Oncogenic Ras Mutations Research Articles

Abstract IA12: Preclinical models for targeting oncogenic Ras signaling in cancer

Abstract Somatic RAS mutations occur in 30% of human cancers, and are common in both melanoma and hematopoietic cancers. Importantly, whereas KRAS mutations predominate in epithelial cancers, NRAS is mutated much more often in melanoma and hematopoietic cancers. The most obvious therapeutic strategy for cancers with RAS mutations – developing specific inhibitors of the mutant protein – is extremely challenging due to the very high affinity of Ras for GTP, structural considerations of the Ras/GTPase activating protein (Ras/GAP) molecular switch, and the loss of normal enzymatic activity as a consequence of oncogenic RAS mutations. Given these issues, most recent drug discovery efforts have focused on inhibiting downstream biochemical targets of mutant Ras such as Raf, MEK, and Akt with many compounds undergoing clinical evaluation. We have exploited strains of mice that recapitulate endogenous expression of cancer-associated RAS alleles or NF1 inactivation to model human hematologic cancers and to conduct biologic and preclinical studies. In particular, we have deployed the MOL4070LTR retrovirus as an insertional mutagen to generate genetically heterogeneous transplantable myeloid and lymphoid leukemias characterized by hyperactive Ras signaling. We utilize cohorts of mice that are engrafted with the same primary leukemias for preclinical testing. In this presentation, I will focus on three biologic and therapeutic questions related to NRAS in tumorigenesis: (1) loss of the normal NRAS allele in cancers with oncogenic NRAS mutations; (2) responses of primary Nras mutant leukemias to MEK and PI3 kinase inhibitors; and, (3) potential strategies for selectively targeting oncogenic N-Ras in cancer. Note: This abstract was presented as part of the Joint Session: Clinical Science Interactions between Melanoma and Hematologic Malignancies with the AACR Special Conference: Hematologic Malignancies: Translating Discoveries to Novel Therapies held September 20-23, 2014 in Philadelphia, PA. Citation Format: Kevin Shannon. Preclinical models for targeting oncogenic Ras signaling in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA12.

Abstract B16: Contributions of Ras and EZH2 in acinar to ductal metaplasia and ductal carcinoma in transgenic mouse pancreas

Abstract Activating mutations of the ras protooncogene is the most frequent and an early genetic alteration associated with pancreatic cancer. To examine the link between mutant ras oncogenes and exocrine pancreatic cancer, and to develop mouse models of PanINs and also of lesions that progress to malignancy, we generated transgenic mice expressing either Kras or Hras in pancreas. The tetracycline transactivator system expression was targeted to the pancreatic acinar cells via the elastase promoter element. The transactivation target ras genes were placed under the control of the tetracycline-response element. Most double transgenic founder mice displayed perinatal pancreatic acinar cell hyperplasia and dysplasia. However, only adult mice expressing Hras displayed preinvasive pancreatic neoplastic lesions with ductal morphology. Therefore, these Kras and Hras models each represent useful models in which either the lesion histotype and agressiveness (Hras) or the initiating genetic alteration overlap with the human disease (Kras). More recently, we have developed and are characterizing the contributions of expressing enhancer of zeste 2 homolog (EZH2), a histone methyl transferase and key regulator of cell differentiation and tissue regeneration, in modifying aggressiveness of mutated ras genes in vivo. Our findings suggest that ras mutations are associated with development of early stage duct-like lesions in pancreas, which vary in severity depending on the mutated ras gene expressed and the stage of development and growth of pancreas tissue. Citation Format: Jayne Ellis, Eric P. Sandgren, Paul J. Grippo, Marxa L. Figueiredo. Contributions of Ras and EZH2 in acinar to ductal metaplasia and ductal carcinoma in transgenic mouse pancreas. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B16.

Oncogenic RAS Mutants Confer Resistance of RMS13 Rhabdomyosarcoma Cells to Oxidative Stress-Induced Ferroptotic Cell Death.

Recent genomic studies revealed a high rate of recurrent mutations in the RAS pathway in primary rhabdomyosarcoma (RMS) samples. In the present study, we therefore investigated how oncogenic RAS mutants impinge on the regulation of cell death of RMS13 cells. Here, we report that ectopic expression of NRAS12V, KRAS12V, or HRAS12V protects RMS13 cells from oxidative stress-induced cell death. RMS13 cells engineered to express NRAS12V, KRAS12V, or HRAS12V were significantly less susceptible to loss of cell viability upon treatment with several oxidative stress inducers including the thioredoxin reductase inhibitor Auranofin, the glutathione (GSH) peroxidase 4 inhibitor RSL3 or Erastin, an inhibitor of the cysteine/glutamate amino acid transporter system that blocks GSH synthesis. Notably, addition of Ferrostatin-1 confers protection against Erastin- or RSL3-induced cytotoxicity, indicating that these compounds trigger ferroptosis, an iron-dependent form of programed cell death. Furthermore, RMS13 cells overexpressing oncogenic RAS mutants are significantly protected against the dual PI3K/mTOR inhibitor PI103, whereas they are similarly sensitive to DNA-damaging drugs such as Doxorubicin or Etoposide. This suggests that oncogenic RAS selectively modulates cell death pathways triggered by cytotoxic stimuli in RMS13 cells. In conclusion, our discovery of an increased resistance to oxidative stress imposed by oncogenic RAS mutants in RMS13 cells has important implications for the development of targeted therapies for RMS.

