Abstract Synthetic lethality occurs when mutations in two otherwise nonessential genes are combined to cause lethality. Because cancer is a disease of genetic alteration, synthetic lethality has been heralded as a method to identity candidate therapeutic targets, e.g., where a target gene could be "synthetic lethal" with a cancer driver mutation. To define synthetic lethal relationships in glioblastoma (GBM), we have performed multiple focused-set and genome-wide CRISPR-Cas9 lethality screens in patient-derived GBM stem-like cells (GSCs) and nontransformed human neural progenitor cells. Because GSCs isolates likely represent a sub-clone of the original tumor and we can determine GSCs' genetic and epigenetic makeup, it is possible to address the concept of synthetic lethality for GBM. To this end, we recently performed comprehensive CRISPR-Cas9 retests of all scoring GBM lethal genes (>900) from screens in three patient isolates with different and overlapping genetic drivers. We then performed secondary retests of high-priority gene targets in 13 GSC harboring various alterations commonly found in GBMs, e.g., EGFRamp, NF1mut, PIK3CAmut, PTENloss/mut, TP53mut, etc. The results were surprising, first in what we did not find. We failed to find synthetic lethal targets for TP53loss/mut, RB1mut, or TERT expression, suggesting that synthetic lethal relationships for these alterations may not exist for GBM. Second, NF1mut interactors defined a broader class of synthetic lethal targets with general overactivity of the RTK/Ras pathway, which can arise from various activating lesions. Third, candidate synthetic lethal relationships can be observed, but, so far, only with EGFRamp, MYC/MYCNamp, and PTEN/PIK3CA alterations. Thus, in general our results suggest that the majority of synthetic lethal relationships in GBM arise from oncogenic activation of the RTK/Ras and PI-3 kinase pathway or amplification of MYC/MYCN. (Synthetic lethal targets will be revealed and discussed at the meeting.) Citation Format: Pia Hoellerbauer, Sonali Arora, Megan Kufeld, Lucas Carter, Emily J. Girard, Heather Feldman, Philip Corrin, James M. Olson, Patrick J. Paddison. Emerging principles in synthetic lethality in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 413.