TAT-RasGAP317–326-mediated tumor cell death sensitization can occur independently of Bax and Bak

The increase of cancer specificity and efficacy of anti-tumoral agents are prime strategies to overcome the deleterious side effects associated with anti-cancer treatments. We described earlier a cell-permeable protease-resistant peptide derived from the p120 RasGAP protein, called TAT-RasGAP_317–326, as being an efficient tumor-specific sensitizer to apoptosis induced by genotoxins in vitro and in vivo. Bcl-2 family members regulate the intrinsic apoptotic response and as such could be targeted by TAT-RasGAP_317–326. Our results indicate that the RasGAP-derived peptide increases cisplatin-induced Bax activation. We found no evidence, using in particular knock-out cells, of an involvement of other Bcl-2 family proteins in the tumor-specific sensitization activity of TAT-RasGAP_317–326. The absence of Bax and Bak in mouse embryonic fibroblasts rendered them resistant to cisplatin-induced apoptosis and consequently to the sensitizing action of the RasGAP-derived peptide. Surprisingly, in the HCT116 colon carcinoma cell line, the absence of Bax and Bak did not prevent cisplatin-induced apoptosis and the ability of TAT-RasGAP_317–326 to augment this response. Our study also revealed that p53, while required for an efficient genotoxin-induced apoptotic response, is dispensable for the ability of the RasGAP-derived peptide to improve the capacity of genotoxins to decrease long-term survival of cancer cells. Hence, even though genotoxin-induced Bax activity can be increased by TAT-RasGAP_317–326, the sensitizing activity of the RasGAP-derived peptide can operate in the absence of a functional mitochondrial intrinsic death pathway.     

A WXW Motif Is Required for the Anticancer Activity of the TAT-RasGAP317–326 Peptide

TAT-RasGAP_317–326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP_317–326 sequence for the anticancer activities of TAT-RasGAP_317–326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP_317–326.     

Revisiting G3BP1 as a RasGAP binding protein: sensitization of tumor cells to chemotherapy by the RasGAP 317-326 sequence does not involve G3BP1

RasGAP is a multifunctional protein that controls Ras activity and that is found in chromosomal passenger complexes. It also negatively or positively regulates apoptosis depending on the extent of its cleavage by caspase-3. RasGAP has been reported to bind to G3BP1 (RasGAP SH3-domain-binding protein 1), a protein regulating mRNA stability and stress granule formation. The region of RasGAP (amino acids 317–326) thought to bind to G3BP1 corresponds exactly to the sequence within fragment N2, a caspase-3-generated fragment of RasGAP, that mediates sensitization of tumor cells to genotoxins. While assessing the contribution of G3BP1 in the anti-cancer function of a cell-permeable peptide containing the 317–326 sequence of RasGAP (TAT-RasGAP_317–326), we found that, in conditions where G3BP1 and RasGAP bind to known partners, no interaction between G3BP1 and RasGAP could be detected. TAT-RasGAP_317–326 did not modulate binding of G3BP1 to USP10, stress granule formation or c-myc mRNA levels. Finally, TAT-RasGAP_317–326 was able to sensitize G3BP1 knock-out cells to cisplatin-induced apoptosis. Collectively these results indicate that G3BP1 and its putative RasGAP binding region have no functional influence on each other. Importantly, our data provide arguments against G3BP1 being a genuine RasGAP-binding partner. Hence, G3BP1-mediated signaling may not involve RasGAP.     

miR-326-Histone Deacetylase-3 Feedback Loop Regulates the Invasion and Tumorigenic and Angiogenic Response to Anti-cancer Drugs

Histone modification is known to be associated with multidrug resistance phenotypes. Cancer cell lines that are resistant or have been made resistant to anti-cancer drugs showed lower expression levels of histone deacetylase-3 (HDAC3), among the histone deacetylase(s), than cancer cell lines that were sensitive to anti-cancer drugs. Celastrol and Taxol decreased the expression of HDAC3 in cancer cell lines sensitive to anti-cancer drugs. HDAC3 negatively regulated the invasion, migration, and anchorage-independent growth of cancer cells. HDAC3 conferred sensitivity to anti-cancer drugs in vitro and in vivo. TargetScan analysis predicted miR-326 as a negative regulator of HDAC3. ChIP assays and luciferase assays showed a negative feedback loop between HDAC3 and miR-326. miR-326 decreased the apoptotic effect of anti-cancer drugs, and the miR-326 inhibitor increased the apoptotic effect of anti-cancer drugs. miR-326 enhanced the invasion and migration potential of cancer cells. The miR-326 inhibitor negatively regulated the tumorigenic, metastatic, and angiogenic potential of anti-cancer drug-resistant cancer cells. HDAC3 showed a positive feedback loop with miRNAs such as miR-200b, miR-217, and miR-335. miR-200b, miR-217, and miR-335 negatively regulated the expression of miR-326 and the invasion and migration potential of cancer cells while enhancing the apoptotic effect of anti-cancer drugs. TargetScan analysis predicted miR-200b and miR-217 as negative regulators of cancer-associated gene, a cancer/testis antigen, which is known to regulate the response to anti-cancer drugs. HDAC3 and miR-326 acted upstream of the cancer-associated gene. Thus, we show that the miR-326-HDAC3 feedback loop can be employed as a target for the development of anti-cancer therapeutics.     

