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Baseline gene expression analysis of B cell lymphoma cell lines with higher (IC50 < 200 nM) or lower sensitive (IC50 > 400 nM) to PQR309.

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.

Supplementary figures, table legends, Table S1, S9 Figure S1. In vitro antitumor activity of the dual PI3K/mTOR inhibitor apitolisib and its correlation with PQR309. Figure S2. Antitumor In vivo activity of PQR309 in the treatment of RI-1 and SU-DHL-6 xenograft model. Figure S3. Apoptosis is induced by co-treatment of PQR309 with venetoclax or panobinostat in primary cells and in the DLBCL SU-DHL-6 cell line. Figure S4. Specific baseline gene expression signatures are associated with higher or lower sensitivity to PQR309. Figure S5. In vitro antitumor activity of the PI3K� inhibitor idelalisib and its correlation with PQR309. Figure S6. PQR309 induced decrease in phosphorylation of AKT-Ser473 and p70S6K-Thr389 in DLBCL cell lines. Figure S7. PQR309 reduces AKT-Ser473 phosphorylation in lymphoma cell lines. Figure S8. Transcriptional expression signature of ABC DLBCL cell lines induced by PQR309. Figure S9. ABC and GCB DLBCL PQR309-treated signatures were highly overlapping. Figure S10. Transcript expression levels of PQR309-treated samples. Figure S11. Changes in protein phosphorylation and RNA expression differently contribute to PQR309 affected biologic pathways in ABC DLBCL. Figure S12. PQR309 can largely regulate the same genes affected by the BTK inhibitor ibrutinib, the PI3K� idelalisib, the dual PI3K�/� inhibitor duvelisib (A), the dual PI3K�/� inhibitor AZD8835 or the AKT inhibitor AZD5363 (B). Figure S13. BCR pathway signature is similarly affected after ibrutinib, idelalisib and duvelisib treatments in ABC DLBCL cell lines. Figure S14. The dual PI3K/mTOR inhibitor PQR309 and the PIM inhibitor AZD1208 synergize in DLBCL cell lines. Supplementary Table 1. List of phosphoresidues investigated by Carna Bioscience to perform RPPA analysis. Supplementary Table 9. Transcripts differentially expressed in ABC DLBCL cell lines treated with ibrutinib (A), idelalisib (B) or duvelisib (C) versus DMSO.

The mechanistic target of rapamycin (mTOR) pathway is hyperactivated in cancer and neurological disorders. Rapalogs and mTOR kinase inhibitors (TORKi) have recently been applied to alleviate epileptic seizures in tuberous sclerosis complex (TSC). Herein, we describe a pharmacophore exploration to identify a highly potent, selective, brain penetrant TORKi. An extensive investigation of the morpholine ring engaging the mTOR solvent exposed region led to the discovery of PQR626 (8). 8 displayed excellent brain penetration and was well-tolerated in mice. In mice with a conditionally inactivated Tsc1 gene in glia, 8 significantly reduced the loss of Tsc1-induced mortality at 50 mg/kg p.o. twice a day. 8 overcomes the metabolic liabilities of PQR620 (52), the first-in-class brain penetrant TORKi showing efficacy in a TSC mouse model. The improved stability in human hepatocytes, excellent brain penetration, and efficacy in Tsc1GFAPCKO mice qualify 8 as a potential therapeutic candidate for the treatment of neurological disorders.

The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is a critical regulator of cell growth and is frequently hyperactivated in cancer. Therefore, PI3K inhibitors represent a valuable asset in cancer therapy. Herein we have developed a novel anticancer agent, the potent pan-PI3K inhibitor PQR514 (4), which is a follow-up compound for the phase-II clinical compound PQR309 (1). Compound 4 has an improved potency both in vitro and in cellular assays with respect to its predecessor compounds. It shows superiority in the suppression of cancer cell proliferation and demonstrates significant antitumor activity in an OVCAR-3 xenograft model at concentrations approximately eight times lower than PQR309 (1). The favorable pharmacokinetic profile and a minimal brain penetration promote PQR514 (4) as an optimized candidate for the treatment of systemic tumors.

The mechanistic target of rapamycin (mTOR) plays a pivotal role in growth and tumor progression and is an attractive target for cancer treatment. ATP-competitive mTOR kinase inhibitors (TORKi) have the potential to overcome limitations of rapamycin derivatives in a wide range of malignancies. Herein, we exploit a conformational restriction approach to explore a novel chemical space for the generation of TORKi. Structure-activity relationship (SAR) studies led to the identification of compound 12b with a ∼450-fold selectivity for mTOR over class I PI3K isoforms. Pharmacokinetic studies in male Sprague Dawley rats highlighted a good exposure after oral dosing and a minimum brain penetration. CYP450 reactive phenotyping pointed out the high metabolic stability of 12b. These results identify the tricyclic pyrimido-pyrrolo-oxazine moiety as a novel scaffold for the development of highly selective mTOR inhibitors for cancer treatment.

Combination therapy is implicated for many oncological indications to circumvent resistance mechanisms that reduce efficacy of targeted drugs. In recent years, mRNA has been successfully utilized preclinically to deliver cytokines to tumors locally at efficacious doses. Versameb has developed a technology platform which enables combinatorial approaches for intra-tumoral application of nucleotide therapeutics that comprise an mRNA element covalently linked to up to three independent siRNA elements targeting various tumor-relevant genes. Such constructs allow a dose-synchronized treatment of different components in parallel, which opens the opportunity to address up to four cancer targets within a single RNA molecule. For proof of concept, we designed a combinatory molecule consisting of mRNA coding for human interleukin 2 (IL-2) linked to a small interfering RNA (siRNA) targeting vascular endothelial growth factor A (VEGFA). Transfection of A549 adenocarcinoma cells led to dose-dependent secretion of efficacious concentrations of IL-2 and simultaneous complete VEGFA knockdown. IL-2 protein functionality was tested in SK-OV-3 cell spheroids co-cultured with human primary peripheral blood mononuclear cells (PBMCs). After treatment, secreted IL-2 protein induced immune cell activation and consecutive spheroid killing by PBMCs from three independent donors. VEGFA functionality was tested in an in vitro vascularization assay in HUVEC cells. Cells were treated with supernatants of vehicle or RNA transfected SCC-4 head & neck cancer cells producing high endogenous VEGFA, and vascular sprouting was analyzed quantitatively. While vehicle-treated cell supernatant induced vessel sprouting, supernatant from RNA treated cells showed much reduced sprouting in vitro consistent with the lower VEGFA levels measured, confirming functionality of the siRNA effect on VEGFA protein expression. We further provide examples of combinatory molecules targeting up to 4 independent key cancer hallmarks and their functional outcomes in various cancer cell types. In conclusion, mRNA combination constructs can be developed into cancer therapeutics that target several pro-tumorigenic pathways simultaneously with a single molecule overcoming hurdles of delivery, toxicity, and regulatory requirements for combination therapies. Citation Format: Petra Hillmann, Justin A. Selvaraj, Sina E. Zimmerli, Pascale G. Birrer, Claudia C. Bohnert, Klaas P. Zuideveld, Stefan J. Scherer, Friedrich Metzger. Design of combinatory RNA molecules to simultaneously activate tumor immunity and target several signaling pathways in solid tumors [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 1081.

