Investigation of the Mechanism of Idelalisib Resistance in the Follicular Lymphoma WSU-Fsccl Cell Line

Idelalisib (Zydelig^®), a first-in-class, selective, oral inhibitor of PI3Kd, is approved in the US and EU for the treatment of patients with follicular lymphoma (FL) who have received at least 2 prior systemic therapies based on the outcome of a phase 2 clinical trial demonstrating an ORR of 54% (Gopal AK, et al. ASH 2014, Abstract 1708;  Zydelig^® SmPC, Mar 2015). Complete responses are rare (6%) and patients ultimately relapse leading to an urgent need to understand mechanisms of resistance (MOR) to idelalisib. We report here the MOR identified from 3 sets of the transformed FL cell line WSU-FSCCL which were made resistant to idelalisib by continuous in vitro exposure. 

Methods:   Idelalisib resistance was established by continuous passaging of a clonal isolate of WSU-FSCCL in the presence of 1 μM idelalisib.  Growth inhibition to idelalisib or other inhibitors was performed using CellTiter Glo viability assay (Promega) at 96 h. Clonal isolates of idelalisib sensitive (FSCCL^S) and idelalisib resistant (FSCCL^R) cell lines were generated through two rounds of single cell limiting dilution. Characterization of mutations and gene expression in FSCCL^S and FSCCL^R clones was done by whole exome sequencing (WES, GENEWIZ) and RNA-Seq (Expression Analysis), respectively. Whole cell lysates were analyzed by Simple Western using Peggy Sue (Protein Simple) or SDS-PAGE and Western blot. 

Results:  WSU- FSCCL were highly sensitive to idelalisib and the pan-PI3K inhibitor GDC-0941 with an EC₅₀ of 140 and 180 nM, respectively, indicating that cell viability is driven by PI3Kd, while profiling of BTK inhibitors showed continued lack of activity.  FSCCL^R showed a loss of sensitivity to idelalisib (1 μM) with a maximal growth inhibition of 16% vs. 85% for parental line (FSCCL^S). WES analysis revealed PIK3CA resistance mutations in 3 independently generated sets of FSCCL^R single cell clones, while no PIK3CD, PIK3CB, or PIK3CG resistance mutations were observed. PIK3CA was mutated at N345K (11/11 clones), P539R (3/3 clones) and E970K (3/3 clones).  PI3K isoform profiling showed no alterations in expression of any of the four p110 isoforms. Idelalisib activity on PIK3CA mutant clone viability was dramatically shifted (EC₅₀ >5 μM). The sensitivity of PIK3CA mutant FSCCL^R to the PI3Kα- specific inhibitor BYL719 was not increased compared to FSCCL^S (EC₅₀ > 1.5 μM). Interestingly the combination of idelalisib treatment with BYL719 (0.5 μM) restored the sensitivity of FSCCL^R to a concentration in the range of parental cells (EC₅₀ = 0.066 μM, Figure 1).  A second set of FSCCL^R clones (set 2) were PI3KCA WT and had a 2.7-fold upregulation of PTEN. A survey of compensatory pathway activation revealed upregulation of Src family kinase (SFK) phosphorylation (p-SFK Y416) and specifically of p-Hck Y411 and p-Lyn Y396. SFK phosphorylation was sensitive to treatment with the SFK inhibitor dasatinib. Set 2 FSCCL^R were slightly less sensitive to dasatinib (EC₅₀ = 0.058 μM) compared to FSCCL^S (EC₅₀ = 0.034 μM).  Interestingly, addition of 30 nM dasatinib (EC₂₅ in FSCCL^S) to FSCCL^R increased the sensitivity of set 2 FSCCL^R  to idelalisib (EC₅₀ = 0.95 μM) (Figure 2).  FSCCL^R clones were more resistant to the Syk inhibitor entospletinib (ENTO) (EC₅₀ > 10 μM) as compared to FSCCL^S (EC₅₀ = 0.18 μM).  Profiling of idelalisib activity on set 2 FSCCL^R  clones showed resistance to idelalisib as expected (EC₅₀ > 10, μM); the addition of entospletinib at a clinically relevant concentration (0.68 μM), resulted in greater sensitivity than use of either single agent alone (EC₅₀ = 2.27 μM ) (Figure 3).  RNA-Seq analysis of the FSCCL^R PIK3CA WT single cell clones additionally revealed that a subset of clones (set 3) upregulated a set of WNT pathway genes.  Western blot analysis of set 3 FSCCL^R showed upregulation of downstream markers of the canonical WNT pathway, including LEF1/TCF, c-Jun, β-catenin, c-Myc and p-GSK3β.  

Conclusions:   Mechanisms of idelalisib resistance in the follicular lymphoma WSU-FSCCL cell line were independent of alterations in p110d but included the likely gain of function mutations in the PIK3CA gene and activation of SFK and WNT pathways. The sensitivity to idelalisib in the resistant clones could be re-established by the combination of idelalisib with inhibitors of compensatory pathways:  BYL719 in PIK3CA mutant clones and dasatinib or entospletinib in SFK activated clones.