Inhibition Of Pathway Activation Research Articles

Bulk integrated single-cell-spatial transcriptomics reveals the impact of preoperative chemotherapy on cancer-associated fibroblasts and tumor cells in colorectal cancer, and construction of related predictive models using machine learning

BackgroundPreoperative chemotherapy (PC) is an important component of Colorectal cancer (CRC) treatment, but its effects on the biological functions of fibroblasts and epithelial cells in CRC are unclear. MethodsThis study utilized bulk, single-cell, and spatial transcriptomic sequencing data from 22 independent cohorts of CRC. Through bioinformatics analysis and in vitro experiments, the research investigated the impact of PC on fibroblast and epithelial cells in CRC. Subpopulations associated with PC and CRC prognosis were identified, and a predictive model was constructed using machine learning. ResultsPC significantly attenuated the pathways related to tumor progression in fibroblasts and epithelial cells. NOTCH3 + Fibroblast (NOTCH3 + Fib), TNNT1 + Epithelial (TNNT1 + Epi), and HSPA1A + Epithelial (HSPA1A + Epi) subpopulations were identified in the adjacent spatial region and were associated with poor prognosis in CRC. PC effectively diminished the presence of these subpopulations, concurrently inhibiting pathway activity and intercellular crosstalk. A risk signature model, named the Preoperative Chemotherapy Risk Signature Model (PCRSM), was constructed using machine learning. PCRSM emerged as an independent prognostic indicator for CRC, impacting both overall survival (OS) and recurrence-free survival (RFS), surpassing the performance of 89 previously published CRC risk signatures. Additionally, patients with a high PCRSM risk score showed sensitivity to fluorouracil-based adjuvant chemotherapy (FOLFOX) but resistance to single chemotherapy drugs (such as Bevacizumab and Oxaliplatin). Furthermore, this study predicted that patients with high PCRSM were resistant to anti-PD1therapy. ConclusionIn conclusion, this study identified three cell subpopulations (NOTCH3 + Fib, TNNT1 + Epi, and HSPA1A + Epi) associated with PC, which can be targeted to improve the prognosis of CRC patients. The PCRSM model shows promise in enhancing the survival and treatment of CRC patients.

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Controllable conformation and reactivity of bicyclic α-methylene cyclopentanones and their NF-κB pathway inhibitory activity.

Tuning the electrophilicities of Michael acceptors is important for the development of targeted covalent drugs. To this end, the electronic effects of electrophilic structures have been well investigated, but not the steric effects. In this work, we synthesized ten α-methylene cyclopentanones (MCPs), screened them for NF-κB inhibitory activity, and analyzed their conformations. We found that MCP-4b, MCP-5b, and MCP-6b are novel NF-κB inhibitors, whereas the corresponding diastereomers MCP-4a, MCP-5a, and MCP-6a are inactive. Conformational analysis suggested that the stereochemistry of the side chain (R) on MCPs dictates the stable conformation of the core bicyclic 5/6 ring system. The conformational preference seemed to influence their reactivity toward nucleophiles. Consequently, a thiol reactivity assay showed that MCP-5b has higher reactivity than MCP-5a. The results indicate that the conformational switching of MCPs may control reactivity and bioactivity in the presence of steric effects.

(+)-Isocryptotanshinone derivatives and its simplified analogs as STAT3 signaling pathway inhibitors

