Mutations In Human Cancers Research Articles

O21. Developmental mechanisms in stem cells and cancer

Our work has focused on understand the signaling pathways that regulate the choice between stem cell renewal and commitment, and define how the same signals are subverted in cancer. We have specifically studied developmental signals such as Wnt, Notch and Hedgehog which are critical regulators of normal development in a variety of systems, and a major target of mutation in human cancer. Our research using knockout and transgenic approaches suggests that these signals are activated in hematopoietic stem cells, and that they functionally contribute to stem cell self-renewal in vivo. In addition our data also shows that inhibition of these signals can block leukemia development and propagation in mouse models of the disease as well as in human leukemia cells.

COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer

COSMIC (http://www.sanger.ac.uk/cosmic) curates comprehensive information on somatic mutations in human cancer. Release v48 (July 2010) describes over 136 000 coding mutations in almost 542 000 tumour samples; of the 18 490 genes documented, 4803 (26%) have one or more mutations. Full scientific literature curations are available on 83 major cancer genes and 49 fusion gene pairs (19 new cancer genes and 30 new fusion pairs this year) and this number is continually increasing. Key amongst these is TP53, now available through a collaboration with the IARC p53 database. In addition to data from the Cancer Genome Project (CGP) at the Sanger Institute, UK, and The Cancer Genome Atlas project (TCGA), large systematic screens are also now curated. Major website upgrades now make these data much more mineable, with many new selection filters and graphics. A Biomart is now available allowing more automated data mining and integration with other biological databases. Annotation of genomic features has become a significant focus; COSMIC has begun curating full-genome resequencing experiments, developing new web pages, export formats and graphics styles. With all genomic information recently updated to GRCh37, COSMIC integrates many diverse types of mutation information and is making much closer links with Ensembl and other data resources.

Oncogenic RAF: a brief history of time

Remarkable clinical activity with the RAF inhibitor PLX-4032 in melanomas containing mutant BRAF has recently been described in the New England Journal of Medicine by Flaherty et al. (2010). In a second manuscript published in Nature, the discovery and preclinical development of the drug is chronicled by the scientist at Plexxicon who led this effort, Gideon Bollag (Bollag et al., 2010). The initial preliminary report of the Phase 1 data revealing the activity of this drug was first presented at the Annual Meeting of the American Society of Clinical Oncology Annual Meeting in 2009, 7 years after the discovery of BRAF mutations in human cancer (Davies et al., 2002). In a disease in which the development of therapies against novel targets is often frustrating and slow, this is an amazing achievement, the origins of which can be traced back over 25 years. In 1983, Rapp et al. cloned v-raf as the transformative factor in a replication-defective type C murine virus (3611-MSV) but no mutations were detected in the homologous human RAF genes (ARAF, BRAF and CRAF) until 2002 (Rapp et al. 1983). At that time, Davies et al. (2002) at the Sanger identified BRAF as commonly mutated in human tumors. These mutations were particularly common in melanoma where they are found in upwards of 50% of patients but were also identified in approximately 10% of colorectal tumors and a third of papillary thyroid cancers. BRAF mutations are typically missense substitutions found clustered in two regions – the glycine rich loop in exon 11 and the activation segment within exon 15. The most common mutations disrupt an inactive conformation of the protein and lead to constitutive hyperactivation of the kinase (Wan et al., 2004). Notably, a single mutation within codon 600 (V600E) is found in over 90% of cases. RAF activation occurs in response to activation of RAS proteins by upstream growth factor-induced signals. Activated RAF in turn phosphorylates and activates MEK1 and 2 [mitogen-activated protein ⁄ extracellular signal-regulated kinase kinase (MAPKK)] which in turn phosphorylate and activate the extracellular signal-related kinases 1 and 2 (ERK1 ⁄2). The activation of this signaling cascade (the ‘classical’ MAP kinase pathway) regulates a number of effector kinases and transcription factors that drive key cellular processes including cell proliferation and survival. Phosphorylation of other RAF substrates may also be important in normal physiology and cellular transformation; however, this remains to be firmly established. Tumor cells with BRAF mutations depend on expression of BRAF for proliferation (Wellbrock et al. 2004). Furthermore, melanoma cell lines with the mutation are almost universally sensitive to inhibition of ERK signaling with MEK inhibitors (Solit et al., 2006). These findings suggested that inhibitors of RAF, MEK or ERK could have major clinical activity in patients whose tumors harbored activating BRAF mutations. Unfortunately, sorafenib (Nexavar, Bayer), the first RAF inhibitor to be tested clinically, lacked meaningful clinical activity in melanoma. This result suggested to some that mutant RAF did not play an important role in these tumors. In retrospect, this conclusion was unwarranted. Sorafenib is an unselective inhibitor of many kinases, and it inhibits tumors with mutant and wild-type RAF with equal potency. It is likely that the toxicity of sorafenib precluded administration of a high enough dose to inhibit ERK signaling adequately. In contrast to sorafenib, allosteric MEK inhibitors are exceptionally selective. These drugs potently inhibit ERK signaling in tumors. In patients, the major dose-limiting toxicity of these agents is skin rash. In clinical trials, the MEK inhibitor AZD6244 exhibited activity in melanoma, with a 13% partial response rate in tumors harboring a BRAF mutation. However, given the expectations from trials of oncoprotein-targeted therapy in other diseases, these results were disappointing. They suggested to many that BRAF ⁄ERK signaling was dispensible for maintenance of most melanomas, possibly because of the cellular heterogeneity and genetic complexity of the disease, as well as to postulated inherent resistance of melanoma stem cells. The two reports by Flaherty et al. (2010) and Bollag et al. (2010) dispel these concerns and validate the dependence of these tumors on BRAF. In the study by Flaherty et al., the authors report the Phase 1 results of the RAF inhibitor PLX4032 (also known as RG7204). PLX4032 is a potent nanomolar, ATP-competitive inhibitor of all three RAF isoforms as well as the V600E mutant form of BRAF. In this trial, a 77% response rate