The mechanistic target of rapamycin (mTOR) pathway is hyperactivated in cancer and neurological disorders. Rapalogs and mTOR kinase inhibitors (TORKi) have recently been applied to alleviate epileptic seizures in tuberous sclerosis complex (TSC). Herein, we describe a pharmacophore exploration to identify a highly potent, selective, brain penetrant TORKi. An extensive investigation of the morpholine ring engaging the mTOR solvent exposed region led to the discovery of PQR626 (

Mechanistic target of rapamycin (mTOR) regulates cell proliferation, growth and survival, and is activated in cancer and neurological disorders, including epilepsy. The rapamycin derivative ("rapalog") everolimus, which allosterically inhibits the mTOR pathway, is approved for the treatment of partial epilepsy with spontaneous recurrent seizures (SRS) in individuals with tuberous sclerosis complex (TSC). In contrast to the efficacy in TSC, the efficacy of rapalogs on SRS in other types of epilepsy is equivocal. Furthermore, rapalogs only poorly penetrate into the brain and are associated with peripheral adverse effects, which may compromise their therapeutic efficacy. Here we compare the antiseizure efficacy of two novel, brain-permeable ATP-competitive and selective mTORC1/2 inhibitors, PQR620 and PQR626, and the selective dual pan-PI3K/mTORC1/2 inhibitor PQR530 in two mouse models of chronic epilepsy with SRS, the intrahippocampal kainate (IHK) mouse model of acquired temporal lobe epilepsy and Tsc1GFAP CKO mice, a well-characterized mouse model of epilepsy in TSC. During prolonged treatment of IHK mice with rapamycin, everolimus, PQR620, PQR626, or PQR530; only PQR620 exerted a transient antiseizure effect on SRS, at well tolerated doses whereas the other compounds were ineffective. In contrast, all of the examined compounds markedly suppressed SRS in Tsc1GFAP CKO mice during chronic treatment at well tolerated doses. Thus, against our expectation, no clear differences in antiseizure efficacy were found across the three classes of mTOR inhibitors examined in mouse models of genetic and acquired epilepsies. The main advantage of the novel 1,3,5-triazine derivatives is their excellent tolerability compared to rapalogs, which would favor their development as new therapies for TORopathies such as TSC.

The mTOR signaling pathway has emerged as a possible therapeutic target for epilepsy. Clinical trials have shown that mTOR inhibitors such as everolimus reduce seizures in tuberous sclerosis complex patients with intractable epilepsy. Furthermore, accumulating preclinical data suggest that mTOR inhibitors may have anti-seizure or anti-epileptogenic actions in other types of epilepsy. However, the chronic use of rapalogs such as everolimus is limited by poor tolerability, particularly by immunosuppression, poor brain penetration and induction of feedback loops which might contribute to their limited therapeutic efficacy. Here we describe two novel, brain-permeable and well tolerated small molecule 1,3,5-triazine derivatives, the catalytic mTORC1/C2 inhibitor PQR620 and the dual pan-PI3K/mTOR inhibitor PQR530. These derivatives were compared with the mTORC1 inhibitors rapamycin and everolimus as well as the anti-seizure drugs phenobarbital and levetiracetam. The anti-seizure potential of these compounds was determined by evaluating the electroconvulsive seizure threshold in normal and epileptic mice. Rapamycin and everolimus only poorly penetrated into the brain (brain:plasma ratio 0.0057 for rapamycin and 0.016 for everolimus). In contrast, the novel compounds rapidly entered the brain, reaching brain:plasma ratios of ∼1.6. Furthermore, they significantly decreased phosphorylation of S6 ribosomal protein in the hippocampus of normal and epileptic mice, demonstrating effective mTOR inhibition. PQR620 and PQR530 significantly increased seizure threshold at tolerable doses. The effect of PQR620 was more marked in epileptic vs. nonepileptic mice, matching the efficacy of levetiracetam. Overall, the novel compounds described here have the potential to overcome the disadvantages of rapalogs for treatment of epilepsy and mTORopathies directly connected to mutations in the mTOR signaling cascade.

One of the pathological hallmarks of Huntington disease (HD) is accumulation of the disease-causing mutant huntingtin (mHTT), which leads to the disruption of a variety of cellular functions, ultimately resulting in cell death. Induction of autophagy, for example by the inhibition of mechanistic target of rapamycin (mTOR) signaling, has been shown to reduce HTT levels and aggregates. While rapalogs like rapamycin allosterically inhibit the mTOR complex 1 (TORC1), ATP-competitive mTOR inhibitors suppress activities of TORC1 and TORC2 and have been shown to be more efficient in inducing autophagy and reducing protein levels and aggregates than rapalogs. The ability to cross the blood-brain barrier of first generation catalytic mTOR inhibitors has so far been limited, and therefore sufficient target coverage in the brain could not be reached. Two novel, brain penetrant compounds - the mTORC1/2 inhibitor PQR620, and the dual pan-phosphoinositide 3-kinase (PI3K) and mTORC1/2 kinase inhibitor PQR530 - were evaluated by assessing their potential to induce autophagy and reducing mHTT levels. For this purpose, expression levels of autophagic markers and well-defined mTOR targets were analyzed in STHdh cells and HEK293T cells and in mouse brains. Both compounds potently inhibited mTOR signaling in cell models as well as in mouse brain. As proof of principle, reduction of aggregates and levels of soluble mHTT were demonstrated upon treatment with both compounds. Originally developed for cancer treatment, these second generation mTORC1/2 and PI3K/mTOR inhibitors show brain penetrance and efficacy in cell models of HD, making them candidate molecules for further investigations in HD.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling cascade is an important therapeutic target for lymphomas. Rapamycin-derivates as allosteric mTOR complex 1 (TORC1) inhibitors have shown moderate preclinical and clinical anti-lymphoma activity. Here, we assessed the anti-tumor activity of PQR620, a novel brain penetrant dual TORC1/2 inhibitor, in 56 lymphoma cell lines. We observed anti-tumor activity across 56 lymphoma models with a median IC50 value of 250 nM after 72 h of exposure. PQR620 was largely cytostatic, but the combination with the BCL2 inhibitor venetoclax led to cytotoxicity. Both the single agent and the combination data were validated in xenograft models. The data support further evaluation of PQR620 as a single agent or in combination with venetoclax.

