TAS2940, a novel brain-penetrable pan-ERBB inhibitor, for tumors with HER2 and EGFR aberrations.

Genetic alterations in human epidermal growth factor receptor type 2 (HER2)/epidermal growth factor receptor (EGFR) are commonly associated with breast and lung cancers and glioblastomas. Cancers with avian erythroblastosis oncogene B (ERBB) deregulation are highly metastatic and can cause primary brain tumors. Currently, no pan-ERBB inhibitor with remarkable brain penetration is available. Here, TAS2940, a novel irreversible pan-ERBB inhibitor with improved brain penetrability, was evaluated for its efficacy against several ERBB aberrant cancer models. The selectivity of TAS2940 was evaluated by enzymatic kinase assays. The inhibitory effects of TAS2940 against ERBB genetic alterations were examined using MCF10A cells expressing various HER2 or EGFR mutations and other generic cell lines harboring deregulated ERBB expression. In vivo efficacy of TAS2940 was examined following oral treatment in subcutaneous or intracranial xenograft cancer models. TAS2940 was highly potent against cells harboring HER2/EGFR alterations. TAS2940 could selectively inhibit phosphorylation of targets and the growth of cancer cells with ERBB aberrations in vitro. TAS2940 also inhibited tumor growth in xenograft mouse models with ERBB aberrations: HER2 amplification, HER2/EGFR exon 20 insertions, and EGFR vIII mutation. TAS2940 was effective in the intracranial xenograft models of HER2/EGFR cancers and improved the survival of these mice. TAS2940 has promising therapeutic effects in preclinical study against cancers harboring HER2/EGFR mutations, especially metastatic and primary brain tumors. Our results highlight potential novel strategies against lung cancers with brain metastases harboring HER2/EGFR exon 20 insertions and glioblastomas with EGFR aberrations.

TAS-119, a novel selective Aurora A and TRK inhibitor, exhibits antitumor efficacy in preclinical models with deregulated activation of the Myc, ß-Catenin, and TRK pathways.

Aurora kinase A, a mitotic kinase that is overexpressed in various cancers, is a promising cancer drug target. Here, we performed preclinical characterization of TAS-119, a novel, orally active, and highly selective inhibitor of Aurora A. TAS-119 showed strong inhibitory effect against Aurora A, with an IC50 value of 1.04 nmol/L. The compound was highly selective for Aurora A compared with 301 other protein kinases, including Aurora kinase B. TAS-119 induced the inhibition of Aurora A and accumulation of mitotic cells in vitro and in vivo. It suppressed the growth of various cancer cell lines harboring MYC family amplification and CTNNB1 mutation in vitro. In a xenograft model of human lung cancer cells harboring MYC amplification and CTNNB1 mutation, TAS-119 showed a strong antitumor activity at well-tolerated doses. TAS-119 induced N-Myc degradation and inhibited downstream transcriptional targets in MYCN-amplified neuroblastoma cell lines. It also demonstrated inhibitory effect against tropomyosin receptor kinase (TRK)A, TRKB, and TRKC, with an IC50 value of 1.46, 1.53, and 1.47 nmol/L, respectively. TAS-119 inhibited TRK-fusion protein activity and exhibited robust growth inhibition of tumor cells via a deregulated TRK pathway in vitro and in vivo. Our study indicates the potential of TAS-119 as an anticancer drug, especially for patients harboring MYC amplification, CTNNB1 mutation, and NTRK fusion.