Isocryptotanshinone (ICTS), a natural product with potential signal transducer and activator of transcription-3 (STAT3) signaling pathway inhibitory activity, shows significant inhibitory activity against several tumors. In this study, a series of ICTS derivatives and simplified analogs containing a 1, 4-naphthoquinone core was designed, synthesized, and evaluated. The results demonstrated that most target compounds were potent STAT3 signaling pathway inhibitors based on their mechanism of inhibition of STAT3 phosphorylation. Moreover, based on the obtained data, the structure–activity relationship (SAR) was rationally deduced. Simultaneously, molecular docking of the compound 16r suggested its possible interaction mode with STAT3. To further verify anticancer activity, all target compounds were tested using HCT116, HepG2, MCF-7, A549, and U251 cell lines. Interestingly, compared with different tumor cell lines, the HCT-116 cell line was determined to be the most sensitive. Furthermore, compounds 21e, 16r, 28a, and 16e showed a dose-dependent inhibition of the growth of HCT116 cells. Thus, the SAR of ICTS derivatives and its simplified analogs was determined, and some of them were discovered to be potential anticancer candidates owing to their ability to inhibit the STAT3 signaling pathway.

Abstract 3030: Different pharmacodynamic profiles of ERK1/2 inhibition can elicit comparable anti-tumor activity

Abstract The MAPK pathway is frequently dysregulated in cancer, resulting in constitutive phosphorylation and activation of its downstream signaling node ERK1/2 (ERK). The direct targeting of activated ERK is being explored in several phase I clinical trials. A good understanding of the relationship between inhibition of pathway activity and inhibition of tumor growth allows dose scheduling in the clinic to maximise response to an inhibitor. We have recently described the development of a novel, potent and selective small molecule inhibitor of both ERK activity and phosphorylation using fragment-based drug discovery. Using this inhibitor as a tool compound, we explored the relationship between duration of ERK inhibition and inhibition of tumor growth. We examined the in vivo activity of our ERK inhibitor in a Colo205 xenograft model. Inhibition of the ERK pathway was monitored by measuring phosphorylation of a direct substrate of ERK, RSK90 (pRSK), and phosphorylated ERK itself (pERK) to determine pharmacodynamic profiles. We observed that pathway inhibition was consistent with the pharmacokinetic profile of the ERK inhibitor and that the duration of pathway inhibition was primarily determined by dosing frequency rather than the total dose of ERK inhibitor. High, single doses (50 or 40 mg/kg) of ERK inhibitor produce strong maximal pathway inhibition at 2 h post dose (pRSK levels 8% of control levels for a single dose of 40 mg/kg) but pRSK levels have returned to levels close to control values by 12 h post dose. In contrast, 3x equally spaced doses of 20 mg/kg have a lower maximal pathway inhibition at 2 h post dose (pRSK levels 29% of control) but extend pathway inhibition beyond 12 h (pRSK levels 21% of control at 14 h). Despite these two different pharmacodynamic profiles, we observed comparable tumor growth inhibition following daily dosing either at 1 × 50 mg/kg (maximal tumor growth inhibition of 21%) or 3 × 20 mg/kg (maximal tumor growth inhibition of 15%). These data suggest that a short duration of ERK inhibition rather than constant suppression can be sufficient to provide anti-tumor activity. This may allow flexibility in clinical dosing schedules to improve tolerability without a loss in efficacy. Citation Format: Christopher Hindley, Luke Bevan, Hannah Braithwaite, Aurélie Courtin, Lynsey Fazal, Roberta Ferraldeschi, Vanessa Martins, Sandra Muench, Nicola Wallis, Nicola Wilsher, John Lyons, Joanne Munck. Different pharmacodynamic profiles of ERK1/2 inhibition can elicit comparable anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3030.

