An Innovative High-Throughput Ex Vivo Drug Assay Incorporating the Native Microenvironment Reveals a Novel Mechanism of Action of Idelalisib in CLL
Authors: Ballesteros Joan , Scarfo Lydia , Mattsson Mattias , Xochelli Aliki , Ranghetti Pamela , Primo Daniel , Robles Alicia , Gorrochategui Julian , Martinez-Lopez Joaquin , De la Serna Javier , González Marcos , Munugalavadla Veerendra , Rosenquist Richard , Stamatopoulos Kostas , Quéva Christophe , Ghia Paolo 
Published: October 2019

Abstract
The microenvironment (ME) critically promotes progression of chronic lymphocytic leukemia (CLL), favoring leukemic cell survival and proliferation as well as inducing drug resistance. We aimed at reproducing the effects of ME stimuli for the development and optimization of an ex vivo assay that would enable to predict in vivo drug efficacy for agents interfering with ME protective effects e.g. the novel BCR inhibitors. To this purpose, we exploited the following 2 new approaches: 1) the Exvitech® proprietary automated flow cytometry-based platform by Vivia Biotech that enables evaluation of up to 20.000 wells and conditions per sample, and 2) Viviaxs Precision Medicine Native Environment approach that utilizes the whole blood sample rather than isolated leukocytes. We present this assay optimization using primary CLL samples and its validation when exposing CLL cells to the registered PI3Kd inhibitor, Idelalisib, for which only a weak pro-apoptotic effect has been reported, besides the known tissue mobilization activity in vivo.
Cryopreserved peripheral blood (PB) mononuclear cells were provided from CLL patients in need of treatment. In order to more closely reproduce the complexity of the in vivo ME, the following elements were evaluated in different combinations: (i) 3 backbone stimulations, previously reported to improve, to different extent, CLL viability and proliferation: CD40L+CpG, CD40L+IL21, CpG+IL2; (ii) "Native Environment", defined as the plasma & erythrocyte/granulocyte fraction of a Ficoll gradient, already shown to improve ex vivo drug testing (Bennet et al. Clin Lymphoma Myeloma Leuk. 2014;14:305-18): the two fractions were added to thawed CLL samples and were obtained from fresh samples of normal donor PB or bone marrow as well as from CLL patients at different stages of disease; (iii) the stroma cell line H5S, added at different ratios (1:10 or 1:100); (iv) both human and bovine fetal serum (at 10 or 20% total volume); (v) stimulatory B cell factors, including IL-21, soluble CD40L, BAFF, and B cell receptor stimulation (anti-IG). CLL cell viability and proliferation was then tested and, although CLL cells from PB are notorious for a low proliferative index and tend to die quickly ex vivo by apoptosis, we achieved a median of 30±3% proliferation (assessed by the CFDA dye) and 60±5% viability (assessed by Annexin V staining) in cryopreserved progressive CLL samples. These results were obtained with the combination of the following assay conditions: CpG+IL2, HS5 (at 1:100 ratio), human serum 10%, and "native environment" from PB of CLL samples (pooled samples to prevent interpatient variability). We then tested the dose responses of Idelalisib in 16 cryopreserved progressive CLL samples and found little effect on the non-proliferative CLL fraction (Fig, 1A), suggesting a limited direct pro-apoptotic activity of the drug. In contrast, potent inhibition of proliferation with median potency (EC50) of 14 nM was observed (Fig 1B). The efficacy was nearly complete leaving a median of 5% resistant CLL cells that proliferated at the highest doses of Idelalisib.
In conclusion, we report a novel ex vivo assay that enables high-throughput pharmacological characterization of compounds and combinations, optimized for CLL cells by incorporating ME stimuli and thereby more accurately simulating in vivo interactions. The increased cell viability and proliferation achieved with this innovative assay offers improved opportunities for ex vivo pharmacology, in particular unraveling a hitherto unknown anti-proliferative mode of action for Idelalisib, a drug interfering with the interaction of CLL cells with the ME.
Figure 1. Dose response curves of Idelalisib incubated for 96 h with 16 CLL samples in the new Microenvironment Native Environment assay. The effect on non-proliferative (A) and proliferative (B) CLL cells identified using flow cytometry as subpopulations with different CFDA staining is shown. Figure 1. Dose response curves of Idelalisib incubated for 96 h with 16 CLL samples in the new Microenvironment Native Environment assay. The effect on non-proliferative (A) and proliferative (B) CLL cells identified using flow cytometry as subpopulations with different CFDA staining is shown.

Disclosures
Ballesteros: Vivia Biotech: Employment. Primo:Vivia Biotech: Employment. Robles:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Munugalavadla:Gilead Sciences: Employment. Stamatopoulos:Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding. Quéva:Gilead Sciences: Employment, Equity Ownership. Ghia:AbbVie: Consultancy; Pharmacyclics: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; Adaptive: Consultancy; Acerta Pharma BV: Research Funding; GSK: Research Funding; Roche: Consultancy, Research Funding; Janssen: Consultancy.