Background. Diffuse large B-cell lymphoma (DLBCL) is the commonest lymphoma in both humans and dogs. Canine DLBCL (cDLBCL) is considered an ideal comparative model for drug development, but a complete genomic characterization of this tumor is still lacking. In this study, we report an integrated analysis to comprehensively define the molecular mechanisms of cDLBCL and possible associations with clinical outcome. Methods. Fifty cDLBCLs were analyzed by RNA-Seq, methyl-CpG-binding sequencing and array comparative genomic hybridization. Normal B-cells derived from lymph nodes of 11 healthy dogs were used as controls.Additionally, immunohistochemistry, in vitroand in vivoexperiments were performed as validation analyses. Results.Compared to normal B-cells, cDLBCL showed a marked up-regulation of genes involved in the PI3K/mTOR and NF-κB pathways, including several TLRs in association with MYD88, indicating mechanisms similar to the human activated B cell-like subtype DLBCL. Both RNA-Seq and methylation sequencing led to the identification of two groups of cDLBCLs bearing different clinical outcome. The two groups did not overlap with the human germinal center B-cell (GCB) and the activated B-cell-like (ABC) DLBCL subtypes or the human DLBCL consensus clusters. The dogs with the poorest outcome presented a signature largely defined by markers of T-cell-mediated immune responses, with a high expression of PDL-1, PD-1 and CTLA-4, also validated in an independent cohort of cDLBCL by immunohistochemistry. These data provide a strong rationale for the use of cDLBCL to study immune checkpoint modulators. The observed high expression of PI3K/mTOR pathway genes was confirmed and validated achieving a clear anti-tumor activity with the use of the PI3K-delta inhibitor idelalisib and of the novel dual PI3K/mTOR inhibitor bimiralisib in the cDLBCL cell line CLBL-1. The cDLBCLs showed an up-regulation of MYC and of its targets, sustained by recurrent gains in the chromosome 13, where the oncogene is located, in approximately half of the cases. Thus, we have exposed the cDLBCL cell line CLBL-1 to the BET inhibitor birabresib (OTX015) and to the BRD4 degrader MZ1. Both compounds caused a significant reduction in the proliferation of tumor cells, and this effect was stronger especially with the second compound. Exposure to MZ1 determined an important downregulation of MYC and also of LIN28B, the most overexpressed transcript in cDLBCL when compared to controls. While LIN28B does not seem to be a relevant gene for human DLBCL, its overexpression causes murine T-cell lymphomas (Beachy et al, Blood 2011), and there is a direct association of MYC with LIN28B promoter resulting in transcriptional transactivation (Chang et al, PNAS 2009). Here, LIN28B genetic silencing in the CLBL-1 lead to a reduction in cell growth, opening new therapeutic target perspectives in canine lymphoma. Conclusions. We have reported the first large next generation sequencing study investigating the cDLBCL transcriptome, methylome and the genome-wide CNVs. We identified deregulated pathways and individual transcripts providing therapeutic targets, including an immune-related signature affecting the outcome of a subgroup of cDLBCL. Our data sustain the use of cDLBCL as comparative models for human DLBCL but also highlight differences that must be kept in consideration. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Wymann:PIQUR Therapeutics AG: Employment, Equity Ownership, Patents & Royalties.

Mechanistic target of rapamycin (mTOR), a key player in cell proliferation, growth and survival, is overactivated in tumors and neurological disorders.[1] Rapalogs have recently been explored to alleviate epileptic seizures in Tuberous Sclerosis Complex (TSC).[2] Herein, we combined pharmacophore features of PQR620[3], the first-in-class brain penetrant ATP-competitive mTOR kinase inhibitor showing efficacy in a TSC mouse model, and PQR617[4], a potent, highly selective mTOR inhibitor. An extensive chemical exploration of the morpholine ring led to the discovery of PQR626, a highly potent, selective, brain penetrant inhibitor of mTORC1/2 kinase. In male Sprague Dawley rats and female C57BL/6J mice, PQR626 displayed an excellent brain penetration compared to everolimus, which possessed a limited ability to cross the blood-brain-barrier (brain/plasma levels ~ 1.4:1 vs 1:92). An additional pharmacokinetic comparative study with everolimus and AZD2014 confirmed the superiority of PQR626 [brain/plasma levels: ~ 1:25 (AZD2014), ~ 1:61 (everolimus), ~ 1.8:1 (PQR626)]. PQR626 showed very good tolerability in mice (MTD 100-150 mg/kg). Efficacy studies, using mice with conditional inactivation of the Tsc1 gene primarily in glia (Tsc1GFAPCKO mice), were performed in a dose-range finding study. PQR626 (50 mg/kg, BID - twice a day) showed a significant effect on survival and significantly prevented/decreased mortality as compared to the vehicle. On the basis of its favorable pharmacological parameters, excellent brain penetration, safety profile and efficacy in Tsc1GFAPCKO mice, PQR626 qualifies as a novel mTOR inhibitor with potential application in the treatment of epilepsy and neurological disorders. [1] Wymann M. P. and Schneiter R. Nat. Rev. Mol. Cell Biol. 2008, 9, 162-176. [2] Krueger D. A. et. al. Neurology 2016, 87, 2408-2415. [3] Rageot D. et. al. J Med Chem. 2018, 61 (22), 10084-10105. [4] Borsari C. et. al. J Med Chem. 2019, 62 (18), 8609-8630. Citation Format: Chiara Borsari, Erhan Keles, Denise Rageot, Anna Melone, Thomas Bohnacker, Lucinda Kate Batchelor, Martina De Pascale, Paul Hebeisen, Petra Hillmann, Doriano Fabbro, Matthias Wymann. Discovery and preclinical characterization of PQR626: A potent, orally available, and brain-penetrant mTOR inhibitor for the treatment of tuberous sclerosis complex [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 665.

Dysregulation of the PI3K/Akt/mTOR pathway has been implicated in several brain disorders, including epilepsy. Rapamycin and similar compounds inhibit mTOR. complex 1 and have been reported to decrease seizures, delay seizure development, or prevent epileptogenesis in different animal models of genetic or acquired epilepsies. However, data for acquired epilepsy are inconsistent, which, at least in part, may be due to the poor brain penetration and long brain persistence of rapamycin and the fact that it blocks only one of the two cellular mTOR complexes. Here we examined the antiepileptogenic or disease-modifying effects of two novel, brain-permeable and well tolerated 1,3,5-triazine derivatives, the ATP-competitive mTORC1/2 inhibitor PQR620 and the dual pan-PI3K/mTORC1/2 inhibitor PQR530 in the intrahippocampal kainate mouse model, in which spontaneous seizures develop after status epilepticus (SE). Following kainate injection, the two compounds were administered over 2 weeks at doses previously been shown to block mTORC1/2 or PI3K/mTORC1/2 in the mouse brain. When spontaneous seizures were recorded by continuous (24/7) video-EEG recording starting 6 weeks after termination of treatment, no effects on incidence or frequency of seizures were observed. Drug treatment suppressed the epilepsy-induced activation of the PI3K/Akt/mTOR pathway in the hippocampus, but granule cell dispersion in the dentate gyrus was not prevented. When epilepsy-associated behavioral alterations were determined 12-14 weeks after kainate, mice pretreated with PQR620 or PQR530 exhibited reduced anxiety-related behavior in the light-dark box, indicating a disease-modifying effect. Overall, the data indicate that mTORC1/C2 or PI3K/mTORC1/C2 inhibition may not be an antiepileptogenic strategy for SE-induced epilepsy.

Mechanistic target of rapamycin (mTOR) promotes cell proliferation, growth, and survival and is overactivated in many tumors and central nervous system disorders. PQR620 (3) is a novel, potent, selective, and brain penetrable inhibitor of mTORC1/2 kinase. PQR620 (3) showed excellent selectivity for mTOR over PI3K and protein kinases and efficiently prevented cancer cell growth in a 66 cancer cell line panel. In C57BL/6J and Sprague–Dawley mice, maximum concentration (Cmax) in plasma and brain was reached after 30 min, with a half-life (t1/2) > 5 h. In an ovarian carcinoma mouse xenograft model (OVCAR-3), daily dosing of PQR620 (3) inhibited tumor growth significantly. Moreover, PQR620 (3) attenuated epileptic seizures in a tuberous sclerosis complex (TSC) mouse model. In conclusion, PQR620 (3) inhibits mTOR kinase potently and selectively, shows antitumor effects in vitro and in vivo, and promises advantages in CNS indications due to its brain/plasma distribution ratio.

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.