Abstract GMM-019: MEK INHIBITOR TRAMETINIB IN HIGH GRADE OVARIAN CANCER PROLIFERATION

Abstract A major challenge in ovarian cancer therapy is very high recurrence that is currently attributed to the presence of cancer stem-like cells (CSCs). CSCs are characterized by pronounced chemoresistance and high clonogenic ability that allow them to survive treatment and trigger relapse. The search for new treatment approaches capable of eradicating CSCs is crucial to prevent recurrent disease. Despite lack of BRAF or KRAS mutations in high grade serous ovarian cancer, MEK1/2-ERK1/2 pathway is activated in ovarian cancer cell lines and primary tumors. The aim of present work was to evaluate MEK inhibitor trametinib on cell proliferation and tumorigenesis. Trametinib is approved by FDA for melanoma and other solid tumors with specific BRAF mutations, thus can be used in a timely manner in clinic if found effective. We used cisplatin-resistant OVCAR8 and PEO4 cell lines displaying very high fraction of CD133-positive cells exhibiting CSC-like properties as main experimental model. Treatment of cells with trametinib caused very efficient inhibition of MEK-ERK pathway activity (up to 100% inhibition of ERK1/2 phosphorylation). Inhibition of pathway activity was confirmed by MEK-ERK-associated genes downregulation as assessed by RT-qPCR. PHLDA1, SPRY2 and DUSP4 genes were identified as early MEK responders whereas SPRY4, ETV4 and ETV5 were responsive to prolonged MEK inhibition. Trametinib treatment resulted in significant reduction of cell growth rate and number of viable cells (5-fold reduction with 100nM trametinib, p<0.05) in both cell lines. Cell cycle assay demonstrated cell cycle arrest in G1/0 -phase indicating that MEK-ERK activation is required for transition from G1 to S-phase. To further elaborate associations between MEK pathway activity and cell proliferation, we studied cells enriched in different stages of S- and G2-phase. MEK pathway activity was subject to prominent activation during S-phase reflected by significant increase in phosphorylation levels of ERK1/2, its direct targets FRA1 and p90RSK and subsequent upregulation of reporter genes PHLDA1, SPRY2 and DUSP4. Our data indicate that trametinib treatment may induce efficient inhibition of ovarian cancer cell proliferation and therefore may have positive impact upon duration of remission for ovarian cancer patients. While trametinib alone is not capable of eradicating ovarian CSCs, its combinations with other drugs may provide a promising tool for ovarian cancer treatment, especially for tumors with high level of MEK-ERK pathway activity. One of such drugs to use in conjunction with trametinib can be Aldehyde Dehydrogenase (ALDH) inhibitor 673A that was previously characterized in our research as a compound capable of inducing cell programed necroptosis in ovarian CSCs. Citation Format: Mikhail Chesnokov, Anmbreen Jamroze, Wootae Ha, Ilana Chefetz. MEK INHIBITOR TRAMETINIB IN HIGH GRADE OVARIAN CANCER PROLIFERATION [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-019.

Engineering varied serine protease inhibitors by converting P1 site of BF9, a weakly active Kunitz-type animal toxin

Although there were a lot of weakly active animal toxins in the venoms, their values and applications are still mysterious, such as BF9, which is a Kunitz-type toxin isolated from the venom of the elapid snake Bungarus fasciatus. Here, we used BF9 to be a molecular scaffold, and engineered eight BF9-derived peptides by changing P1 site Asn17 of BF9, such as BF9-N17Y and BF9-N17T designed from the polar subfamily, BF9-N17L and BF9-N17G designed from the Non-polar subfamily, BF9-N17D designed from acidic subfamily, and BF9-N17H, BF9-N17K and BF9-N17R designed from basic subfamily. Through enzyme inhibitor experiment assays, we found a potent and selective chymotrypsin inhibitor BF9-N17Y, a potent and selective coagulation factor XIa inhibitor BF9-N17H, and two highly potent coagulation factor XIa inhibitors BF9-N17K and BF9-N17. APTT and PT assays further showed that BF9-N17H, BF9-N17K and BF9-N17R were three novel anticoagulants with selectively intrinsic coagulation pathway inhibitory activity. Considering that natural weakly active animal toxins are also a huge peptide resource, our present work might open a new window about pharmacological applications of weakly active animal toxins, which might be good templates for potent and selective molecular probe and lead drug designs.

