N-ras Oncogene Research Articles

Mutated N-ras does not induce p19arf in CO25 cell line

The mouse cell line (CO25) used in this study was transfected with a glucocorticoid inducible mutated human N-ras oncogene under transcriptional control of the steroid-sensitive promoter of the mouse mammary tumors virus long terminal repeat MMTV-LTR. This study was aimed to investigate the expression of p19 arf and MDM2 genes under the effect of N-ras oncogene induction and to invent the role of p19 arf , MDM2 in N-ras pathway during various periods (12, 24, 48, 72, 96 h) using western blotting method. The levels of â-actin proteins in the same periods were our control group. The observations showed no increase of p19 arf protein expression in normal, cancer and differentiated CO25 cells. MDM2 was accumulated until 72 h and after 96 h, it showed a dramatical decrease while β-actin levels were increased correlated to the volume of protein loaded to the gel. Because of the role of p19 arf as tumor suppressor and p53-MDM2 linker, it is highly recommended to investigate the relationship between N-ras and p53 and MDM2 in the same system to recognize the molecule that may play a linker molecule between p53 and MDM2 in p19 arf lack system. Key words : Oncogene, N-ras, p19 arf , myoblast, CO25 cells, differentiation, MDM2.

A High-Resolution Melting Protocol for Rapid and Accurate Differential Diagnosis of Thyroid Nodules

A large majority of thyroid nodules are benign, and only 5% have malignant features on cytological examination. Unfortunately, fine-needle aspiration is inconclusive in approximately 30% of all thyroid biopsies, because the cytological features are indeterminate (suspicious for malignancy but not completely diagnostic or nondiagnostic). Wide panels of somatic mutations have been identified in thyroid cancers, and detection of genetic alterations in fine-needle aspirate has been demonstrated to improve diagnostic accuracy. Nevertheless, the relatively high number of genetic targets to be investigated, in comparison with the low percentage of malignant samples, makes the usual diagnostic protocol both time-consuming and expensive. We developed a reliable and sensitive protocol based on high-resolution melting analysis for the rapid screening of mutations of KRAS, HRAS, NRAS, and BRAF oncogenes in thyroid fine-needle aspirations. The entire procedure can be completed in approximately 48 hours, with a dramatic reduction in costs. The proposed protocol was applied to the analysis of 260 consecutive fine-needle aspiration biopsy (FNAB) samples. In 35 of 252 samples, 36 sequence variants were detected for BRAF (17 samples), NRAS (6 samples), HRAS (3 samples), KRAS codon 12 (9 samples), and KRAS codon 61 (1 sample).

Marked genetic differences between BRAF and NRAS mutated primary melanomas as revealed by array comparative genomic hybridization

Somatic mutations of BRAF and NRAS oncogenes are thought to be among the first steps in melanoma initiation, but these mutations alone are insufficient to cause tumor progression. Our group studied the distinct genomic imbalances of primary melanomas harboring different BRAF or NRAS genotypes. We also aimed to highlight regions of change commonly seen together in different melanoma subgroups. Array comparative genomic hybridization was performed to assess copy number changes in 47 primary melanomas. BRAF and NRAS were screened for mutations by melting curve analysis. Reverse transcription PCR and fluorescence in-situ hybridization were performed to confirm the array comparative genomic hybridization results. Pairwise comparisons revealed distinct genomic profiles between melanomas harboring different mutations. Primary melanomas with the BRAF mutation exhibited more frequent losses on 10q23-q26 and gains on chromosome 7 and 1q23-q25 compared with melanomas with the NRAS mutation. Loss on the 11q23-q25 sequence was found mainly in conjunction with the NRAS mutation. Primary melanomas without the BRAF or the NRAS mutation showed frequent alterations in chromosomes 17 and 4. Correlation analysis revealed chromosomal alterations that coexist more often in these tumor subgroups. To find classifiers for BRAF mutation, random forest analysis was used. Fifteen candidates emerged with 87% prediction accuracy. Signaling interactions between the EGF/MAPK-JAK pathways were observed to be extensively altered in melanomas with the BRAF mutation. We found marked differences in the genetic pattern of the BRAF and NRAS mutated melanoma subgroups that might suggest that these mutations contribute to malignant melanoma in conjunction with distinct cooperating oncogenic events.

Raf Inhibitors Target Ras Spatiotemporal Dynamics

The lateral segregation of Ras proteins into transient plasma membrane nanoclusters is essential for high-fidelity signal transmission by the Ras mitogen-activated protein kinase (MAPK) cascade. In this spatially constrained signaling system, the dynamics of Ras nanocluster assembly and disassembly control MAPK signal output. We show here that BRaf inhibitors paradoxically activate CRaf and MAPK signaling in Ras transformed cells by profoundly dysregulating Ras nanocluster dynamics. Specifically, BRaf inhibitors selectively enhance the plasma membrane nanoclustering of oncogenic K-Ras and N-Ras but have no effect on H-Ras nanoclustering. Raf inhibitors are known to drive the formation of stable BRaf-CRaf and CRaf-CRaf dimers. Our results demonstrate that the presence of two Ras-binding domains in a single Raf dimer is sufficient and required to increase Ras nanoclustering, indicating that Raf dimers promote K- and N-Ras nanocluster formation by crosslinking constituent Ras proteins. Ras crosslinking increases the fraction of K-Ras and N-Ras in their cognate nanoclusters, leading to an increase in MAPK output from the plasma membrane. Intriguingly, increased MAPK signalingin BRaf inhibited cells is accompanied by significantly decreased Akt activation. We show that this signal pathway crosstalk results from a novel mechanism of competition between stabilized Raf dimers and p110α for recruitment to Ras nanoclusters. Our findings reveal that BRaf inhibitors disrupt Ras nanocluster dynamics with significant, yet divergent, consequences for MAPK and PI3K signaling.