ARHGEF15 overexpression worsens the prognosis in patients with pancreatic ductal adenocarcinoma through enhancing the motility and proliferative activity of the cancer cells.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplastic diseases, associated with a remarkably poor prognosis. However, the molecular mechanisms underlying the development of PDAC remain elusive. The aim of this study was to identify genes whose expressions are correlated with a poor prognosis in PDAC patients, and to unravel the mechanisms underlying the involvement of these genes in the development of the cancer. METHODS: Global gene expression profiling was conducted in 39 specimens obtained from Japanese patients with PDAC to identify genes whose expressions were correlated with a shorter overall survival. The effect of gene silencing or overexpression of ARHGEF15 in pancreatic cancer cell lines was examined by introducing siRNAs of ARHGEF15 or the ARHGEF15 expression vector. After assessing the effect of ARHGEF15 deregulation on the Rho-family proteins by pull-down assay, wound healing, transwell and cell viability assays were carried out to investigate the cellular phenotypes caused by the perturbation. RESULTS: The global mRNA expression profiling revealed that overexpression of ARHGEF15, a Rho-specific GEF, was significantly associated with a poor prognosis in patients with PDAC. We also found that the depletion of ARHGEF15 by RNA interference in pancreatic cancer cell lines downregulated the activities of molecules of the Rho signaling pathway, including RhoA, Cdc42 and Rac1. Then, we also showed that ARHGEF15 silencing significantly reduced the motility and viability of the cells, while its overexpression resulted in the development of the opposite phenotype in multiple pancreatic cancer cell lines. CONCLUSION: These data suggest that upregulation of ARHGEF15 contributes to the development of aggressive PDAC by increasing the growth and motility of the pancreatic cancer cells, thereby worsening the prognosis of these patients. Therefore, ARHGEF15 could serve as a novel therapeutic target in patients with PDAC.

3'-ethynylcytidine, an RNA polymerase inhibitor, combined with cisplatin exhibits a potent synergistic growth-inhibitory effect via Vaults dysfunction.

BACKGROUND: We previously reported that 3'-ethynylcytidine (ECyd, TAS-106), an RNA polymerases inhibitor, enhances the anti-tumor efficacy of platinum in several tumor types in both in vitro and in vivo tumor models. However, the molecular mechanisms underlying the ECyd-induced enhancement remain elusive. METHODS: Cisplatin (CDDP)-resistant head and neck cancer KB cells were established by stepwise dose escalation with CDDP. The combination effect of ECyd and CDDP were assessed using isobologram analysis. The transcriptional and post-translational statuses of several molecules were detected using real-time PCR, immunoblot analysis and immunocytochemistry. Xenograft assays were used to confirm the mechanisms underlying the ECyd induced enhancement of CDDP anti-tumor efficacy in vivo. RESULTS: ECyd sensitized KB to CDDP by inhibiting the drug transporter Vault complex (Vaults). First, we showed that Vaults were overexpressed in CDDP-resistant KB cells. The suppression of major vault protein (MVP) by RNA interference restored the sensitivity to CDDP. Next, we showed that ECyd significantly sensitized the resistant cells to CDDP, compared with the parental paired cell line. A molecular analysis revealed that ECyd inhibited the synthesis of vRNAs as well as the induction of MVP, both of which are critical components of Vaults as a drug transporter. Furthermore, we found that the synergistic effect of ECyd and CDDP was correlated with the MVP expression level when the effect was analyzed in additional cancer cell lines. Finally, we demonstrated that ECyd decreased the vRNAs expression level in xenograft tumor. CONCLUSIONS: Our data indicated the ability of ECyd to cancel the resistance of cancer cells to CDDP by inhibiting the Vaults function and the decrease of Vaults expression itself, and the ability of the combination therapy with CDDP and ECyd to offer a new strategy for overcoming platinum resistance. Moreover, the study results suggest that Vaults could be a biomarker for stratifying patients who may benefit from the combination therapy with ECyd and platinum.

Expression ratio of CCND1 to CDKN2A mRNA predicts RB1 status of cultured cancer cell lines and clinical tumor samples.