Loss of Dnmt3a and endogenous Kras(G12D/+) cooperate to regulate hematopoietic stem and progenitor cell functions in leukemogenesis.

Oncogenic NRAS and KRAS mutations are prevalent in human juvenile and chronic myelomonocytic leukemia (JMML/CMML). However, additional genetic mutations cooperating with oncogenic RAS in JMML/ CMML progression and/or their transformation to acute myeloid leukemia (AML) remain largely unknown. Here we tested the potential genetic interaction of DNMT3A mutations and oncogenic RAS mutations in leukemogenesis. We found that Dnmt3a(-/-) induces multiple hematopoietic phenotypes after a prolonged latency, including T-cell expansion in the peripheral blood, stress erythropoiesis in the spleen and myeloid malignancies in the liver. Dnmt3a(-/-) significantly promoted JMML/CMML progression and shortened the survival of Kras(G12D/+) mice in a cell-autonomous manner. Similarly, downregulating Dnmt3a also promoted myeloid malignancies in Nras(G12D/+) mice. Further studies show that Dnmt3a deficiency rescues Kras(G12D/+)-mediated depletion of hematopoietic stem cells and increases self-renewal of Kras(G12D/+) myeloid progenitors (MPs). Moreover, ~33% of animals developed an AML-like disease, which is driven by Kras(G12D/+); Dnmt3a(-/-) MPs. Consistent with our result, COSMIC database mining demonstrates that the combination of oncogenic RAS and DNMT3A mutations exclusively occurred in patients with JMML, CMML or AML. Our results suggest that DNMT3A mutations and oncogenic RAS cooperate to regulate hematopoietic stem and progenitor cells and promote myeloid malignancies.

Pharmacogenomic Characterization of Cytotoxic Compounds from Salvia officinalis in Cancer Cells.

Salvia officinalis is used as a dietary supplement with diverse medicinal activity (e.g. antidiabetic and antiatherosclerotic effects). The plant also exerts profound cytotoxicity toward cancer cells. Here, we investigated possible modes of action to explain its activity toward drug-resistant tumor cells. Log10IC50 values of two constituents of S. officinalis (ursolic acid, pomolic acid) were correlated to the expression of ATP-binding cassette (ABC) transporters (P-glycoprotein/ABCB1/MDR1, MRP1/ABCC1, BCRP/ABCG2) and epidermal growth factor receptor (EGFR) or mutations in RAS oncogenes and the tumor suppressor gene TP53 of the NCI panel of cell lines. Gene expression profiles predicting sensitivity and resistance of tumor cells to these compounds were determined by microarray-based mRNA expressions, COMPARE, and hierarchical cluster analyses. Furthermore, the binding of both plant acids to key molecules of the NF-κB pathway (NF-κB, I-κB, NEMO) was analyzed by molecular docking. Neither expression nor mutation of ABC transporters, oncogenes, or tumor suppressor genes correlated with log10IC50 values for ursolic acid or pomolic acid. In microarray analyses, many genes involved in signal transduction processes correlated with cellular responsiveness to these compounds. Molecular docking indicated that the two plant acids strongly bound to target proteins of the NF-κB pathway with even lower free binding energies than the known NF-κB inhibitor MG-132. They interacted more strongly with DNA-bound NF-κB than free NF-κB, pointing to inhibition of DNA binding by these compounds. In conclusion, the lack of cross-resistance to classical drug resistance mechanisms (ABC-transporters, oncogenes, tumor suppressors) may indicate a promising role of the both plant acids for cancer chemotherapy. Genes involved in signal transduction may contribute to the sensitivity or resistance of tumor cells to ursolic and pomolic acids. Ursolic and pomolic acid may target different steps of the NF-κB pathway to inhibit NF-κB-mediated functions.

Phosphorylation of ERK5 on Thr732 Is Associated with ERK5 Nuclear Localization and ERK5-Dependent Transcription

Extracellular signal-regulated kinases (ERKs) play critical roles in numerous cellular processes, including proliferation and differentiation. ERK5 contains a kinase domain at the N-terminal, and the unique extended C-terminal includes multiple autophosphorylation sites that enhance ERK5-dependent transcription. However, the impact of phosphorylation at the various sites remain unclear. In this study, we examined the role of phosphorylation at the ERK5 C-terminal. We found that a constitutively active MEK5 mutant phosphorylated ERK5 at the TEY motif, resulting in the sequential autophosphorylation of multiple C-terminal residues, including Thr732 and Ser769/773/775. However, when ERK1/2 was selectively activated by an oncogenic RAS mutant, ERK5 phosphorylation at Thr732 was induced without affecting the phosphorylation status at TEY or Ser769/773/775. The Thr732 phosphorylation was U0126-sensitive and was observed in a kinase-dead mutant of ERK5 as well, suggesting that ERK1/2 can phosphorylate ERK5 at Thr732. This phosphorylation was also promoted by epidermal growth factor and nerve growth factor in HEK293 and PC12 cells, respectively. The ERK5–T732A mutant was localized in the cytosol under basal conditions. In contrast, ERK5 phosphorylated at Thr732 via the RAS-ERK1/2 pathway and ERK5–T732E, which mimics the phosphorylated form, were localized in both the nucleus and cytosol. Finally, ER–32A and U0126 blocked ERK5-dependent MEF2C transcriptional activity. Based on these findings, we propose a novel cross-talk mechanism in which ERK1/2, following activation by growth factor stimulation, phosphorylates ERK5 at Thr732. This phosphorylation event is responsible for ERK5 nuclear localization and ERK5-dependent transcription.