A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells

The global impairment of mature microRNAs (miRNAs) is emerging as a common feature of human tumors. One interesting scenario is that defects in the nuclear export of precursor miRNAs (pre-miRNAs) might occur in transformed cells. Exportin 5 (XPO5) mediates pre-miRNA nuclear export and herein we demonstrate the presence of XPO5-inactivating mutations in a subset of human tumors with microsatellite instability. The XPO5 genetic defect traps pre-miRNAs in the nucleus, reduces miRNA processing, and diminishes miRNA-target inhibition. The XPO5 mutant form lacks a C-terminal region that contributes to the formation of the pre-miRNA/XPO5/Ran-GTP ternary complex and pre-miRNAs accumulate in the nucleus. Most importantly, the restoration of XPO5 functions reverses the impaired export of pre-miRNAs and has tumor-suppressor features.

Frameshift Mutation of MARS Gene Encoding an Aminoacyl-tRNA Synthetase in Gastric and Colorectal Carcinomas with Microsatellite Instability

To the editor, Gene translation is one of the most fundamental biologic processes for cells. Aminoacyl-tRNA synthetases (ARSs) are enzymes that charge tRNAs with their cognate amino acids during the translation.1 Of the ARSs, nine ARSs form macromolecular complexes with ARS-interacting multifunctional proteins (AIMP1, 2 and 3). Many of the complex-forming ARSs and AIMPs play roles not only in gene translation, but also in other biological processes such as apoptosis, angiogenesis and inflammation.1 It has been known that AIMP genes may act as tumor suppressor genes.2 Although non-translational roles of the complex-forming ARSs are known, their roles in cancer biology remain unknown. Microsatellite instability (MSI) is defined by length alterations in repeated DNA sequences, and 10-30% of colorectal cancer (CRC) and gastric cancer (GC) are classified as MSI-positive cancers.3 Many tumor suppressor genes in cancers with MSI harbor frameshift mutations at the repeats in coding sequences.3 By analyzing public database, we found a T9 repeat in exon 3 of methionyl-tRNA synthetase (MARS) gene (nucleotides 204-212) that had not been analyzed for the mutations in cancers. To see whether the T9 is mutated in GC and CRC with MSI, we analyzed them by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP). We analyzed 30 high-MSI (MSI-H) and 15 low-MSI (MSI-L) GC, and 40 MSI-H and nine MSI-L CRC. Malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle by microdissection as described previously.4 Tumor and normal DNA were amplified with by PCR using a primer pair that could amplify the T9 in exon 3 (product size 118 bps). Radioisotope ([32P]dCTP) was incorporated into the PCR for detection by SSCP. After SSCP, mobility shifts compared to wild-type bands were analyzed by visual inspection. Direct DNA sequencing was performed in the cancers with mobility shifts in the SSCP. We repeated the experiments twice to ensure the specificity of the results. PCR-SSCP analysis identified aberrant bands in two of the GC with MSI-H (2/30; 6.7%) and one of the CRC with MSI-H (1/40; 2.5%), but not in those with MSI-L. DNA from normal tissue showed no shifts in SSCP, indicating the mutations had risen somatically. Direct DNA sequencing of the cancers with the aberrant bands led to identification of a recurrent MARS mutation in the T9 repeat sequences (Fig. 1). The mutation was c.212delT, which would result in premature stops of the amino acid synthesis (p.Leu71CysfsX33). Fig. 1 Frameshift mutation of MARS in a gastric carcinoma with MSI. Direct DNA sequencing analysis of MARS exon 3 from a gastric adenocarcinoma with MSI-H shows a heterozygous T deletion in tumor tissue as compare to normal tissue. Human MARS exists as a component of the multi-ARS macromolecular complex, but this protein can dissociate from the complex and move to nucleus.5 After entering the nucleus, MARS plays a role in biogenesis of rRNA in nucleoli.5 Human MARS (about 900 amino acids long) contains N-terminal 267 amino acids and C-terminal 40 amino acids that are involved in protein-protein interactions. The intervening sequences are for its catalytic functions. Also, there are two nuclear localization signals (NLSs) (amino acids 897-900 and 724-730).5 The frameshift mutation of MARS identified in this study would lead to premature stops of amino acid syntheses in MARS protein (p.Leu71CysfsX33) and would abolish most of the domains of MARS (the catalytic domain, the NLSs and parts of the protein interaction domains). The MARS mutation may alter the structure of MARS, and might inactivate the functions of MARS. However, the sequencing data of the mutation (heterozygous mutation) suggest that the second allele may be intact. It is possible that the catalytic function of MARS for the protein translation that is essential for viability of cells may be preserved by the intact second allele. The mutant allele may alter the non-catalytic functions of MARS and might play a role in cancer pathogenesis. However, to date, the background data on non-catalytic functions of MARS is not enough to interpret the role of the mutant MARS in cancers. In this study, we for the first time identified an ARS-encoding gene mutation in human cancers. To our knowledge, there has been no report on functions of MARS protein related to MSI. To address consequences of such down-regulations in cancer development (especially related to cancers with MSI-H), functional studies on the mutated genes and their products should be further performed. Also, to see whether mutation of MARS gene is a common feature of cancers, the gene status should be further analyzed in other cancers.

Caveolin-1 P132L Mutation in Human Cancers: 1 CAVeat to be Voiced

Caveolae are 50–100 nm flask-shaped invaginations of the plasma membrane that function as membrane organizing centers and play important roles in intracellular trafficking of cellular components (eg, lipid and cholesterol) and signal transduction.1 The CAV1 gene maps to 7q31.1 and encodes the ubiquitously expressed caveolin-1, which is an essential component of caveolae. Caveolin-1 is believed to regulate the activity of a plethora of signaling molecules highly represented in caveolae, such as H-ras, c-src, and src-like kinases; endothelial nitric oxide synthase; G-proteins and membrane-associated receptors coupled to G-proteins; or tyrosine kinase receptors including epidermal growth factor receptor.1 In normal breast tissue, caveolin-1 is expressed at high levels in adipocytes, endothelial cells, fibroblasts, and myoepithelial cells of the breast.1 Initial studies have suggested that caveolin-1 would have properties consistent with those of a tumor suppressor protein.2,3,4 A dominant negative mutation of the CAV1 gene was reported to be found in up to 20% of estrogen receptor (ER)-positive breast cancers.2,4 Surprisingly, however, CAV1 knock-out mice failed to develop any tumor.1,3 Furthermore, the presence of CAV1 gene mutations could not be independently validated.5,6,7,8,9 Concurrent with the controversies about the role of CAV1 as a tumor suppressor gene, evidence of caveolin-1 as a potential oncogene has been mounting.7,8,9,10,11,12,13,14,15 In fact, multiple independent groups have reported overexpression of caveolin-1 in aggressive subtypes of cancer,10,11,13,14,15 and even CAV1 gene amplification has been documented,14,16 although this has been shown to be a rare phenomenon. Furthermore, expression of caveolin-1 in stromal cells of breast cancer has also been shown to be associated with the outcome of breast cancer patients.17,18 Despite the interest in the tumor suppressive and oncogenic functions of caveolin-1, numerous issues remain unanswered. For instance, caveolin-1 expression in normal luminal epithelial cells of normal breast lobules and ducts remains a matter of controversy, as does the existence and prevalence of the CAV1 P132L dominant-negative mutation. In this issue of The Journal of Molecular Diagnostics, Koike et al19 have addressed the question of the prevalence of the CAV1 P132L mutation in human cancer. Using three different methods for mutation detection (ie, reverse-transcriptase direct sequencing, DNA-based direct sequencing, and cycleave PCR assay), the authors failed to detect the CAV1 P132L mutation in a large series of 409 human tumors, including 139 breast cancers [114 estrogen receptor (ER)-positive and 25 ER-negative tumors] and 270 cancers from other anatomical sites (ie, gastrointestinal tract, lung, ovary, and pancreas). Taken together, the findings reported by Koike et al19 and those by others5,6,7,8,9 call into question the presence of the CAV1 P132L mutation in breast cancers and other cancer types and heat up the debate about the roles of caveolin-1 in human cancer.