BACKGROUND: The retinoblastoma product (RB1) is frequently deregulated in various types of tumors by mutation, deletion, or inactivation through association with viral oncoproteins. The functional loss of RB1 is recognized to be one of the hallmarks that differentiate cancer cells from normal cells. Many researchers are attempting to develop anti-tumor agents that are preferentially effective against RB1-negative tumors. However, to identify patients with RB1-negative cancers, it is imperative to develop predictive biomarkers to classify RB1-positive and -negative tumors. RESULTS: Expression profiling of 30 cancer cell lines composed of 16 RB1-positive and 14 RB1-negative cancers was performed to find genes that are differentially expressed between the two groups, resulting in the identification of an RB1 signature with 194 genes. Among them, critical RB1 pathway components CDKN2A and CCND1 were included. We found that microarray data of the expression ratio of CCND1 and CDKN2A clearly distinguished the RB1 status of 30 cells lines. Measurement of the CCND1/CDKN2A mRNA expression ratio in additional cell lines by RT-PCR accurately predicted RB1 status (12/12 cells lines). The expression of CCND1/CDKN2A also correlated with RB1 status in xenograft tumors in vivo. Lastly, a CCND1/CDKN2A assay with clinical samples showed that uterine cervical and small cell lung cancers known to have a high prevalence of RB1-decifiency were predicted to be 100% RB1-negative, while uterine endometrial or gastric cancers were predicted to be 5-22% negative. All clinically normal tissues were 100% RB1-positive. CONCLUSIONS: We report here that the CCND1/CDKN2A mRNA expression ratio predicts the RB1 status of cell lines in vitro and xenograft tumors and clinical tumor samples in vivo. Given the high predictive accuracy and quantitative nature of the CCND1/CDKN2A expression assay, the assay could be utilized to stratify patients for anti-tumor agents with preferential effects on either RB1-positive or -negative tumors.

Synthetic lethal interactions for the development of cancer therapeutics: biological and methodological advancements.

Synthetic lethal interaction is defined as a combination of two mutations that is lethal when present in the same cell; each individual mutation is non-lethal. Synthetic lethal interactions attract attention in cancer research fields since the discovery of synthetic lethal genes with either oncogenes or tumor suppressor genes (TSGs) provides novel cancer therapeutic targets. Due to the selective lethal effect on cancer cells harboring specific genetic alterations, it is expected that targeting synthetic lethal genes would provide wider therapeutic windows compared with cytotoxic chemotherapeutics. Here, we review the current status of the application of synthetic lethal screening in cancer research fields from biological and methodological viewpoints. Very recent studies seeking to identify synthetic lethal genes with K-RAS and p53, which are known to be the most frequently occurring oncogenes and TSGs, respectively, are introduced. Among the accumulating amount of research on synthetic lethal interactions, the synthetic lethality between BRCA1/2 and PARP1 inhibition has been clinically proven. Thus, both preclinical and clinical data showing a preferential anti-tumor effect on BRCA1/2 deficient tumors by a PARP1 inhibitor are the best examples of the synthetic lethal approach of cancer therapeutics. Finally, methodological progress regarding synthetic lethal screening, including barcode shRNA screening and in vivo synthetic lethal screening, is described. Given the fact that an increasing number of synthetic lethal genes for major cancerous genes have been validated in preclinical studies, this intriguing approach awaits clinical verification of preferential benefits for cancer patients with specific genetic alterations as a clear predictive factor for tumor response.

Expression levels of NF-Y target genes changed by CDKN1B correlate with clinical prognosis in multiple cancers.

CDK inhibitors CDKN1B (p27) and CDKN2A (p16) inhibit cell cycle progression. A lower expression level of only p27 has been correlated with poorer prognosis in various types of clinical cancers. The difference may be the result of distinct genes downstream of these CDK inhibitors. Here, we report that NF-Y transcription factor-targeted genes specifically down-regulated by p27 correlate with poor prognosis in multiple tumor types. We performed mRNA expression profiling in HCT116 cells over-expressing either p16 or p27 and identified their regulatory genes. In silico transcription factor prediction indicated that most of the genes specifically down-regulated by p27 are controlled by NF-Y. Under the hypothesis that NF-Y-targeted genes are responsible for poor prognosis, we predicted prognosis in four types of cancer based on genes with the NF-Y motif, and found a significant association between the expression of NF-Y-targeted genes and poor prognosis.

