Vladimir Cmiljanovic

Targeting melanoma with dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitors.

Phosphoinositide 3-kinase (PI3K)/protein kinase B/Akt and Ras/mitogen-activated protein kinase pathways are often constitutively activated in melanoma and have thus been considered as promising drug targets. Exposure of melanoma cells to NVP-BAG956, NVP-BBD130, and NVP-BEZ235, a series of novel, potent, and stable dual PI3K/mammalian target of rapamycin (mTOR) inhibitors, resulted in complete G1 growth arrest, reduction of cyclin D1, and increased levels of p27KIP1, but negligible apoptosis. In contrast, treatment of melanoma with the pan-class I PI3K inhibitor ZSTK474 or the mTORC1 inhibitor rapamycin resulted only in minor reduction of cell proliferation. In a syngeneic B16 mouse melanoma tumor model, orally administered NVP-BBD130 and NVP-BEZ235 efficiently attenuated tumor growth at primary and lymph node metastatic sites with no obvious toxicity. Metastatic melanoma in inhibitor-treated mice displayed reduced numbers of proliferating and significantly smaller tumor cells. In addition, neovascularization was blocked and tumoral necrosis increased when compared with vehicle-treated mice. In conclusion, compounds targeting PI3K and mTOR simultaneously were advantageous to attenuate melanoma growth and they develop their potential by targeting tumor growth directly, and indirectly via their interference with angiogenesis. Based on the above results, NVP-BEZ235, which has entered phase I/II clinical trials in patients with advanced solid tumors, has a potential in metastatic melanoma therapy. (Mol Cancer Res 2009;7(4):601–13)

5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a Potent, Brain-Penetrant, Orally Bioavailable, Pan-Class I PI3K/mTOR Inhibitor as Clinical Candidate in Oncology

Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, buparlisib). Compound 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compounds safety profile, identifies 1 as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.

Abstract 4514: PQR309: A potent, brain-penetrant, dual pan-PI3K/mTOR inhibitor with excellent oral bioavailability and tolerability

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently activated in tumors and promotes oncogenic cell transformation, proliferation and tumor growth. PQR309, a novel dual inhibitor of PI3K and mTOR, is currently in Phase I clinical development in cancer patients. PQR309 binds potently and specifically to the ATP binding pocket of all PI3K class I isoforms and mTORC1/2, attenuates PI3K signaling and inhibits tumor cell growth. The preclinical pharmacological and toxicological characterization of PQR309 is presented here. Methods: PQR309 pharmacokinetics/-dynamics (PK/PD) were investigated in rats and mice. Tissue samples from plasma, brain and liver were analyzed by LC/MS detecting PQR309 distribution as well as blood insulin and glucose. Toxicological studies were performed in rats and dogs. Effects on neurological, hematopoietic, respiratory, lymphoid, reproductive and cardiovascular system as well as general health were monitored. The metabolic fate of PQR309 was analyzed in rat, dog and human hepatocytes. Results: PQR309 PK studies in rats, mice and dogs revealed dose-proportional PK, both PO and IV, with a half-life of 5-8 hours in plasma, brain and liver, allowing for once a day oral application. As on-target effect, increase of blood insulin and glucose could be observed within hours after oral dosage in rats, which makes both molecules suitable as PD markers. In in vivo PC-3 rat tumor xenograft models, PQR309 effectively inhibited PI3K signaling in tumors and reduced tumor growth at 10 mg/kg oral dosing. Preclinical toxicity testing showed no signs of cardiotoxicity (including lack of hERG binding), phototoxicity (3T3 NRU test) or mutagenicity (AMES test) for PQR309. No marked effect on CYP450 activity was observed making PQR309 a good combination partner in cancer therapy. As for other PI3K inhibitors, PQR309 leads at elevated doses to a fully reversible loss of body weight and appetite in rats and dogs. No further significant adverse events were observed when testing PQR309 for 28 days in these species. Conclusions: PQR309 potently inhibits class I PI3K isoforms and mTORC1/2 and shows anti-tumor effects in vitro and in vivo. The physico-chemical properties of PQR309 result in good oral bioavailability and equal distribution between plasma and brain. Pre-clinical data led to initiation of a Phase I clinical study of PQR309 in solid tumors. Citation Format: Vladimir Cmiljanovic, Robert A. Ettlin, Florent Beaufils, Walter Dieterle, Petra Hillmann, Juergen Mestan, Anna Melone, Thomas Bohnacker, Marc Lang, Natasa Cmiljanovic, Bernd Giese, Paul Hebeisen, Matthias P. Wymann, Doriano Fabbro. PQR309: A potent, brain-penetrant, dual pan-PI3K/mTOR inhibitor with excellent oral bioavailability and tolerability. [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 4514. doi:10.1158/1538-7445.AM2015-4514

