https://openworks.mdanderson.org/sumexp22/1081/thumbnail.jp

Amero, Paola

Jansen, Joseph

Karadeniz-Saygili, Suna

Lopez-Berestein, Gabriel

Rodriguez-Aguayo, Cristian

English

OpenWorks @ MD Anderson

AbstractReferences1. St. Jude Children’s Research Hospital2. Zhu C, Wei Y, Wei X. AXL receptor tyrosine kinase as a promising anti-cancer approach: functions, molecular mechanisms and clinical applications. Mol Cancer. 2019 Nov 43. Cesarini V, Scopa C, Silvestris DA, Scafidi A, Petrera V, Del Baldo G, Gallo A. Aptamer-Based In Vivo Therapeutic Targeting of Glioblastoma. Molecules. 2020 Sep 174. Amero P, et al. Conversion of RNA Aptamer into Modified DNA Aptamers Provides for Prolonged Stability and Enhanced Antitumor Activity. J Am Chem Soc. 2021 May 265. Tang, Z. et al. (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res, 10.1093/nar/gkx2476. Cancer Cell Line Encyclopedia, https://sites.broadinstitute.org/ccle/Axl Inhibition in Pediatric Medulloblastoma Cell Lines with the GLB-A04 DNA AptamerJoseph Jansen1, Suna Karadeniz-Saygili2, Cristian Rodriguez-Aguayo2, Gabriel Lopez-Berestein2, Paola Amero2The University of Alabama1, MD Anderson Cancer Center, Department of Experimental Therapeutics2Figure 4. Cells were incubated with varying concentration of GLB-A04 orscramble aptamer for 72 hours and then collected, labeled with Annexin Vand PI and visualized via flow cytometry. No difference in viability or apoptoticcell count was found between any of the concentrations of GLB-A04 and thescramble control.Figure 1. GLB-A04 Structure, Binding, and Stability. (A) Predicted 3Dstructure of GLB-A04 according to MFold Software. (B)) Binding curve ofGLB-A04 (blue) compared to scramble aptamer. (C) Interaction of GLB-A04 aptamer and scramble aptamer (D) with Axl. (E) Stability of GLB-A04in 95% human serum.Figure 2. (A) High expression of AXL is associated with lower survival in braintumor patients (Center for Cancer Research, BioDiscovery Portal). (B) Axl isoverexpressed in brain tumors compared to normal tissue5. CCLE data6 (C)and western blot analysis (D) indicate high expression of Axl in DAOY anduw426 cell lines. (E) Gas6-stimulated (200ng/mL) medulloblastoma cell linewas treated with 400nM GLB-A04 and scramble aptamers to determine p-Axlexpression.MethodsCell lines : DAOY and uw426 cell lines were maintained in high-glucose DMEM supplemented with 10% FBS and 1% penicillin. Celllines were maintained at 37ºC with 5% CO2 and 95% air.Western Blotting: Cells were lysed in RIPA buffer with protease andphosphatase inhibitors. Protein was quantified using BCA analysisand samples were run on 4-15% polyacrylamide SDS gel. Proteinwas transferred to PVDF membrane and blocked for 30min in 6%milk. Antibodies were prepared in 5% milk and incubated for 48hr (p-Axl) or overnight. Secondary antibodies were prepared in 5% milkand incubated overnight (p-AXL) or for 90min. Bound antibodieswere visualized using ECL lighting reagents.Flow Cytometry: Apoptosis assay was carried out using FITCAnnexin V Apoptosis detection kit (BD Pharmingen). Cells weretreated with GLB-A04 at varying concentrations for 96 or 120 hours.Cells were then collected, washed with PBS, and treated with 4µL ofAnnexin V and propidium iodide. After 10min incubation in the dark,samples analyzed by flow cytometry.Conclusion• AXL is highly expressed in medulloblastoma cell lines andassociated with poor patient survival.• The GLB-A04 modified DNA aptamer downmodulated phospho-AXL in vitro.