non-mutant models (P < 0.05). MEK inhibition (cobimetinib) but not BRAF inhibition (vemurafenib) also inhibited tumor growth regardless of V600E mutation (P < 0.05). Temozolomide, CPT 11, and bevacizumab also prolonged survival in a V600E-non-mutant intracranial model (median overall survival (OS) 68.5, 62.5, and 42.5 days, respectively) in contrast to controls (31.5 days, P < 0.001). CONCLUSIONS: These findings suggest that when adjuvant treatment is clinically indicated for PXA, temozolomide, CPT 11, or bevacizumab may be considered. Additionally, a trial of a MEK inhibitor or tyrosine kinase inhibitor could be considered for PXA regardless of V600E mutation status.

The incidence and management of cutaneous adverse events of the epidermal growth factor receptor inhibitors.

Overexpression of the epidermal growth factor receptor (EGFR) is found in many cancers, including those of the head and neck area, non-small-cell lung cancer, and colorectal, cervical, prostate, breast, ovary, stomach, and pancreatic cancer. The EGFR inhibitors are used at present in the treatment of such cancers. Skin lesions that develop during and after cancer treatment may be due to specific cytostatics, molecular-targeted drugs, radiation therapy, complementary therapy, or the cancer itself, and hence knowledge is essential to distinguish between them. The mechanism through which skin toxicity arises during treatment with EGFR inhibitors is not well known, but seems to be due to the modification of the RAS/RAF/MEK/ERK signal path associated with its activation, which results in the similarity between the adverse effects of EGFR inhibitors and the treatment of melanoma with BRAF and MEK inhibitors. The most common side effects are pruritus, xerosis, papulopustular rash, hand-foot skin reaction, alopecia and dystrophy of the hair, and paronychia. This work presents options for prevention and suggestions for managing these adverse events, which are of importance in the care of patients undergoing oncological treatment.

Trametinib for the treatment of melanoma.

Advanced melanoma traditionally has had poor prognosis with limited, modestly effective and relatively toxic systemic treatment options like cytotoxic chemotherapy (dacarbazine) and immunomodulating agents (high-dose interleukin-2 and ipilimumab) which have response rates of 6-20%. With the identification of BRAF mutations found to be present in 50% of melanomas and the clinical success of serine/threonine-protein kinase B-raf inhibitors the prognostic landscape of melanoma has changed considerably. Vemurafenib and dabrafenib have been at the forefront of antimelanoma-targeted agents with a tolerable side effect profile and efficacy that compared well with the standard chemotherapy. These characteristics have led to the regulatory approval of both agents for the treatment of melanoma. However, these agents are not curative and have a short life span primarily due to rapidly occurring drug resistance. More recently, mitogen-activated protein kinase kinase (MEK) inhibitors have been found to have strong anticancer activity independently as well as when combined with other agents like B-raf inhibitors due to their activity downstream of RAF. Preclinical data and limited clinical data suggest that MEK inhibitors may be a component of effective therapy for a broad spectrum of cancers with other oncogenic drivers.

Prion formation correlates with activation of translation-regulating protein 4E-BP and neuronal transcription factor Elk1.

Cellular mechanisms play a role in conversion of the normal prion protein PrP(C) to the disease-associated protein PrP(Sc). The cells provide not only PrP(C), but also still largely undefined factors required for efficient prion replication. Previously, we have observed that interference with ERK and p38-JNK MAP kinase pathways has opposing effects on the formation of prions indicating that the process is regulated by a balance in intracellualar signaling pathways. In order to obtain a "flow-chart" of such pathways, we here studied the activation of MEK/ERK and mTORC1 downstream targets in relation to PrP(Sc) accumulation in GT1-1 cells infected with the RML or 22L prion strains. We show that inhibition of mTORC1 with rapamycin causes a reduction of PrP(Sc) accumulation at similar low levels as seen when the interaction between the translation initiation factors eIF4E and eIF4G downstream mTORC1 is inhibited using 4EGI-1. No effect is seen following the inhibition of molecules (S6K1 and Mnk1) that links MEK/ERK signaling to mTORC1-mediated control of translation. Instead, stimulation (high [KCl] or [serum]) or inhibition (MEK-inhibitor) of prion formation is associated with increased or decreased phosphorylation of the neuronal transcription factor Elk1, respectively. This study shows that prion formation can be modulated by translational initiating factors, and suggests that MEK/ERK signaling plays a role in the conversion of PrP(C) to PrP(Sc) via an Elk1-mediated transcriptional control. Altogether, our studies indicate that prion protein conversion is under the control of intracellular signals, which hypothetically, under certain conditions may elicit irreversible responses leading to progressive neurodegenerative diseases.