Background: The PI3K/AKT/mTOR pathway represents an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor under clinical investigation for patients affected by lymphomas and solid tumors. Here, we present novel data obtained with PQR309 in combination with other targeted agents in lymphoma models. Materials and Methods: Established lymphoma cell lines derived from mantle cell lymphoma (MCL) and from diffuse large B cell lymphoma (DLBCL) of the activated B cell like (ABC) or the germinal center B cell type (GCB) were exposed to combinations of PQR309 with selected compounds for 72 hrs. Synergy was assessed with Chou-Talalay combination index (CI): synergism ( 1.1). Results: PQR309 was evaluated in combination with additional targeted agents. The combination with the BTK inhibitor ibrutinib was synergistic in 3/3 ABC-DLBCL (HBL1, median CI 0.26, 95%C.I. 0.22-0.29; OCI-LY-10, median CI 0.67, 95%C.I. 0.43-0.75; OCI-LY-3, median CI 0.68, 95%C.I. 0.6-0.73) and in 4/4 MCL (JEKO1, median CI 0.53, 95%C.I. 0.47-0.64; REC1, median CI 0.6, 95%C.I. 0.37-0.69; SP49, median CI 0.68, 95%C.I. 0.55-0.81; UPN1, median CI 0.69, 95%C.I. 0.64-0.75) cell lines tested. When PQR309 was assayed in combination with the BCL2 inhibitor venetoclax in MCL, 3/4 were synergistic (JEKO1, median CI 0.82, 95% CI 0.75-1.01; REC1, median CI 0.49, 95% CI 0.35-0.78; SP49, median CI 0.8, 95% CI 0.67-0.93) while one had no benefit (UPN1, CI >3). The combination with the novel anti-BRD4 Protac ARV-825 was synergistic in 4/8 cell lines (RI1, median CI 0.61, 95% CI 0.51-0.66; KARPAS422, median CI 0.68, 95% CI 0.56-0.81; OCI-LY-18, median CI 0.78, 95% CI 0.71-1.04; SU-DHL-6, median CI 0.71, 95% CI 0.65-0.88), additive in 2/8 (U2932, median CI 0.98, 95% CI 0.9-1.14; DOHH2, median CI 0.97, 95% CI 0.67-1.09) and with no benefit in 2/8 (OCI-LY-3, median CI 1.29, 95% CI 1.02-1.39; TMD8, median CI 1.35, 95% CI 1.33-1.4) cell lines. Finally, following the previous demonstration of an upregulation of PIM1 kinase after exposure to PQR309 and synergism combining PQR309 with the PIM inhibitor AZD1208 in a few DLBCL cell lines (Tarantelli et al, ASH 2015), we tested the efficacy of this combination in a larger panel of DLBCL cell lines. Seven out of seven DLBCL lymphoma cell lines benefited from the combination, achieving synergism to PQR309 plus AZD1208 (OCI-LY-10, median CI 0.51, 95% CI 0.47-0.57; OCI-LY-3, median CI 0.52, 95% CI 0.49-0.66; RI1, median CI 0.2, 95% CI 0.16-0.22; U2932, median CI 0.37, 95% CI 0.34-0.44; DOHH2, median CI 0.34, 95% CI 0.31-0.41; KARPAS422, median CI 0.86, 95% CI 0.8-0.92; OCI-LY-18, median CI 0.54, 95% CI 0.46-0.66). Conclusions: PQR309 showed synergistic activity in B cell lymphomas when combined with inhibitors of BTK, BCL2, BRD4, and PIM, suggesting these combinations are worthy of being further investigated. Citation Format: Chiara Tarantelli, Elena Bernasconi, Filippo Spriano, Petra Hillmann, Eugenio Gaudio, Luciano Cascione, Ivo Kwee, Doriano Fabbro, Emanuele Zucca, Vladimir Cmiljanovic, Francesco Bertoni. PQR309-containing combinations show synergistic antilymphoma activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B041.

The P2Y(2) receptor, which is activated by UTP, ATP, and dinucleotides, was studied as a prototypical nucleotide-activated GPCR. A combination of receptor mutagenesis, determination of its effects on potency and efficacy of agonists and antagonists, homology modeling, and chemical experiments was applied. R272 (extracellular loop EL3) was found to play a gatekeeper role, presumably responsible for recognition and orientation of the nucleotides. R272 is also directly involved in binding of dinucleotides, which behaved as partial agonists. Y118A (3.37) mutation led to dramatically reduced efficacy of agonists; it is part of the entry channel as well as the triphosphate binding site. While the Y114A (3.33) mutation did not have any effect on agonist activities, the antagonist Reactive Blue 2 (6) was completely inactive at that mutant. The disulfide bridge Cys25-Cys278 was found to be important for agonist potency but neither for agonist efficacy nor for antagonist potency.

High blood levels of ammonium/ammonia (NH(4)(+)/NH(3)) are associated with severe neurotoxicity as observed in hepatic encephalopathy (HE). Astrocytes are the main targets of ammonium toxicity, while neuronal cells are less vulnerable. In the present study, an astrocytoma cell line 1321N1 and a neuroblastoma glioma hybrid cell line NG108-15 were used as model systems for astrocytes and neuronal cells, respectively. Ammonium salts evoked a transient increase in intracellular calcium concentrations ([Ca(2+)](i)) in astrocytoma (EC(50)=6.38 mM), but not in NG108-15 cells. The ammonium-induced increase in [Ca(2+)](i) was due to an intracellular effect of NH(4)(+)/NH(3) and was independent of extracellular calcium. Acetate completely inhibited the ammonium effect. Ammonium potently reduced calcium signaling by G(q) protein-coupled receptors (H(1) and M3) expressed on the cells. Ammonium (5 mM) also significantly inhibited the proliferation of 1321N1 astrocytoma cells. While mRNA for the mammalian ammonium transporters RhBG and RhCG could not be detected in 1321N1 astrocytoma cells, both transporters were expressed in NG108-15 cells. RhBG and RhBC in brain may promote the excretion of NH(3)/NH(4)(+) from neuronal cells. Cellular uptake of NH(4)(+)/NH(3) was mainly by passive diffusion of NH(3). Human 1321N1 astrocytoma cells appear to be an excellent, easily accessible human model for studying HE, which can substitute animal studies, while NG108-15 cells may be useful for investigating the role of the recently discovered Rhesus family type ammonium transporters in neuronal cells. Our findings may contribute to the understanding of pathologic ammonium effects in different brain cells, and to the treatment of hyperammonemia.

A library of anilinoanthraquinone derivatives was synthesized by parallel Ullmann coupling reaction of bromaminic acid with aniline derivatives in solution using a compact parallel synthesizer. The products were purified by HPLC and evaluated as antagonists at mouse and human P2Y2 receptors. 4-Phenylamino-substituted 1-amino-2-sulfoanthraquinones, for example, 1-amino-4-(2-methoxyphenyl)-2-sulfoanthraquinone (PSB-716), were potent P2Y2 antagonists with IC50 values in the low micromolar range.