Photo-Regulation of Small G Protein Normal and Oncogenic K-Ras using Photochromic Molecules

Ras is one of the small G-proteins known as a molecular switch mediating cellular signalling. Switching ON state of Ras is induced by exchange of bound GDP for GTP and OFF state is by hydrolysis of GTP to GDP. Interestingly, the core nucleotide-binding motif of Ras is considerably conserved with the ATP driven motor proteins, myosin and kinesin. Therefore, it is believed that these nucleotide requiring proteins share common molecular mechanism utilizing nucleotide hydrolysis cycle. Previously, we have incorporated photochromic molecules, 4-phenylazophenyl maleimide (PAM), into the functional site of kinesin as a photo-switching nano device and succeeded to regulate kinesin ATPase activities reversibly upon visible light (VIS) and ultra-violet (UV) light irradiation. Therefore, it is expected that Ras can be also regulated using photochromic molecules.In this study, we performed basic study to control the function of Ras using photochromic molecules upon VIS and UV light irradiations. We prepared normal and oncogenic Ras mutants which have a single cysteine at functional sites and modified with photochromic molecules of azobenzene and spiropyran derivatives stoichiometrically. The GTPase activities of PAM-Ras were reversibly altered upon VIS and UV light irradiations. In order to monitor the effect on GTPase kinetic pathway by the photoisomerization of PAM, we synthesized fluorescent GTP analogue, NBD-GTP. The Kinetic studies suggested that the initial binding step of NBD-GTP to Ras and the dissociation step of NBD-GDP from Ras were regulated by the photoisomerization of PAM.

Thyroid nodules in children - rules of management

In children, thyroid lesions both palpable (nodules) and non-palpable, but visible during ultrasound (incidentaloma), require a full diagnostic procedure in accordance with the nodular goiter management algorithm. It is especially important because in children the risk of neoplastic process in thyroid nodule is 4-5 times higher than in adult patients, and reaches between 9 and 50% (on average 25%). The pathogenesis of a goiter is not entirely known. Among the causative factors for thyroid nodules or focal lesions the literature mentions: environmental factors i.e. iodine excess or insufficiency, exposure to radiation, natural goitrogens, chemical substances (lithium carbonate), pollution (phenols, phthalates, pyridines and aromatic carbohydrates), hormonal factors (TSH, IGF-I, EGF, FGF), neuropeptides, cytokines (TGF- β), immunoglobulins (TGI) and genetic factors – TSH receptor mutation, RAS oncogene mutation (mainly responsible for follicular adenoma), mutations and polymorphisms of several genes including thyroid peroxidase gene, thymoglobulin gene, sodium-iodide symporter gene, BRAF gene, as well as mutation of genes encoding microRNA. The DICER 1 mutation and germinal mutation of KEAP 1 have recently been discovered. It is also worth mentioning that during adolescence, when the requirement for thyroid hormones is increased, the thyroid gland is particularly susceptible to enlargement. Hyperglobulinemia resulting from augmented estrogen secretion in girls is another factor. It causes an increase in the pool of globulin-bound hormones and therefore relative free thyroxin (FT4) deficiency. That, in turn, leads to increased TSH levels, which is the most important factor stimulating thyroid growth. Thyroid nodules are present in 0.2-5% of children, more frequently in girls. Morbidity increases with age. The rate of malignancy in thyroid nodules is greater in children before the age of 10 and in boys. Similarly, as in adults, papillary carcinoma is the most common thyroid malignancy. Thyroid nodules in children demonstrate particular characteristics. Most neoplastic and non-neoplastic lesions tend to form singular nodules. Children present clinical signs such as dyspnea, dysphagia or hoarseness more rarely than adults. Patients are usually asymptomatic. Thyroid nodules in children comprise neoplastic and non-neoplastic masses, among the latter the nodular type of Hashimoto disease is the most frequent. However it is important to remember about the possibility of thyroid congenital disorders. The main goal of thyroid nodule diagnostic process is to detect patients with malignancy risk factors. Primarily, the medical history should be obtained to gain information about the occurrence of thyroid diseases, including cancers, in patient’s family. Especially, the family history in first-degree relatives should be carefully determined for MEN (multiple endocrine neoplasm) type 1 and 2, familiar medullary thyroid cancer and familial non-medullary thyroid cancer. Medical history should also include information about exposition to therapeutic or environmental radiation, about symptoms which may indicate hyper- or hypothyroidism, the history of nodule growth (rapid growth), neck pain or any symptoms of compression. Palpation allows estimation of the size and consistency of the thyroid gland, nodules and local lymph nodes. Among complementary diagnostic methods, the main role is reserved for ultrasound-guided fine needle aspiration (FNA). Moreover, thyroid function tests should always be performed, including measuring concentrations of TSH, thyroid hormones, and anti-thyroid autoantibodies. Thyroid scintigraphy is recommended in some cases, such as preparation for radioiodine therapy of hyperthyroidism, examination of ectopic thyroid gland or diagnosis of congenital hypothyroidism. It is important to evaluate calcitonin level in the following cases: clinical suspicion of medullary cancer or confirmed RET mutation, non-diagnostic FNA with no further plans for surgery, FNA indicating follicular neoplasm, coexistence of thyroid nodules and adrenal gland lesions, and – finally – before each thyroid surgery, as the knowledge of calcitonin level enhances pre- and intraoperative diagnosis of medullary cancer and helps to determine the extent of the surgery. In benign thyroid nodules that do not enlarge, it is acceptable to follow a “watch and wait” approach. Pharmacotherapy with levothyroxine (L-T4) is recommended in small, benign nodules presenting in children inhabiting iodide-deficient regions. The aim is to suppress TSH secretion. The dose of L-T4 is regulated based on TSH level, which should remain in lower limit of normal range. If the environmental iodide supply is adequate, this kind of therapy ought to be considered when anti-thyroid autoantibodies are detected. Nevertheless, thyroidectomy (subtotal or – less frequently – lobectomy) remains the most efficient therapeutic measure in thyroid nodules in children.