The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer

Fast and reliable tests to detect mutations in human cancers are required to better define clinical samples and orient targeted therapies. KRAS mutations occur in 30-50% of colorectal cancers (CRCs) and represent a marker of clinical resistance to cetuximab therapy. In addition, the BRAF V600E is mutated in about 10% of CRCs, and the development of a specific inhibitor of mutant BRAF kinase has prompted a growing interest in BRAF (V600E) detection. Traditional methods, such as PCR and direct sequencing, do not detect low-level mutations in cancer, resulting in false negative diagnoses. In this study, we designed a protocol to detect mutations of KRAS and BRAF(V600E) in 117 sporadic CRCs based on coamplification at lower denaturation temperature PCR (COLD-PCR) and high-resolution melting (HRM). Using traditional PCR and direct sequencing, we found KRAS mutations in 47 (40%) patients and BRAF(V600E) in 10 (8.5%). The use of COLD-PCR in apparently wild-type samples allowed us to identify 15 newly mutated CRCs (10 for KRAS and 5 for BRAF (V600E)), raising the percentage of mutated CRCs to 48.7% for KRAS and to 12.8% for BRAF (V600E). Therefore, COLD-PCR combined with HRM permits the correct identification of less represented mutations in CRC and better selection of patients eligible for targeted therapies, without requiring expensive and time-consuming procedures.

Recent Progress in Phosphoinositide 3-Kinases: Oncogenic Properties and Prognostic and Therapeutic Implications

Phosphatidylinositol 3-kinases (PI3Ks) are a group of lipid kinases that coordinate various fundamental cellular responses including mitogenic signaling, cell survival and proliferation, vesicular trafficking, cytoskeletal rearrangement and metabolism. Overexpression caused by genetic alterations such as the amplification or gain of copy numbers was frequently observed for various PI3Ks in different human cancers. In recent years, the high-frequency somatic mutations of PIK3CA observed in different human cancers further strengthen the notion that PI3Ks are optimal targets for therapeutic intervention. In this review, I summarize current experimental evidence about the transformation capability and oncogenic properties of different PIK3CA mutations identified in human cancers. I also discuss the association of PIK3CA mutations with other genetic markers, as well as the prognostic value of PIK3CA somatic mutations in human cancers. Finally, I discuss the application of PI3Ks as therapeutic targets in human cancers, including targeted and combinational therapies and the potential challenges that confront their clinical applications.

