FTI-277 and GGTI-289 induce apoptosis via inhibition of the Ras/ERK and Ras/mTOR pathway in head and neck carcinoma HEp-2 and HSC-3 cells.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is a major malignancy worldwide. Ras overexpression in HNSCC is known to promote tumor cell growth; therefore, inhibition of Ras activation could lead to tumor growth suppression in HNSCC patients. Here, we investigated the effect of FTI-277, a farnesyl transferase inhibitor, and GGTI-287, a geranyltransferase 1 inhibitor, on the Ras signaling pathway in HNSCC cell lines-HEp-2 and HSC-3. METHODS: Cell viability was analyzed using the trypan blue staining exclusion assay. The apoptosis of cells was assessed by flow cytometry and caspase activation analysis. The expression levels of proteins were examined using western blot analysis. RESULTS: FTI-277 and GGTI-287 induced cell death, enhanced caspase 3 activity, and increased the number of annexin V-positive cells in HEp-2 and HSC-3 cells. FTI-277 and GGTI-287 induced apoptosis in HSC-3 cells at much lower concentrations than that in HEp-2 cells. FTI-277 and GGTI-287 decreased the concentration of phosphorylated ERK1/2 and mTOR via membrane localization of Ras and enhanced Bim expression. Furthermore, FTI-277 and GGTI-287 induced cell death in v-H-Ras-transfected NIH3T3 (NW7) cells and not in empty vector-transfected NIH3T3 (NV20) cells. CONCLUSION: FTI-277 and GGTI-287 may be useful as potential therapeutic agents for treating HNSCC patients; moreover, farnesyl transferase and geranylgeranyltransferase 1 inhibitors can be further developed as anticancer agents.

ERK Inhibitor LY3214996-Based Treatment Strategies for RAS-Driven Lung Cancer.

RAS gene mutations are the most frequent oncogenic event in lung cancer. They activate multiple RAS-centric signaling networks among them the MAPK, PI3K, and RB pathways. Within the MAPK pathway, ERK1/2 proteins exert a bottleneck function for transmitting mitogenic signals and activating cytoplasmic and nuclear targets. In view of disappointing antitumor activity and toxicity of continuously applied MEK inhibitors in patients with KRAS-mutant lung cancer, research has recently focused on ERK1/2 proteins as therapeutic targets and on ERK inhibitors for their ability to prevent bypass and feedback pathway activation. Here, we show that intermittent application of the novel and selective ATP-competitive ERK1/2 inhibitor LY3214996 exerts single-agent activity in patient-derived xenograft (PDX) models of RAS-mutant lung cancer. Combination treatments were well tolerated and resulted in synergistic (ERKi plus PI3K/mTORi LY3023414) and additive (ERKi plus CDK4/6i abemaciclib) tumor growth inhibition in PDX models. Future clinical trials are required to investigate if intermittent ERK inhibitor-based treatment schedules can overcome toxicities observed with continuous MEK inhibition and-equally important-to identify biomarkers for patient stratification.

New advances in the clinical management of RAS and BRAF mutant colorectal cancer patients.

INTRODUCTION: In colorectal carcinogenesis, genetic alterations in RAS and BRAF oncogenes play an important role for cancer initiation and/or progression and represent a key focus in the search for targeted therapies. Despite many years of research and a great amount of studies, until very recently this pathway was considered extremely hard to downregulate to obtain a significant clinical impact in colorectal cancer patients. But better times are coming with the advent of new promising drugs and combinations strategies. AREAS COVERED: In this review, we go over the biological characteristics of the MAPK pathway in colorectal tumors, while illustrating the clinical correlation of RAS and BRAF mutations, particularly its prognostic and predictive value. We also present newly data about recent improvements in the treatment strategy for patients harboring these types of tumors. EXPERT COMMENTARY: With great advances in the knowledge of molecular basis of RAS and BRAF mutant colorectal cancer in conjunction with biotechnology development and the constant effort for improvement, in the near future many new therapeutic options would be available for the management of this group of patient with dismal prognosis.

Photodynamic Therapy for ras-Driven Cancers: Targeting G-Quadruplex RNA Structures with Bifunctional Alkyl-Modified Porphyrins.

