Abstract 671: BKM120-mediated G2 arrest: Structural and functional segregation of off-target action and PI3K inhibition

Due to its central role in growth, proliferation, survival and migration, phosphoinositide 3-kinase (PI3K) is considered as an important drug target in oncology (1). BKM120 is one of the clinically most advanced PI3K inhibitors (PI3Ki), and is currently listed in more than 80 clinical studies aimed at attenuating tumour progression. As an off-target effect, BKM120 was reported to disrupt microtubules (MT) at concentrations around 1 μM (2). Here, we elucidate in detail the structural factors defining PI3K- and tubulin-binding of BKM120, and present a pure PI3K inhibitor (PQR309) and a potent MT disruptor (MTD147) differing from BKM120 by only 1 Dalton. Separation of PI3Ki and MT disruption activities of BKM120 allowed profiling of BKM120 against PQR309 and MTD147: cellular growth profiles of PQR309 clustered with other PI3Ki such as GDC0941/GDC0980, while BKM120 matched MTD147. Both yielded a G2/M cell cycle arrest with typical histone3 phosphorylation. Accumulation of G2/M arrested cells was already evident at concentrations yielding 50% growth inhibition. Interestingly, BKM120 concentrations for 50% cell growth inhibition (with evident G2/M arrest) ranged below or within its reported AUC0-24 levels at day 8 in patient plasma (3,4). This result implies that the two activities of BKM120 cannot be separated, thus complicating the understanding of drug action and impacting on the rational of combination therapies at relevant drug doses. Using X-ray crystallography we found that BKM120 binds to the colchicine pocket on β-tubulin. This study further highlights the importance of the pyrimidine core orientation for tight tubulin binding. Interestingly, activities of regio-isomers of the pyrimidine core are inversed for PI3Ki and tubulin association, and modulate binding by a factor of >30x. Finally, a combination of biochemical, cellular and structural data suggests an inverted orientation of BKM120 in the catalytic cleft of PI3K as previously proposed (6). In summary, the dissection of BKM120 functions allows reassessment of its dominant activity, to increase drug safety, and to flexibly control PI3K and/or MT targeting in combination therapy. 1. M. P. Wymann, R. Schneiter, Nat Rev Mol Cell Biol 9, 162 (2008).; 2. S. M. Brachmann et al., Mol Cancer Ther 11, 1747 (2012). 3. J. C. Bendell et al., J Clin Oncol 30, 282 (2012). 4. C. Saura et al., Clin Cancer Res 20, 1935 (2014). 5. A. E. Prota et al., Science 339, 587 (2013). 6. S. M. Maira et al., Mol Cancer Ther 11, 317 (2012). Citation Format: Thomas Bohnacker, Florent Beaufils, Andrea E. Prota, John E. Burke, Anna Melone, Alison J. Inglis, Ludovico Fusco, Vladimir Cmiljanovic, Natasa Cmiljanovic, Denise Rageot, Katja Bargsten, Gonzalo Saez-Calvo, Olivier Pertz, Amol B. Aher, Anna Akhmanova, Fernando J. Diaz, Doriano Fabbro, Marketa Zvelebil, Roger L. Williams, Michel O. Steinmetz, Matthias P. Wymann. BKM120-mediated G2 arrest: Structural and functional segregation of off-target action and PI3K inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 671. doi:10.1158/1538-7445.AM2015-671