The Scatter Factor Signaling Pathways as Therapeutic Associated Target in Cancer Treatment

Receptor tyrosine kinases (RTKs) are key regulators of critical cellular processes such as proliferation, differentiation, neo-vascularization, and tissue repair. In addition to their importance in the regulation of normal physiology, aberrant expression of certain RTKs has also been associated to the development and progression of many types of cancer. c-Met and RON are two RTKs with closely related sequences, structural homology, and similar functional properties. Both these receptors, once activated by their respective ligands, the Hepatocyte Growth Factor/Scatter Factor (HGF/SF1) and the Macrophage Stimulating Protein/Scatter Factor 2 (MSP/SF2), can induce cell migration, invasion and proliferation. Soon after its discovery in the mid-1980s, c-Met attracted a great interest because of its role in modulating cell motility. Moreover, the causal role for c-Met activating mutations in human cancer propelled an intensive drug discovery effort throughout academic institutions and pharmaceutical companies. While c-Met is now a well-accepted target for anticancer drug design, less is known about the role of RON in cancer and less has been done to target this receptor. In this review we will discuss the biological relevance of c-Met and RON, their deregulation in human cancers and the progress, so far, in identifying c-Met and RON signaling inhibitors. Finally, we will focus on the development of therapeutic strategies and drug efficacy studies based on interfering the scatter factor signaling pathways.

Genomic and Biological Characterization of Exon 4 KRAS Mutations in Human Cancer

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3.

Early FDG-PET responses to PLX4032 in BRAF-mutant advanced melanoma.

8529 Background: FDG-PET is an effective imaging modality at assessing early response to a variety of agents that target key driver mutations in human cancer. Reduction in FDG uptake reflects inhib...

Abstract 3837: PI-3 kinase acts upstream of oncogenic Ras in hematopoietic cells through a cytokine-PLCγ-RasGRP pathway

Abstract Introduction: KRAS is the most common gain-of-function mutation in human cancer and occur in some myeloid malignancies. Despite extensive data implicating aberrant responsiveness to cytokines in the pathogenesis of myeloid leukemias with hyperactive Ras, the underlying mechanisms are largely unknown. We investigated the biochemical basis of hypersensitivity to granulocyte macrophage colony stimulating factor (GM-CSF), IL-3, and stem cell factor (SCF) in primary cells from Mx1-Cre, KrasG12D mice. Methods: We utilized conventional biochemical techniques, phospho-specific flow cytometry, and small molecule inhibitors to interrogate signaling downstream of the activated GM-CSF and c-Kit receptors. Results: We found that endogenous KrasG12D expression deregulates multiple effectors in primary myeloid lineage cells. Although Ras-GTP levels are markedly elevated in bone marrow cells from KrasG12D mice, these cells only fully activate the Raf/MEK/ERK cascade upon cytokine stimulation. We used a “chemical genetic” strategy to explore the biochemical mechanism linking the activated GM-CSF receptor to oncogenic K-RasG12D and ERK. Surprisingly, inhibitors of type I PI3 kinases (PI-90, PI-103) as well as a p110 γ/α isoform inhibitor (AS605240) interfered with GM-CSF and SCF induced Ras/MAPK activation in wild-type and KrasG12D myeloid cells. These effects of PI3 kinase are mediated through activation of PLC-γ and the RasGRP guanine nucleotide exchange factors. Functional studies confirmed that inhibiting PI3 kinase or PLC-γ blocked KrasG12D-mediated myeloid progenitor colony growth. This PI3 kinase/PLC-γ module also functions downstream of the activated c-kit receptor in lineage- cKit+ Sca1+ (LSK) cells in SCF-induced ERK activation. Conclusions: PI3 kinase, PLC-γ, and RasGRPs function upstream of normal and oncogenic K-Ras to efficiently activate the Raf/MEK/ERK cascade in primary hematopoietic cells. These data infer that PI3 kinase is both an important upstream regulator as well as a major downstream effector of Ras-GTP. Significance: Targeting PI3 kinase in hematologic cancers characterized by hyperactive Ras may have unanticipated biochemical and functional consequences in malignant and normal hematopoietic cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3837.