A phase I dose-escalation and dose-expansion study of brontictuzumab in subjects with selected solid tumors

BackgroundBrontictuzumab is a monoclonal antibody that targets Notch1 and inhibits pathway activation. The purpose of this first-in-human study was to determine the maximum tolerated dose (MTD), safety, pharmacokinetics, immunogenicity and preliminary efficacy of brontictuzumab in patients with solid tumors. Patients and methodsSubjects with selected refractory solid tumors were eligible. Brontictuzumab was administered intravenously at various dose levels and schedule during dose escalation, and at 1.5mg/kg every 3 weeks (Q3W) during expansion. Evidence of Notch1 pathway activation as determined by an immunohistochemistry assay was required for entry in the expansion cohort. Adverse events were graded according to the NCI-CTCAE v 4.03. Efficacy was assessed by RECIST 1.1. ResultsForty-eight subjects enrolled (33 in dose escalation and 15 in the expansion phase). The MTD was 1.5mg/kg Q3W. Dose-limiting toxicities were grade 3 diarrhea in two subjects and grade 3 fatigue in one subject. The most common drug-related adverse events of any grade were diarrhea (71%), fatigue (44%), nausea (40%), vomiting (21%), and AST increase (21%). Brontictuzumab exhibited nonlinear pharmacokinetics with dose-dependent terminal half-life ranging 1–4days. Clinical benefit was seen in 6 of 36 (17%) assessable subjects: 2 had unconfirmed partial response (PR) and 4 subjects had prolonged (≥6months) disease stabilization (SD). Both PRs and three prolonged SD occurred in adenoid cystic carcinoma (ACC) subjects with evidence of Notch1 pathway activation. Pharmacodynamic effects of brontictuzumab were seen in patients’ blood and tumor. ConclusionBrontictuzumab was well tolerated at the MTD. The main toxicity was diarrhea, an on-target effect of Notch1 inhibition. An efficacy signal was noted in subjects with ACC and Notch1 pathway activation. ClinicalTrials.gov identifierNCT01778439

3D QSAR Studies of Tricyclic Compounds as Porcupine Inhibitors for Wnt Pathway Inhibitory Activity

3D QSAR Studies of Tricyclic Compounds as Porcupine Inhibitors for Wnt Pathway Inhibitory Activity

The Notch inhibitor cowanin accelerates nicastrin degradation

Aberrant activation of Notch signaling contributes to the pathogenesis of several different types of cancer, and Notch pathway inhibitors may have significant therapeutic potential. Using a unique cell-based assay system, we isolated twelve compounds, including one new natural product from Garcinia speciosa, that inhibit the Notch signaling pathway. HES1 and HES5 are target genes of the Notch cascade, and compound 2, referred to as cowanin, decreased the protein levels of HES1 and HES5 in assay cells. Furthermore, cowanin (2) showed potent cytotoxicity against human leukemic HPB-ALL cells. The Notch signaling inhibitory activity of cowanin (2) is linked to the increased degradation of nicastrin, which is one of the components of the γ-secretase complex. To the best of our knowledge, this is the first example of a compound with Notch pathway inhibitory activity mediated by nicastrin degradation.

Atom based linear index descriptors in QSAR-machine learning classifiers for the prediction of ubiquitin-proteasome pathway activity

This report showed the use of the atom-based linear index together with different classic and machine learning classification techniques in a QSAR (quantitative structure-activity relationship) study. A PubChem BioAssay DataSet composed by 705 compounds with inhibitory (258 chemicals) and non-inhibitory (447 compounds) activity against the ubiquitin-proteasome pathway were used. The classification models were developed using the linear discriminant analysis, support vector machine, Bayesian networks, k-nearest neighbor, and random forest techniques. In this sense, all the QSAR models show accuracies above 85% in the training set and values of the Matthews correlation coefficient ranging from 0.68 to 0.83. The external validation set shows adequate classifications between 81.25 and 86.36% and Matthews’s correlation coefficient values ranging from 0.59 to 0.70. The present approach contributes as a useful tool for the early detection of novel UPP inhibitors for the treatment of the multiple myeloma and related diseases. A dataset of 705 compounds was extracted from PubChem, with 258 active and 447 non-active compounds in ubiquitin-proteasome pathway inhibitory activity. Later this dataset was divided in training and set, consisting of 529 and 176 compounds, respectively. The quality of the QSAR models developed was proved using the validation set and also checking the applicability domain.