Designing small molecules able to break down G4 structures in mRNA (RG4s) offers an interesting approach to cancer therapy. Here, we have studied cationic porphyrins (CPs) bearing an alkyl chain up to 12 carbons, as they bind to RG4s while generating reactive oxygen species upon photoirradiation. Fluorescence-activated cell sorting (FACS) and confocal microscopy showed that the designed alkyl CPs strongly penetrate cell membranes, binding to KRAS and NRAS mRNAs under low-abundance cell conditions. In Panc-1 cells, alkyl CPs at nanomolar concentrations promote a dramatic downregulation of KRAS and NRAS expression, but only if photoactivated. Alkyl CPs also reduce the metabolic activity of pancreatic cancer cells and the growth of a Panc-1 xenograft in SCID mice. Propidium iodide/annexin assays and caspase 3, caspase 7, and PARP-1 analyses show that these compounds activate apoptosis. All these data demonstrate that the designed alkyl CPs are efficient photosensitizers for the photodynamic therapy of ras-driven cancers.

MEK inhibition suppresses B regulatory cells and augments anti-tumor immunity.

Mitogen-activated protein kinase (MAPK) kinase (MEK) is an integral component of the RAS pathway and a therapeutic target in RAS-driven cancers. Although tumor responses to MEK inhibition are rarely durable, MEK inhibitors have shown substantial activity and durable tumor regressions when combined with systemic immunotherapies in preclinical models of RAS-driven tumors. MEK inhibitors have been shown to potentiate anti-tumor T cell immunity, but little is known about the effects of MEK inhibition on other immune subsets, including B cells. We show here that treatment with a MEK inhibitor reduces B regulatory cells (Bregs) in vitro, and reduces the number of Bregs in tumor draining lymph nodes in a colorectal cancer model in vivo. MEK inhibition does not impede anti-tumor humoral immunity, and B cells contribute meaningfully to anti-tumor immunity in the context of MEK inhibitor therapy. Treatment with a MEK inhibitor is associated with improved T cell infiltration and an enhanced response to anti-PD1 immunotherapy. Together these data indicate that MEK inhibition may reduce Bregs while sparing anti-tumor B cell function, resulting in enhanced anti-tumor immunity.

RASpecting the oncogene: New pathways to therapeutic advances.

RAS is the most commonly mutated driver of tumorigenesis, seen in about 30% of all cancer cases. There is a subset of tumors termed RAS-driven cancers in which RAS mutation or overactivation is evident, including as much as 95% in pancreatic and 50% in colon cancer. RAS is a family of small membrane bound GTPases that act as a signaling node to control both normal and cancer biology. Since the discovery of RAS' overall prominence in many tumor types and specifically in RAS-dependent cancers, it has been an obvious therapeutic target for drug development. However, RAS has proved a very elusive target, and after a few prominent RAS targeted drugs failed in clinical trials after decades of research, RAS was termed "undruggable" and research in this field was greatly hampered. An increase in knowledge about basic RAS biology has led to a resurgence in the generation of novel therapeutics targeting RAS signaling utilizing various and distinct approaches. These new drugs target RAS activation directly, block downstream signaling effectors and inhibit proper post-translational processing and trafficking/recycling of RAS. This review will cover how these new drugs were developed and how they have fared in preclinical and early phase clinical trials.

The inhibitory effect of Cordycepin on the proliferation of cisplatin-resistant A549 lung cancer cells.

The goal of this study is to determine the anti-cancer mechanism of Cordycepin in A549 Cisplatin-Resistance (CR) lung cancer cells. Cordycepin inhibited the viability of A549CR cells in a dose-dependent manner. The cell inhibition was due to induction of apoptosis in the cells treated with Cordycepin by activation of caspase -3, -8 and -9 activities. The cell cycle analysis showed that accumulation of Sub G1 was observed in Cordycepin-treated with A549CR lung cancer cells. Based on the data of expression profile analysis of cell signaling proteins using IPS-FPAA, H-Ras was down-regulated in Cordycepin-treated A549CR cells. Collectively, anti-proliferative function of Cordycepin was due to stimulation of the cell apoptosis and the cell cycle arrest via caspases activation and down-regulation of H-Ras.