Abstract 93: COSMIC: The catalogue of somatic mutations in cancer receives full genome variant annotations

Abstract COSMIC, the Catalogue Of Somatic Mutations In Cancer (http://www.sanger.ac.uk/cosmic), is one of the most comprehensive web systems describing the impact of somatic mutations in human cancer. The information is sourced directly from the published scientific literature and the Cancer Genome Project's high throughput sequencing laboratories at the Wellcome Trust Sanger Institute UK (CGP), allowing a high data quality to be maintained. The v44 release (Nov. 2009) contained the curation of 8336 papers covering 71 known cancer genes by point mutation and 30 by gene fusion. The mutation data for all these genes is being maintained up-to-date and new genes are regularly added to this list. Additionally, 4871 genes and 2764 samples have been scrutinized by the CGP laboratories. As cancer genetics moves toward the analysis of whole genomes, COSMIC is being updated to handle and display these data and increase the functionality of the website. With the rapid development of sequencing technologies, the quantity of data that can be put into COSMIC is increasing fast. We have extended our curation process to include large exome-wide candidate gene screens, recently including the first such screen from Sjoblom et al (2006), and the first TCGA screen (2008) with other large scale screening datasets being curated. In addition, whole-genome screens are becoming available with many non-coding variations. From the CGP laboratories, we already have two low-coverage genome rearrangement screens in COSMIC and this will rapidly expand, beginning with the imminent release of rearrangement scans in a set of 24 breast tumours. High-coverage genome analyses are also beginning to become available; with full genome coverage, the majority of somatic mutations in a tumour can be identified and COSMIC is being prepared to receive and display this data. With the inclusion of these new data, the COSMIC website has evolved to allow more easy access to required data. New specialization filters are in place to provide methods to search for subsets of COSMIC data in the usual user-friendly web pages. More extensive investigations can be performed using the new COSMIC Biomart, an industry-standard data mining system allowing access through web pages or programmatic interfaces. As the inclusion of new genomic data accelerates and the systems through which it can be accessed evolve, COSMIC is well placed to remain a central resource in human cancer genetics. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 93.

Abstract 1651: The use of ‘X-MAN’ isogenic cell lines to define PI3-kinase inhibitor activity profiles

Abstract The tumour microenvironment promotes selective outgrowth of cells carrying mutations in oncogenes or tumour suppressors, enabling the activated pathways to drive tumour growth. Two such pathways are the PI3-K/AKT and RAS/RAF/MEK signaling pathways, which demonstrate a high frequency of mutations in human cancer. These pathways represent important therapeutic targets, and a number of targeted agents are now in clinical trials. Despite being pertinent to the clinical situation, the role that the tumour microenvironment plays in modifying responsiveness to therapeutic agents targeted against these pathways is poorly understood. A major challenge faced during the development of PI3-K/AKT and RAS/RAF/MEK targeted agents is to demonstrate inhibitor selectivity and efficacy in a patient-relevant cell based system. Isogenic ‘X-MAN’ paired cell lines help address this by providing a system in which the endogenous wild-type or mutant alleles of the parental cell line are selectively activated or silenced via targeted homologous integration, providing a pair of human cell-lines in which the only difference between the cells is the mutation of interest. This approach therefore overcomes the issues that may occur when over-expression methods are employed. We therefore set out to investigate how changes in the tumour microenvironment altered cell responses to PI3-K and MEK inhibitors. We employed isogenic HCT116 human colon cancer cells where the parental cells harbor pre-existing H1047R PIK3CA and G13D KRAS alleles and compared these to isogenic clones that retain either the PIK3CA or KRAS wild-type alleles. Inhibitors were profiled under a range of defined conditions chosen to model the tumour microenvironment, including low glucose, low serum and hypoxia, and the activity of the compounds investigated. Under standard culture conditions, we found that PI3-K and MEK inhibitors showed minimal selectivity for cells with activating mutations compared to wild-type cells. When grown under defined culture conditions which more closely mimic the tumour microenvironment, the effects of the driving mutations became dominant and a differential between wild-type and mutant cell responses to inhibitors was observed. The selective PI3-K inhibitors GDC-0941 and PI-103 showed a >10-fold increase in potency when tested in anti-proliferative assays in mutant vs, wild-type cell lines. However, the dual PI3-K/mTOR inhibitor BEZ-235 did not show selectivity in this system. We show that by using isogenic cell lines under conditions which model the tumour microenvironment, differences in activity profiles between specific inhibitors can be quickly and definitively identified, highlighting the advantages of using this system in profiling molecularly targeted agents. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1651.