Inhibition of p70S6K2 down-regulates Hedgehog/GLI pathway in non-small cell lung cancer cell lines.

BACKGROUND: The Hedgehog (HH) pathway promotes tumorigenesis in a diversity of cancers. Activation of the HH signaling pathway is caused by overexpression of HH ligands or mutations in the components of the HH/GLI1 cascade, which lead to increased transactivation of GLI transcription factors. Although negative kinase regulators that antagonize the activity of GLI transcription factors have been reported, including GSK3beta, PKA and CK1s, little is known regarding positive kinase regulators that are suitable for use on cancer therapeutic targets. The present study attempted to identify kinases whose silencing inhibits HH/GLI signalling in non-small cell lung cancer (NSCLC). RESULTS: To find positive kinase regulators in the HH pathway, kinome-wide siRNA screening was performed in a NSCLC cell line, A549, harboring the GLI regulatory reporter gene. This showed that p70S6K2-silencing remarkably reduced GLI reporter gene activity. The decrease in the activity of the HH pathway caused by p70S6K2-inhibition was accompanied by significant reduction in cell viability. We next investigated the mechanism for p70S6K2-mediated inhibition of GLI1 transcription by hypothesizing that GSK3beta, a negative regulator of the HH pathway, is activated upon p70S6K2-silencing. We found that phosphorylated-GSK3beta (Ser9) was reduced by p70S6K2-silencing, causing a decreased level of GLI1 protein. Finally, to further confirm the involvement of p70S6K2 in GLI1 signaling, down-regulation in GLI-mediated transcription by PI3KCA-inhibition was confirmed, establishing the pivotal role of the PI3K/p70S6K2 pathway in GLI1 cascade regulation. CONCLUSION: We report herein that inhibition of p70S6K2, known as a downstream effector of the PI3K pathway, remarkably decreases GLI-mediated transactivation in NSCLC by reducing phosphorylated-GSK3beta followed by GLI1 degradation. These results infer that p70S6K2 is a potential therapeutic target for NSCLC with hyperactivated HH/GLI pathway.

Discovery of gene expression-based pharmacodynamic biomarker for a p53 context-specific anti-tumor drug Wee1 inhibitor.

BACKGROUND: Wee1 is a tyrosine kinase regulating S-G2 cell cycle transition through the inactivating phosphorylation of CDC2. The inhibition of Wee1 kinase by a selective small molecule inhibitor significantly enhances the anti-tumor efficacy of DNA damaging agents, specifically in p53 negative tumors by abrogating S-G2 checkpoints, while normal cells with wild-type p53 are not severely damaged due to the intact function of the G1 checkpoint mediated by p53. Since the measurement of mRNA expression requires a very small amount of biopsy tissue and is highly quantitative, the development of a pharmacodynamic (PD) biomarker leveraging mRNA expression is eagerly anticipated in order to estimate target engagement of anti-cancer agents. RESULTS: In order to find the Wee1 inhibition signature, mRNA expression profiling was first performed in both p53 positive and negative cancer cell lines treated with gemcitabine and a Wee1 inhibitor, MK-1775. We next carried out mRNA expression profiling of skin samples derived from xenograft models treated with the Wee1 inhibitor to identify a Wee1 inhibitor-regulatory gene set. Then, the genes that were commonly modulated in both cancer cell lines and rat skin samples were extracted as a Wee1 inhibition signature that could potentially be used as a PD biomarker independent of p53 status. The expression of the Wee1 inhibition signature was found to be regulated in a dose-dependent manner by the Wee1 inhibitor, and was significantly correlated with the inhibition level of a direct substrate, phosphorylated-CDC2. Individual genes in this Wee1 inhibition signature are known to regulate S-G2 cell cycle progression or checkpoints, which is consistent with the mode-of-action of the Wee1 inhibitor. CONCLUSION: We report here the identification of an mRNA gene signature that was specifically changed by gemcitabine and Wee1 inhibitor combination treatment by molecular profiling. Given the common regulation of expression in both xenograft tumors and animal skin samples, the data suggest that the Wee1 inhibition gene signature might be utilized as a quantitative PD biomarker in both tumors and surrogate tissues, such as skin and hair follicles, in human clinical trials.