Abstract PL04-02: Ras and Raf signaling: From biology to therapeutics

Abstract The protein kinase BRAF is mutated in about 50% of human melanomas and the small G-protein NRAS is mutated in another 20% of cases. We have developed mouse models of melanoma driven by oncogenic BRAF, NRAS and KRAS. These studies have established that acquisition of BRAF, NRAS or KRAS mutations can be founder events in melanomagenesis and we are currently using these models to investigate gene-gene and gene-environment interactions in cutaneous melanoma. We are also investigating other targets of BRAF in melanoma cells and have shown that BRAF drives melanoma cell migration and invasion by down-regulating expression of the cyclic GMP phosphodiesterase PDE5A. 2011 was a landmark year in melanoma research with the registration of vemurafenib and ipilimumab, drugs that show clinical benefit in patients with advanced disease. Critically, BRAF inhibitory drugs achieve objective clinical responses in about 80% of melanomas that harbour mutations in BRAF. Surprisingly however, these drugs also drive paradoxical activation of CRAF in melanoma cells that harbour mutations in NRAS, or in cells in which RAS is activated due to increased upstream signalling. This is due to the induction of BRAF/CRAF heterodimers and CRAF homodimers and importantly, activation of the pathway by this paradox can drive tumourigenesis in some circumstances. We have used a two-stage skin carcinogenesis model to show that BRAF inhibitors are not tumour promoters pre se, but rather that they work by accelerating the growth of tumours carrying pre-existing mutations in HRAS. Thus, we conclude that BRAF inhibitors appear to accelerate the inevitable in susceptible patients, but do not induce these lesions de novo. Notably, concurrent administration of a MEK inhibitor with a BRAF drug blocks the development of these skin lesions, providing a rationale for combining BRAF and MEK inhibitors in melanoma patients. Unexpectedly, we have also found that the ABL/CKIT inhibitor nilotinib also drives paradoxical activation of the RAF pathway because it possesses weak off-target activity against BRAF and CRAF. The consequence of this paradoxical activation is that nilotinib provides a survival advantage to chronic myeloid leukaemia (CML) cells that are resistant to nilotinib, but also provides an Achilles heel to these cells because they become dependent on MEK signalling in the presence of nilotinib. Thus, MEK inhibitors cooperate with nilotinib to drive synthetic lethality in nilotinib-resistant CML cells. Thus, paradoxical activation of the RAF proteins by small molecular weight drugs can have important consequences to patient responses, creating specific clinical problems in some circumstances, but therapeutic opportunities in others. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr PL04-02. doi:1538-7445.AM2012-PL04-02

Spliceosomal gene aberrations are rare, coexist with oncogenic mutations, and are unlikely to exert a driver effect in childhood MDS and JMML

AbstractSomatic mutations of the spliceosomal machinery occur frequently in adult patients with myelodysplastic syndrome (MDS). We resequenced SF3B1, U2AF35, and SRSF2 in 371 children with MDS or juvenile myelomonocytic leukemia. We found missense mutations in 2 juvenile myelomonocytic leukemia cases and in 1 child with systemic mastocytosis with MDS. In 1 juvenile myelomonocytic leukemia patient, the SRSF2 mutation that initially coexisted with an oncogenic NRAS mutation was absent at relapse, whereas the NRAS mutation persisted and a second, concomitant NRAS mutation later emerged. The patient with systemic mastocytosis and MDS carried both mutated U2AF35 and KIT in a single clone as confirmed by clonal sequencing. In the adult MDS patients sequenced for control purposes, we detected previously reported mutations in 7/30 and a novel SRSF2 deletion (c.284_307del) in 3 of 30 patients. These findings implicate that spliceosome mutations are rare in pediatric MDS and juvenile myelomonocytic leukemia and are unlikely to operate as driver mutations.

Targeting oncogenes in advanced melanoma

The ability to target oncogenes in malignancies such as CML, GIST, APML and ERBB2-positive breast cancer has revolutionized the management of those diseases. Interesting over 70% of melanomas contain genomic amplification or mutations in one of the oncogenes BRAF, NRAS, KIT, CCND1 or CDK4 that may induce an oncogene addicted state. Inhibition of BRAF with Vemurafenib or GSK2118436 in BRAF-mutant melanoma have shown responses in over 50% of patients with advanced disease and in the case of Vemurafenib striking improvements in survival compared to DTIC. These data suggest that there are therapeutically targetable oncogenes in melanoma. However emergence of resistance is common. A number of mechanisms of resistance have been identified including reactivation of the RAS/RAF/MEK/ERK pathway. Current focus is the development of combination strategies including the addition of MEK-inhibitors to BRAF-inhibitors and the combining targeted agents with immunological agents such as Ipilimumab. Together these data indicate that targeting oncogenes in melanoma offers significant therapeutic opportunities in one of the most challenging of human malignancies for systemic therapy.