Synergistic Anti-Cancer Effect of Phenformin and Oxamate

Phenformin (phenethylbiguanide; an anti-diabetic agent) plus oxamate [lactate dehydrogenase (LDH) inhibitor] was tested as a potential anti-cancer therapeutic combination. In in vitro studies, phenformin was more potent than metformin, another biguanide, recently recognized to have anti-cancer effects, in promoting cancer cell death in the range of 25 times to 15 million times in various cancer cell lines. The anti-cancer effect of phenformin was related to complex I inhibition in the mitochondria and subsequent overproduction of reactive oxygen species (ROS). Addition of oxamate inhibited LDH activity and lactate production by cells, which is a major side effect of biguanides, and induced more rapid cancer cell death by decreasing ATP production and accelerating ROS production. Phenformin plus oxamate was more effective than phenformin combined with LDH knockdown. In a syngeneic mouse model, phenformin with oxamate increased tumor apoptosis, reduced tumor size and ¹⁸F-fluorodeoxyglucose (FDG) uptake on positron emission tomography/computed tomography compared to control. We conclude that phenformin is more cytotoxic towards cancer cells than metformin. Furthermore, phenformin and oxamate have synergistic anti-cancer effects through simultaneous inhibition of complex I in the mitochondria and LDH in the cytosol, respectively.     

Anticancer Activity of Amauroderma rude

More and more medicinal mushrooms have been widely used as a miraculous herb for health promotion, especially by cancer patients. Here we report screening thirteen mushrooms for anti-cancer cell activities in eleven different cell lines. Of the herbal products tested, we found that the extract of Amauroderma rude exerted the highest activity in killing most of these cancer cell lines. Amauroderma rude is a fungus belonging to the Ganodermataceae family. The Amauroderma genus contains approximately 30 species widespread throughout the tropical areas. Since the biological function of Amauroderma rude is unknown, we examined its anti-cancer effect on breast carcinoma cell lines. We compared the anti-cancer activity of Amauroderma rude and Ganoderma lucidum, the most well-known medicinal mushrooms with anti-cancer activity and found that Amauroderma rude had significantly higher activity in killing cancer cells than Ganoderma lucidum. We then examined the effect of Amauroderma rude on breast cancer cells and found that at low concentrations, Amauroderma rude could inhibit cancer cell survival and induce apoptosis. Treated cancer cells also formed fewer and smaller colonies than the untreated cells. When nude mice bearing tumors were injected with Amauroderma rude extract, the tumors grew at a slower rate than the control. Examination of these tumors revealed extensive cell death, decreased proliferation rate as stained by Ki67, and increased apoptosis as stained by TUNEL. Suppression of c-myc expression appeared to be associated with these effects. Taken together, Amauroderma rude represented a powerful medicinal mushroom with anti-cancer activities.     

Design,synthesis and biological evaluation of 2-methyl-(1,1′-biphenyl)-pyrimidine conjugates

Small molecule inhibitors of biphenyl structure as core backbone have shown a significant effect on PD-1/PD-L1 axis, and 2-amino-pyrimidine structure is a promising privileged scaffold in medicinal chemistry and drug discovery. We designed by combination principles and synthesized 27 novel compounds with N-((2-methyl-[1,1′-biphenyl]-3-yl)methyl)pyrimidin-2-amine as a basic skeletal structure, and their anti-cancer activity was evaluated. Among compounds, 15a-d and 16b displayed strong anti-cancer effects on 9 tested cancer cell lines, in particular, the 16b did the highest inhibitive activity, but against HepG2 cells, and possessed the lowest IC₅₀ value of 2.08 μΜ towards HT-29 cells.     