Background. Pharmacogenomics studies the role of genomics in drug response. By measuring the individual genome, it is hoped that pharmaceutical drug treatments can deviate from a "one-dose-fits-all" approach to a more "personalized" treatment. To achieve this goal, we need to explain which genomic differences cause the lack of response and to be able to predict the response from baseline omics data, that is from data available before starting treatment. Here, we present an integrated omics approach to analyze drug sensitivity based on in vitro experiments. Methods. We measured the drug response in 61 lymphoma cell lines for a number of anti-cancer drugs using a standard MTT cell proliferation assay. The baseline genomics of these cell lines were fully profiled for gene expression, copy number and methylation. Pathway and gene set signatures were computed using GO, KEGG, Biocarta and lymphoma databases to provide gene set level features. Additionally, we computed high level "biological concept" features. These multi-scale features were directly correlated with drug sensitivity but also correlated between the multiple data types. Using a novel parallel clustering approach, we integrated our data for the different omics types and at different scales: gene, gene set and concept level. Finally, a shortest path algorithm extracted the most probable explanation between genotype and response phenotype. Results. As a proof of concept, we first applied our method to the classification of activated B- cell like (ABC) and germinal center B (GCB) subtypes in diffuse large B-cell lymphomas (DLBCL). In accordance to previous knowledge, our method showed the MYD88/CD40/NFKB axis as strongly upregulated in the ABC, while, albeit less pronounced, the PI3K/MTOR and NOTCH signalling pathways were more enriched in the GCB subtype. We then applied our method to the drug sensitivity data of our lymphoma cell line panel. The analysis showed that drug response was largely driven by differential methylation rather than copy number aberrations. We found that B cell receptor signaling (BCR) and CD40 signalling were among the most correlated to drug sensitivity in a drug-specific manner. Genes in these pathways (such as SYK, SPIB and CD79A) appeared as good biomarkers for drug-specific response in lymphoma. Conclusion. Using an integrated analysis of multiple omics data at multiple scales, we successfully identified functional modules related with drug sensitivity in lymphoma cell lines. The same method can be applied to study the sensitivity to other drugs in either hematological and/or solid cancer types. Citation Format: Ivo Kwee, Andrea Rinaldi, Alberto J. Arribas, Eugenio Gaudio, Chiara Tarantelli, Filippo Spriano, Petra Hillmann, Francesco Bertoni. Multi-scale omics integration using parallel heatmap clustering for the systemic analysis and biomarker discovery of drug sensitivity in lymphoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 552. doi:10.1158/1538-7445.AM2017-552

The PI3K/AKT/mTOR signaling pathway plays a fundamental role in cell proliferation, growth and survival and aberrant activation of this signaling pathway has been shown to drive the progression of malignant tumors.[1] Drugs targeting the pathway at multiple points, such as dual PI3K/mTOR inhibitors appear to have the broadest activity profile to address cancer therapeutic strategies and are currently being explored in numerous clinical studies. Recently, we presented PQR309, a novel, brain-penetrant pan-PI3K/mTOR inhibitor, which entered phase II clinical trials in 2016.[2] Here, we report the lead optimization of PQR530, a potent and brain-penetrant follow-up compound as pan-PI3K/mTORC1/2 inhibitor. The development of a follow-up compound concentrated on the improvement of both, the potency and the selectivity for all targeted kinases, namely the class IA PI3K isoforms as well as mTOR. We present a detailed ligand-based structure-activity relationship study which was obtained by systematic modifications of the hinge region as well as the affinity binding substituents. This study led to the identification of PQR530, a dual pan-PI3K/mTORC1/2 inhibitor showing excellent activities in cellular assays as well as in PI3Kα and mTOR enzymatic binding assays. In A2058 melanoma cells PQR530 inhibited protein kinase B (PKB, pSer473) and ribosomal protein S6 (pS6, pSer235/236) phosphorylation with IC50 values of 0.07 µM. PQR530 showed excellent selectivity over a wide panel of kinases, as well as excellent selectivity versus unrelated receptor enzymes and ion channels. Moreover, PQR530 displayed potency in a panel of 44 cancer cell lines (NTRC OncolinesTM) to prevent cancer cell growth (mean value for GI50 of 426 nM). Oral application of PQR530 to mice resulted in a dose-proportional PK and demonstrated good oral bioavailability and excellent brain penetration.[3] An optimized, robust synthetic route allowed rapid access to multi-gram quantities of PQR530 for pre-clinical development in only 4 steps. In conclusion, PQR530 inhibits all PI3K isoforms and the mammalian target of rapamycin (mTOR) complexes C1/2 potently and selectively, and shows anti-tumor effects in vitro and in vivo. [1] M. P. Wymann, M. Zvelebil, M. Laffargue (2003). Phosphoinositide 3-kinase signalling – which way to target? Trends Pharmacol Sci.; 24, 366-376. [2] V. Cmiljanovic et. al. “PQR309: Structure-Based Design, Synthesis and Biological Evaluation of a Novel, Selective, Dual Pan-PI3K/mTOR Inhibitor” presented at AACR Annual Meeting 2015, April 18-22, Philadelphia, Pennsylvania, USA. [3] P. Hillmann et al. “Pharmacological Characterization of the Selective, Orally Bioavailable, Potent Dual PI3K/mTORC1/2 Inhibitor PQR530” abstract submitted for AACR Annual Meeting 2017, April 1-5, Washington, D. C., USA. Citation Format: Denise Rageot, Florent Beaufils, Anna Melone, Alexander M. Sele, Thomas Bohnacker, Marc Lang, Jürgen Mestan, Petra Hillmann, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Discovery and biological evaluation of PQR530, a highly potent dual pan-PI3K/mTORC1/2 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 140. doi:10.1158/1538-7445.AM2017-140

Introduction: The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a fundamental role in many cellular processes like growth, survival, proliferation, differentiation and motility. In cancers several mutations have been identified that lead to constitutive activation of PI3K. PQR530 is a novel, ATP site directed inhibitor of all PI3K isoforms and the mammalian target of rapamycin (mTOR) complexes C1/2 that is currently in pre-clinical development. PQR530 potently binds to its targets, inhibits cell proliferation and shows excellent selectivity versus related and unrelated kinases [1]. Results: PQR530 inhibits PI3K signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been found for PQR530 during GLP toxicological testing in rats and dogs. Increase in insulin and blood glucose, a treatable class effect of PI3K inhibitors, has been observed after PQR530 administration to mice. Investigation of mutagenicity and hERG binding resulted in a clean profile. PQR530 exhibited dose-proportional pharmacokinetics (PK) in male C57BL/6J mice. A maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (7.8 μg/ml and 112.6 μg/ml, respectively) indicating that efficacious concentrations were reached in both tissues. The calculated half-life (t1/2) for plasma and brain was approximately 5 hours. PQR530 potently inhibited PI3K signaling in vivo for several hours after administration of a single oral dose of 50 mg/kg. Tumor growth was significantly decreased in SUDHL-6 lymphoma, RIVA lymphoma and OVCAR-3 ovarian cancer mouse xenografts using daily, oral administration. Conclusion: PQR530 is a potent, ATP competitive pan-PI3K and mTORC1/2 inhibitor. The physico-chemical properties of PQR530 result in good oral bioavailability and excellent brain penetration. PQR530 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound. [1] Rageot D, et al., Discovery and biological evaluation of PQR530, a highly potent dual pan-PI3K/mTORC1/2 inhibitor, abstract submitted for AACR Annual Meeting 2017, April 1-5, Washington, D. C., USA. Citation Format: Petra Hillmann, Denise Rageot, Florent Beaufils, Anna Melone, Alexander Sele, Robert A. Ettlin, Jürgen Mestan, Vladimir Cmiljanovic, Marc Lang, Elisabeth Singer, Carolin Walter, Hoa HP Nguyen, Paul Hebeisen, Matthias P. Wymann, Doriano Fabbro. Pharmacological characterization of the selective, orally bioavailable, potent dual PI3K/mTORC1/2 inhibitor PQR530 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 159. doi:10.1158/1538-7445.AM2017-159