Long-term treatment with chaethomellic acid A reduces glomerulosclerosis and arteriolosclerosis in a rat model of chronic kidney disease.

The high prevalence of end-stage renal disease emphasizes the failure to provide therapies to effectively prevent and/or reverse renal fibrosis. Therefore, the aim of this study was to evaluate the effect of long-term treatment with chaethomellic acid A (CAA), which selectively blocks Ha-Ras farnesylation, on renal mass reduction-induced renal fibrosis. Male Wistar rats were sham-operated (SO) or subjected to 5/6 renal mass reduction (RMR). One week after surgery, rats were placed in four experimental groups: SO:SO rats without treatment (n=13); SO+CAA: SO rats treated with CAA (n=13); RMR:RMR rats without treatment (n=14); and RMR+CAA:RMR rats treated with CAA (n=13). CAA was intraperitoneally administered in a dose of 0.23µg/kg three times a week for six months. Renal fibrosis was evaluated by two-dimensional ultrasonography and histopathological analysis. The kidneys of the RMR animals treated with CAA showed a significantly decrease in the medullary echogenicity (p<0.05) compared with the RMR rats that received no treatment. Glomerulosclerosis and arteriolosclerosis scores were significantly lower (p<0.001) in the RMR+CAA group when compared with the RMR group. There were no significant differences in interstitial fibrosis, interstitial inflammation and tubular dilatation scores between the RMR+CAA and RMR groups. These data suggest that CAA can be a potential future drug to attenuate the progression of chronic kidney disease.

Continuous treatment with FTS confers resistance to apoptosis and affects autophagy.

High percentage of human cancers involves alteration or mutation in Ras proteins, including the most aggressive malignancies, such as lung, colon and pancreatic cancers. FTS (Salirasib) is a farnesylcysteine mimetic, which acts as a functional Ras inhibitor, and was shown to exert anti-tumorigenic effects in vitro and in vivo. Previously, we have demonstrated that short-term treatment with FTS also induces protective autophagy in several cancer cell lines. Drug resistance is frequently observed in cancer cells exposed to prolonged treatment, and is considered a major cause for therapy inefficiency. Therefore, in the present study, we examined the effect of a prolonged treatment with FTS on drug resistance of HCT-116 human colon cancer cells, and the involvement of autophagy in this process. We found that cells grown in the presence of FTS for 6 months have become resistant to FTS-induced cell growth inhibition and cell death. Furthermore, we discovered that the resistant cells exhibit altered autophagy, reduced apoptosis and changes in Ras-related signaling pathways following treatment with FTS. Moreover we found that while FTS induces an apoptosis-related cleavage of p62, the FTS-resistant cells were more resistant to apoptosis and p62 cleavage.

Doubling Down on Mutant RAS Can MEK or Break Leukemia.

Targeting of the RAS pathway has long been a critical therapeutic challenge in oncology. Burgess et al. examine how the relative expression of mutant and wild-type KRAS modulates clonal fitness and sensitivity to MEK inhibitors in a model of KrasG12D mutant acute myeloid leukemia and propose its use as a predictive biomarker.

The RAS-Effector Interaction as a Drug Target.

About a third of all human cancers harbor mutations in one of the K-, N-, or HRAS genes that encode an abnormal RAS protein locked in a constitutively activated state to drive malignant transformation and tumor growth. Despite more than three decades of intensive research aimed at the discovery of RAS-directed therapeutics, there are no FDA-approved drugs that are broadly effective against RAS-driven cancers. Although RAS proteins are often said to be "undruggable," there is mounting evidence suggesting it may be feasible to develop direct inhibitors of RAS proteins. Here, we review this evidence with a focus on compounds capable of inhibiting the interaction of RAS proteins with their effectors that transduce the signals of RAS and that drive and sustain malignant transformation and tumor growth. These reports of direct-acting RAS inhibitors provide valuable insight for further discovery and development of clinical candidates for RAS-driven cancers involving mutations in RAS genes or otherwise activated RAS proteins. Cancer Res; 77(2); 221-6. ©2017 AACR.

K-Ras and its inhibitors towards personalized cancer treatment: Pharmacological and structural perspectives.

