TERT Promoter Mutations and Cancer (2016)

The vast majority of cancers depend on the aberrant expression of the enzyme telomerase reverse transcriptase (TERT) to enable their continuous replication and escape from senescence. Therefore, inhibiting this enzyme's action represents a potential cancer therapy. Two point mutations in the promoter region that regulates TERT expression have been linked to aberrant TERT expression in cancer cells. These mutations, located 124 and 146 base pairs upstream of the ATG translation start site for TERT, are both single nucleotide substitutions from cytosine to thymine. Our project sought to determine whether introducing the more common -124 mutation into otherwise normal human fibroblast cells is sufficient to cause aberrant TERT expression. A CRISPR/CRISPR-associated systems 9 (CRISPR/Cas9) genome editing experiment was performed in an attempt to introduce the TERT promoter mutation introduced into healthy human fibroblast cells. CRISPR/Cas9 utilizes a guide RNA coupled to the Cas9 nuclease to create sequence-specific single-stranded or double-stranded breaks in genomic DNA. Of the roughly 100 colonies of fibroblasts which contained significant numbers of cells following the CRISPR/Cas9 experiment, Sanger sequencing revealed that only one colony of fibroblasts contained the intended mutation. However, this successfully mutated colony experienced hampered growth and eventually senesced. There are many possible explanations for this phenotype. Off-target genome editing could have occurred which was toxic to the cells. Another explanation is that TERT may have been turned on by the mutation, but oncogene-induced senescence occurred. Yet another explanation is that the cells may have had short telomeres and senesced at a normal time. The colony of fibroblasts did not contain enough cells to perform experiments which would evaluate their levels of TERT expression and telomerase activity, therefore there were insufficient data to assess the reason for the phenotype. Difficulties with CRISPR/Cas9 genome editing prevented us from efficiently introducing the -124 TERT promoter mutation into fibroblasts. These difficulties likely stem from the chromatin environment of the TERT promoter in healthy somatic cells. In primary, mortal human fibroblasts, TERT expression is off, and the promoter is thought to be surrounded by condensed chromatin. Thus, the TERT promoter DNA of primary, mortal human fibroblasts may be largely inaccessible to CRISPR/CAS9 machinery. Support or Funding Information Funding for this project was provided by an NIH R01 grant to T.R.C, an NHMRC Postdoctoral Fellowship to J.S and an HHMI EXROP Fellowship for R.N. The Sanger sequencing chromatograms to the left correspond to the photographs on the right. The wild type fibroblasts (top right photo) far outnumber those containing the mutation (bottom right photo, individual fibroblast cells indicated by red arrows) The Sanger sequencing chromatograms to the left correspond to the photographs on the right. The wild type fibroblasts (top right photo) far outnumber those containing the mutation (bottom right photo, individual fibroblast cells indicated by red arrows)