Liposome-Induced Conformational Changes of an Epitopic Peptide and its Palmitoylated Derivative of Influenza Virus Hemagglutinin

The conformation of synthetic HA_317-329-NH₂representing the major B- and T-cell epitopic region of influenza virus hemagglutinin, its palmitoylated derivative (HA_317-329-Thr(Pal)-NH₂), and the intersubunit peptide (HA_317-341-NH₂) comprising also the fusion peptide, were studied in aqueous buffer and in the presence of neutral and negatively charged liposomes. The free peptide is unordered in aqueous solution, even in the presence of liposomes. However, grafting the palmitic acid or the fusion peptide onto the C-terminus of the peptide enables the hydrophilic HA_317-329to adopt folded (turn) and β-strand structure on the surface of neutral and negatively charged liposomes, respectively. The results emphasize the importance of some kind of anchor for achieving a specific conformation of epitopic peptide HA_317-329-NH₂on the surface of liposomes.     

Wee1 Inhibition by MK-1775 Leads to Tumor Inhibition and Enhances Efficacy of Gemcitabine in Human Sarcomas

Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10–20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible and new agents are needed. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by MK1775 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo both alone and in combination with gemcitabine, which is frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of gemcitabine in sarcoma cells lines with different p53 mutational status. In patient-derived bone and soft tissue sarcoma samples we showed that MK1775 alone and in combination with gemcitabine causes significant apoptotic cell death. Magnetic resonance imaging (MRI) and histopathologic studies showed that MK1775 induces significant cell death and terminal differentiation in a patient-derived xenograft mouse model of osteosarcoma in vivo. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients.     

Preclinical small molecule WEHI-7326 overcomes drug resistance and elicits response in patient-derived xenograft models of human treatment-refractory tumors

Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.Subject terms: Breast cancer, Cancer models, Cancer therapeutic resistance, Drug development, Mitosis     

Mapping of a protective helper T cell epitope of human influenza A virus hemagglutinin

The synthetic peptide comprising the 317-341 region of human influenza A virus (H1N1 subtype) hemagglutinin elicits peptide-specific antibody and helper T cell responses and confers protection against lethal virus infection. Molecular mapping of the 317-329 region, which encompasses the epitope recognized by peptide-specific T cells, revealed that the minimal size required for T cell activation was the 317-326 segment. The most likely peptide alignment, which placed 320Leu to pocket 1 of the I-E(d) peptide binding groove, was predicted by molecular mechanics calculations performed with the parental and with the Ala-substituted analogs. In line with the prediction data, the results of the peptide binding assay, where the relative binding efficiency to I-E(d) molecules expressed on the surface of antigen-presenting cells was monitored, identified the 320-326 core sequence interacting with the major histocompatibility class II peptide binding groove. Functional analysis of Ala-substituted variants by functional assays and by calculating the surface-accessible areas of the single peptidic amino acids in the I-E(d)-peptide complexes demonstrated that 324Pro is a primary contact residue for the T cell receptor. Our results show that this type of analysis offers a suitable tool for molecular mapping of helper T cell epitopes and thus provides valuable data for subunit vaccine design.     

Lymphocyte-induced production of prostaglandin E2 by tumor cells in vitro: Requirements for direct contact and lymphocyte viability

The production of prostaglandin E₂ by tumor cell lines in response to exposure to purified lymphocytes has prompted the suggestion that this phenomenon may represent a defense mechanism whereby tumors may subvert an immune response mounted against them. To further characterize this phenomenon, cell lines derived from carcinogen-induced bladder tumors and embryo fibroblasts in Fischer rats were incubated with purified lymphocytes from peripheral blood, spleen, thymus, and lymph nodes from Fischer rats under a variety of conditions, and the amount of prostaglandin E₂ (PGE₂) production was determined by radioimmunoassay. Increased numbers of blood or splenic lymphocytes were associated with the induction of increased levels of PGE₂ production by the tumor cells. However, no prostaglandin was produced by the tumor cells after exposure to thymus or lymph node lymphocytes. Irradiation of lymphocytes prior to exposure to the tumor cells led to lower levels of PGE₂ production by the tumors, as did sonication of the lymphocyte preparations prior to addition to the tumor monolayers. Separation of lymphocytes from direct contact with the tumor cells resulted in less PGE₂ production by the tumor cell lines; however, when these lymphocytes were later layered onto fresh tumor cell monolayers, PGE₂ production occurred. Results in the present study suggest that direct contact between intact, viable, functionally active lymphocytes and tumor cells is necessary for tumor cell prostaglandin production to occur. Moreover, PGE₂ production only appears to occur in response to exposure to particular populations of lymphocytes, and this may correlate with the number of specific effector or attacker lymphocytes that are present. This specificity of response to effector cell challenge may be important in probing the defense mechanisms tumor cells may have to lymphocyte challenge, as well as in gauging the efficacy of a particular cellular immune response as it may be regulated both by cells involved in effecting this response as well as by the targets in lymphocyte/tumor cell interactions.     