Abstract 2664: PQR309: Structure-based design, synthesis and biological evaluation of a novel, selective, dual pan-PI3K/mTOR inhibitor

Phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling is key to the control of many physiological and pathophysiological processes, and promotes cancer and inflammatory disease. Therefore, targeting of PI3K and/or mTOR pathways is currently explored in numerous clinical studies. PQR309 is a novel, brain penetrant, potent and selective pan-PI3K/mTOR inhibitor with PK properties suitable for once a day oral dosing in humans. Structure activity relationship studies for PI3K and mTOR interactions are presented, including X-Ray analysis of PI3Kgamma co-crystal structures, modeling of PI3Kalpha and mTOR structures, and chemical derivatization. This led to the identification of PQR309 as a potent pan-PI3K and moderate mTOR inhibitor. PQR309 displays excellent selectivity versus PI3K-related lipid kinases (PIKKs) and protein kinases (KINOMEscan), as well as excellent selectivity versus unrelated targets (Cerep expresSProfile). PQR309 features excellent cell permeability, and was characterized as a BCS class II compound due to its limited water solubility (40 μM). Moreover, PQR309 is not a substrate for P-glycoprotein 1 (P-gp). In A2058 melanoma cells PQR309 demonstrated inhibition of protein kinase B (PKB/Akt; pS473) and ribosomal protein S6 (S6, pSer235/236) phosphorylation with IC50 values of 0.13 μM and 0.58 μM, respectively. In IGF-stimulated MCF7 breast cancer cells, PQR309 at 1 μM inhibited phosphorylation of downstream substrates of PI3K including PKB/Akt, S6, p70S6 kinase, GSK3 and Bad by 60-95%. PQR309 inhibited proliferation of all 58 cell lines of the NCI60 panel (GI50 from 50 to 3300 nM), of the NTRC Oncoline panel (44 cell lines, GI50 from 100-6700 nM) and of a lymphoma cell line panel (40 lymphoma cell lines, GI50 from 25-1740 nM). A concise 4-step synthetic process utilizing a novel protective group strategy provides a robust and scalable supply of PQR309 for clinical trials. In summary, PQR309 is a novel, potent, dual pan-PI3K/mTOR inhibitor with a balanced PI3K vs. mTOR profile, and displays excellent physico-chemical and pharmacological properties. The safety profile of PQR309 is currently addressed in Phase I clinical studies. Citation Format: Vladimir Cmiljanovic, Natasa Cmiljanovic, Romina Marone, Florent Beaufils, Xuxiao Zhang, Marketa Zvelebil, Paul Hebeisen, Marc Lang, Juergen Mestan, Anna Melone, Thomas Bohnacker, Eugenio Gaudio, Chiara Tarantelli, Francesco Bertoni, Reto Ritschard, Vincent Pretre, Andreas Wicki, Doriano Fabbro, Petra Hillmann, Roger Williams, Bernd Giese, Matthias P. Wymann. PQR309: Structure-based design, synthesis and biological evaluation of a novel, selective, dual pan-PI3K/mTOR inhibitor. [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 2664. doi:10.1158/1538-7445.AM2015-2664

PQR309 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy

Purpose: Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacologic inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the in vitro and in vivo activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clinical evaluation, in preclinical lymphoma models. Experimental Design: This study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, validation experiments on in vivo models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors. Results: PQR309 had in vitro antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was associated with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib. Conclusions: On the basis of these results, PQR309 appeared as a novel and promising compound that is worth developing in the lymphoma setting. Clin Cancer Res; 24(1); 120–9. ©2017 AACR.

Abstract 2652: Pre-clinical activity and mechanism of action of the novel dual PI3K/mTOR inhibitor PQR309 in B-cell lymphomas

PQR309 is a novel oral PI3K/mTOR inhibitor, being now evaluated as single agent in a phase I study for solid tumors patients (NCT01940133). Here, we present the activity of the compound in lymphoma pre-clinical models, also integrating response data with genomic features. Methods. IC50s were calculated with the MTT assay at 72h on 40 cell lines [27 diffuse large B-cell lymphoma (DLBCL), 10 mantle cell lymphoma (MCL), 3 splenic marginal zone lymphoma (SMZL)] treated with increasing doses of PQR309, a second dual PI3K/mTOR inhibitor (GDC0980) and the PI3Kdelta inhibitor Idelalisib. Gene expression profiling (GEP) was performed with Illumina HumanHT-12 Expression BeadChips. Results. PQR309 had potent anti-proliferative activity in most of the cell lines. Median IC50 were 166 nM in DLBCL (95%CI 128-343 nM), 235 in MCL (155-381), 214 in SMZL (188-304). Activated B-cell like (ABC) and germinal center B-cell like (GCB) DLBCL subtypes were equally sensitive. PQR309 and GDC0980 presented a highly correlated pattern of anti-proliferative activity (R = 0.9). Idelalisib appeared significantly less active, with a pattern of sensitive cell lines less correlated with PQR309 (R = 0.6) or GDC0980 (R = 0.6). Apoptosis after PQR309 (500 nM, 72h) was limited to 1/7 of cell lines. GEP was performed on 8 DLBCL cell lines (4 GCB, 4 ABC) after treatment with DMSO or PQR309 (1 mcM) for 4, 8 and 12h. PQR309 affected, in a time-dependent manner, relevant biologic pathways in both subtypes. Down-regulated transcripts were enriched of MYC targets, genes involved in NFKB/MYD88/BCR/IFN signaling, apoptosis, DNA damage and proteasome. Transcripts up-regulated were enriched of genes involved in cell cycle and senescence, up-regulated after MYD88 silencing, down-regulated by PI3K, involved in packaging of telomere, in autophagosome, up-regulated by inhibitors of HDAC, BET Bromodomain and JAK2. CXCR4, PIM1, PIM2, YPEL5 (up-regulated), LYAR, CCDC86, DDX21, HSPA8, STIP1, and PAK1IP1 (down-regulated) were among the genes changing after PQR309 treatment in more than one time-point or DLBCL cell-type. To identify markers of resistance we looked for transcripts differently regulated between cell lines with higher or lower sensitivity to PQR309 and we also compared baseline GEP between very sensitive (IC50 400 nM). Transcripts more expressed in sensitive cells were significantly enriched of genes involved in BCR pathway/signaling, kinases regulation, and immune system. Transcripts associated with less sensitive cells were enriched of members of proteasome pathway, oxidative phosphorylation, translation initiation. PQR309 (1 mcM) was able to inhibit IgM-stimulation induced p-AKT(Ser 473) in 3/3 DLBCL and 3/3 MCL cells. Conclusions. PQR309 showed strong anti-proliferative activity in lymphomas and GEP identified affected biologic pathways and features possibly associated with response to the molecule. Citation Format: Chiara Tarantelli, Eugenio Gaudio, Ivo Kwee, Andrea Rinaldi, Elena Bernasconi, Luciano Cascione, Petra Hillmann, Anastasios Stathis, Laura Carrassa, Massimo Broggini, Georg Stussi, Doriano Fabbro, Florent Beaufils, Anna Melone, Thomas Bohnacker, Matthias P. Wymann, Andreas Wicki, Emanuele Zucca, Vladimir Cmiljanovic, Francesco Bertoni. Pre-clinical activity and mechanism of action of the novel dual PI3K/mTOR inhibitor PQR309 in B-cell lymphomas. [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 2652. doi:10.1158/1538-7445.AM2015-2652

Abstract 1336: Structure-activity relationship studies, synthesis, and biological evaluation of PQR620, a highly potent and selective mTORC1/2 inhibitor