Integrated genomic and pharmacological approaches to identify synthetic lethal genes as cancer therapeutic targets.

Various types of cancers are generated through mutations or dysregulations of oncogenes/tumor suppressor genes involved in cell cycles and signaling transduction pathways. To identify cancer therapeutic targets whose inhibition selectively kills cancer cells, synthetic lethal screening is being developed to identify genes whose intervention suppresses tumor progression only when combined with the dysregulation of the genes. The recent emergence of genomic technologies, including microarray, RNA interference and chemogenomics, provides platforms to realize this concept. This review introduces the research that could successfully identify synthetic lethal genes in cancer cells harboring major gene alterations such as p53, RB, K-Ras, or Myc. We also illustrate remarkable candidate targets that were identified by synthetic lethal screening to find chemosensitizers for paclitaxel and cisplatin. Next, we introduce the chemogenomics approaches that explore chemical compounds that exhibit synthetic lethality to cancer gene alterations. Although the synthetic lethal compounds are of great interest in terms of cancer drug development, a method of identifying target proteins for the phenotypic compounds has been elusive. Finally, we demonstrate several noteworthy techniques to identify target proteins for the compounds: a Connectivity Map that compares expression profiles of compound-treated cells by pattern-matching algorithms; an siRNA/compound co-treatment assay to find enhancer genes for the phenotypes of compounds; and a state-of-the-art proteomics approach that modifies classical compound-immobilized affinity chromatography. The integration of genomic and pharmacological analyses would significantly accelerate the identification of cancer-specific synthetic lethal targets.

Role of the Toll-like receptor 4/NF-kappaB pathway in saturated fatty acid-induced inflammatory changes in the interaction between adipocytes and macrophages.

OBJECTIVE: Previous studies demonstrated that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. Using an in vitro coculture system composed of 3T3-L1 adipocytes and RAW264 macrophages, we previously demonstrated that saturated fatty acids (FAs) and tumor necrosis factor (TNF)-alpha derived from adipocytes and macrophages, respectively, play a major role in the coculture-induced inflammatory changes. METHODS AND RESULTS: Coculture of adipocytes and macrophages resulted in the activation of nuclear factor-kappaB (NF-kappaB), a primary regulator of inflammatory responses, in both cell types. Pharmacological inhibition of NF-kappaB markedly suppressed the coculture-induced production of proinflammatory cytokines and adipocyte lipolysis. Peritoneal macrophages obtained from Toll-like receptor 4 (TLR4) mutant mice exhibited marked attenuation of TNFalpha production in response to saturated FAs. Notably, coculture of hypertrophied adipocytes and TLR4-mutant macrophages resulted in marked inhibition of proinflammatory cytokine production and adipocyte lipolysis. We also observed that endogenous FAs, which are released from adipocytes via the beta3-adrenergic stimulation, resulted in the activation of the TLR4/NF-kappaB pathway. CONCLUSIONS: These findings suggest that saturated FAs, which are released in large quantities from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for TLR4, thereby inducing the inflammatory changes in both adipocytes and macrophages through NF-kappaB activation.

Can systems biology understand pathway activation? Gene expression signatures as surrogate markers for understanding the complexity of pathway activation.

Cancer is thought to be caused by a sequence of multiple genetic and epigenetic alterations which occur in one or more of the genes controlling cell cycle progression and signaling transduction. The complexity of carcinogenic mechanisms leads to heterogeneity in molecular phenotype, pathology, and prognosis of cancers.Genome-wide mutational analysis of cancer genes in individual tumors is the most direct way to elucidate the complex process of disease progression, although such high-throughput sequencing technologies are not yet fully developed. As a surrogate marker for pathway activation analysis, expression profiling using microarrays has been successfully applied for the classification of tumor types, stages of tumor progression, or in some cases, prediction of clinical outcomes. However, the biological implication of those gene expression signatures is often unclear. Systems biological approaches leverage the signature genes as a representation of changes in signaling pathways, instead of interpreting the relevance between each gene and phenotype. This approach, which can be achieved by comparing the gene set or the expression profile with those of reference experiments in which a defined pathway is modulated, will improve our understanding of cancer classification, clinical outcome, and carcinogenesis. In this review, we will discuss recent studies on the development of expression signatures to monitor signaling pathway activities and how these signatures can be used to improve the identification of responders to anticancer drugs.

