The Novel PI3K/mTOR Dual Inhibitor PQR309 in Pre-Clinical Lymphoma Models: Demonstration of Anti-Tumor Activity As Single Agent and in Combination and Identification of Gene Expression Signatures Associated with Response
Authors: Tarantelli Chiara , Gaudio Eugenio , Kwee Ivo , Rinaldi Andrea , Stifanelli Matteo , Bernasconi Elena , Barassi Chiara , Hillmann Petra , Stathis Anastasios , Carrassa Laura , Broggini Massimo , Stussi Georg , Fabbro Doriano , Hebeisen Paul , Betts Frances , Wicki Andreas , Zucca Emanuele , Cmiljanovic Vladimir , Bertoni Francesco 
Published: October 2019

Abstract
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.