Mammalian target of rapamycin (mTOR) signaling pathway plays a fundamental role in cell proliferation, differentiation, growth and survival.[1] As a consequence, various tumors and central nervous system (CNS) disorders share aberrant activation of the mTOR pathway. Drugs targeting the mTOR pathway represent therefore a valuable path to address multiple therapeutic areas.[1-2] Here, we report the lead optimization of PQR620, a novel potent and selective brain penetrant inhibitor of mTORC1/2. The development of selective mTOR inhibitors is particularly challenging due to extensively conserved amino acid residues in the ATP binding pocket within the PI3K and PI3K-related protein kinase family. Here, we present a detailed ligand-based structure activity relationship study allowing selective targeting of mTOR kinase activity without the interference of other PI3K family members. Systematic variation of the hinge region and affinity binding motifs led to the identification of PQR620, a morpholino-triazinyl derivative, as potent and selective mTOR inhibitor. Substitution of the morpholine binding to the hinge region and introduction of a 2-aminopyridine, substituted with a difluoromethyl group, induced a >1000-fold selectivity towards mTOR over PI3Kα in enzymatic binding assays. In A2058 melanoma cells PQR620 demonstrated inhibition of protein kinase B (pSer473) and ribosomal protein S6 (pSer235/236) phosphorylation with IC50 values of 0.2 μM and 0.1 μM, respectively. The physico-chemical properties of PQR620 result in good oral bioavailability and excellent brain penetration. PQR620 showed excellent selectivity over a wide panel of kinases, as well as excellent selectivity versus unrelated receptor enzymes and ion channels. Moreover, PQR620 demonstrated its potency to prevent cancer cell growth in an NTRC 44 cancer cell line panel, resulting in a 10log(IC50) of 2.86 (nM). Further pharmacological properties and in vivo efficacy of PQR620 are presented in detail in Ref. [3]. The preparation of PQR620 was optimized towards a robust synthetic route involving only 4 steps, allowing for a rapid access to quantities required for pre-clinical testing. In conclusion, PQR620 inhibits mTOR potently and selectively, and shows anti-tumor effects in vitro and in vivo. PQR620 is currently in pre-clinical development. [1] M. Laplante, D. Sabatini, Cell 2012, 149, 274-293. [2] Z. Z. Chong, Y. C. Shang, L. Zhang, S. Wang, K. Maiese, Oxid. Med. Cell. Longev. 2010, 3, 374–391. [3] F. Beaufils, D. Rageot, A. Melone, A. M. Sele, M. Lang, J. Mestan, R. A. Ettlin, P. Hillmann, V. Cmiljanovic, C. Walter, E. Singer, H. P. Nguyen, P. Hebeisen, D. Fabbro, M. P. Wymann, “Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620” presented at AACR Annual Meeting 2016, April 16-20, New Orleans, Louisiana, USA. Citation Format: Florent Beaufils, Denise Rageot, Anna Melone, Marc Lang, Jurgen Mestan, Vladimir Cmiljanovic, Petra Hillmann, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Structure-activity relationship studies, synthesis, and biological evaluation of PQR620, a highly potent and selective mTORC1/2 inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1336.