Caspase dependent apoptotic activity of polycyclic cage-like heterocyclic hybrids

A small library of cage-like heterocyclic hybrids encompassing pyrroloisoquinolines, pyridinone and acenaphthene structural moieties have been synthesized and tested for their potential as anticancer agents against HCT116 and JURKAT cell lines. The results revealed that these cell lines are more sensitive towards compound 1g and it showed dose dependent cytotoxic effect at 48 hrs of incubation. The IC₅₀ values of compound 1g against HCT116 and JURKAT cell lines are 12.14 ± 1.53 and 10.68 ± 0.68 µM, respectively. Further studies on the determination of mechanism of action of compound 1g discovered that it brought the cell death by inducing Caspase 3 dependent apoptosis and also by arresting the cell cycle at S phase. These studies revealed that compound 1g can be recommended as a potential anti-cancer agent.     

Synthesis of retinoid enhancers based on 2-aminobenzothiazoles for anti-cancer therapy

Indole-3-amides and dipeptides were produced from 2-aminobenzothiazoles using the PyBop peptide coupling reagent. These analogues were tested in anti-cancer cell viability assays against SH-SY5Y neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines, and were found to exhibit cytotoxic activities at concentrations ranging from 0.1 to 20 μM. These compounds were also found to act additively with a low dosage of 13-cis-retinoic acid in neuroblastoma cells. Then, using neuroblastoma cells transfected to stably overexpress the RARβ₂ gene, a SAR was developed for the indole-3-amides. Real-time PCR was also used to demonstrate their RARβ₂ agonistic activity.     

Generation of human-melanoma-specific T lymphocyte clones defining novel cytolytic targets with panels of newly established melanoma cell lines

Melanoma is a cancer where the immune system is believed to play an important role in the control of malignant cell growth. To study the variability of the immune response in melanoma patients, we derived melanoma cell lines from several HLA-A2⁺ and HLA-A2^– patients. The melanoma cell lines studied were designated FM3, FM6, FM9, FM28, FM37, FM45, FM55_P, FM55_M1 and FM55_M2 and were established from eight metastatic tumors as well as from one primary tumor from a total of seven different patients. On the basis of the ability of tumor cells to induce specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes (PBL) in mixed lymphocyte/tumor culture with HLA-A2⁺ melanoma cells, the FM3 cell line was characterized as highly immunogenic. To investigate the expression of different melanoma-associated antigens recognized by CTL on different melanoma cell lines, we selected the cell line FM3 for restimulation and further T cell cloning experiments. The lytic activity of CTL clones with good proliferative activity was examined using a panel of HLA-A2⁺ and HLA-A2^– melanoma cell lines. None of the tested HLA-A2^– melanoma cell lines were susceptible to lysis by the CTL clones, whereas allogeneic HLA-A2⁺ melanoma cell lines were lysed only by a few CTL clones. On the basis of their reactivity with different melanoma cell lines, it was possible to divide the present CTL clones into at least four groups suggesting the recognition of at least four different antigens. Three of these target structures probably are different from already-described HLA-A2-restricted melanoma-associated antigens, because their expression in the different melanoma cell lines do not correlate with the recognition of melanoma cells by these CTL. The results first indicate that poorly immunogenic melanoma cells may express melanoma-associated antigens, and also suggest that, by using CTL clones obtained against different HLA-class-I-matched melanoma cells, it is possible to define such antigens.     