The Endocannabinoid/Endovanilloid N-Arachidonoyl Dopamine Suppresses the Plasma Membrane Translocation and Neoplastic Transforming Activity of NRAS and KRAS4A

The RAS family includes three RAS genes, which encode four highly homologous proteins: H-, N-, and KRAS4A and 4B, the latter two being alternatively spliced forms differing only at the carboxyl terminus. RAS oncogenic mutations, mostly KRAS and NRAS, are common in human cancers. Since the enzymatic activity of RAS is used to turn itself off and its GTP binding affinity is very high, RAS proteins have been difficult to target. Identification of alternative means to block the RAS oncogenic signaling is critical for developing therapies against RAS-driven cancer. We took a chemical biology approach to discover novel therapeutic strategies for RAS-related cancer. Since the biological activity of RAS proteins relies upon lipid modifications and RAS regulates lipid metabolisms in cancer cells, we used a RAS specific cell viability assay to screen a bioactive lipid library for potential therapeutics against RAS-driven cancer. We found that endocannabinoid/endovanilloid N-arachidonoyl dopamine (NADA) could effectively kill RAS-transformed cells. The structure-activity analysis showed that the anti-RAS activity of NADA relied on both the dopamine and arachidonoyl moieties in the same molecule and was independent of its ability to engage cannabinoid or vanilloid receptors. Further analysis showed that NADA was more active in killing cells transformed by oncogenic NRAS or KRAS4A than that of KRAS4B. Consistently, NADA was more active in inhibiting the phosphorylation of ERK in NRAS-transformed cells than that in KRAS4B-transformed cells. Interestingly, NADA blocked the plasma membrane translocation of NRAS and KRAS4A, but not that of KRAS4B. Significant effect of NADA on palmitoylated NRAS levels was not detected, suggesting that NADA inhibits NRAS and KRAS4A plasma membrane translocation through a novel mechanism. These results demonstrate that NADA possesses a novel property of inhibiting the plasma membrane translocation and neoplastic transforming activity of NRAS and KRAS4A. Since NRAS mutations are common in hematological malignancies as well as in solid tumors such as melanoma and colorectal carcinoma, and KRAS4A is required for carcinogen-induced lung cancer, the new findings would help to develop novel therapies for a broad range of human cancers. DisclosuresNo relevant conflicts of interest to declare.

Biochemical and Functional Analysis of Novel KRAS Insertions in MPN and Other Cancers