In vitro functional analysis of HER2 variants in lung cancers to evaluate their oncogenic activity and predict clinical response to HER2 targeted therapies.

e23150 Background: HER2 variants are implicated as oncogenic drivers in a variety of cancers. However, not all HER2 variants found on next-generation sequencing (NGS) have been studied, raising the question if these variants of unknown significance (VUS) are oncogenic. This study explores the use of an in-vitro functional assay on HER2 and other variants found on NGS in patients (pts) with lung cancer, and its predictive value for response to HER2 targeted therapy. Methods: Pts with lung cancer at Memorial Sloan Kettering identified to have a HER2 variant by NGS on MSK IMPACT are eligible. NGS data on HER2 variants and co-mutations were analyzed. Functional status was determined by the NovellusDx FACT platform. Mutations were synthesized and transfected into HeLa cells with a fluorescently tagged signaling pathway reporter (MAPK, NFKB, JAK-STAT). Fluorescent microscopy quantified the translocation of reporters to the nucleus, inferring gain of function. Afatinib was added and its effect on pathway activation analyzed. Functional information was correlated with clinical outcomes. Results: As of 2/1/17, 13 pts were included, 9 unique HER2 variants analyzed. Known activating HER2 mutations analyzed (exon 20 insertions (A775_G776insYVMA, G778_P780insGSP, G776delinsVC), point mutations (V659E, L755P, S301F)) showed in vitro gain of function. HER2 VUS analyzed included D277Y showed no gain of function; exon 16 splice variant c.1889 showed gain of function. Afatinib was added to 4 cell lines with gain of function variants (G778_P780insGSP, V659E, L755P, exon 16 splice variant c.1889) all had inhibited pathway activation suggesting in vitro sensitivity to afatinib. Clinically, no response was seen in 2 pts treated with afatinib but more pts are planned to be treated. Objective responses to ado-trastuzumab emtansine (NCT02675829) were seen in 5 pts, all of which had HER2 variants with gain of function. Conclusions: In vitro functional analysis of HER2 variants based on NGS reports is feasible, and has the potential to identify novel actionable oncogenic drivers from VUS. Its predictive value for clinical response to targeted therapy requires further study.

Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols

Cellular lipids are speculated to act as key intermediates in Hedgehog signal transduction, but their precise identity and function remain enigmatic. In an effort to identify such lipids, we pursued a Hedgehog pathway inhibitory activity that is particularly abundant in flagellar lipids of Chlamydomonas reinhardtii, resulting in the purification and identification of ergosterol endoperoxide, a B-ring oxysterol. A mammalian analog of ergosterol, 7-dehydrocholesterol (7-DHC), accumulates in Smith-Lemli-Opitz syndrome, a human genetic disease that phenocopies deficient Hedgehog signaling and is caused by genetic loss of 7-DHC reductase. We found that depleting endogenous 7-DHC with methyl-β-cyclodextrin treatment enhances Hedgehog activation by a pathway agonist. Conversely, exogenous addition of 3β,5α-dihydroxycholest-7-en-6-one, a naturally occurring B-ring oxysterol derived from 7-DHC that also accumulates in Smith-Lemli-Opitz syndrome, blocked Hedgehog signaling by inhibiting activation of the essential transduction component Smoothened, through a mechanism distinct from Smoothened modulation by other lipids.