The discovery of genetic, genomic and clinical biomarkers have revolutionized the treatment option in the form of personalized medicine which allows to accurately predict a person's susceptibility/progression of disease, the patient's response to therapy, and maximize the therapeutic outcome in terms of low/no toxicity for a particular patient. Recently, the U.S. Food and Drug Administration has realized the contribution of pharmacogenomics in better healthcare and advocated the consideration of pharmacogenomic principles in making safer and more effective drug. Many anticancer drugs show reduced or no response in cancer patients with tumor specific gene mutations such as B-Raf and K-Ras. The high incidence of K-Ras mutation has been reported in pancreatic, colon, and lung carcinomas. The identification of K-Ras as a clinical biomarker and potential therapeutic target has attracted the scientific community to develop effective and precise anticancer drug. Inhibitors which block farnesylation of Ras have been developed or under clinical trial studies. Tipifarnib, approved by USFDA for the treatment of elderly acute leukemia is a Ras pathway inhibitor. Some peptidomimetics and bi-substrate inhibitors like FTI 276, FTI 277, B956, B1086, L731, L735, L739, L750, BMS-214662, L778123, and L778123 are under clinical trials. Recently mutant K-Ras has been considered as potential biomarker and target for precise cancer therapy. This review focuses primarily on the Ras/Raf/MEK/ERK signaling pathway including K-Ras mutation as therapeutic target, inhibitors and their structure activity relationships (SARs) for the design and development of anticancer agents.

Oncogenic RAS: From Its Activation to Its Direct Targeting.

The RAS oncogenes are the most commonly mutated genes across human cancers. This review is a look back at the discovery of the RAS genes, how we came to understand their function, and how these oncogenes are aberrantly activated, driving cancer development and growth. It also highlights the comprehensive efforts made during the last 30 years to develop therapeutic strategies against the oncogenic mutant RAS. Although there have been many lost battles in the war against mutant RAS-bearing cancer cells, and we have yet to determine which is the "best" path for inhibition of the mutated RAS, a better understanding of the protein's structure combined with technological advances in the field has allowed the development of new strategies to make anti-RAS therapies a reality. The state-of-the-art for each strategy is discussed, focusing on the promises and potential pitfalls of each of them.

XRP44X, an Inhibitor of Ras/Erk Activation of the Transcription Factor Elk3, Inhibits Tumour Growth and Metastasis in Mice.

Transcription factors have an important role in cancer but are difficult targets for the development of tumour therapies. These factors include the Ets family, and in this study Elk3 that is activated by Ras oncogene /Erk signalling, and is involved in angiogenesis, malignant progression and epithelial-mesenchymal type processes. We previously described the identification and in-vitro characterisation of an inhibitor of Ras / Erk activation of Elk3 that also affects microtubules, XRP44X. We now report an initial characterisation of the effects of XRP44X in-vivo on tumour growth and metastasis in three preclinical models mouse models, subcutaneous xenografts, intra-cardiac injection-bone metastasis and the TRAMP transgenic mouse model of prostate cancer progression. XRP44X inhibits tumour growth and metastasis, with limited toxicity. Tumours from XRP44X-treated animals have decreased expression of genes containing Elk3-like binding motifs in their promoters, Elk3 protein and phosphorylated Elk3, suggesting that perhaps XRP44X acts in part by inhibiting the activity of Elk3. Further studies are now warranted to develop XRP44X for tumour therapy.

Isolation of Bioactive Rotenoids and Isoflavonoids from the Fruits of Millettia caerulea.

Three new rotenoids (1-3), two new isoflavonoids (4 and 5), and six known analogues (6-11) were isolated from an n-hexane partition of a methanol extract of the fruits of Millettia caerulea, with the structures of the new compounds elucidated by analysis of their spectroscopic data. The relative configurations of the rotenoids were determined by interpretation of their NMR spectroscopic data, and their absolute configurations were established using electronic circular dichroism spectra and specific rotation values. All compounds isolated were evaluated for their cell growth inhibitory activity against the HT-29 human colon cancer cell line, and the known compounds, (-)-3-hydroxyrotenone (6) and (-)-rotenone (7), were found to be potently active. When tested in an NF-κB inhibition assay, compound 6 showed activity. This compound, along with the new compound, (-)-caeruleanone D (1), and the known compound, ichthynone (8), exhibited K-Ras inhibitory potency. Further bioactivity studies showed that the new compounds, (-)-3-deoxycaeruleanone D (2) and (-)-3-hydroxycaeruleanone A (3), and the known compounds 8 and 11 induced quinone reductase in murine Hepa 1c1c7 cells.