The somatic KRAS mutations found in ~25% of human cancers commonly encode amino acid substitutions at codons 12, 13, and 61. Each of these mutations lead to elevated levels of active Ras-GTP by reducing intrinsic Ras GTPase activity and conferring resistance to GTPase activating proteins (GAPs). Amino acids 12, 13, and 61 play key roles in binding the γ phosphate of GTP and facilitating GAP-mediated catalysis. The germline KRAS point mutations found in Noonan syndrome encode different amino acid substitutions that have less severe biochemical consequences. Together, these observations and the structural conservation of the Ras/GAP switch through evolution suggest that a limited spectrum of mutations can constitutively activate Ras and cause disease. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm characterized by driver Ras pathway mutations in 85% of cases, including common somatic KRAS and NRAS substitutions. We unexpectedly identified a novel somatic KRAS insertion, 66GQEEYSA67, in the bone marrow of a patient who meets most, but not all diagnostic criteria, for JMML and lacks any other Ras pathway mutation. This seven amino acid insertion is located in the conserved Switch II domain of Ras, which binds to and activates PI3 kinase and other Ras effectors. We characterized the functional and biochemical consequences of this mutation, and of a similar five amino acid insertion identified in two lung cancers and one colon cancer archived in the COSMIC database (66EEYSA67). We find that unlike WT K-Ras, the expression of both insertion mutants supports cytokine independent growth of IL-3 dependent pro-B Ba/F3 cells similar to oncogenic K-RasG12D. Ba/F3 cells expressing K-Ras66GQEEYSA67 or K-Ras66EEYSA67 mutant proteins have markedly elevated levels of Ras-GTP that are insensitive to IL-3 stimulation, and result in constitutive ERK and Akt phosphorylation. Confirming that these Switch II insertion mutants have oncogenic potential in the hematopoietic system, fetal liver cells transduced with KRas66GQEEYSA67, KRas66EEYSA67, or KRasG12D formed cytokine independent colony forming unit granulocyte macrophage (CFU-GM) colonies and also exhibited GM-CSF hypersensitivity, which is a cellular hallmark of JMML. Biochemical characterization of recombinant K-Ras66GQEEYSA67 and K-Ras66EEYSA67 proteins showed them to have reduced intrinsic GTP hydrolysis, and to accumulate in the GTP-bound conformation when compared to oncogenic K-RasG12D. Furthermore, whereas recombinant p120 GAP markedly accelerated the rate of GTP hydrolysis by WT K-Ras, K-Ras66GQEEYSA67 and K-Ras66EEYSA67 were completely resistant. The most likely structural explanation is that these insertions perturb critical contacts between glutamine 61 in the Switch II domain of Ras and GAPs. We conclude that KRAS66GQEEYSA67 and KRAS66EEYSA67 are oncogenic driver mutations in JMML and other cancers. These data illuminate a new class of oncogenic RAS mutations and are of immediate diagnostic relevance. Furthermore, these data reveal unanticipated structural flexibility in a domain of Ras that plays a critical role in effector binding, which has implications for potential mechanism of resistance to emerging therapeutic approaches. DisclosuresBollag:Plexxikon: Employment. Zhang:Plexxikon: Employment.

Abstract A03: Modeling the therapeutic benefit of Ras-family inhibition in vivo

Abstract Cancers depend upon co-opting the core machinery that allows a normal cell to convert extrinsic signals into a cellular response such as proliferation, arrest, or apoptosis. Inhibition of the protein families at the core of this machinery may cause the regression of tumors; however, this inhibition could disrupt normal tissues that depend upon continuous proliferation. One such protein family that holds a key place in this regulatory machinery is the Ras family of small GTPases, comprised of N-Ras, K-Ras and H-Ras. The Ras proteins integrate cell surface signals from Receptor Tyrosine Kinases (RTKs) and transduce their consolidated signal via a cascade of downstream intracellular kinases that regulate transcription factors, cell architecture, cell cycle and metabolism. In order to model the potential therapeutic index of Ras-family inhibition in vivo, we have generated a novel transgenic mouse in which the expression of a pan-Ras dominant negative protein (H-RasN17) is regulated by doxycycline. These mice enable us to model inhibition of the Ras pathway in vivo and thus determine the likely therapeutic efficacy of Ras inhibition. Expression of H-RasN17 reduces the expression of phospho-Erk in mouse embryonic fibroblasts (MEFs). Furthermore, persistent expression delays entry into the cell cycle from G0 and slows asynchronously proliferating cells. These effects are completely reversible when expression of H-RasN17 is switched off, through removal of doxycycline. H-RasN17 sequesters Ras-specific guanine exchange factors (GEFs) and therefore may not inhibit active, oncogenic Ras mutants that are constitutively associated with GTP. However, we find that H-RasN17 also reduces proliferation of MEFs that express oncogenic forms of K-Ras. Further studies will explore the ability of H-RasN17 to inhibit transformation induced by oncogenic forms of K-Ras, and determine the mechanism of inhibition. Preliminary in vivo data suggest that the effects of systemic H-RasN17 expression depend upon its level of expression in different tissues. High levels of H-RasN17 generally result in increased apoptosis in several tissues, particularly the pancreas, within a week of expression, whereas lower levels are well tolerated by adult mice for up to two months. To complement our in vitro analysis of Ras inhibition in cells expressing oncogenic K-ras, we have expressed H-RasN17 in KrasG12D driven non-small cell lung tumors. Early results suggest that expression of H-RasN17 in these tumors reduces tumor burden, most likely by slowing tumor growth rather than by inducing cell death. Currently, we are further investigating the effects of both high and low levels of H-RasN17 on normal tissues and also in additional tumor models such as LSL-BrafV619E melanoma, MMTV-Her2 mammary gland tumors, and Eμ-Myc lymphomas. Citation Format: Deborah L. Burkhart, Ivonne Gamper, Ana P. Rebocho, Haixi Yan, Trevor D. Littlewood, Gerard I. Evan. Modeling the therapeutic benefit of Ras-family inhibition in vivo. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A03. doi: 10.1158/1557-3125.RASONC14-A03

Persistent ERK/MAPK activation promotes lactotrope differentiation and diminishes tumorigenic phenotype.