Background. The PI3K/AKT/mTOR pathway is an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor that has shown in vitroanti-lymphoma activity (Tarantelli et al, ASH2015) and is in phase 2 trial (NCT02249429, , NCT02723877, NCT02669511). PQR620 is a novel mTORC1/2 inhibitor that has shown preclinical activity in solid tumor models (Beaufils et al, AACR 2016). Here, we present the in vitro and in vivo anti-lymphoma activity of PQR620 as single agent and also the in vivo results of PQR620 or PQR309 containing combinations with the BCL2 inhibitor venetoclax. Materials and Methods. The drug concentration causing 50% inhibition of cell proliferation (IC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well plates. For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/J) mice were subcutaneously inoculated with 10 x106 (RIVA) or with 5 x106(SU-DHL-6) cells. Results. PQR620 had a median IC50 of 250 nM (95%CI, 200-269 nM) when tested on 44 lymphoma cell lines. Activity was higher in B cell (no.=36) than in T cell tumors (no.=8) (median IC50s: 250 nM vs 450 nM; P=0.002). At 72h, anti-tumor activityof PQR620 was mostly cytostatic and apoptosis induction was seen only in 6/44 cell lines (13%), Sensitivity to PQR620 or apoptosis induction did not differ between DLBCL and MCL, and they were not affected by the DLBCL cell of origin, by TP53 status or by the presence of MYC or BCL2 translocations. The activity of PQR620 as single agent underwent in vivo evaluation in two DLBCL models, the germinal center B cell type DLBCL (GCB-DLBCL) SU-DHL-6 and the acivated B cell-like DLBCL (ABC-DLBCL) RIVA. Treatments with PQR620 (100mg/kg dose per day, Qdx7/w) started with 100-150 mm3 tumors and were carried for 14 (SU-DHL-6) or 21 days (RIVA). In both models, PQR620 determined a 2-fold decrease of the tumor volumes in comparison with control, with significant differences in both SU-DHL-6 (D7, D9, D11, D14; P < 0.005) and RIVA (D14, D16, D19, D21; P < 0.005). Based on the previously reported synergy between the dual PI3K/mTOR inhibitor PQR309 and venetoclax (Tarantelli et al, ASH 2015), we evaluated the combination of the PQR620 or PQR309 with the BCL2 inhibitor venetoclax (100 mg/kg, Qdx7/w) in the SU-DHL-6 model. Both the venetoclax combination with the dual PI3K/mTOR inhibitor and the venetoclax combination with mTORC1/2 inhibitor were superior to the compounds given as single agents, leading to the eradication of the xenografts. The combination of PQR620 with venetoclax showed highly significant differences either versus control or single agents during all days of the experiment (D4, D7, D9, D11, D14; P < 0.001). Similarly, the combination of PQR309 with venetoclax showed highly significant differences versus venetoclax (D7, D9, D11, D14; P < 0.001) and PQR309 (D7, D9, D11; P < 0.005) alone. Conclusions. The novel mTORC1/2 inhibitor PQR620 had in vitro and in vivo anti-lymphoma activity as single agent. In vivo experiments showed that both PQR620 and the dual PI3K/mTOR inhibitor PQR309 can strongly benefit from the combination with the BCL2 inhibitor venetoclax. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Fabbro:PIQUR Therapeutics AG: Employment. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees.

Background The pathogenesis of HD is not entirely understood, but it is clear that the expression of the disease-causing mutant huntingtin protein leads to the disruption of a variety of cellular functions, which ultimately results in cell death. A hallmark of HD is the accumulation of intracellular inclusions of mHTT, the mHTT aggregates. Although the toxicity of mHTT aggregates is still debated, various studies have demonstrated that induction of autophagy, the major pathway for the degradation of mHTT aggregates, leads to increased cell viability. ATP- competitive mTOR inhibitors have been shown to be more efficient in induction of autophagy and reduction of protein aggregates than allosteric mTOR inhibitors, like Rapamycin. Aim The ability of catalytic mTOR inhibitors to cross the blood-brain barrier, however, is limited and mTOR inhibition in brain is insufficient. Therefore two novel mTOR inhibitors were evaluated for their efficacy in mTOR inhibition in an HD context, by assessing their potential for inducing autophagy and reducing HTT levels. Methods Expression levels of autophagic markers and well-studied mTOR targets were analysed by western blot analysis in STHdhQ7/Q7 and STHdhQ111/Q111 cells and brain samples from mice treated with both compounds. Cytotoxic effects were monitored in STHdh cells. For studying HTT levels HEK293T cells were transiently transfected with HTT- Exon 1- eGFP constructs with either 19 or 51 Q and analysed by fluorescent imaging and filter trap assay. Results Treatment with both compounds led to decreased cytotoxicity in STHdh cells. The phosphorylation level of mTOR targets 4EBP1 and ribosomal protein S6 were found to be reduced, while LC3B – II levels increased in vitro and in vivo, indicating inhibition of mTOR activity and induction of autophagy. Furthermore reduction of aggregates was demonstrated upon treatment with both substances. Conclusion Originally derived from cancer treatment, these second generation mTOR inhibitors show brain penetrance and effectiveness in cell models of HD, which suggests them as potentially relevant in treatment of neurodegenerative disorders.

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 (t1/2) for plasma and brain was approximately 5 hours. After 8 hours, the total exposure (expressed as AUC0-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.

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, Jürgen 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.

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.

Cancer cells exhibit several unique metabolic phenotypes that are critical for cell growth and proliferation(1). Specifically, they overexpress the M2 isoform of the tightly regulated enzyme pyruvate kinase (PKM2), which controls glycolytic flux, and are highly dependent on de novo biosynthesis of serine and glycine(2). Here we describe a new rheostat-like mechanistic relationship between PKM2 activity and serine biosynthesis. We show that serine can bind to and activate human PKM2, and that PKM2 activity in cells is reduced in response to serine deprivation. This reduction in PKM2 activity shifts cells to a fuel-efficient mode in which more pyruvate is diverted to the mitochondria and more glucose-derived carbon is channelled into serine biosynthesis to support cell proliferation.

Addition of short (6 to 16 amino acids) peptide sequences to the N-terminus of p110α induces a gain of function. Such sequences include the common Flag, His and VSV tags as well as random sequences. An N-terminal myristylation signal generally believed to activate p110α by providing a constitutive membrane address is also activating, even if myristylation is mutationally abolished. The gain of function seen with N-terminally tagged (NTT) p110α constructs extends to signaling, oncogenic transformation and stimulation of cell growth. The activating effect of N-terminal tags requires a functional Rasbinding domain in p110α. Mutations in the RBD domain (T208D and K227A) abolish the gains of function in oncogenicity and signaling. The dominant negative mutant of Ras, RasN17, interferes with transformation induced by NTT p110α. In contrast, binding to p85 activity is not required for cellular transformation and enhanced signaling by NTT p110α.