• GLB-A04 modified DNA aptamer does not induce in vitro apoptosisat the concentrations and times tested in the medulloblastoma celllines.AcknowledgmentsThis work was supported by the Summer Undergraduate ResearchProgram (SURP) at MD Anderson Cancer CenterTargeting AXL with a Highly Stable Modified Aptamer in Medulloblastoma Cell LinesJoseph Jansen1, Suna Karadeniz-Saygili2, Cristian Rodriguez-Aguayo2, Gabriel Lopez-Berestein2, Paola Amero2The University of Alabama1,  MD Anderson Cancer Center, Department of Experimental Therapeutics2Medulloblastomas are the most common types of pediatric braintumors, that start in the cerebellum. They represent approximately 20% ofpediatric brain tumors, with an average age of diagnosis between 5 and 9years old1. Surgery followed by radiation, and chemotherapy is the standardtreatment, however, it can be ineffective due to the location of the tumor andthe age of the patient. Thus, patients would benefit from non-toxic, targetedtherapeutics.AXL, a receptor tyrosine kinase, is overexpressed in many types ofcancers and associated with poor patient survival. AXL is activated by itsligand, Gas6. AXL/Gas6 signaling pathway has been shown to enhanceproliferation, invasion, metastasis, and drug resistance2.Aptamers are synthetic, single-stranded RNA or DNAoligonucleotides that selectively bind their target proteins with high affinity.Aptamers are considered chemical antibodies with the advantage to benon-immunogenic and easy tumor penetration due to their small size.The lower survival in patients expressing high levels of AXL makes ita potentially viable therapeutic target in the treatment of brain cancer.Additionally, the enhanced stability of the GLB-A04 aptamer and strongbinding to AXL have been shown to inhibit AXL in vivo. Here, although wefind no difference in cell viability or apoptotic protein expression,experiments need to be repeated with higher concentrations of aptamer andlonger treatment times.Here, we demonstrate that overexpression of AXL in GBM patients isassociated with poor survival. We then tested the ability of a highly modifiedDNA aptamer, targeting AXL to inhibit phospho-AXL and produce effectsapoptosis by Western Blot and Annexin V/PI flow cytometry analysis. Thus,aptamer-induced inhibition of phospho-AXL may be a viable therapeutictarget. Results obtained so far by us provide the strongest evidence and laythe foundation for the next stage of development to advance towards thetranslation of the aptamer candidates and companion biomarkers to theclinic.Introduction• AXL has been identified as an important driver of mesenchymal and stem-like phenotype in diffuse pontine glioma (DIPG)• Our group has generated a new molecule DNA aptamer targeting phospho-AXL with better features, more stable and higher activity, and bioavailability.• Here, we examined the effect of the modified DNA aptamer GLB-A04 on twomedulloblastoma cell lines expressing high levels of AXL. DAOY and UW426cells were treated with the DNA aptamer GLB-A04 and the effects on viabilityand apoptosis were assessed.HypothesisTargeting AXL leads to anti-tumor activity in medulloblastoma cell lines.Objectives• To determine the expression of AXL in medulloblastoma cell lines andexamine the effect on survivability.• To determine the ability of GLB-A04 to inhibit phospho-AXL inmedulloblastoma cell lines.• To examine the effects of GLB-A04 to induce apoptosis.ResultsFigure 3. Cells treated with concentrations of 400nM, 800nM, and 1.6µM GLB-A04 and 1.6µM scramble aptamer showed no difference in expression of pro-death apoptotic proteinsGLB-A04 Preliminary DataAxl expression is correlated with poor survival, is highly expressed in brain tumor and medulloblastoma cell lines, and Axl activation is inhibited by GLB-A04Treatment with GLB-A04 fails to impact cell viability in Annexin V/PI Flow CytometryTreatment with GLB-A04 does not impactexpression of apoptotic proteins in DAOY and uw426 cell lines

Targeting AXL with a Highly Stable Modified Aptamer in Medulloblastoma Cell Lines

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1081&context=sumexp22

Targeting AXL with a Highly Stable Modified Aptamer in Medulloblastoma Cell Lines

Abstract

Similar works

Full text

Available Versions

OpenWorks @ MD Anderson