Endogenous Oncogenic Nras Signaling Promotes Proliferative Exhaustion of Hematopoietic Stem Cells Through a Dose-Dependent Hyperactivation of ERK,

Abstract 3997Both monoallelic and biallelic oncogenic NRAS mutations are identified in human leukemias, suggesting a dose-dependent role of oncogenic NRAS in leukemogenesis. We recently characterized conditional mouse models that express one or two copies of constitutively active Nras G12D in the hematopoietic compartment (Nras G12D/+ and Nras G12D/G12D). Nras G12D/G12D results in stronger downstream signaling than Nras G12D/+ and consequently distinct hematopoietic phenotypes. In particular, we found that somatic expression of Nras G12D/G12D but not Nras G12D/+ leads to an acute myeloproliferative neoplasm (MPN). The development of acute MPN is associated with cytokine-evoked hyperactivation of ERK but not Stat5 and Akt in hematopoietic stem/progenitor cells (HSPCs). Interestingly, genetically altered HSCs appear to be required for initiation and maintenance of chronic and acute MPD phenotypes mediated by Nras G12D/+ and Nras G12D/G12D, respectively. Furthermore, Nras G12D/G12D-mediated signaling promotes excessive HSC proliferation and leading to HSC exhaustion. Exhaustion of HSCs is tightly associated with diminished MPN phenotypes in non-engineered recipient mice into which Nras G12D/G12D HSCs are transferred by bone marrow transplantation. To investigate the molecular mechanisms underlying the exhaustion of Nras G12D/G12D HSCs, we performed a microarray analysis using highly purified control, Nras G12D/+, and Nras G12D/G12D HSCs. To our surprise, neither p16INK4a and p15INK4b, critical cell senescence genes, or other genes or pathways known to involve in HSC self-renewal were differentially expressed in Nras G12D/G12D HSCs. In contrast, gene ontology analysis revealed significant differential expression of genes in the MEK/ERK pathway in Nras G12D/G12D HSCs. This result is consistent with our phospho-flow study showing that Nras G12D/G12D selectively hyperactivates ERK in HSPCs. It has been documented that prolonged ERK signaling is a potent inducer of differentiation in cultured PC12 cells and inhibition of normal ERK signaling by a chemical inhibitor increases self-renewal in mouse embryonic stem cells. Thus, we are currently testing whether hyperactivation of ERK promotes depletion of Nras G12D/G12D HSCs through promoting HSC differentiation and whether blocking hyperactivation of ERK restores the normal HSC function and prevents MPN development. Disclosures:No relevant conflicts of interest to declare.

Inhibition of RNA Polymerase I Transcription by CX-5461 As a Therapeutic Strategy for the Cancer-Specific Activation of p53 in MLL-Rearranged Acute Myeloid Leukemias