Intratumoral peptide injection enhances tumor cell antigenicity recognized by cytotoxic T lymphocytes: a potential option for improvement in antigen-specific cancer immunotherapy

Antigen-specific cancer immunotherapy is a promising strategy for improving cancer treatment. Recently, many tumor-associated antigens and their epitopes recognized by cytotoxic T lymphocytes (CTLs) have been identified. However, the density of endogenously presented antigen-derived peptides on tumor cells is generally sparse, resulting in the inability of antigen-specific CTLs to work effectively. We hypothesize that increasing the density of an antigen-derived peptide would enhance antigen-specific cancer immunotherapy. Here, we demonstrated that intratumoral peptide injection leads to additional peptide loading onto major histocompatibility complex class I molecules of tumor cells, enhancing tumor cell recognition by antigen-specific CTLs. In in vitro studies, human leukocyte antigen (HLA)-A*02:01-restricted glypican-3_144–152 (FVGEFFTDV) and cytomegalovirus_495–503 (NLVPMVATV) peptide-specific CTLs showed strong activity against all peptide-pulsed cell lines, regardless of whether the tumor cells expressed the antigen. In in vivo studies using immunodeficient mice, glypican-3_144–152 and cytomegalovirus_495–503 peptides injected into a solid mass were loaded onto HLA class I molecules of tumor cells. In a peptide vaccine model and an adoptive cell transfer model using C57BL/6 mice, intratumoral injection of ovalbumin_257–264 peptide (SIINFEKL) was effective for tumor growth inhibition and survival against ovalbumin-negative tumors without adverse reactions. Moreover, we demonstrated an antigen-spreading effect that occurred after intratumoral peptide injection. Intratumoral peptide injection enhances tumor cell antigenicity and may be a useful option for improvement in antigen-specific cancer immunotherapy against solid tumors.     

Synthesis,characterization, in vitro SAR and in vivo evaluation of N,N′bisnaphthylmethyl 2-alkyl substituted imidazolium salts against NSCLC

Alkyl- and N,N′-bisnaphthyl-substituted imidazolium salts were tested in vitro for their anti-cancer activity against four non-small cell lung cancer cell lines (NCI–H460, NCI–H1975, HCC827, A549). All compounds had potent anticancer activity with 2 having IC₅₀ values in the nanomolar range for three of the four cell lines, a 17-fold increase in activity against NCI-H1975 cells when compared to cisplatin. Compounds 1–4 also showed high anti-cancer activity against nine NSCLC cell lines in the NCI-60 human tumor cell line screen. In vitro studies performed using the Annexin V and JC-1 assays suggested that NCI-H460 cells treated with 2 undergo an apoptotic cell death pathway and that mitochondria could be the cellular target of 2 with the mechanism of action possibly related to a disruption of the mitochondrial membrane potential. The water solubilities of 1–4 was over 4.4 mg/mL using 2-hydroxypropyl-β-cyclodextrin as a chemical excipient, thereby providing sufficient solubility for systemic administration.     

A novel diquinolonium displays preclinical anti-cancer activity and induces caspase-independent cell death

Quinolines are a class of chemical compounds with emerging anti-cancer properties. Here, we tested the activity of series of quinolines and quinoline-like molecules for anti-cancer activity and identified a novel diquinoline, 1-methyl-2-[3-(1-methyl-1,2-dihydroquinolin-2-yliden)prop-1-enyl]quinolinium iodide (Q²). Q² induced cell death in leukemia, myeloma, and solid tumor cell lines with LD50s in the low to submicromolar range. Moreover, Q² induced cell death in primary acute myeloid leukemia (AML) cells preferentially over normal hematopoietic cells. In a mouse model of leukemia, Q² delayed tumor growth. Mechanistically, Q² induced cell death through caspase independent mechanisms. By electron microscopy, Q² increased cytoplasmic vacuolization and mitochondrial swelling. Potentially consistent with the induction of autophagic cell death, Q² treatment led to a punctate distribution of LC3 and increased MDC staining. Thus, Q² is a novel quinolinium with preclinical activity in malignancies such as leukemia and myeloma and warrants further investigation.     

Downregulation of Axl in non-MYCN amplified neuroblastoma cell lines reduces migration

Neuroblastomas (NBL) are common pediatric solid tumors with a variable clinical course. At diagnosis half of all neuroblastoma patients presents with metastatic disease. The mechanisms of metastasis are largely unknown. Gene expression profiles (HU133plus2.0 arrays, Affymetrix) of 17 NBL and 5 peripheral neuro-ectodermal cell lines were used to identify a subgroup of non-MYCN amplified (non-NMA) NBL cell lines with a distinct gene expression profile and characterized by high expression of AXL. Axl is a tyrosine kinase receptor which plays a role in the metastatic process of several types of cancer. We hypothesized that Axl contributes to the metastasizing potential of non-NMA NBL and tested if AXL silencing diminishes malignant properties of high Axl expressing cell lines.