Abstract A117: An integrated phosphoproteomic approach to dissect the mechanism of action of the novel dual PI3K/mTOR inhibitor PQR309 in B-cell lymphomas
Authors: Tarantelli Chiara , Gaudio Eugenio , Kwee Ivo , Cascione Luciano , Bernasconi Elena , Hillmann Petra , Stussi Georg , Fabbro Doriano , Zucca Emanuele , Wicki Andreas , Cmiljanovic Vladimir , Bertoni Francesco 
Published: May 2016

Introduction. PQR309 is a novel oral dual PI3K/mTOR inhibitor (Cmiljanovic et al, AACR 2015) that has shown pre-clinical activity in lymphomas (Tarantelli et al, ASH 2014). Positive results from the first-in-human Phase 1 trial in patients with advanced solid tumors have just been presented (NCT01940133; Kristeleit et al, ASCO 2015). A phase I is now open in patients with relapsed or refractory lymphomas (NCT02249429). Here, we present results from extensive proteomic studies to dissect the PQR309 mechanism of action in lymphomas.

Methods. 8 cell lines derived from mantle cell lymphoma (MCL, n = 3), activated B-cell like diffuse large B cell lymphoma (ABC-DLBCL, n = 2), germinal center B cell DLBCL (GCB-DLBCL, n = 2), prolymphocytic leukemia (n = 1), exposed to DMSO or PQR309, were analyzed with the PathScan Akt Signaling Antibody Array (Cell Signaling Technology). Six of the cell lines (2 MCL, 2 ABC-DLBCL, 2 GCB-DLBCL) were also analyzed via Reverse Phase Protein Array (RPPA) (Carna Biosciences, Inc., Kobe Japan) and mass spectrometry (Biognosys, Schlieren, Switzerland). Changes at individual phosphoresidues were validated by immunoblotting or flow cytometry.

Results. Lymphoma cell lines derived from mature lymphoid neoplasms were exposed to PQR309 or to DMSO and analyzed for phosphorylation changes using different proteomic approaches. We first performed an analysis using an antibody-based array exploring phosphorylation changes in 14 proteins involved in the AKT signaling pathway, detecting reduced phosphoresidues after PQR309 in 10 of these proteins (RPS6, PDK1, GSK3B, AKT1S1, BAD, RPS6KA1, PTEN, PRKAA1/PRKAA2, AKT-Thr308, P70S6K-Thr421/Ser424). Then, we assessed 180 phospho-residues via RPPA, detecting a down-regulation of phosphoresidues in 7 proteins (RPS6, EIF4G, EIF4EBP1, MAPK1/MAPK3, SMAD3, AKT1S1, MTOR). Finally, phosphopeptides were enriched and prepared for mass spectrometry. LC-MS/MS shotgun measurements were carried out on protein libraries enriched for phosphopeptides, identifying a total of 4,349 unique phosphopeptide sequences. A functional analyses of the observed changes in phosphopeptides showed that these were enriched in proteins involved in transcription and metabolism regulation, RAS signaling, G2/M checkpoint, mitotic spindle assembly, unfolded protein response, NF-kB signaling, PI3K/AKT/mTOR pathway, DNA repair, and in proteins encoded by target genes of E2F, MYC and SP1. CXCR4, T2FA, SRRM2, SUGP2 were among the top up-regulated phosphopeptides, while RPS6, PDCD4, IF4B were among the top down-regulated phosphopeptides.

Conclusions. The use of multiple proteomic approaches allowed the identification of pathway and proteins affected by PQR309 in lymphoma cells, providing insights of the drug mechanism of action and new potential pharmacodynamics markers.

Citation Format: Chiara Tarantelli, Eugenio Gaudio, Ivo Kwee, Luciano Cascione, Elena Bernasconi, Petra Hillmann, Georg Stussi, Doriano Fabbro, Emanuele Zucca, Andreas Wicki, Vladimir Cmiljanovic, Francesco Bertoni. An integrated phosphoproteomic approach to dissect the mechanism of action of the novel dual PI3K/mTOR inhibitor PQR309 in B-cell lymphomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A117.