Abstract 1548Increased transcription of the ribosomal genes (rDNA) by RNA Polymerase I (Pol I) is a common feature of human cancer1. Inhibition of Pol I transcription causes nucleolar stress that leads to the release of ribosomal proteins from the nucleolus into the nucleoplasm where they can sequester the p53 inhibitory protein MDM2, causing activation of p53 and induction of apoptosis2. We have developed a potent and selective small molecule inhibitor of Pol I transcription (CX-5461) that is non-genotoxic3. When evaluated for anti-proliferative activity against genetically diverse cancer cell lines, CX-5461 exhibited its greatest potency against wild-type (wt) p53 cells derived from hematological malignancies (median IC50 = 12 nM), while the median IC50s in cells derived from p53 mutated hematological, p53wt and p53 mutated solid tumors and normal cells were less sensitive to CX-5461 (median IC50s = 94 nM, 164 nM, 265 nM and 5 mM respectively), indicating CX-5461 selectively kills p53-wild type malignant hematopoietic cells. Consistent with the nucleolar stress model, p53 wt human leukemia cell lines exhibited robust activation of p53 signaling and apoptotic death in response to low nanomolar doses of CX-5461. To explore the therapeutic potential of Pol I transcription inhibition in vivo in hematological malignancies wt for p53 that are refractory to cytotoxic therapies we tested CX-5461 in mouse models of human acute myeloid leukemia (AML) expressing MLL fusion proteins4. Mice transplanted with 5×106 leukemia cells expressing MLL/ENL or MLL/AF9 together with oncogenic NRAS linked to GFP and luciferase biomarkers by virtue of bicistronic retroviral vectors, rapidly developed aggressive leukemia characterized by anemia, leukocytosis, hepatosplenomegaly and within 7–10 days (Mac1+, Gr-1) leukemic cell counts in peripheral blood. Treatment of recipient mice harboring MLL/ENL+NRAS leukemia with CX-5461 (40mg/kg Q3D) significantly increased median survival (17 days for vehicle vs 36 days for drug, P <0.0001), and reduced leukemic burden, as determined by leukemic cell luminescence imaging, peripheral white blood cell counts (20.77×103cells/ml±2.25 for vehicle vs 6.2×103cells/ml±0.68 for drug, P <0.0001) at sacrifice and FACS analysis of the peripheral blood (24.36% GFP leukemic cells±1.84 for vehicle vs 6.30% leukemic cells±0.5 for drug, P <0.0001) after 3 doses of treatment. The reduction in tumour burden was associated with in vivo activation of p53 signaling and apoptosis. In marked contrast, treatment of MLL/ENL+NRAS leukemic mice with a combination regimen of cytarabine (50 mg/kg 5XQD IP) and doxorubicin (1.5 mg/kg 3XQD IP) at the combined MTD dose failed to provide a significant survival advantage. Treatment of recipient mice harboring the highly aggressive MLL/AF9 + NRAS leukemia with CX-5461 (40mg/kg Q3D) was also able to increased overall survival (15 days for vehicle vs 23 days for drug, P 0.0009), with this delay accompanied by a significant decrease in leukemic burden as determined by peripheral white blood cell counts (114×103cells/ml±12.24 for vehicle vs 4.68×103cells/ml±0.7 for drug) and FACS analysis of the peripheral blood (65% GFP leukemic cells±4.9 for vehicle vs 0.26% leukemic cells±0.05 for drug) after 2 respectively 3 doses of treatment. Critically, in murine xenograft of human AML MV 4;11 that carries the MLL/AF4 fusion, treatment with CX-5461 (125 mg/kg Q7D IP) inhibited tumour growth by 93% and extended time to sacrifice from 21 to 36 days. Together this work demonstrates that CX-5461, which selectively inhibits Pol I transcription and non-genotoxically activates p53, may be used to therapeutically treat aggressive p53-wild type AMLs that are highly refractory to standard cytotoxic therapies. The fact that mutations or deletions of the p53 gene are relatively rare in AML, coupled with our data that p53-wt AML cancer cells are particularly sensitive to CX-5461, provides a compelling rationale for evaluating CX-5461 in this patient population. Disclosures:Drygin:Cylene Pharmaceuticals Inc: Employment. Huser:Cylene Pharmaceuticals Inc: Employment. Bliesath:Cylene Pharmaceuticals Inc: Employment. Ryckman:Cylene Pharmaceuticals Inc: Employment. Rice:Cylenen Pharmaceuticals Inc: Employment. Hannan:Cylene Pharmaceuticals Inc: Consultancy.

Endogenous oncogenic Nras mutation initiates hematopoietic malignancies in a dose- and cell type-dependent manner

AbstractBoth monoallelic and biallelic oncogenic NRAS mutations are identified in human leukemias, suggesting a dose-dependent role of oncogenic NRAS in leukemogenesis. Here, we use a hypomorphic oncogenic Nras allele and a normal oncogenic Nras allele (Nras G12Dhypo and Nras G12D, respectively) to create a gene dose gradient ranging from 25% to 200% of endogenous Nras G12D/+. Mice expressing Nras G12Dhypo/G12Dhypo develop normally and are tumor-free, whereas early embryonic expression of Nras G12D/+ is lethal. Somatic expression of Nras G12D/G12D but not Nras G12D/+ leads to hyperactivation of ERK, excessive proliferation of myeloid progenitors, and consequently an acute myeloproliferative disease. Using a bone marrow transplant model, we previously showed that ∼ 95% of animals receiving Nras G12D/+ bone marrow cells develop chronic myelomonocytic leukemia (CMML), while ∼ 8% of recipients develop acute T-cell lymphoblastic leukemia/lymphoma [TALL] (TALL-het). Here we demonstrate that 100% of recipients transplanted with Nras G12D/G12D bone marrow cells develop TALL (TALL-homo). Although both TALL-het and -homo tumors acquire Notch1 mutations and are sensitive to a γ-secretase inhibitor, endogenous Nras G12D/+ signaling promotes TALL through distinct genetic mechanism(s) from Nras G12D/G12D. Our data indicate that the tumor transformation potential of endogenous oncogenic Nras is both dose- and cell type-dependent.

Abstract 867: Somatic BRAF and NRAS mutations in familial melanoma: a GenoMEL study

