PI3Kδ inhibitor idelalisib in combination with BTK inhibitor ONO/GS-4059 in diffuse large B cell lymphoma with acquired resistance to PI3Kδ and BTK inhibitors

Abstract

Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton's tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton's tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of PIK3CD. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton's tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (TNFAIP3 Q143*), which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton's tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to evaluate the combination of idelalisib and ONO/GS-4059 (NCT02457598).

Conflict of interest statement

All authors were employees of the study sponsor, Gilead Sciences, Inc, at the time the study was conducted. Additionally, we adhere to the PLOS ONE guidelines on sharing data and materials.

Figures

Fig 1. TMD8 as a model for evaluation of PI3Kδ and BTK inhibition.

(A) Cell viability with isoform-specific PI3K inhibitors was assessed by CellTiter Glo after 96 hours, mean ± SEM, n = 4. (B) PI3K isoform expression in TMD8 cells using Simple Western, concentration (pg/μL) quantitated by recombinant protein standards, representative experiment shown of n = 3. (C) Cell viability of TMD8 was assessed with idelalisib in combination with ONO/GS-4059 at fixed concentrations (3, 6, or 12 nM) or with ONO/GS-4059 in combination with idelalisib at fixed concentrations (20, 100 or 600 nM) after 96 hours, mean ± SEM, n = 8. (D) Cells were treated with idelalisib (420 nM), ONO/GS-4059 (320 nM) or in combination for 48 hours and apoptosis was assessed by FITC Annexin V staining, and measured by flow cytometry, mean values ± SD, n = 4. (E) Cells were treated with idelalisib (420 nM), ONO/GS-4059 (320 nM) or in combination for 2 and 24 hours; lysates were analyzed by western blot (p-AKT S473) and Simple Western, representative experiment shown of n = 3.

Fig 2. In vivo inhibition of PI3Kδ and BTK leads to TMD8 tumor regression.

(A) Response of randomized TMD8 xenograft tumors (n = 13 per group) treated with PI3Kδ inhibitor (GS-649443, 1 or 5 mg/kg, BID), BTK inhibitor (ONO/GS-4059, 3 or 10 mg/kg, BID) or combination. Tumor volumes are expressed as mean ± SEM with P

Fig 3. Combination of idelalisib and ONO/GS-4059 can overcome TMD8 acquired idelalisib resistance.

(A) Cell viability of vehicle control line (TMD8IDELA-S) or idelalisib resistant line (TMD8IDELA-R) in response to idelalisib treatment, 96 hour CellTiterGlo assay, mean ± SEM, n = 4. (B) Cell viability of TMD8IDELA-S (top graph) or TMD8IDELA-R cells (bottom graph) treated with idelalisib, ONO/GS-4059 or ONO/GS-4059 in combination with idelalisib (1 μM), n = 4, mean ± SEM. Combination curve is normalized to the single agent alone. (C) Protein lysates from TMD8IDELA-S (vehicle treated) and TMD8IDELA-R treated with vehicle, idelalisib (420 nM), ONO/GS-4059 (320 nM) or in combination for 2 hours were generated and analyzed in a single run using Simple Western, representative image of a single run of n = 3. (D) Quantification of Fig 3C showing AUC normalization to actin, and values normalized to TMD8IDELA-S DMSO treated control for all treatment groups from a single run.

Fig 4. PI3K upregulation in acquired resistance is sensitive to idelalisib and AKT or PDK1 inhibitor combinations.