Abstract A24: Neuropilin 1 (NRP1) as a potential biomarker for tivozanib + mFOLFOX6 versus bevacizumab + mFOLFOX6 in metastatic colorectal cancer (mCRC): Post-hoc biomarker analysis of BATON-CRC phase 2 trial

Abstract Purpose: Tivozanib (T), is a potent oral selective inhibitor of VEGF receptors 1, 2, and 3. Bevacizumab (B) is an antibody targeting VEGF A mediated signaling. The randomized, open label phase 2 BATON-CRC (Biomarker Assessment of Tivozanib in ONcology), trial compared the progression free survival (PFS) and safety of T + mFOLFOX6 vs. B + mFOLFOX6 in mCRC patients. Data from the pre-specified interim analysis demonstrated futility for superiority; however the addition of T to mFOLFOX6 resulted in comparable PFS (9.4 vs 10.7 months, p=0.706), overall response rate (45.2% vs 43.2%, p=0.718), and safety to B + mFOLFOX6 and resulted in discontinuation of the study. At the time of the interim analysis (September, 2013) 62 patients out of 163 progressed to generate a PFS event and none of the pre-specified biomarkers demonstrated statistically significant association with PFS (ESMO 2014). At the close of the study (February, 2014), there were 91 PFS events out of 164 patients with baseline serum samples. The final analysis included the updated events which led to the observation that serum NRP1 may be a predictive biomarker for T. Serum NRP1, a pre-specified biomarker in BATON CRC, is associated with improved PFS and overall survival (OS) in multiple T studies in renal cell carcinoma (RCC). This strong association and clinical benefit in RCC prompted a post-hoc analysis of the final BATON-CRC PFS data to examine the differential association between base line serum NRP1 levels and outcomes in the two arms. Methods: Similar to the RCC trials, baseline serum from patients in BATON-CRC had NRP1 quantified using the Myriad RBM ELISA assay. The median NRP1 level from this dataset was used to define the cut off for NRP1 high and NRP1 low. A Cox proportional hazard model was used to assess the association between serum NRP1 levels and PFS. Results: In BATON-CRC, 265 patients were enrolled and randomized 2:1 to T + mFOLFOX6 (n = 177) or B + mFOLFOX6 (n = 88). A total of 164 baseline serum samples (T, n = 109 and B, n = 55) were available for NRP1 analysis. In both the T and B arms, patients with NRP1 low showed an improved PFS vs patients with NRP1 high; for T HR (95% CI): 0.224 [0.120, 0.42], p=3.56e-7 and for B HR [95% CI]: 0.487 [0.237, 1.00], p=0.046) supporting the value of NRP1 as a potential prognostic marker for angiogenesis inhibitors. T treated NRP1 low patients demonstrated a statistically significantly improvement in PFS vs B, PFS =17.9 vs 11.2 mos (multivariate analysis stratified for cancer origin, metastatic sites, LDH levels HR [95% CI]: 0.325 [0.147, 0.717], p=0.005). In NRP1 high patients, there was no difference in PFS between the two arms (HR (95% CI): 0.889 [0.639, 1.980], p=0.683). The interaction p-value is 0.027. Conclusions: The data suggest that NRP1 might be a predictive biomarker of T PFS benefit over B in combination with mFOLFOX 6 in advanced CRC. This finding needs to be further validated in another prospective trial. The differential activity observed with T vs B, in NRP1 low patients is potentially due to the broader VEGF pathway inhibitory activity of T. Citation Format: Al Benson, III, Andrew K. Krivoshik, Chip Van Sant, Jeno Gyuris, Bin Feng. Neuropilin 1 (NRP1) as a potential biomarker for tivozanib + mFOLFOX6 versus bevacizumab + mFOLFOX6 in metastatic colorectal cancer (mCRC): Post-hoc biomarker analysis of BATON-CRC phase 2 trial. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A24.

Cycloartane Glycosides from the Roots of Cimicifuga foetida with Wnt Signaling Pathway Inhibitory Activity.

Four new 9,19-cycloartane triterpenoids, cimilactone E (1), cimilactone F (2), 2′-O-(E)-butenoyl-23-epi-26-deoxyactein (3), and 2′,12β-O-diacetylcimiracemonol-3-O-β-d-xylopyranoside (4), together with four known constituents (5–8) were isolated from the roots of Cimicifuga foetida. The new structures were elucidated by extensive spectroscopic analysis. In addition, compounds 7 and 8 showed significant Wnt signaling pathway inhibitory activity, with IC50 values of 3.33 and 13.34 μM, respectively, using the luciferase reporter gene assay.