Ifenprodil Attenuates Methamphetamine-Induced Behavioral Sensitization and Activation of Ras-ERK-∆FosB Pathway in the Caudate Putamen.

Addiction is a debilitating, chronic psychiatric disorder that is difficult to cure completely owing to the high rate of relapse. Behavioral sensitization is considered to may underlie behavioral changes, such as relapse, caused by chronic abuse of psychomotor stimulants. Thus, its animal models have been widely used to explore the etiology of addiction. Recently, increasing evidence has demonstrated that N-methyl-D-aspartate receptors (NMDARs) play an important role in addiction to psychomotor stimulants. However, the role of GluN2B-containing receptors and their downstream signaling pathway(s) in behavioral sensitization induced by methamphetamine (METH) have not been investigated yet. In this study, we used different doses of ifenprodil (2.5, 5, 10 mg/kg), a selective antagonist of the GluN2B subunit, to investigate the role of GluN2B-containing NMDARs in METH-induced behavioral sensitization. We then examined changes in the levels of Ras, phosphorylated extracellular signal-regulated kinase (pERK)/ERK, and ∆FosB in the caudate putamen (CPu) by western blot. We found that 2.5 or 10 mg/kg ifenprodil significantly attenuated METH-induced behavioral sensitization, whereas the mice treated with a moderate dose of ifenprodil (5 mg/kg) displayed no significant changes. Further results of western blot experiments showed that repeated administration of METH caused the increases in the levels of Ras, pERK/ERK and ∆FosB in the CPu, and these changes were inhibited by only the 2.5 mg/kg dose of ifenprodil. In conclusion, these results demonstrated that 2.5 mg/kg ifenprodil could attenuate METH-induced behavioral sensitization. Moreover, GluN2B-containing NMDARs and their downstream Ras-ERK-∆FosB signaling pathway in the CPu might be involved in METH-induced behavioral sensitization.

Anti-EGFR Therapy for Metastatic Colorectal Cancer in the Era of Extended RAS Gene Mutational Analysis.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. In the past 2 decades, advances in cancer therapeutics allowed for a remarkable improvement in terms of survival for patients with metastatic CRC. The advent of targeted therapy, coupled with more efficient chemotherapy regimens, was the pillar achievement that contributed to the success of CRC therapy. Cetuximab and panitumumab, monoclonal antibodies targeting the epidermal growth factor receptor pathway, are the focus of this review since their mechanism of action and efficiency are closely related to the mutational status of a predictive biomarker, the Kristen rat Sarcoma viral oncogene (KRAS). More recently, another biomarker, the neuroblastoma rat sarcoma viral oncogene (NRAS), was found to be as valuable for the refinement of this targeted therapy. The arguments for the use of extended analysis of the RAS gene are thoroughly reviewed because they directly affect the choice of targeted agents and potentially the choice of backbone chemotherapy.

Aspafilioside B induces G2/M cell cycle arrest and apoptosis by up-regulating H-Ras and N-Ras via ERK and p38 MAPK signaling pathways in human hepatoma HepG2 cells.