(A) Protein lysates were generated from TMD8IDELA-S and TMD8IDELA-R cells, and analyzed by western blot (p-AKT S473, p-AKT T308, AKT, p-S6RP S235/236, S6RP, p-GSK3β S9, GSK3β) and Simple Western (PTEN). (B) Cell viability of TMD8IDELA-S (left) and TMD8IDELA-R (right) cells treated with idelalisib, MK-2206 or combination of idelalisib and MK-2206 (1 μM), 96 hours CellTiterGlo assay, mean ± SEM, n = 4. Combination curve is normalized to the single agent alone. (C) Cell viability of TMD8IDELA-S (left) and TMD8IDELA-R (right) cells treated with idelalisib, GSK2334470 or combination of idelalisib and GSK2334470 (3 μM), 96 hours CellTiterGlo assay, mean ± SEM, n = 4. Combination curve is normalized to the single agent alone. (D) TMD8IDELA-R cells were treated with vehicle, idelalisib (1 μM), MK-2206 (1 μM), and idelalisib plus MK-2206, or (E) Vehicle, idelalisib (1 μM), GSK2334470 (1 μM), and idelalisib plus GSK2334470 for 48 hours. Apoptosis was assessed by 7AAD and PE annexin V staining, and analyzed by flow cytometry, mean ± SD, n = 3. (F) TMD8IDELA-R cells were treated with vehicle, idelalisib (1 μM), MK-2206 (1 μM), or idelalisib plus MK-2206 and (G) Vehicle, idelalisib (1 μM), GSK2334470 (1 μM), or idelalisib plus GSK2334470 for 2 hours. Protein lysates were generated and analyzed by western blot, representative experiment of n = 3. TMD8IDELA-S vehicle control was included as a reference on the same membrane.

Fig 5. Loss of A20 and BTK C481F mutation as mechanisms of BTK inhibitor resistance in TMD8.

(A) Cell viability of BTK inhibitor (BTKi)-sensitive (TMD8BTKi-S) and BTKi-resistant (TMD8BTKi-R) cells to ibrutinib, 96 hour CellTiterGlo assay, mean ± SEM, n = 4. (B) Protein lysates were generated for TMD8 (DMS0 control), TMD8A20-Q143*and TMD8BTK-C481F cell lines, and protein expression of A20, p-IκBα, total IκBα, p-BTK and total BTK was analyzed using Simple Western. (C) Cell viability of BTKi-resistant TMD8A20-Q143* (left) or BTKi-resistant TMD8BTK-C481F (right) treated with ONO/GS-4059, idelalisib, or a combination of idelalisib plus ONO/GS-4059 (50 nM), mean ± SEM, n = 4. (D) Lysates of TMD8 DMSO control and TMD8A20-Q143* lines were analyzed by western blot (p-ERK 1/2, p-AKT S473, AKT) and Simple Western (p-BTK, p-IκBα S32, IκBα). Cells were treated with idelalisib (420 nM), ONO/GS-4059 (320 nM) or in combination for 24 hours. (E) Quantification of p-ERK1/2 and p-IκBα S32 blot results from Fig 5D. Values are normalized to actin and graphed as percent of DMSO vehicle control. Simple Western and western blot images are representative experiments from n = 2–3 experiments.

Fig 6. Schematic summarizing the proposed mechanisms of PI3Kδ and BTK inhibitor resistance.

Upon antigen binding to the B-cell receptor (BCR), the PI3K, MAPK and NF-kB pathways are activated through a cascade of phosphorylation events. BCR stimulation recruits SYK to the membrane, where it is phosphorylated and then subsequently phosphorylates BTK. BTK activation leads to subsequent dual phosphorylation and activation of PLCγ2, which leads to downstream generation of the second messengers IP and Dag. Active PI3Kδ is recruited to the membrane and converts PIP2 to PIP3, leading to subsequent activation and phosphorylation of PDK1 and AKT. PTEN is a phosphatase which negatively regulates the PI3K pathway. PKC can be activated downstream of both PI3K and BTK leading to NF-kB and MAPK pathway activation [27]. In our idelalisib resistance model, PTEN is lost which results in the hyperactivation of the PI3K pathway. Resistance can be overcome by inhibiting PI3Kδ and the downstream phosphoproteins PDK1 and AKT. In the BTK resistance model, A20 is lost which leads to hyperactivation of the NF-κB pathway which could be overcome by inhibiting BTK and PI3Kδ. Additionally, the BTK inhibitor binding site at C481 was mutated resulting in ONO/GS-4059 resistance.