The signaling pathways that govern the lactotrope-specific differentiated phenotype, and those that control lactotrope proliferation in both physiological and pathological lactotrope expansion, are poorly understood. Moreover, the specific role of MAPK signaling in lactotrope proliferation vs differentiation, whether activated phosphorylated MAPK is sufficient for prolactinoma tumor formation remain unknown. Given that oncogenic Ras mutations and persistently activated phosphorylated MAPK are found in human tumors, including prolactinomas and other pituitary tumors, a better understanding of the role of MAPK in lactotrope biology is required. Here we directly examined the role of persistent Ras/MAPK signaling in differentiation, proliferation, and tumorigenesis of rat pituitary somatolactotrope GH4 cells. We stimulated Ras/MAPK signaling in a persistent, long-term manner (over 6 d) in GH4 cells using two distinct approaches: 1) a doxycycline-inducible, oncogenic V12Ras expression system; and 2) continuous addition of exogenous epidermal growth factor. We find that long-term activation of the Ras/MAPK pathway over 6 days promotes differentiation of the bihormonal somatolactotrope GH4 precursor cell into a prolactin-secreting, lactotrope cell phenotype in vitro and in vivo with GH4 cell xenograft tumors. Furthermore, we show that persistent activation of the Ras/MAPK pathway not only fails to promote cell proliferation, but also diminishes tumorigenic characteristics in GH4 cells in vitro and in vivo. These data demonstrate that activated MAPK promotes differentiation and is not sufficient to drive tumorigenesis, suggesting that pituitary lactotrope tumor cells have the ability to evade the tumorigenic fate that is often associated with Ras/MAPK activation.

Abstract A47: Targeting Ras - is there Ras-specific palmitoylation in human cells that can be targeted for cancer treatment?

Abstract Ras GTPases act on the plasma membrane (PM) to mediate extracellular signaling and tumorigenesis. PM association for many Ras proteins depends on palmitoylation, which in budding yeast is catalyzed by the Erf2-Erf4 palmitoyl transferase. Despite this, there was no evidence that its human ortholog DHHC9 can similarly control Ras in vivo, and some studies even proclaimed that there was no Ras-specific palmitoylation. We performed an unbiased screen to seek fission yeast (Schizosaccharomyces pombe, sp) mutants with reduced PM Ras. Mutations affecting sp-erf2 to varying degrees were isolated 5 times; in contrast, deleting several other S. pombe palmitoyl transferases did not affect Ras. Sp-Erf2 localizes to the trans-Golgi, mediated by its third transmembrane domain and the Erf4 cofactor. In fission yeast, the human ortholog zDHHC9 fully rescues the phenotypes of sp-erf2 null cells, while expressing zDHHC14, another sp-Erf2-like human protein, did not rescue Ras1 mislocalization in these cells. Importantly, ZDHHC9 is widely overexpressed in human cancers, including several types that do not contain frequent oncogenic RAS mutations. Overexpressing ZDHHC9 in mammalian cells promotes, while repressing it diminishes, Ras PM localization, as well as transformation. These data strongly demonstrate that sp-Erf2/zDHHC9 palmitoylates Ras proteins in a highly selective manner in the trans-Golgi to facilitate PM targeting via the trans Golgi network. zDHHC9 overexpression can apparently increase Ras signaling outputs at the PM, such that tumor formation can occur in many tissues with no oncogenic mutations in RAS genes themselves. zDHHC9 may thus be a good drug target for the treatment of a wide range of cancers. Citation Format: Evelin C. Young, Ze-Yi Zheng, Angela D. Wilkins, Hee-Tae Jeong, Min Li, Lichtarge Olivier, Eric C. Chang. Targeting Ras - is there Ras-specific palmitoylation in human cells that can be targeted for cancer treatment? [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A47. doi: 10.1158/1557-3125.RASONC14-A47

Oncogenic Ras stimulates Eiger/TNF exocytosis to promote growth.

Oncogenic mutations in Ras deregulate cell death and proliferation to cause cancer in a significant number of patients. Although normal Ras signaling during development has been well elucidated in multiple organisms, it is less clear how oncogenic Ras exerts its effects. Furthermore, cancers with oncogenic Ras mutations are aggressive and generally resistant to targeted therapies or chemotherapy. We identified the exocytosis component Sec15 as a synthetic suppressor of oncogenic Ras in an in vivo Drosophila mosaic screen. We found that oncogenic Ras elevates exocytosis and promotes the export of the pro-apoptotic ligand Eiger (Drosophila TNF). This blocks tumor cell death and stimulates overgrowth by activating the JNK-JAK-STAT non-autonomous proliferation signal from the neighboring wild-type cells. Inhibition of Eiger/TNF exocytosis or interfering with the JNK-JAK-STAT non-autonomous proliferation signaling at various steps suppresses oncogenic Ras-mediated overgrowth. Our findings highlight important cell-intrinsic and cell-extrinsic roles of exocytosis during oncogenic growth and provide a new class of synthetic suppressors for targeted therapy approaches.