Mutant p53 induces the GEF-H1 oncogene, a guanine nucleotide exchange factor-H1 for RhoA, resulting in accelerated cell proliferation in tumor cells.

The tumor suppressor gene p53 is known to induce G1-S and G2-M cell cycle arrest and apoptosis by transactivating various wild-type (WT) p53 regulatory genes. Mutational inactivation of p53 is detected in more than half of human cancers, depriving the p53 protein of its tumor-suppressive functions. Recent studies have shown that mutant p53 provides tumor cells with gain-of-function properties, such as accelerated cell proliferation, increased metastasis, and apoptosis resistance. However, the mechanism underlying the elevated tumorigenicity by p53 mutation remains to be elucidated. In the present study, we showed that GEF-H1, a guanine exchange factor-H1 for RhoA, is transcriptionally activated by the induction of mutant p53 proteins, thereby accelerating tumor cell proliferation. Osteosarcoma U2OS cell lines, which express inducible p53 mutants (V157F, R175H, and R248Q), were established, and the expression profiles of each cell line were then analyzed to detect genes specifically induced by mutant p53. We identified GEF-H1 as one of the consensus genes whose expression was significantly induced by the three mutants. The GEF-H1 expression level strongly correlated with p53 status in a panel of 32 cancer cell lines, and GEF-H1 induction caused activation of RhoA. Furthermore, growth of mutant p53 cells was dependent on GEF-H1 expression, whereas that of WT p53 cells was not. These results suggest that increased GEF-H1 expression contributes to the tumor progression phenotype associated with the p53 mutation.

Biomarker discovery by integrated joint non-negative matrix factorization and pathway signature analyses.

Predictive biomarkers are important for selecting appropriate patients for particular treatments. Comprehensive genomic, transcriptomic, and pharmacological data provide clues for understanding relationships between biomarkers and drugs. However, it is still difficult to mine biologically meaningful biomarkers from multi-omics data. Here, we developed an approach for mining multi-omics cell line data by integrating joint non-negative matrix factorization (JNMF) and pathway signature analyses to identify candidate biomarkers. The JNMF detected known associations between biomarkers and drugs such as BRAF mutation with PLX4720 and HER2 amplification with lapatinib. Furthermore, we observed that tumours with both BRAF mutation and MITF activation were more sensitive to BRAF inhibitors compared to tumours with BRAF mutation without MITF activation. Therefore, activation of the BRAF/MITF axis seems to be a more appropriate biomarker for predicting the efficacy of a BRAF inhibitor than the conventional biomarker of BRAF mutation alone. Our biomarker discovery scheme represents an integration of JNMF multi-omics clustering and multi-layer interpretation based on pathway gene signature analyses. This approach is also expected to be useful for establishing drug development strategies, identifying pharmacodynamic biomarkers, in mode of action analysis, as well as for mining drug response data in a clinical setting.

Crystal structures of the N-terminal kinase domain of human RSK1 bound to three different ligands: Implications for the design of RSK1 specific inhibitors.