Abstract 393A: Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620

Introduction: The mammalian target of rapamycin (mTOR) signaling pathway is an integrating factor in cell physiology that influences many processes like growth, metabolism and proliferation. mTOR signaling is constitutively activated in many cancers. Rapamycin is an allosteric inhibitor of mTOR that targets a subset of mTOR functions via inhibition of the mTORC1 complex. An ATP site-directed mTORC1/2 inhibitor that fully blocks all mTOR functions is desirable as cancer therapeutic. PQR620 is a novel, ATP site directed inhibitor of mTOR that is currently in pre-clinical development. PQR620 potently binds to its target (Kd = 6 nM) and shows excellent selectivity versus related and unrelated kinases [1]. Results: PQR620 inhibits mTOR signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been observed in mice (MTD = 150 mg/kg). A 14 day GLP toxicological study in rats showed very good tolerability (MTD = 30 mg/kg). Only minor toxicities such as dose-related changes in body weight and blood count were observed. PQR620 was administered to male C57BL/6J mice for a pharmacokinetic (PK) and pharmacodynamics (PD) evaluation. After oral application PQR620 exhibited dose-proportional PK, a maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (4.8 μg/ml and 7.7 μg/ml, respectively). In muscle, Cmax (7.6 μg/ml) was reached after 2 hours. The calculated half-life (t 1/2 ) for plasma and brain was approximately 5 hours. After 8 hours, the total exposure (expressed as AUC 0-tz (area under the curve)) was 20.5 μg*h/ml in plasma, while it was approximately 30% higher in both, brain and thigh muscle (30.6 and 32.3 μg*h/ml, respectively). PQR620 potently inhibited mTOR signaling in vivo after administration of a single oral dose of 50 mg/kg. Importantly, no effect on plasma insulin levels was observed. In an OVCAR-3, ovarian carcinoma mouse xenograft, PQR620 effectively attenuated tumor growth using daily, oral dosing. Conclusion: PQR620 potently inhibits mTORC1/2 in vitro and in vivo. The physico-chemical properties of PQR620 result in good oral bioavailability and excellent brain penetration. PQR620 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound. [1] Beaufils F, Rageot D, et al., Structure-Activity Relationship Studies, Synthesis and Biological Evaluation of PQR620, a Highly Potent and Selective mTORC1/2 Inhibitor, AACR annual meeting 2016 Citation Format: Florent Beaufils, Denise Rageot, Anna Melone, Alexander Sele, Marc Lang, Juergen Mestan, Robert A. Ettlin, Petra Hillmann, Vladimir Cmiljanovic, Carolin Walter, Elisabeth Singer, Hoa HP Nguyen, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 393A.

Abstract 380: The dual PI3K/MTOR inhibitor PQR309 is active in mature B cell lymphoma cell lines bearing resistance to the PI3K-delta inhibitor idelalisib and specific gene expression features

Introduction The PI3K-delta idelalisib is among the most promising novel agents for lymphomas, but certain patients present a primary resistant disease or relapse after an initial response. Here, we studied the dual PI3K/MTOR inhibitor PQR309 in lymphoma cell lines that were resistant to idelalisib. Methods IC50 values were calculated with the MTT assay (72h) for 40 lymphoma cell lines. Gene expression profiling was done with Illumina HumanHT12 Expression BeadChips, data mining with Limma, GSEA and LINCSCLOUD. Results PQR309 and idelalisib showed only a partially overlapping pattern of activity (R = 0.6). While no cell line was sensitive to idelalisib and not to PQR309, 10 cell lines were sensitive to both compounds and 7 were sensitive only to PQR309. Idelalisib median IC50 was 28 nM (95%CI; 13-147) in the PQR309/idelalisib group and 13.4 μM (7.2-16.7 μM) in the PQR309-only (P 0.0006). PQR309 median IC50 was 113 nM (33-166 nM) in PQR309/idelalisib sensitive and 193 nM (111-372 nM) in the PQR309-only sensitive cells (P 0.03). No differences between the two groups were observed in terms of histology, diffuse large B cell lymphoma cell of origin, MYC deregulation, TP53 inactivation, BCL6 or BCL2 translocations. The gene expression signature of the PQR309/idelalisib sensitive cell lines was enriched of genes involved in IFN signaling, IL6/Jak/Stat3 signaling, oxidative phosphorylation, PI3K/MTOR signaling, chemokines signaling and proteasome (GSEA FDR Conclusions. The dual PI3K/MTOR inhibitor PQR309 was active in a subset of mature B cell lymphoma cell lines bearing a primary resistance to the PI3K-delta inhibitor idelalisib and specific gene expression features. Expression of SOX4 and CD24 appear worth of validation on clinical specimens derived from prospectively treated patients. Citation Format: Tarantelli Chiara, Eugenio Gaudio, Ivo Kwee, Alberto Arribas, Andrea Rinaldi, Elena Bernasconi, Luciano Cascione, Petra Hillmann, Anastasios Stathis, Laura Carrassa, Massimo Broggini, Georg Stussi, Doriano Fabbro, Emanuele Zucca, Vladimir Cmiljanovic, Francesco Bertoni. The dual PI3K/MTOR inhibitor PQR309 is active in mature B cell lymphoma cell lines bearing resistance to the PI3K-delta inhibitor idelalisib and specific gene expression features. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 380.

Abstract A117: An integrated phosphoproteomic approach to dissect the mechanism of action of the novel dual PI3K/mTOR inhibitor PQR309 in B-cell lymphomas

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.