Abstract 740: Phenotypic profiling and selectivity optimization of ERK inhibitors using a high-throughput imaged-based cell cycle assay

Abstract The extracellular-signal-regulated kinases (ERK-1 and ERK-2) represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated due to oncogenic mutations in RAF or RAS or due to upstream oncogenic signaling, such as through receptor tyrosine kinase (RTK) signaling. We have discovered and developed potent and selective small molecule inhibitors of both ERK-1 and 2. Optimization of kinase selectivity was a critical goal in order for ATP-competitive ERK inhibitors to maintain parity with the safety profile of highly selective allosteric MEK inhibitors. As a complement to enzymatic kinase panel profiling, we employed an imaged-based cell cycle assay, based on the expected on-target phenotype of cytostatic G1 arrest in B-Raf-mutant cell lines. We were thus able to simultaneously measure on-target and off-target activities. Inhibition of Cdk2, one of the primary off-targets, was associated with a distinct G2 cell cycle arrest, which enabled us to quantify the functional selectivity margin between ERK and Cdk2 in a single assay, and select ERK inhibitors with acceptable selectivity margins for further development. Note: This abstract was not presented at the meeting. Citation Format: Grace Ka-Yan Chan, Tracy Kleinheinz, Matthew Martinson, Hok-Sum Cheung, John G. Moffat. Phenotypic profiling and selectivity optimization of ERK inhibitors using a high-throughput imaged-based cell cycle assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 740. doi:10.1158/1538-7445.AM2014-740

Abstract DDT02-03: Discovery of GDC-0994, a potent and selective ERK1/2 inhibitor in early clinical development

Abstract The extracellular-signal-regulated kinases (ERK1 and ERK2) represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that commonly is activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling, such as receptor tyrosine kinase (RTK) activation. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Here, we present the discovery and characterization of GDC-0994, an orally bioavailable, small molecule inhibitor of ERK kinase activity. GDC-0994 is highly selective for ERK1 and ERK2, with biochemical potency of 1.1 nM and 0.3 nM, respectively. Daily, oral dosing of GDC-0994 results in significant single-agent activity in multiple in vivo cancer models, including KRAS-mutant and BRAF-mutant human xenograft tumors in mice. PD biomarker inhibition of phospho-p90RSK in these tumors correlates with potency in vitro and in vivo. In contrast to other published ERK inhibitors, GDC-0994 neither increases nor decreases phospho-ERK, suggesting that different ERK inhibitors have alternative mechanisms of action with respect to feedback signaling. Furthermore, we demonstrate a novel approach for targeting the oncogenic signaling through the RAS pathway by combining ERK and MEK inhibitors. GDC-0994 is currently in Phase I clinical development. Citation Format: Kirk Robarge, Jacob Schwarz, Jim Blake, Michael Burkard, Jocelyn Chan, Huifen Chen, Kang-Jye Chou, Dolores Diaz, John Gaudino, Stephen Gould, Jonas Grina, Xin Linghu, Lichuan Liu, Matthew Martinson, David A. Moreno, Christine Orr, Patricia Pacheco, Ann Qin, Kevin Rasor, Li Ren, Sheerin Shahidi-Latham, Jeffrey Stults, Francis Sullivan, Weiru Wang, Peter Yin, Aihe Zhou, Marcia Belvin, Mark Merchant, John G. Moffat. Discovery of GDC-0994, a potent and selective ERK1/2 inhibitor in early clinical development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr DDT02-03. doi:10.1158/1538-7445.AM2014-DDT02-03

Abstract 2928: AZD6244 inhibits cisplatin induced ERK1/2 activation and potentiates its cytotoxic effects in NSCLC preclinical models

Abstract Introduction; Lung cancer is still the leading cause of cancer death worldwide, indicating that there are urgent needs for elucidating the mechanisms of resistance and developments of strategies to overcome it. Cisplatin is still one of the most commonly used drugs for the treatment of advanced lung cancer and, however, the underlying mechanism of activation of MAPK/ERK1/2 after cisplatin administration is still complex and elusive. It is considered one of the main mechanisms mediating resistance to cisplatin based doublet chemotherapy. In this study we aimed to evaluate the effectiveness of combination of MAPK inhibitor, AZD6244, with cisplatin in NSCLC models. Materials and Methods; NSCLC cells harbouring oncogenic RAS mutation, NCI-H1299, -H2009, -H460, and A549 cells, and LSL K-Ras G12D mouse model was used. Protein expression was evaluated by immunoblotting and cytotoxicity was measured by FACS followed by annexin-V and PI staining. LSL Kras G12D mouse were inhaled 5X 107 PFU AdCre virus at 8 weeks after birth and, in 24 weeks after inhalation, were taken microCT, randomized into 4 groups, and then treated with AZD6244 (25mg/kg bid by oral gavage) and/or cisplatin (5 mg/kg weekly) for two weeks. Results; There was a dose dependent increase of pERK1/2, which is most prominent at 24 hr after cisplatin treatment. Expression of proapoptotic protein, BIM, was decreased whereas that of antiapoptotic proteins, BCL-2 and MCL-1, was increased with cisplatin treatment. Treatment of AZD6244 prevented phosphorylation mediated degradation of BIM after cisplatin treatment. AZD6244 itself did not show cytotoxic effects up to 10 uM whereas combination of 1 uM of AZD6244 potentiates cytotoxic effects of cisplatin. Combined treatment of AZD6244 with cisplatin showed significant tumor reduction in 2 weeks in LSL K-ras G12D mouse model when compared those treated with single agents or vehicle. Conclusion; Combination of AZD6244 with cisplatin potentiates cytotoxic effects and anti-tumor activities in NSCLC preclinical models. These findings suggest that clinical trials using combination with AZD6244 and cisplatin may be beneficial in advanced NSCLC patients. Citation Format: Eun Young Kim, Arum Kim, Min Kwang Byun, Se Kyu Kim, Hyung Jung Kim, Joon Chang, Chul Min Ahn, Yoon Soo Chang. AZD6244 inhibits cisplatin induced ERK1/2 activation and potentiates its cytotoxic effects in NSCLC preclinical models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2928. doi:10.1158/1538-7445.AM2014-2928