The p90 ribosomal S6 kinases (RSKs) also known as MAPKAP-Ks are serine/threonine protein kinases that are activated by ERK or PDK1 and act as downstream effectors of mitogen-activated protein kinase (MAPK). RSK1, a member of the RSK family, contains two distinct kinase domains in a single polypeptide chain, the regulatory C-terminal kinase domain (CTKD) and the catalytic N-terminal kinase domain (NTKD). Autophosphorylation of the CTKD leads to activation of the NTKD that subsequently phosphorylates downstream substrates. Here we report the crystal structures of the unactivated RSK1 NTKD bound to different ligands at 2.0 A resolution. The activation loop and helix alphaC, key regulatory elements of kinase function, are disordered. The DFG motif of the inactive RSK1 adopts an "active-like" conformation. The beta-PO(4) group in the AMP-PCP complex adopts a unique conformation that may contribute to inactivity of the enzyme. Structures of RSK1 ligand complexes offer insights into the design of novel anticancer agents and into the regulation of the catalytic activity of RSKs.

Muscle RAS oncogene homolog (MRAS) recurrent mutation in Borrmann type IV gastric cancer.

The prognosis of patients with Borrmann type IV gastric cancer (Type IV) is extremely poor. Thus, there is an urgent need to elucidate the molecular mechanisms underlying the oncogenesis of Type IV and to identify new therapeutic targets. Although previous studies using whole-exome and whole-genome sequencing have elucidated genomic alterations in gastric cancer, none has focused on comprehensive genetic analysis of Type IV. To discover cancer-relevant genes in Type IV, we performed whole-exome sequencing and genome-wide copy number analysis on 13 patients with Type IV. Exome sequencing identified 178 somatic mutations in protein-coding sequences or at splice sites. Among the mutations, we found a mutation in muscle RAS oncogene homolog (MRAS), which is predicted to cause molecular dysfunction. MRAS belongs to the Ras subgroup of small G proteins, which includes the prototypic RAS oncogenes. We analyzed an additional 46 Type IV samples to investigate the frequency of MRAS mutation. There were eight nonsynonymous mutations (mutation frequency, 17%), showing that MRAS is recurrently mutated in Type IV. Copy number analysis identified six focal amplifications and one homozygous deletion, including insulin-like growth factor 1 receptor (IGF1R) amplification. The samples with IGF1R amplification had remarkably higher IGF1R mRNA and protein expression levels compared with the other samples. This is the first report of MRAS recurrent mutation in human tumor samples. Our results suggest that MRAS mutation and IGF1R amplification could drive tumorigenesis of Type IV and could be new therapeutic targets.

MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts.

PURPOSE: Investigate the efficacy and pharmacodynamic effects of MK-1775, a potent Wee1 inhibitor, in both monotherapy and in combination with gemcitabine (GEM) using a panel of p53-deficient and p53 wild-type human pancreatic cancer xenografts. EXPERIMENTAL DESIGN: Nine individual patient-derived pancreatic cancer xenografts (6 with p53-deficient and 3 with p53 wild-type status) from the PancXenoBank collection at Johns Hopkins were treated with MK-1775, GEM, or GEM followed 24 hour later by MK-1775, for 4 weeks. Tumor growth rate/regressions were calculated on day 28. Target modulation was assessed by Western blotting and immunohistochemistry. RESULTS: MK-1775 treatment led to the inhibition of Wee1 kinase and reduced inhibitory phosphorylation of its substrate Cdc2. MK-1775, when dosed with GEM, abrogated the checkpoint arrest to promote mitotic entry and facilitated tumor cell death as compared to control and GEM-treated tumors. MK-1775 monotherapy did not induce tumor regressions. However, the combination of GEM with MK-1775 produced robust antitumor activity and remarkably enhanced tumor regression response (4.01-fold) compared to GEM treatment in p53-deficient tumors. Tumor regrowth curves plotted after the drug treatment period suggest that the effect of the combination therapy is longer-lasting than that of GEM. None of the agents produced tumor regressions in p53 wild-type xenografts. CONCLUSIONS: These results indicate that MK-1775 selectively synergizes with GEM to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts.

MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor efficacy of various DNA-damaging agents, including 5-fluorouracil.

MK-1775 is a potent and selective small molecule Wee1 inhibitor. Previously we have shown that it abrogated DNA damaged checkpoints induced by gemcitabine, carboplatin, and cisplatin and enhanced the anti-tumor efficacy of these agents selectively in p53-deficient tumor cells. MK-1775 is currently in Phase I clinical trial in combination with these anti-cancer drugs. In this study, the effects of MK-1775 on 5-fluorouracil (5-FU) and other DNA-damaging agents with different modes of action were determined. MK-1775 enhanced the cytotoxic effects of 5-FU in p53-deficient human colon cancer cells. MK-1775 inhibited CDC2 Y15 phosphorylation in cells, abrogated DNA damaged checkpoints induced by 5-FU treatment, and caused premature entry of mitosis determined by induction of Histone H3 phosphorylation. Enhancement by MK-1775 was specific for p53-deficient cells since this compound did not sensitize p53-wild type human colon cancer cells to 5-FU in vitro. In vivo, MK-1775 potentiated the anti-tumor efficacy of 5-FU or its prodrug, capecitabine, at tolerable doses. These enhancements were well correlated with inhibition of CDC2 phosphorylation and induction of Histone H3 phosphorylation in tumors. In addition, MK-1775 also potentiated the cytotoxic effects of pemetrexed, doxorubicin, camptothecin, and mitomycin C in vitro. These studies support the rationale for testing the combination of MK-1775 with various DNA-damaging agents in cancer patients.

Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents.

Wee1 is a tyrosine kinase that phosphorylates and inactivates CDC2 and is involved in G(2) checkpoint signaling. Because p53 is a key regulator in the G(1) checkpoint, p53-deficient tumors rely only on the G(2) checkpoint after DNA damage. Hence, such tumors are selectively sensitized to DNA-damaging agents by Wee1 inhibition. Here, we report the discovery of a potent and selective small-molecule inhibitor of Wee1 kinase, MK-1775. This compound inhibits phosphorylation of CDC2 at Tyr15 (CDC2Y15), a direct substrate of Wee1 kinase in cells. MK-1775 abrogates G(2) DNA damage checkpoint, leading to apoptosis in combination with DNA-damaging chemotherapeutic agents such as gemcitabine, carboplatin, and cisplatin selectively in p53-deficient cells. In vivo, MK-1775 potentiates tumor growth inhibition by these agents, and cotreatment does not significantly increase toxicity. The enhancement of antitumor effect by MK-1775 was well correlated with inhibition of CDC2Y15 phosphorylation in tumor tissue and skin hair follicles. Our data indicate that Wee1 inhibition provides a new approach for treatment of multiple human malignancies.

Identification of dicarboxylate carrier Slc25a10 as malate transporter in de novo fatty acid synthesis.

Mitochondrial solute carrier family 25 member 10 (Slc25a10) transports dicarboxylates such as malate or succinate across the mitochondrial inner membrane. Although fatty acid synthesis in adipose tissue or the liver is initiated by citrate transport in exchange for malate across the mitochondrial membrane, the transporter responsible for supplying malate during citrate transport has not been identified. In the present study, we clarified the role of Slc25a10 in supplying malate for citrate transport and examined the effect of Slc25a10 suppression on the lipogenic pathway and lipid accumulation. We have reported an Slc25a10 increase in white adipose tissue in obese mouse models and a decrease in a fasted mouse model using expression profiles. Next, we examined the effect of Slc25a10 suppression by small interfering RNA on citrate transport in the lipogenic cell lines HepG2 and 3T3-L1. We observed that inhibition of malate transport by Slc25a10 suppression significantly reduced the citrate transport from the mitochondria to the cytosol. We also found that suppression of Slc25a10 down-regulated the lipogenic pathway, indicated by decreases in ACC1 expression and malonyl-CoA level. Furthermore, suppression of Slc25a10 decreased triglyceride lipid accumulation in adipose-differentiated 3T3-L1 cells. These results suggested that Slc25a10 plays an important role in supplying malate for citrate transport required for fatty acid synthesis and indicated that inhibition of Slc25a10 might effectively reduce lipid accumulation in adipose tissues.