Lentiviral-based mutagenesis to identify mutations that confer resistance to anti-cancer drugs [lenti]

Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT) and we show this property can be leveraged to identify mutations that confer resistance to targeted anti-cancer drugs, a technique we term “LentiMutate”. First, we improved LentiMutate by making the lentiviral RT more error-prone. Next, we applied this technique to two anti-cancer drugs, imatinib and AMG 510. We find novel deletions in BCR-ABL that confer resistance to BCR-ABL inhibitors and point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations, in KRAS-G12C or wild-type KRAS, respectively, that confer resistance to AMG 510. LentiMutate may prove highly valuable to clinical and preclinical cancer drug development The overall design of all these experiments is a technique known as LentiMutate. Here a cDNA of a drug target is packaged into a lentiviral particle, which then infects cells, generates mutations at a low frequency, and then the cells are treated with an anti-cancer drug that targets the cDNA. Cells that have mutations in the cDNA that cause resistance to the drug are able to survive drug treatment, and the cDNA in these cells is sequenced and compared to the parental population to identify drug resistnace mutations (since drug resistance mutations will be enriched in this surviving population)