Project description: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). We optimized and leveraged this property to identify drug resistance mutations, a technique we term LentiMutate. After validating this technique by identifying clinically relevant EGFR resistance mutations, we applied this technique to two additional anti-cancer drugs, imatinib and AMG 510. We find novel deletions in BCR-ABL1 that confer resistance to BCR-ABL1 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.
Project description:Therapies targeting signaling molecules mutated in cancers can often have striking short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures. Resistance can sometimes result from a secondary mutations in rare cells, but other times, there is no clear genetic cause, raising leaving the possibility of non-genetic rare cell variability. Here, we show that melanoma cells can display profound transcriptional variability at the single cell level that predicts which cells will ultimately resist drug treatment. This variability involves semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces an epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming begins withis a progressive process consisting of a loss of SOX10-mediated differentiation followed by activation of new signaling pathways, partially mediated by activity of Jun-AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics. We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general rare-cell expression program.
Project description:Therapies targeting signaling molecules mutated in cancers can often have striking short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures. Resistance can sometimes result from a secondary mutations in rare cells, but other times, there is no clear genetic cause, raising leaving the possibility of non-genetic rare cell variability. Here, we show that melanoma cells can display profound transcriptional variability at the single cell level that predicts which cells will ultimately resist drug treatment. This variability involves semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces an epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming begins with a progressive process consisting of a loss of SOX10-mediated differentiation followed by activation of new signaling pathways, partially mediated by activity of Jun-AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics. We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general rare-cell expression program.