Synthesis and evaluation of novel N-3-benzimidazolephenylbisamide derivatives for antiproliferative and Hedgehog pathway inhibitory activity

7m, as a novel Hedgehog inhibitor, interacted closely with the smoothened receptor at the co-crystallized ligand (taledegib) site.

Novel ATP-Competitive MEK Inhibitor E6201 Is Effective against Vemurafenib-Resistant Melanoma Harboring the MEK1-C121S Mutation in a Preclinical Model

Many clinical cases of acquired resistance to the BRAF inhibitor vemurafenib have recently been reported. One of the causes of this acquired resistance is the BRAF downstream kinase point mutation MEK1-C121S. This mutation confers resistance to not only vemurafenib, but also to the allosteric MEK inhibitor selumetinib (AZD6244). Here, we investigated the pharmacologic activities and effectiveness of the novel MEK inhibitor E6201 against BRAF (v-raf murine sarcoma viral oncogene homolog B1)-V600E mutant melanoma harboring the MEK1-C121S mutation. A cell-free assay confirmed that E6201 is an ATP-competitive MEK inhibitor, meaning it has a different binding mode with MEK compared with allosteric MEK inhibitors. E6201 is more effective against BRAF-V600E mutant melanoma compared with BRAF wild-type melanoma based on MEK inhibition. We found that the acquired MEK1-C121S mutation in BRAF-V600E mutant melanoma conferred resistance to both vemurafenib and selumetinib but not E6201. The effectiveness of E6201 in this preclinical study is a result of its binding with MEK1 far from the C121S point mutation so the mutation is unable to influence the MAPK pathway inhibitory activity. These results support further clinical investigation of E6201.

Synthesis and cytotoxicity studies of Hedgehog enzyme inhibitors SANT-1 and GANT-61 as anticancer agents

Cancer-related death is one of the most common causes of mortality in society. Small molecules have the capability to disrupt aberrant signaling pathways in tumors, leading to anticancer activities. Therefore the search for new molecules for cancer treatment continues to draw attention to the scientific research community. Synthesis and biological evaluation of hedgehog (Hh) pathway inhibitors SANT-1 and GANT-61 are disclosed. These molecules have been synthesized from common precursors using simple conversions, our synthesis features Vils-Meier-Haack reaction, imine formation reaction and N-arylation reaction. These drugs were evaluated using a Hh reporter assay to confirm pathway inhibitory activity, and tested for cell viability against pancreatic and prostate cancer cells. These methodologies can be applied to make potent analogs of both inhibitors.

Abstract C65: First-in-human Phase I dose-escalation study of a MET/AXL/FGFR inhibitor, S 49076, in patients with advanced solid tumors.

