453 Plasma metabolomic signature of novel signal transduction inhibitors from preclinical identification to clinical validation

function aMOI and a NF1 loss of function aMOI, while SW694 has a NF1 loss of function aMOI, but these mutations do not correlate with sensitivity to trametinib. Interestingly, HT1080 has a heterozygous mutation in NRAS (Q61K) and mutations in NRAS have been shown to correlate with sensitivity to trametinib in cell lines with BRAF wild type background. To validate whether NRAS Q61K determines sensitivity to trametinib in HT1080, we used the CRISPR/Cas9 system to repair NRAS Q61K by homologous recombination. The system utilizes the type II prokaryotic CRISPR/Cas9 adaptive immune system and targets the Cas9 nuclease by a 20 nucleotides guide sequence cloned upstream of a 50-NGG “protospacer adjacent motif” (PAM). The induced site-specific strand breaks are repaired either by nonhomologous end-joining (NHEJ) to yield indels or by homologous recombination (HR) if homologous donor templates are available, thereby enhancing the efficiency of HR-based gene targeting. We designed several different small guide RNA sequences and we verified that all sequences were able to introduce indels in the NRAS gene using a control cell line. We therefore used either wild type Cas9 or Cas9D10A (nickase) with donor DNAs to perform NRAS gene repair. Clones are actively being screened for NRAS repair. Results for these experiments will be presented in the poster. The combination of drug sensitivity data, together with the mutation sequencing and precise gene editing using the CRISPR/Cas9 system may lead to precise targeting of genomic errors and to the identification of more effective therapeutic strategies for sarcoma. Funded by NCI Contract No. HHSN261200800001E. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute.