Status Of RAS Research Articles

Mutations in the Fms-Related Tyrosine Kinase 3 (FLT3) Gene Independently Predict Poor Outcome in Acute Myeloid Leukemia (AML) with t(8;21): A Study of the German-Austrian AML Study Group (AMLSG).

Abstract 825▪▪This icon denotes an abstract that is clinically relevant. Background:Acute myeloid leukemia (AML) with t(8;21)(q22;q22) is considered as a prognostically favorable subgroup of AML. However, outcome is heterogeneous and almost half of adult patients (pts) cannot be cured by current treatment. Candidate molecular markers have been assessed in an effort to predict outcome in AML with t(8;21) at the time of diagnosis and to potentially guide the development of genotype-specific approaches. In most, but not all studies, KIT mutations were associated with adverse prognosis in AML with t(8;21). However, no larger study has elucidated the independent prognostic impact of various gene mutations in a comprehensive molecular analysis. Methods:Bone marrow and/or blood specimens from 146 adult pts diagnosed with de novo (n=137), therapy-related (n=5) or unknown history (n=4) t(8;21) AML were studied for the presence of additional chromosome abnormalities and for mutations in FLT3 [internal tandem duplications (ITD) and tyrosine kinase domain mutations (TKD)], N-/K-RAS, KIT and JAK2 (V617F) genes. All pts were treated on one of 7 prospective protocols of the German-Austrian AML Study Group (AMLSG). For induction pts received anthracycline-and cytarabine-based therapy regimens; pts achieving a complete remission (CR) were assigned to postremission therapy incorporating higher doses of cytarabine in various settings or to autologous stem cell transplantation. Multivariable analyses were performed to assess the prognostic value of gene mutations on relapse-free (RFS) and overall survival (OS) and were stratified for treatment protocols. Results:Mutations were identified in 56% of the pts with the highest frequency observed in KIT (30%), followed by mutations in RAS (21%), FLT3 (13%) [ITD (9.5%) and TKD (3.5%)] and JAK2 (3.5%) genes. When correlating gene mutations with clinical features, pts with RAS mutations had a higher WBC (P=0.003) and a lower frequency of the most common secondary chromosome abnormality represented by the loss of a sex chromosome (LOS; P=0.03) when compared to pts with wild-type RAS; for the other genes studied no differences in pretreatment characteristics were observed. The median age of the study cohort at diagnosis was 46 years (yrs; range, 17-73 yrs), and the median white blood count (WBC) was 8.7 × 109/l (range, 0.9-152 × 109/l). Median follow-up for survival according to Korn was 3.4 yrs [95%-confidence interval (CI), 2.6.-5.2 yrs]. The CR rate in the entire study cohort was 89% and none of the gene mutations impacted as single marker on the CR rate. In univariable and multivariable analyses, only FLT3 mutations significantly affected relapse-free survival (RFS) and overall survival (OS). No significant difference in RFS and OS was observed with respect to the mutational status of KIT, RAS and JAK2 genes. In univariable analyses, pts with FLT3 mutations relapsed more frequently (P=0.03; 3-yr RFS rates, 22% vs 58%) and had a shorter survival time (P=0.006; 3-yr OS rates, 26% vs 63%) than those without FLT3 mutations. Multivariable analyses revealed the mutational status of FLT3 as independent prognostic variable for RFS and OS. Age was a significant risk factor for OS. Additional variables that were also included in multivariable models were mutational status of KIT and RAS, log10(WBC), and presence of LOS. Pts harboring FLT3 mutations relapsed more frequently (HR, 3.20, P=0.01) than pts with FLT3 wild-type. In addition, the risk of death in pts with FLT3 mutations was more than four times higher (HR, 4.24, P=0.004) than in pts lacking these mutations. Conclusions:In conclusion, we show here in a large group of adult AML pts with t(8;21) that the presence of activating FLT3 mutations independently predicts for poor outcome within this favorable subset of AML. Thus, adults with t(8;21)-positive AML and FLT3 mutations require alternative treatment strategies. Our data support the rationale of evaluating FLT3 tyrosine kinase inhibitors in these pts. Disclosures:No relevant conflicts of interest to declare.

Oncogenic KRAS Activates Hedgehog Signaling Pathway in Pancreatic Cancer Cells

Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAF(E600), but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression.

Melanoma Differentiation Associated Gene-7 (mda-7): A Novel Anti-Tumor Gene for Cancer Gene Therapy

The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.

No association between radiosensitivity and TP53 status, G1 arrest or protein levels of p53, myc, ras or raf in human melanoma lines

Purpose: First, to investigate whether TP53 status and/or radiation-induced G1 arrest are associated with radiosensitivity, and, second, to detect possible associations between protein levels of p53, myc, ras or raf and radiosensitivity and to investigate whether hypoxia-induced changes in the levels of these proteins are related to hypoxia-induced changes in radiosensitivity in human melanoma lines. Materials and methods: Radiosensitivity was assessed by clonogenic assays. TP53 status was investigated at the genomic level by constant denaturant gel electrophoresis and at the cDNA level by sequencing. G1 arrest was investigated by flow cytometric analysis of DNA. Protein expression of hypoxia-treated and untreated cells was assessed by flow cytometric measurements and Western blotting. Results: Considerable differences in radiosensitivity were detected among melanoma lines with wild-type TP53. Only a fraction of the melanoma cells, differing between the lines, was arrested in G1. No association between the fraction of arrested cells and radiosensitivity was detected. Protein levels of p53, myc, ras or raf were not associated with radiosensitivity. Hypoxia-induced changes in p53, ras and raf levels were detected in all cell lines. Changes in the level of myc protein were detected for two of the four cell lines, while hypoxia-induced changes in radiosensitivity were observed only for one. Conclusions: Differences in radiosensitivity among melanoma lines cannot be elucidated by TP53 status, differences in G1 arrest or different levels of p53, myc, ras or raf proteins. Hypoxia-induced changes in p53, myc, ras or raf levels do not seem to be related to hypoxia-induced changes in radiosensitivity.