Abstract 806: Selective killing of cancer cells with mitochondrial respiratory dysfunction induced by activation of oncogenic Ras

Abstract Mitochondrial dysfunction is an important mechanism by which cancer cells become resistant to chemotherapeutic agents. Oncogenic Ras activation is often observed in human cancer and was reported to cause mitochondrial dysfunction by compromising mitochondrial electron transport chain. Therefore, identification of new agents capable of effectively killing mitochondrial defective cancer cells would have important therapeutic implications. Our previous work has discovered that mitochondrial respiratory dysfunction could cause increased NADPH oxidase (NOX) activity to support cell survival in the metabolic switch from mitochondrial oxidative phosphorylation to glycolysis. Mitochondrial respiratory dysfunction can also cause profound redox alteration to adapt ROS stress. Here, we report that targeted inhibition of NOX and glutathione synthesis has synergy effect in killing mitochondrial respiratory dysfunction cells, including cancer cells harboring oncogenic Ras activation. Activation of NOX and increased glutathione antioxidants are always observed in Ras activated cells. Treatment of these cancer cells with DPI and BSO to inhibit NOX activity and antioxidant adaptation function resulted in an elevated ROS stress, increased gamma-H2AX expression, leading to massive death of the cancer cells, such as Panc-1 and HCT116 cells. Antioxidant NAC and catalase attenuated the BSO and DPI induced cell death in mitochondrial respiratory dysfunction cells. The synergistic effect of BSO and DPI to kill oncogenic Ras bearing HCT116 cells was further confirmed using colony formation assay. Our results suggest that DPI, in synergy with BSO, is a promising compound capable of killing cancer cells bearing oncogenic Ras mutations through a ROS-mediated mechanism and warrants further investigations. Citation Format: Weiqin Lu, Yaying Yang, Craig Logsdon, Peng Huang. Selective killing of cancer cells with mitochondrial respiratory dysfunction induced by activation of oncogenic Ras. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 806. doi:10.1158/1538-7445.AM2014-806

Abstract 5314: A proteomic signature predicts response to a therapeutic vaccine in pancreas cancer; analysis from the GI-4000-02 trial

Abstract Background: We have previously reported that adjuvant treatment with a therapeutic vaccine targeting the mutated Ras oncogene product generated mutation-specific T cell responses associated with a trend toward improved survival in patients with post-operative residual disease (R1 resections) but no improvement in the overall population1. Initial analysis of 90 pretreatment plasma samples using matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) showed the potential to predict improved RFS and OS for treatment with GI-4000/gemcitabine, but not placebo/gemcitabine. Methods: We have developed a novel technique, combining methods used in recent advances in learning theory (‘deep learning’) with newly-refined MS techniques that allow exploration deeper into the proteome to create diagnostic tests. Using 500,000 laser shot Deep MALDI spectra2 more than 700 mass spectral features were identified. A subset of these was used to create many multivariate classifiers that were filtered for performance and combined using dropout regularization. This method allows the use of smaller training sets and so left a test set with which performance of the signature could be independently assessed. This new methodology was used to create a test (BDX-001) to identify patients likely to benefit from the addition of GI-4000 to gemcitabine. Results: Using BDX-001 for stratification, subjects who are BDX-001(+) demonstrated a 499 day advantage in median OS when treated with GI-4000/gemcitabine vs. placebo/gemcitabine. Additionally, these subjects demonstrated a 351 day improvement in median RFS. BDX-001 did not predict response for placebo/gemcitabine treated subjects. These results were obtained using only test set data, and although the small sample size prohibited statistical significance, it should give an unbiased test performance estimate to be validated independently. Conclusions: BDX-001 is a test developed using novel proteomic and learning theory methods that appears to predict treatment response to GI-4000 in resected pancreas cancer patients, potentially identifying patients with improved RFS and OS in the GI-4000/gemcitabine arm. We plan to prospectively validate BDX-001 as a companion diagnostic in a future study of GI-4000 in pancreas cancer.

Evaluation of allelic strength of human TET2 mutations and cooperation between Tet2 knockdown and oncogenic Nras mutation

Evaluation of allelic strength of human <i><scp>TET</scp>2</i> mutations and cooperation between <i>Tet2</i> knockdown and oncogenic <i>Nras</i> mutation