Abstract Up to 10% of all melanoma patients show a family history of disease. One main predisposing gene for familial melanoma development is CDKN2A, a tumor suppressor gene that encodes two distinct proteins, p16INK4A and p14ARF. CDKN2A germline mutations have been found in 20-40% of melanoma-prone families. So far, BRAF and NRAS are the most frequently altered oncogenes described in cutaneous melanoma. To date, little is known about the somatic changes that occur in familial melanoma. The aim of this study was to determine the prevalence of BRAF and NRAS somatic mutations in familial melanomas and to evaluate the associations between these mutations and clinicopathological factors. The study was performed as a collaboration within GenoMEL (The Melanoma Genetics Consortium). A total of 135 primary familial cutaneous melanomas (88 tumors from CDKN2A mutation-carriers and 47 from CDKN2A non-carriers) were investigated. Paraffin-blocks of tumors were collected from 8 GenoMEL centers in Australia, Italy, Latvia, the Netherlands, Spain, Sweden, and UK. Tumor cells were isolated by laser capture microdissection and mutation screening (BRAF exon 15 and NRAS exon 3) was performed by direct sequencing. Mutation analysis revealed BRAF and NRAS mutations in 41.6% and 11.4% of tumors, respectively. In no case were BRAF and NRAS mutations found to coexist in the same tumor. There was no difference in BRAF mutation frequency between tumors from patients with CDKN2A alterations and tumors from patients wild-type for CDKN2A (41.0% vs. 42.9%). Similarly, the frequency of NRAS mutations did not differ markedly when tumors from CDKN2A mutation-carriers and non-carriers were compared (9.0% vs. 15.6%). There was also no difference in the frequency of BRAF mutations between tumors with CDKN2A mutations disrupting both p16INK4A and p14ARF and tumors with CDKN2A mutations affecting p16INK4A only (40.6% vs. 36.4%). When tumors with different CDKN2A founder mutations were compared, BRAF mutations were seen in 50.0%, 45.8%, 37.5% and 35.0% of p.M53I, p.R112_L113insR, p.G101W and c.225_243del19 altered tumors, respectively. NRAS mutations were observed in 14.3% 12.5% and 10.5% of p.R112_L113insR, p.M53I and c.225_243del19 altered tumors, respectively, whereas no NRAS mutations were seen in tumors with the p.G101W founder mutation. Patients with BRAF-mutated tumors had a significantly lower median age at diagnosis compared to patients with NRAS-mutated or BRAF/NRAS wild-type tumors (43.5, 54 and 51.5 years, respectively). Moreover, melanomas with mutated BRAF or NRAS were thicker than BRAF/NRAS wild-type cases (median thickness 1.0, 1.4 and 0.7mm, respectively). Our results suggest a role for the BRAF and NRAS oncogenes in familial melanoma. Somatic changes of these genes occur in tumors from both CDKN2A mutation carriers and non-carriers. Presence of BRAF or NRAS mutations associated with patient and tumor characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 867. doi:10.1158/1538-7445.AM2011-867

Abstract 3780: Autophagy targeting therapies in NSCLC: Autophagy induction vs. inhibition in combination with erlotinib shows Torin1 can sensitize NSCLCs with KRAS mutations to Erlotinib

Abstract In non-small cell lung cancer (NSCLC), therapies targeting the epidermal growth factor receptor (EGFR) are effective in the about 10% of cases that express mutant EGFR. The limited response of such therapies indicates the need to uncover additional therapeutic targets to increase the efficacy of EGFR targeted agents in a broader range of cases. One potential new targeted that may have therapeutic implications is the process of autophagy, which cells use to recycle proteins other cellular components. Currently, two opposing hypotheses exist concerning the role of autophagy in cancer. Autophagy may have tumor suppressive properties such as controlling cell growth, degrading damaged organelles, and serving as an alternate death mechanism. Conversely, it may function in tumor growth by enabling survival in conditions of low nutrients and oxygen, and protecting tumor cells against chemotherapy induced damage. The role of autophagy in cancer may also vary depending on cell type, tumor histology, oncogenotype, stage of tumor progression, or other factors. The type of treatment that will be most useful for the treatment of lung cancer will depend on which of these scenarios is most accurate in NSCLC cases. Approach: This study aims to analyze the role of autophagy in NSCLC by comparing autophagy inducing agents with an autophagy inhibitor for their ability to sensitize lung cancer cells to EGFR targeted therapy. A panel of NSCLC cell lines has been screened with an autophagy inhibitor (chloroquine) and autophagy inducers (Torin1 [Whitehead, DFCI] and rapamycin) alone and in combination with EGFR inhibitor erlotinib. Response to drug treatments was tested using a five day MTS assay for both single agent and combination treatments. Liquid colony formation assays confirmed the sensitization to erlotinib by autophagy inducers seen by MTS assay. The effects of drug treatments on autophagic flux were determined using a ‘tandem fluorescent’ LC3 protein reporter assay (ptfLC3, Yoshimori), in which the percent co-localization of mRFP and EGFP signals can be used to determine the relative levels of autophagosome formation and degradation (Kimura et al.(2007) Autophagy 3(5):452-60). Conclusions: Chloroquine, had little impact on sensitizing the lung cancer cell lines to erlotinib. Combinations with Torin1 or rapamycin resulted in 10-20 fold sensitization to erlotinib in a subset of NSCLC cell lines which had wild type EGFR and oncogenic KRAS or NRAS and wild type or mutant p53. These results indicate that autophagy induction and not autophagy inhibition, may be an effective targeted therapy in combination with erlotinib for certain cases of NSCLC. However, because sensitization occurred in only a small subset of lung cancer cell lines, there is a need to determine biomarkers that may be used to predict in which cases sensitization will most likely occur. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3780. doi:10.1158/1538-7445.AM2011-3780

Abstract 2860: Defining the role of NRAS in melanoma maintenance using a novel inducible mouse model

Abstract As treatments move into the era of targeted therapy, there is a growing need to understand the alterations that result in melanoma formation, maintenance, and progression. To this end, we have developed a somatic cell gene transfer model of melanoma based on the RCAS/TVA system that allows for tissue- and cell-specific targeted infection of mammalian cells through ectopic expression of the viral receptor. This system utilizes a retroviral vector, RCASBP(A), derived from the avian leukosis virus (ALV). The receptor for RCASBP(A) is encoded by the tv-a gene and is normally expressed in avian cells; infection results in integration of the virus into the genome of dividing cells and expression of delivered genes. Since the virus is replication-defective in mammalian cells, these cells can be infected with multiple ALV-derived viruses. This allows efficient modeling of melanoma because multiple oncogenic alterations can be introduced into the same cell. The dopachrome tautomerase (DCT) promoter was chosen to drive expression of the viral receptor TVA since this gene is expressed early in melanocyte development when the cells are mitotically active. A significant percentage of both familial and sporadic melanomas have mutations that functionally inactivate both INK4a and ARF; therefore, DCT-TVA mice were crossed to Ink4a/Arflox/lox mice. Activated NRAS oncogenes have also been detected in approximately 20% of human melanomas. Therefore, as a proof-of-principle, newborn mice were injected subcutaneously with retroviruses containing Cre-recombinase and NRASQ61R. Whereas no tumors were detected in TVA-negative mice, melanomas were visible in DCT-TVA;Ink4a/Arflox/lox mice as early as three weeks of age. Within three months, more than two-thirds of DCT-TVA;Ink4a/Arflox/lox mice developed melanoma that was histologically similar to the human disease. To allow for regulation of NRAS expression post-delivery using the tetracycline (tet)-regulated system, a tet-responsive element (TRE) was inserted upstream of the NRAS gene in the viral vector. Expression from the TRE requires the presence of a tetracycline transcriptional activator (tTA) such as Tet-off or a reverse tTA (rtTA) such as Tet-on. In the context of Tet-off, the Tet-responsive gene is repressed in the presence of Dox. Doxycycline (Dox) was administered to suppress NRAS expression once tumors reached 1,000 mm3. Within four days, Dox induced substantial regression of NRAS and Cre induced melanomas in DCT-TVA/Ink4a/Arf lox/lox mice. Tumors regressed in all mice treated with Dox. Interestingly, the tumors did not disappear completely but regressed to ∼5 mm3 and became dormant. This model will be used to study tumor dormancy, define mechanisms of resistance, and test novel combination therapies. Our long-term goals are to translate the knowledge gained from these studies into improvements in molecular targeted therapies for the treatment of advanced melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2860. doi:10.1158/1538-7445.AM2011-2860

Hematopoiesis and leukemogenesis in mice expressing oncogenic NrasG12D from the endogenous locus

AbstractNRAS is frequently mutated in hematologic malignancies. We generated Mx1-Cre, Lox-STOP-Lox (LSL)-NrasG12D mice to comprehensively analyze the phenotypic, cellular, and biochemical consequences of endogenous oncogenic Nras expression in hematopoietic cells. Here we show that Mx1-Cre, LSL-NrasG12D mice develop an indolent myeloproliferative disorder but ultimately die of a diverse spectrum of hematologic cancers. Expressing mutant Nras in hematopoietic tissues alters the distribution of hematopoietic stem and progenitor cell populations, and Nras mutant progenitors show distinct responses to cytokine growth factors. Injecting Mx1-Cre, LSL-NrasG12D mice with the MOL4070LTR retrovirus causes acute myeloid leukemia that faithfully recapitulates many aspects of human NRAS-associated leukemias, including cooperation with deregulated Evi1 expression. The disease phenotype in Mx1-Cre, LSL-NrasG12D mice is attenuated compared with Mx1-Cre, LSL-KrasG12D mice, which die of aggressive myeloproliferative disorder by 4 months of age. We found that endogenous KrasG12D expression results in markedly elevated Ras protein expression and Ras-GTP levels in Mac1+ cells, whereas Mx1-Cre, LSL-NrasG12D mice show much lower Ras protein and Ras-GTP levels. Together, these studies establish a robust and tractable system for interrogating the differential properties of oncogenic Ras proteins in primary cells, for identifying candidate cooperating genes, and for testing novel therapeutic strategies.

Dominant Negative Effect of Palmitoylation-Deficient NRAS In Suppression of BCR/ABL Leukemogenesis

