Abstract 154: Novel AKT isoform-preferred substrates as potentially targetable drivers of metastatic prostate cancer
Authors: Degan Seamus , Holtz Renae , Withers Henry G. , Qu Jun , Rasam Sailee Suryakant , Gelman Irwin H. 
Published: June 2022

PI3K/AKT pathway activating mutations are the most prevalent in all of human cancers, and yet drugs that target the PI3K lipid kinase or AKT serine/threonine kinase families have performed poorly in clinical trials on solid cancers. PTEN loss, one of the most frequent mutations in prostate cancer (PC), results in AKT activation, an assumed driver of aggressive metastatic disease (mPC). The poor efficacy of PI3K/AKT inhibitors in PC likely relate to multiple PI3K and AKT isoforms which have compensatory or even oppositional roles. Whereas PI3K-p110 isoform-specific inhibitors are being used in clinical trials, no clinical AKT isoform-specific inhibitors exist. Data from our lab and others indicate that progression to aggressive mPC is dependent on AKT2-, and possibly AKT3-specific pathways. However, previous screens could not distinguish between direct vs. indirect AKT isoform-preferred substrates, and they only focused on substrates with canonical phosphorylation motifs (RxRxxS*/T*). We addressed this gap using a novel phosphoproteomics analysis that identifies direct AKT isoform substrates from PC lysates in which substrates are in endogenous protein-protein complexes. Scores of novel and previously known substrates were identified that are preferred by individual or combinations of AKT isoforms, conforming to both the canonical and four novel non-canonical motifs; &gt;95% of these sites exist in phosphoproteome databases. By comparing data from PC cell lines isogenic for PTEN loss or expression, we identified PTEN-influenced substrate phosphorylation or AKT isoform preference. However, most of these cases did not correlate with changes in substrate abundance, suggesting novel roles for PTEN in regulating substrate modifications or cellular compartmentalization. We hypothesize that AKT2 and AKT3 substrate phosphorylation drives parameters of in vitro and in vivo mPC progression, suggesting that these AKT isoforms may be therapeutic targets if specific inhibitors can be produced. Data will be shown i) confirming AKT isoform preference of specific substrates, ii) identifying novel mechanisms by which PTEN influences substrate usage or isoform preference via altering cellular compartmentalization or post-translational modification, and iii) validating our identified non-canonical AKT isoform-specific phosphorylation sites. We also show that the phosphorylation of AKT2/3-preferred substrates drives metastatic progression in vitro and in vivo. Our data identify a much broader array of AKT isoform-specific, -preferred and -shared substrates, including those encoding previously unidentified non-canonical phosphorylation motifs. We also show novel function for PTEN, namely, to influence AKT isoform substrate usage and preference. These data will allow us to develop AKT isoform-specific inhibitors that could be used to suppress mPC progression.

Citation Format: Seamus Degan, Renae Holtz, Henry G. Withers, Jun Qu, Sailee Suryakant Rasam, Irwin H. Gelman. Novel AKT isoform-preferred substrates as potentially targetable drivers of metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 154.