Poster Presentation Clinical Oncology Society of Australia Annual Scientific Meeting 2019

Novel human cancer cell-line genomic model using CRISPR/Cas9 for assessing methotrexate pharmacogenomics: MiSSK Study – Phase I (#157)

Zeyad Ibrahim 1 , Evan Ingley 2 , Julie Marsh 3 , Rhonda Clifford 4
  1. PCH and UWA, Shelley, WA, Australia
  2. Cell Signaling Group, Harry Perkins Institute of Medical Research, Nedlands, WA, Australia
  3. Biostatistics, Telethon Kids Institute, Nedlands, WA, Australia
  4. School of Allied Health and Medical Sciences, University of Western Australia, Nedlands, WA, Australia

Background

 Methotrexate (MTX) is an antimetabolite that interferes with folic-acid cycle enzymes (primarily Dihydrofolate Reductase), inhibiting cell growth. Thus, MTX is used to treat various cancers and other autoimmune diseases. Folic-acid cycle enzymes and protein-transporters exhibit extensive polymorphisms that strongly impact individual patients’ ability to handle MTX, especially at high doses. To delineate the effects of these polymorphisms, appropriate genomic models are needed.

 Aim (Phase I of Methotrexate in Seriously Sick Kids (MiSSK) Study):

 Design and build different in-vitro models for human cancer cell-lines that express polymorphisms of folic-acid cycle enzymes, using CRISPR/Cas9 genomic engineering.

  1. Test the effect of each polymorphism on cellular sensitivity to different MTX doses.

 Methodology and results:

 Cellular models were generated using polymorphisms with the most significant effect on MTX kinetics; methylene tetrahydrofolate reductase (MTHFR) enzyme and reduced-folate carrier (RFC/SLC19A1) transporter-protein. Human osteosarcoma cells (143B) and lymphoblastic-leukaemia cells (CCRF-CEM119) were used to quantify the effect in cancer cells whilst human embryonic-kidney cells (HEK293) were utilised as a non-cancer-control. Each cell-line underwent the following modifications:

 

  1. MTHFR and/or RFC gene-knockout (KO) using CRISPR/Cas9 gene-editing techniques. KO clones were isolated by antibiotic selection followed by multiple rounds of flow-cytometric cell sorting.
  2. Transfection of each KO cell-line with either wild-type genes (MTHFR or SLC19A1), or variants generated by site-directed mutagenesis (MTHFR_C677T, MTHFR_A1298C or SLC19A1_G>A80). Variant-expressing clones were isolated by antibiotic selection followed by multiple rounds of flow-cytometric cell sorting.

After generation, the polymorphic-variant cell models were tested for MTX biological cytotoxic effects, using proliferation/viability assays (IncuCyteZOOM real-time cell monitoring). Metabolic effects were investigated by determining residual MTX concentrations in media and cell-lysates via a novel High-Performance Liquid Chromatography (HPLC) methodology.

 Conclusion:

 Polymorphic-variant cell models were successfully generated to examine the relationship between genomics and MTX metabolism in an attempt to individualise MTX treatment in practice.

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