AbstractAbstract 3157RAS mutations are found in ∼30% of all human cancers, with NRAS being the RAS isoform found most frequently mutated in hematological malignancies including acute myelogenous leukemia (AML) and chronic myelomonocytic leukemia (CMML). We have previously shown that expression of oncogenic NRAS efficiently induces a CMML- or AML-like disease in mice. Like all RAS proteins, NRAS must undergo a series of post-translational modifications for differential targeting to distinct cellular membrane microdomains. We have previously found that palmitoylation, in addition to prenylation, is an essential process for NRAS leukemogenesis. The results suggest that targeting RAS palmitoylation may be an effective therapy for hematological malignancies as well as other NRAS related cancers.In addition to the direct activation by mutations, RAS can also be functionally activated by other oncogenic mutations, including activated protein tyrosine kinases such as BCR/ABL. While significant advances have been made in the treatment of chronic myelogenous leukemia (CML) by targeting the kinase activity of BCR/ABL, these treatments do not constitute a cure, as BCR/ABL-positive cells persist and eventually develop mutations that cause resistance to the kinase inhibitors. In addition, although advances have also been achieved in the treatment of BCR/ABL+ B-acute lymphoblastic lymphoma (B-ALL) through the use of combination chemotherapy along with the ABL kinase inhibitor imatinib, BCR/ABL+ B-ALL continues to pose a huge challenge; they are highly refractory to imatinib, and are still the least treatable subtype of ALL. The identification of molecular targets that impede the progression of BCR/ABL+ leukemias not relying on targeting the kinase activity of BCR/ABL is likely beneficial. Previous studies have shown that RAS is a critical downstream transducer of the oncogenic signaling by BCR/ABL. We have shown that palmitoylation-deficient oncogenic NRAS is mislocalized away from the plasma membrane yet still capable of binding GTP in cells. This finding suggests that palmitoylation-deficient, activated RAS may have a dominant negative effect in RAS signaling. As a proof-of-concept study for treating cancers driven by RAS regulators through blocking RAS palmitoylation, we evaluated the effect of blocking RAS palmitoylation in BCR/ABL leukemogenesis. We found that expression of palmitoylation-deficient oncogenic NRAS or palmitoylation-deficient wild type NRAS significantly impeded progression of BCR/ABL induced B-ALL- and CML-like diseases in mice. The palmitoylation-deficient NRAS suppresses the activation of downstream effectors of RAS, possibly by sequestrating RAS effectors away from the plasma membrane. The results suggest that targeting RAS palmitoylation may also constitute an effective therapy in cancers driven by oncogenes upstream of RAS. Disclosures:No relevant conflicts of interest to declare.

An RNAi Screen Reveals a Pattern of Addiction to Epigenetic Pathways That Distinguishes MLL-AF9 Leukemia From Normal Hematopoietic Cells

AbstractAbstract 65Epigenetic pathways act to control gene expression in a heritable fashion without altering DNA sequence, typically involving the control of chromatin structure. Several lines of evidence implicate the involvement of epigenetics in the pathogenesis of human cancer, however it remains uncertain to what extent manipulating epigenetic pathways can fully rectify malignant cellular states for therapeutic benefit. To systematically explore this issue, we have developed a novel RNAi screening pipeline that can distinguish the epigenetic requirements for normal and malignant hematopoiesis. This was achieved by first constructing a custom shRNA library targeting all known enzymatic complexes that regulate chromatin structure (1100 shRNAs in total). Next, each shRNA was delivered systematically to cells derived from a mouse model of chemotherapy-resistant acute myeloid leukemia driven by the oncogenes MLL-AF9 and NRAS, or to several non-transformed hematopoietic cell lines of different lineages. shRNAs were scored for their capacity to differentially inhibit growth of leukemic cells without influencing growth of non-leukemic cells. Each of the identified genes was then evaluated in vivo for its influence on normal reconstitution of the hematopoietic system following transplantation of shRNA-infected hematopoietic stem and progenitor cells into lethally irradiated recipient mice. Each gene was likewise suppressed in leukemia cells in vivo using both constitutive and conditional RNAi vectors. The net result of the in vivo testing was identification of 6 genes encoding different regulators of chromatin structure whose suppression provides therapeutic benefit in a mouse model of therapy-resistant AML, without significantly influencing the production of normal blood lineages. In support of the accuracy of our screening protocol, one of the identified genes from the screen encodes the protein Menin, a known MLL-AF9 cofactor essential for disease initiation and shown to be dispensable for steady-state hematopoiesis in knockout mice.Two of the identified hits in our screen are the genes Eed and Suz12, which encode two subunits of the PRC2 Polycomb complex (Eed and Suz12), which catalyzes histone H3K27 methylation to suppress gene expression. Inhibiting PRC2 function in MLL-AF9 leukemia cells leads to monocytic differentiation, as revealed by FACS, RT-qPCR, and cell morphology analysis. Microarray experiments coupled with chromatin immunoprecipitation identified a core program of myeloid fate determinants that are suppressed by PRC2 via H3K27 methylation. In addition, we observe that MLL-AF9 directly occupies several Polycomb gene promoters to upregulate their expression in leukemic cells. Our findings highlight an unexpected alliance between the MLL-AF9 oncogene (a Trithorax protein) and PRC2 (a Polycomb complex), which act together to block myeloid differentiation in AML. Our findings also highlight the utility of employing RNAi in vivo to identify novel therapeutic targets in otherwise chemotherapy-resistant disease models. Disclosures:No relevant conflicts of interest to declare.