Cancer-specific mutations in the iSH2 (inter-SH2) and nSH2 (N-terminal SH2) domains of p85alpha, the regulatory subunit of phosphatidylinositide 3-kinase (PI3K), show gain of function. They induce oncogenic cellular transformation, stimulate cellular proliferation, and enhance PI3K signaling. Quantitative determinations of oncogenic activity reveal large differences between individual mutants of p85alpha. The mutant proteins are still able to bind to the catalytic subunits p110alpha and p110beta. Studies with isoform-specific inhibitors of p110 suggest that expression of p85 mutants in fibroblasts leads exclusively to an activation of p110alpha, and p110alpha is the sole mediator of p85 mutant-induced oncogenic transformation. The characteristics of the p85 mutants are in agreement with the hypothesis that the mutations weaken an inhibitory interaction between p85alpha and p110alpha while preserving the stabilizing interaction between p85alpha iSH2 and the adapter-binding domain of p110alpha.

Introduction. PQR309 is a novel oral dual PI3K/mTOR inhibitor (Cmiljanovic et al, AACR 2015), being now evaluated as single agent in a phase I study in patients with relapsed or refractory lymphomas (NCT02249429). We previously reported activity of PQR309 as single agent in a large panel of lymphoma cell lines, and early combination data in two diffuse large B-cell lymphoma (DLBCL) cell lines (Tarantelli et al, ASH 2014). Here, we present extended combination data that might enhance the anti-proliferative effect of the single drug. Methods. A panel of DLBCL cell lines derived from activated B-cell like (ABC) or germinal center B (GCB) subtype, with or without BCL2 or MYC deregulation, were used: RIVA (ABC, BCL2 amplification), SU-DHL-2 (ABC), TMD8 (ABC) and U2932 (ABC, BCL2 amplification); DOHH2 (GCB, BCL2 and MYC translocations), SU-DHL-6 (GCB, BCL2 translocation), KARPAS422 (GCB, BCL2 translocation), OCI-LY18 (GCB, BCL2 and MYC translocations), SU-DHL-10 (GCB), OCI-LY1 (GCB, BCL2 translocation). Cell lines were exposed to increasing doses of PQR309 alone or in combination with increasing doses of other agents for 72h and synergism was assessed with the Chou-Talalay combination index (CI): 1.1, no benefit. Results. The combination of PQR309 with the BCL2 inhibitor venetoclax (ABT199) gave positive results in 6/8 cell lines, with strong synergism in three (U2932, median CI=0.1, SU-DHL-6, 0.14, Karpas422, 0.14) and synergism in the remaining three (TMD8, 0.5, RIVA, 0.56, DOHH2, 0.65). No benefit was observed in SU-DHL-2 and OCI-LY18. The combination of PQR309 with the BTK inhibitor ibrutinib was synergistic in 3/4 ABC-DLBCL (RIVA, 0.42; U2932, 0.6; TMD8, 0.57), while it was of no benefit in SU-DHL-2. The combination of PQR309 with the immunomodulator lenalidomide was of benefit in 4/4 ABC-DLBCL: synergistic in three (RIVA, 0.38; U2932, 0.4; TMD8, 0.5) and additive effect SU-DHL-2 (1.01). The combination of PQR309 with the HDAC-inhibitor Panobinostat was synergistic in 5/6 (KARPAS422, 0.21; U2932, 0.65; SU-DHL-6, 0.67; DOHH2, 0.8; SU-DHL-2, 0.83), but not in the TMD8 (1.14). In our models, synergism was observed in only 2/5 cell lines (TMD8, 0.6; DOHH2, 0.89) exposed to both PQR309 and anti-CD20 monoclonal antibody, while not in KARPAS422, SU-DHL-6, or U2932. Finally, since we had previously observed an up-regulation of the PIM1 kinase after exposure to PQR309 (Tarantelli et al, ICML 2015), potentially acting as a mechanism of adaptive resistance, we evaluated the addition of the PIM inhibitor AZD1208 to PQR309 in four DLBCL cell lines. The combination showed synergism in two (OCI-LY1, 0.29; SU-DHL-10, 0.57), an additive effect in TMD8 (0.95) and no benefit in the SU-DHL-2 (1.15). Conclusions. The novel dual PI3K/mTOR inhibitor PQR309 showed synergism when combined with additional targeted agents in different DLBCL models, including some derived from double hit lymphomas. In particular, the combination with the BCL2 inhibitor venetoclax showed very good results, especially in cell lines bearing BCL2 gene deregulation due to chromosomal translocation or genomic amplification, and the combination with a PIM inhibitor might overcome early adaptive resistance to PQR309. These data provide the basis for future pre-clinical and clinical studies. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Stathis:PIQUR Therapeutics AG: Research Funding. Fabbro:PIQUR Therapeutics AG: Employment. Wicki:PIQUR Therapeutics AG: Employment, Research Funding. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees. Bertoni:PIQUR Therapeutics AG: Research Funding; Oncology Therapeutic Development: Research Funding.

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.

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

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

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

Dual PI3K/mTOR inhibitors represent a promising class of anti/cancer compounds, of potential interest in lymphoid neoplasms which present activation of both targeted pathways. PQR309 is a novel, oral, member of this class of compounds and, as single agent, is currently being evaluated in a phase I for patients with solid tumors (NCT01940133). Here, we present the activity of the compound in pre-clinical models of mature lymphoid tumors, also integrating response data with genomic features. Methods. 48 cell lines [27 derived from diffuse large B-cell lymphoma (DLBCL), 10 from mantle cell lymphoma (MCL), 3 from splenic marginal zone lymphoma (SMZL), 8 from anaplastic large cell lymphoma (ALCL)] were treated with increasing doses of PQR309 and MTT assays were performed after 72 hrs exposure. A second dual PI3K/mTOR inhibitor, GDC0980, and the PI3Kdelta inhibitor Idelalisib were also used on all the cell lines. IC50, GI50, LC50, and TGI values were used to estimate the cytotoxic and cytostatic effects. PQR309-induced cytotoxic activity was tested by AnnexinV assay. Synergy was assessed by the Chou-Talalay combination index (CI) on 2 DLBCL cell lines (TMD8, U2932) exposed to increasing doses of PQR309 alone or in combination with increasing doses of other drugs for 72 hrs. Baseline gene expression profiling (GEP) was obtained on the cell lines with the Illumina HumanHT-12 Expression BeadChips and integrated with the anti-proliferative effect. Results. PQR309 showed potent anti-proliferative activity in most of the cell lines tested. The median IC50 was 242 nM (18nM-3.6 mcM), GI50 141 nM (25 nM-1.7 mcM), LC50 2.7 mcM (306 nM->10 mcM), TGI 711 nM (69 nM - >10 mcM). DLBCL (median IC50=166 nM), MCL (234 nM) and SMZL (214 nM) were all more sensitive than ALCL (664 nM) (P=0.005). Activated B-cell like (ABC) and germinal center B-cell like (GCB) DLBCL subtypes were equally sensitive. Across the 48 cell lines, PQR309 and GDC0980 presented a highly correlated pattern of anti-proliferative activity (R=0.95). Idelalisib appeared significantly less active and its pattern of sensitive cell lines was less correlated with PQR309 (R=0.67) or GDC0980 (R=0.71). In DLBCL cell lines, PQR309 (1 mcM) was able to inhibit IgM-stimulation induced p-AKT(Ser 473) in 2/2 cells and the baseline p-AKT(Ser 473) levels in 1/1. PQR309 (500 nM, 72 hrs) caused apoptosis in 1/7 cell lines. Synergism or additive effected were observed in 2/2 cells combining PQR309 with the BCL2 inhibitor ABT199 (CI = 0.1 and 0.5), the immunomodulatory drug lenalidomide (0.5 and 0.4), the BTK-inhibitor ibrutinib (0.6 and 0.57) or the proteasome inhibitor bortezomib (0.9 and 0.9), and in 1/2 with the anti-CD20 monoclonal antibody rituximab (0.6), the BET inhibitor JQ1 (0.7) and the chemotherapy agent bendamustine (0.7). We then looked for baseline GEP features associated with sensitivity to PQR309, by comparing very sensitive (IC50 < 200 nM) versus less sensitive DLBCL cell lines (IC50 > 400 nM). Transcripts more expressed in sensitive cells were significantly enriched of genes involved in B-cell receptor pathway/signaling, kinases regulation, immune system. Transcripts associated with less sensitive cells were enriched of members of proteasome pathway, oxidative phosphorylation, translation initiation. Genes coding for individual proteins involved in PI3K signaling cascade were differentially expressed between the two groups of cells. Conclusions. PQR309 showed promising activity as single agent and in combination providing the basis for phase I/II studied dedicated for lymphoma patients. Baseline features associated with response were identified and are worth of being validated in the context of the next clinical trials. (CT and EG contributed equally to this work) Disclosures Hillmann: Piqur Therapeutics AG: Employment. Fabbro:Piqur Therapeutics AG: Employment. Hebeisen:Piqur Therapeutics AG: Employment. Betts:Piqur Therapeutics AG: Consultancy. Wicki:Piqur Therapeutics AG: Research Funding; University Hospital Basel: Employment. Cmiljanovic:Piqur Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees. Bertoni:Piqur Therapeutics AG: Research Funding.