Mutational analysis of mononucleotide repeats in dual specificity tyrosine phosphatase genes in gastric and colon carcinomas with microsatellite instability

Coordinated protein phosphorylation and dephosphorylation are crucial in the regulation of cell signaling, and disruption of the coordination is known to play important roles in cancer development. Recent reports revealed that classical protein tyrosine phosphatase (PTP)-encoded genes are somatically mutated in human colorectal cancer. However, data on dual specificity phosphatase (DPTP) gene mutations in human cancers are lacking. By analyzing a public genomic database, we found that five DPTP genes, CDC14A, MTM1, MTMR3, SSH1, and SSH2, have mononucleotide repeats in their coding DNA sequences. To see whether these genes are mutated in cancers with microsatellite instability (MSI), we analyzed the mononucleotide repeats in 26 gastric cancers (GC) with MSI (MSI-H), 12 GC with low MSI (MSI-L), 45 GC with stable MSI (MSS), 33 colorectal cancers (CRC) with MSI-H, 14 CRC with MSI-L, and 45 CRC with MSS by single-strand conformation polymorphism (SSCP). We found CDC14A and MTMR3 mutations in five and one cancer (s), respectively. These mutations were detected in MSI-H cancers, but not in MSI-L or MSS cancers. The GC and CRC with MSI-H harbored the mutations in 15% and 6%, respectively. The CDC14A and MTMR3 mutations detected in the GC and CRC were deletion or duplication mutations of one base in the nucleotide repeats that would result in premature stops of the amino acid syntheses. Our data show that frameshift mutations of DPTP genes in MSI-H cancers occur at moderate frequencies. The data suggested that alterations in the CDC14A and MTMR3 genes may play a role in the development of GC and CRC with MSI-H by deregulating phosphatase functions possibly together with mutations of classical PTP genes.

Somatic mutations in human cancer—moving towards large scale resequencing of whole cancer genomes

Somatic mutations in human cancer—moving towards large scale resequencing of whole cancer genomes

Activating ROCK1 somatic mutations in human cancer

Cancer cells acquire characteristics of deregulated growth, survival and increased metastatic potential. Genetic mutations that provide a selective advantage by promoting these characteristics have been termed 'drivers,' whereas mutations that do not contribute to disease initiation/progression are termed 'passengers.' The advent of high-throughput methodologies has facilitated large-scale screening of cancer genomes and the subsequent identification of novel somatic mutations. Although this approach has generated valuable results, the data remain incomplete until the functional consequences of these mutations are determined to differentiate potential drivers from passengers. ROCK1 is an essential effector kinase downstream of Rho GTPases, an important pathway involved in cell migration. The Cancer Genome Project identified three nonsynonymous mutations in the ROCK1 gene. We now show that these somatic ROCK1 mutations lead to elevated kinase activity and drive actin cytoskeleton rearrangements that promote increased motility and decreased adhesion, characteristics of cancer progression. Mapping of the kinase-interacting regions of the carboxy terminus combined with structural modeling provides an insight into how these mutations likely affect the regulation of ROCK1. Consistent with the frequency of ROCK1 mutations in human cancer, these results support the conclusion that there is selective pressure for the ROCK1 gene to acquire 'driver' mutations that result in kinase activation.

In vivo analysis of p53 tumor suppressor function using genetically engineered mouse models

p53 is a crucial tumor suppressor, as evidenced by the high propensity for p53 mutation during human cancer development. Already more than a decade ago, p53 knockout mice confirmed that p53 is critical for preventing tumorigenesis. More recently, a host of p53 knock-in mouse strains has been generated, with the aim of either more precisely modeling p53 mutations in human cancer or better understanding p53's regulation and downstream activities. In the first category, several mouse strains expressing mutant p53 proteins corresponding to human-tumor-derived mutants have demonstrated that mutant p53 is not equivalent to loss of p53 but additionally exhibits gain-of-function properties, promoting invasive and metastatic phenotypes. The second class of p53 knock-in mouse models expressing engineered p53 mutants has also provided new insight into p53 function. For example, mice expressing p53 mutants lacking specific posttranslational modification sites have revealed that these modifications serve to modulate p53 responses in vivo in a cell-type- and stress-specific manner rather than being absolutely required for p53 stabilization and activation as suggested by in vitro experiments. Additionally, studies of p53 mouse models have established that both p53-driven cell-cycle arrest and apoptosis responses contribute to tumor suppression and that activation of p53 by oncogenic stress imposes an important barrier to tumorigenesis. Finally, the use of mouse strains expressing temporally regulatable p53 has demonstrated that p53 loss is not only required for tumor development but also required for tumor maintenance, suggesting that p53 restoration in human cancer patients may be a promising therapeutic strategy. These sophisticated p53 mouse models have taught us important lessons, and new mouse models will certainly continue to reveal interesting and perhaps surprising aspects of p53's complex biology.