Endogenous Oncogenic Nras Mutation Initiates T-Cell Leukemia/Lymphoma In a Dose-Dependent Manner

AbstractAbstract 4183The oncogenic NRAS mutations are frequently identified in myeloid diseases but rare in lymphoid diseases. They occur in 4% of acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) patients and 22% of human T-ALL cell lines. Its differential roles in myeloid versus lymphoid disease development remain unclear. Here we examine the tumorigenic potential of oncogenic Nras in T-cells using two conditional Nras G12D murine knock-in models that either hypomorphically (NrasG12D Hypo) or normally (NrasG12D Norm) expresses oncogenic Nras G12D from its endogenous locus. Mice expressing monoallelic or biallelic NrasG12D Hypo develop normally and are tumor free. However, NrasG12D Norm leads to acute T-cell leukemia/lymphoma (TAL/L) in a bone marrow transplantation model, with a low incidence (∼8%) when expressing one allele (TAL/L-het) and a complete penetrance when expressing two alleles (TAL/L-homo). TAL/L-het tumors are associated with spontaneous up-regulation of oncogenic Nras in ∼67% of animals, and tumor cells are TdT positive, suggesting that they are transformed at an immature stage. In contrast, TAL/L-homo tumors express comparable levels of Nras to control thymocytes, and tumor cells are TdT negative, suggesting that they are transformed at a more mature stage. Both TAL/L-het and TAL/L-homo tumors are oligoclonal or polyclonal. Above 70% of these tumors contain clonal Notch1 mutations and are sensitive to gamma-secretase inhibitor. These data indicate that Notch1 mutations are acquired at an early stage and play an important role in the development of TAL/L-het and TAL/L-homo tumors. Together, our results show that engdogenous oncogenic Nras mutation leads to TAL/L in a dose-dependent manner, and thus explain the low incidence of oncogenic NRAS mutations in human T-cell diseases. Disclosures:No relevant conflicts of interest to declare.

Metastatic Melanoma to the Mandible: Case Report and Review of the Literature

Melanoma is the most serious form of skin cancer. It is a neoplasm of dysplastic pigment-producing melanocytes, and its development is related to the amount of ultraviolet B rays in sunlight that a person is exposed to over his or her lifetime. 1 Fears T.R. Bird C.C. Guerry D. et al. Mean UVB flux and time outdoors predict melanoma risk. Cancer Res. 2002; 62: 3992 PubMed Google Scholar At the molecular level, mutations in the tumor suppressor gene CDKN2A and in the oncogenes N-ras and H-ras seem to play significant roles in its development. 2 Ortonne J.P. Photobiology and genetics of malignant melanoma. Br J Dermatol. 2002; 146: 11 Crossref PubMed Scopus (35) Google Scholar Unlike some other forms of skin cancer, such as basal cell or squamous cell carcinoma, melanomas are highly aggressive and unpredictable, and they can metastasize to almost anywhere in the body.

Effects of AKR1B10 gene silence on the growth and gene expression of HCC cell line MHCC97H

To explore the biological function and possible underlying mechanism of aldo-keto reductase family 1 member B10 (AKR1B10) gene during hepatocarcinogenesis. A pair of chemically synthesized small interfering RNA (siRNA) targeting on AKR1B10 was transfected into liver cancer cell line MHCC97H by LipofectamineTM 2000. After confirming the interfering effects of AKR1B10-siRNAs through Quant SYBR Green polymerase chain reaction (Real-time PCR), Western blot and enzymatic activity assay, the capabilities of proliferation and apoptosis of the transfected cells were observed by CCK-8 assay and flow cytometry analysis, and the expressions of a group of tumor-related gene such as c-myc, c-fos, N-ras were observed through Real-time PCR. The expressions of AKR1B10 and the enzymatic activity were down-regulated significantly in AKR1B10-siRNA-transfected cells. Compared with mock and blank control groups, cell growth in AKR1B10-siRNA-transfected group was inhibited by 26.6%+/-3.1% at 72h after transfection. The ratio of apoptotic cells was 37.3%+/-1.0% in AKR1B10-siRNA-transfected group, which was significantly higher than that in mock and blank control groups (P < 0.01). Real-time PCR showed that the expressions of oncogene c-myc, c-fos and N-ras, and the proliferation-associated gene ki-67 were down-regulated in AKR1B10-siRNA-transfected cells, while the expressions of apoptosis-promoting gene caspas-3 and bax were up-regulated. AKR1B10 might promote proliferation, inhibit apoptosis and then induce malignant transformation of hepatocytes by regulating the expression level of some tumor-related genes.

Ral activation promotes melanomagenesis

Up to one third of human melanomas are characterized by an oncogenic mutation in the gene encoding the small GTPase NRAS. Ras proteins activate three primary classes of effectors: Rafs, PI3Ks, and RalGEFs. In melanomas lacking NRAS mutations, the first two effectors can still be activated vis-à-vis oncogenic BRAF mutations coupled with a loss of the PI3K negative regulator PTEN. This suggests that Ras effectors promote melanoma, regardless of whether they are activated by oncogenic NRas. The only major Ras effector pathway not explored for its role in melanoma is the RalGEF-Ral pathway, in which the Ras activation of RalGEFs converts the small GTPases RalA and RalB to an active GTP-bound state. We report that RalA is activated in a number of human melanoma cancer cell lines harboring an oncogenic NRAS allele, an oncogenic BRAF allele, or wild type NRAS and BRAF alleles. Furthermore, shRNA-mediated knock down of RalA, and to a lesser extent RalB, inhibited the tumorigenic growth of melanoma cell lines having these three genotypes. Thus, as is the case for Raf and PI3K signaling, Rals also contribute to melanoma tumorigenesis.