Melanoma is a highly aggressive malignancy with an exceptional ability to develop resistance and no curative therapy is available for patients with metastatic disease. Inhibitor of apoptosis proteins (IAP) play a key role in preventing cell death by apoptosis. In normal cell, IAPs are highly regulated by endogenous antagonists (e.g. SMAC) but in melanoma cell lines and in patient samples expression levels of IAPs are generally high and depleting IAPs by siRNA tended to reduce cell viability, with XIAP reduction being the most efficient [1]. Small molecule IAP antagonists have the ability to switch IAP-controlled pro-survival pathways towards apoptosis and cell death. Recent evidence suggests that a true dual antagonist of both cIAP1 and XIAP will promote an effective apoptotic response through generation of death-inducing ripoptosome complexes, with resultant caspase activation [2, 3]. We have used our fragment-based drug discovery technology PyramidTM to derive a non-peptidomimetic IAP antagonist, AT-IAP, which does not have an alanine warhead and has nanomolar cellular potency for both XIAP and cIAP1. Initial pharmacokinetic and pharmacodynamic modeling of AT-IAP in mice bearing the MDA-MB-231 cell line indicated that daily oral dosing of AT-IAP at 30 mg/kg ensures high concentrations of compound in tumor and plasma over a 24 h period with resultant inhibition of both XIAP and cIAP1 and induction of apoptosis markers (cleaved PARP and cleaved caspase-3). In this paper, we describe the characterization of AT-IAP in melanoma models. An in vitro cell line proliferation screen demonstrated that 36% of melanoma cell lines exhibited enhanced sensitivity to AT-IAP, which was improved on addition of exogenous 1 ng/ml TNF-α (92% of cell lines were sensitive to AT-IAP + TNF-α). Sensitivity of melanoma cells to AT-IAP has also been confirmed in a panel of 20 primary melanoma tumors in colony formation assays set up in the presence and absence of added TNF-α. Finally, a set of biomarkers has been identified and used to predict single agent activity of AT-IAP in a range of melanoma cell line and patient derived xenograft models. [1] Engesaeter et al., Cancer Biology & Therapy, 2011, 12 (1), 47 [2] Ndubaku et al., ACS Chem Biol., 2009, 4 (7), 557 [3] Meier, P., Nat Rev. Cancer, 2010, 10 (8), 561 Citation Format: Gianni Chessari, Ahn Maria, Ildiko Buck, Elisabetta Chiarparin, Joe Coyle, James Day, Martyn Frederickson, Charlotte Griffiths-Jones, Keisha Hearn, Steven Howard, Tom Heightman, Petra Hillmann, Aman Iqbal, Christopher N. Johnson, Jon Lewis, Vanessa Martins, Joanne Munck, Mike Reader, Lee Page, Anna Hopkins, Alessia Millemaggi, Caroline Richardson, Gordon Saxty, Tomoko Smyth, Emiliano Tamanini, Neil Thompson, George Ward, Glyn Williams, Pamela Williams, Nicola Wilsher, Alison Woolford. AT-IAP, a dual cIAP1 and XIAP antagonist with oral antitumor activity in melanoma models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2944. doi:10.1158/1538-7445.AM2013-2944

Nature 491, 458–462 (2012); doi:10.1038/nature11540 In Fig. 1a of this Letter, the western blot originally published in the PKM2 panel was a re-probe of the blot used in the PKM1 panel and the signal picked up was from the first antibody (PKM1) and not from PKM2. We have repeated the experiment on three different membranes with the antibodies used in the original paper for PKM1, PKM2 and total PKM, using actin as the loading control (Fig.

XIAP and cIAP1 are members of the inhibitor of apoptosis (IAP) protein family. Both proteins have the ability to attenuate apoptosis induced through intrinsic and extrinsic stimuli via inhibition of caspase-3, -7, -8 and -9. The defining feature of both XIAP and cIAP1 is the presence in their protein sequence of 3 Baculoviral IAP Repeat (BIR) domains, which are necessary for their antiapoptotic activity. The mitochondrial protein SMAC uses its N-terminal region (AVPI) to interact with BIR domains and deactivate the antiapoptotic function of IAPs. Several companies and academic groups have active programs developing SMAC peptidomimetic compounds based on the AVPI motif. In general, those compounds have the tendency to be cIAP1 selective like their tetrapeptide progenitor (AVPI IC50 values for XIAP-BIR3 and cIAP1-BIR3 are 0.3 uM and 0.016 uM respectively). Using our fragment-based screening approach, PyramidTM, we identified a non-peptidomimetic chemotype which binds with similar potency to the BIR3 domain of both XIAP and cIAP1. Hit optimisation using a structure based approach led to the discovery of potent true dual XIAP and cIAP1 antagonists with good in vivo physico-chemical profile and no P450 or hERG liabilities. Dual XIAP/cIAP1 inhibitors have potential for more effective apoptosis and less toxicity associated with cytokine production. Compounds were initially characterised in fluorescence polarisation binding assays using XIAP-BIR3 or cIAP1-BIR3 domains. Robust induction of apoptosis was observed in two sensitive breast cancer cell lines (EC50s well below 0.1 uM in EVSA-T and MDA-MB-231); whilst HCT116 cells (colon cancer) were insensitive (unless exogenous TNF-α was added). This in vitro cell line killing was demonstrated to correlate closely with cIAP1 antagonism and hence a parallel cell assay was established to measure XIAP antagonism. An engineered HEK293 cell line was stably co-transfected with full length FLAG-tagged human XIAP cDNA and full length (untagged) human caspase-9 cDNA. Inhibition of caspase-9 binding to XIAP was measured in immunoprecipitation assays. This gave us a sensitive read-out for XIAP antagonism in cells which could be plotted against the most sensitive cell killing read-out (from the EVSA-T cell line) to establish relative XIAP vs cIAP1 selectivities and to select dual antagonists of both IAPs. Potent compounds (HEK293-EC50 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2018. doi:1538-7445.AM2012-2018

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 compound’s 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.