Abstract C63: Biological characterization of GSK2126458, a novel and potent inhibitor of phosphoinositide 3-kinase and the mammalian target of rapamycin (mTOR)

Abstract The phosphoinositide 3-kinase (PI3K) pathway is among the most commonly activated pathways in human cancer. The biological role of PI3K in growth and survival of cancer cells and the prevalence of activating mutations in human cancers are well documented and a significant proportion of tumors would be predicted to benefit from inhibition of this pathway. Here we report on the characterization of the pan-PI3K pyridylsulfonamide inhibitor GSK2126458: a very potent (PI3K app Ki = 19 pM), reversible, ATP-competitive inhibitor of wild-type PI3K and the ‘hotspot’ activating mutants of p110 found in human cancer. GSK2126458 demonstrated good selectivity for the PI3K family of enzymes including the mTORC1 and mTORC2 complexes, when evaluated in a large panel of protein kinases. Consistent with potent PI3K enzyme inhibition, GSK2126458 decreased the cellular levels of phosphorylated AKT, p70S6K, PRAS40 and ERK in a concentration and time dependent manner, the IC50 for pAKT in the HCC1954 breast carcinoma cell line was 2 nM. GSK2126458 induced the nuclear translocation of the FOXO3a transcription factor in a concentration dependent manner that mechanistically appeared bimodal. Growth inhibition was time and concentration dependent with a 3 day exposure resulting in a growth IC50 (gIC50) of 9 nM in HCC1954 cells with evidence of cell death. Cell death correlated with induction of caspase 3 and 7 activity suggesting apoptosis was the mechanism of cell death. GSK2126458 had a breadth of activity for potent cell growth inhibition and induction of cell death in a variety of human cancer cells. GSK2126458 demonstrated robust, dose dependent in vivo pharmacodynamic activity as measured by inhibition of phospho-AKT in advanced BT474 breast cancer cell xenograft tumors. Inhibition of AKT phosphorylation was rapid and lasted for several hours after a single oral administration of GSK2126458. Transient increases in plasma insulin and blood glucose were observed. Evaluation of in vivo tumor growth effects of GSK2126458 in xenograft models demonstrated dose dependent tumor growth delay in several models of diverse tumor lineage and tumor regression in a breast cancer xenograft. The biological profile of GSK2126458 supports the clinical advancement of this compound and GSK2126458 has entered Phase I human clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C63.

Abstract A79: Genome-wide RNAi screen for tetraploid-specific lethality in cancer cells

Abstract Cancer cells are often genetically unstable, resulting in aneuploid genomes. The consequences of aneuploidy are poorly understood, but have been suggested to play roles in tumor aggressiveness, chemotherapy resistance, and metastasis. One important route to an aneuploid genome may be through doubling of the genome, leading to unstable tetraploid cells. Many cell division errors are ultimately manifest as a failure of cytokinesis. Especially in cells defective in p53 function, tetraploid cells proliferate, but generate both high rates of whole chromosome aneuploidy and defects in the maintenance of chromosome structural integrity. Our laboratory has demonstrated that, in p53-primary cells, tetraploidy promotes tumor development and the resulting tumors display markedly abnormal genomes (Fujiwara T. et al. Nature 2005, 437: 1043). It is now known that several human cancer-mutations — e.g. Rb loss or loss of the adenomatous polyposis coli tumor suppressor — also predispose cytokinesis failure and tetraploidy. These findings highlight a need to understand in detail the physiological changes that accompany tetraploidy. To gain a comprehensive understanding of physiological alterations associated with tetraploidy in cancer cells, we have established tetraploid cell lines derived from a near diploid human colorectal cancer cell line HCT116. Using these paired tetraploid and diploid cell lines, we have performed a genome-wide RNAi screen to identify genes whose knock down selectively affect the viability of either diploids or tetraploid cells. We are currently validating the result of our ploidy-specific lethality screen and we would like to update our progress at this conference. Citation Information: Cancer Res 2009;69(23 Suppl):A79.