Targeting central B cell tolerance checkpoints in acute lymphoblastic leukemia
Final Report Abstract
Next generation sequencing generates increasing amounts of data, which comprise gross gene aberrations as well as distinct single nucleotide substitutions. Substitutions are classified into being non-synonymous (replacement mutations), which leads to a change in the respective amino acid, while in case the corresponding amino acid remains the same, the substitution is referred to as being synonymous (silent mutation). When analyzing sequencing data, silent mutations are often disregarded and overlooked, since most of the time, no functional consequence is to be expected. The here described project is based on the hypothesis that while malignant cells might tolerate silent mutations in tumor relevant genes, replacement mutations that could negatively alter essential gene functions should be selected against during tumor evolution. In this scenario, tumor relevant genes would display an increase in silent mutations as compared to replacements. This can be determined with the replacement/silent (R/S)-ratio, by calculating the deviation for a specific gene from the R/S ratio of 2.9, which represents the ratio expected in an unbiased setting. To proof this concept, for each gene, R/S ratios for 25447 primary patient samples taken from the online data platform COSMIC were generated and a total of around 5.5 million substitutions (replacement and silent) were analyzed. The top 25 highly replacement enriched genes across all cancers constituted well known tumor suppressors e.g. TP53 and RB1 (inactivating replacements) as well as oncogenes, including KRAS and NRAS (activating replacements). As expected, the top 25 genes that showed silent enrichment in this PAN cancer analysis represented a variety of essential proteins for a cell, like TBP, MLLT3 and UBB. While these results strengthen the hypothesis that R/S ratios can be used to predict the importance of a gene, ongoing studies on this data currently analyze the R/S ratios for each gene on individual cancer subtype level, rather than across all cancers taken together. This more refined approach will give insights into tumor relevant genes and how they differ within specific cancer types. In a second step, the project was directed towards a functional validation of the data retrieved from comparing R/S ratios in different tumor subtypes. To establish a method that allows the targeted introduction of mutations into genes that show high silent mutation enrichment in the respective cancer, a CRISPR-Cas9 based pilot experiment was designed. In this approach, a dCas9-AID fusion construct was paired with an inducible sgRNA system, allowing the controlled introduction of AID-mediated mutations on specific DNA sequences. First success was achieved by using this system to generate mutations within the variable immunoglobulin heavy chain locus of the Burkitt Lymphoma cell line DAUDI. Nevertheless, in the future, additional refinements of the method are needed to properly utilize this technique to efficiently target genes of interest.
Publications
- Loss of Pax5 Exploits Sca1-BCR-ABL(p190) Susceptibility to Confer the Metabolic Shift Essential for pB-ALL. Cancer Res, 2018. 78(10): p. 2669-2679
Martin-Lorenzo, A., et al.
(See online at https://doi.org/10.1158/0008-5472.CAN-17-3262) - Histone H3 trimethylation at lysine 36 guides m(6)A RNA modification co-transcriptionally. Nature, 2019. 567(7748): p. 414-419
Huang, H., et al.
(See online at https://doi.org/10.1038/s41586-019-1016-7) - JAK2 p.G571S in B-cell precursor acute lymphoblastic leukemia: a synergizing germline susceptibility. Leukemia, 2019
Lin, M., et al.
(See online at https://doi.org/10.1038/s41375-019-0459-z)