Abstract Background: S 49076 is a novel ATP-competitive tyrosine kinase inhibitor which displays a unique profile targeting MET, AXL and FGFR-1/2/3 (IC50 for inhibition of pathway activation was <200nM) but not VEGFR-2. Considering the strong implication of these kinases in tumor progression and metastasis formation combined to the ability of S 49076 to inhibit known MET and FGFR mutated variants, efficacy is expected in treatment of patients (pts) with primary cancer and metastasis. Based on the non-clinical studies showing a favorable and novel pharmacological profile, an oral formulation of S 49076 is evaluated in a phase I dose-escalation study in pts with advanced solid tumors. This first-in-human study aims to determine the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), pharmacokinetics (PK), pharmacodynamics (PD) and potential efficacy of S 49076. Methods: Pts are enrolled on dose-escalation cohorts, using a “3+3” design. Two dosing regimens of S 49076 administration (once a day, QD, or twice a day, BID) are tested in this study, in which pts receive oral capsule(s) of S 49076 during a continuous 21-day cycle. The PK measurements are performed at D1 and at steady state. Tumor biopsies for genomic and proteomic analyses are taken predose and on treatment. Tumor response is measured once every 2 cycles. Results: To date 49 pts have been treated at doses ranging from 15 to 270 mg QD or 7.5 to 225 mg BID. Median age is 59 years, and the most common tumor types are colorectal, NSCLC and uveal melanoma. The most frequent adverse events reported as treatment-related by the investigator have been hypomagnesemia, peripheral edema and hypoalbuminemia mostly grade (G) 1-2. Three DLTs were observed, including failure to restart S 49076 within 1 week due to G2 asymptomatic ejection fraction decrease in 30 mg QD and 225 mg BID cohorts and G3 hypotension in the 180 mg BID cohort. MTD has not been reached and the dose escalation is ongoing. Among 37 evaluable pts, 20 pts had stable disease (SD) ranging from 6 to 36 weeks of whom 3 pts with uveal melanoma had prolonged SD from 12 to 24 weeks. Preliminary PK analyses showed that the maximal plasma concentration (Cmax) occurred between 2h and 6h after oral administration. The bioavailability was estimated to be around 30% and the effective half-life around 15h. There was no evidence of non-linearity since Cmax and AUC24h increased proportionally over the range of doses tested and no evidence of time dependent PK. Based on PK data, the plasma concentrations reached in human at the higher doses are consistent with inhibition of the S 49076 targets MET, AXL and FGFR. Conclusions: The general safety profile of oral treatment with S 49076 is good at the doses tested. PK data suggest dose proportionality in exposure without marked drug accumulation. S 49076 has demonstrated preliminary anti-tumor activity, especially in uveal melanoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C65. Citation Format: Antoine Hollebecque, Jean-Charles Soria, Ratislav Bahleda, Natividad Lopez-Busto, Anne Jacquet-Bescond, Mike F. Burbridge, Cattan Valérie, Jeanne Pauly, Maria Herranz, Analia Azaro, Stéphane Depil, Jordi Rodon. First-in-human Phase I dose-escalation study of a MET/AXL/FGFR inhibitor, S 49076, in patients with advanced solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C65.

Abstract A289: Novel ATP-competitive MEK inhibitor E6201 is effective against vemurafenib-resistant melanoma harboring the MEK1-C121S mutation.

Abstract Many clinical cases of acquired resistance to the BRAF inhibitor vemurafenib have recently been reported. One of the causes of this acquired resistance is the BRAF downstream kinase point mutation MEK1-C121S. This mutation confers resistance to not only vemurafenib, but also to the allosteric MEK inhibitor selumetinib (AZD6244). Here, we investigated the pharmacological activities and effectiveness of the novel MEK inhibitor E6201 against BRAF-V600E mutant melanoma harboring the MEK1-C121S mutation. A cell-free assay confirmed that E6201 is an ATP-competitive MEK inhibitor meaning it has a different binding mode with MEK compared with allosteric MEK inhibitors. E6201 is more effective against BRAF-V600E mutant melanoma compared with BRAF wild-type melanoma based on MEK inhibition. We found that the acquired MEK1-C121S mutation in BRAF-V600E mutant melanoma conferred resistance to both vemurafenib and selumetinib but not E6201. The effectiveness of E6201 is a result of its binding with MEK1 far from the C121S point mutation so the mutation is unable influence the MAPK pathway inhibitory activity. We conclude that E6201 is a potential therapeutic choice for improving the clinical outcome of vemurafenib-based therapy. However, further validation is needed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A289. Citation Format: Yusuke Narita, Kiyoshi Okamoto, Megumi Ikemori Kawada, Kazuma Takase, Yukinori Minoshima, Kotaro Kodama, Masao Iwata. Novel ATP-competitive MEK inhibitor E6201 is effective against vemurafenib-resistant melanoma harboring the MEK1-C121S mutation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A289.