We recently establish that aspafilioside B, a steroidal saponin extracted from Asparagus filicinus, is an active cytotoxic component. However, its antitumor activity is till unknown. In this study, the anticancer effect of aspafilioside B against HCC cells and the underlying mechanisms were investigated. Our results showed that aspafilioside B inhibited the growth and proliferation of HCC cell lines. Further study revealed that aspafilioside B could significantly induce G2 phase cell cycle arrest and apoptosis, accompanying the accumulation of reactive oxygen species (ROS), but blocking ROS generation with N-acetyl-l-cysteine (NAC) could not prevent G2/M arrest and apoptosis. Additionally, treatment with aspafilioside B induced phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAP kinase. Moreover, both ERK inhibitor PD98059 and p38 inhibitor SB203580 almost abolished the G2/M phase arrest and apoptosis induced by aspafilioside B, and reversed the expression of cell cycle- and apoptosis-related proteins. We also found that aspafilioside B treatment increased both Ras and Raf activation, and transfection of cells with H-Ras and N-Ras shRNA almost attenuated aspafilioside B-induced G2 phase arrest and apoptosis as well as the ERK and p38 activation. Finally, in vivo, aspafilioside B suppressed tumor growth in mouse xenograft models, and the mechanism was the same as in vitro study. Collectively, these findings indicated that aspafilioside B may up-regulate H-Ras and N-Ras, causing c-Raf phosphorylation, and lead to ERK and p38 activation, which consequently induced the G2 phase arrest and apoptosis. This study provides the evidence that aspafilioside B is a promising therapeutic agent against HCC.

Impairing effects of angiotensin-converting enzyme inhibitor Captopril on bone of normal mice.

There are contradicting results about the effects of angiotensin-converting enzyme inhibitors (ACEIs) on bones. This study was aimed to investigate the effect of ACEI, Captopril, on bone metabolism and histology as well as the action of Captopril on skeletal renin-angiotensin system (RAS) and bradykinin receptor pathway in normal male mice. The urine, serum, tibias and femurs from normal control mice and Captopril-treated (10mg/kg) mice were collected for biochemical, histological and molecular analyses after drug administration for eight weeks. The mice after the treatment with Captopril had a significant decrease of serum testosterone level. The histological measurements showed the loss of trabecular bone mass and trabecular bone number, and the breakage of trabecular bone network as well as the changes of chondrocyte zone at epiphyseal plate in Captopril-treated mice. The defect of Captopril on trabecular bone was reflected by the quantitative bio-parameters from micro-CT. The expression of renin receptor and bradykinin B2 receptor (B2R) was significantly up-regulated in tibia of mice upon to the Captopril treatment, which decreased the ratio of OPG/RANKL and the expression of osteoblastic factor RUNX2. Furthermore, Captopril treatment resulted in the increase of pAkt/Akt and pNFκB expression in tibia. The present study revealed the impairing effects of Captopril on bone via interfering with the circulating sex hormone level and B2R pathway, which suggests that the bone metabolism of patients need to be carefully monitored when being prescribed for ACEIs.

Phase I study of S-trans, trans-farnesylthiosalicylic acid (salirasib), a novel oral RAS inhibitor in patients with refractory hematologic malignancies.

BACKGROUND: Rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase activation (mutational or nonmutational) is a key pathway for survival and proliferative advantage of leukemic cells. Salirasib (Concordia Pharmaceuticals) is an oral RAS inhibitor that causes dislocation of RAS by competing directly with farnesylated RAS in binding to its putative membrane-binding proteins. Salirasib does not inhibit farnesyl transferase enzyme. PATIENTS AND METHODS: We report on a phase I study of Salirasib in patients with relapsed/refractory hematologic malignancies. Salirasib was administered orally twice daily on days 1 to 21 of a 28-day cycle in a "3+3" dose escalation design. RESULTS: Seventeen patients with relapsed/refractory leukemia were treated for a median of 4 cycles (range, 1-29). Three patients each were enrolled at a dose level of 100, 200, 400, 600, and 800 mg twice daily and 2 patients at a dose level of 900 mg twice daily. No dose-limiting toxicities were encountered. Grade 1/2 diarrhea was the only frequent nonhematologic toxicity observed in 14 of 17 (82%) patients and was resolved with oral antidiarrheal agents. Eight (47%) patients (4 with myelodysplastic syndrome, 2 with acute myeloid leukemia, 1 with chronic myelomonocytic leukemia, and 1 with chronic myeloid leukemia) had hematological improvement; 1 in 3 lineages, 1 in 2 lineages, and 6 in 1 lineage. None of the patients achieved complete remission. The responses lasted for a median of 10 weeks (range, 5-115). The study was discontinued because of financial constraints. CONCLUSION: Salirasib was well tolerated and showed modest activity in relapsed/refractory hematological malignancies. The safety profile of Salirasib and its hematological malignancy relevant target makes it a potential drug to be used in combination therapy.