Combination of Idelalisib and ONO/GS-4059 in Lymphoma Cell Lines Sensitive and Resistant to BTK Inhibitors

Abstract

Idelalisib (Zydelig™), a first-in-class, selective, oral inhibitor of PI3Kδ, is approved for the treatment of chronic lymphocytic leukemia (CLL) in combination with rituximab and as monotherapy for patients with follicular lymphoma who have received at least 2 prior therapies. Despite remarkable clinical efficacy, complete responses are rare, highlighting the need to identify more effective therapies, including combinations of novel agents. GS-4059 (ONO-4059) is an investigational next generation BTK inhibitor with improved selectivity compared to ibrutinib. We report here on the results of the combination of idelalisib and GS-4059 in lymphoma cell lines. Methods: Growth inhibition was assessed using CellTiter-Glo™ Assay (Promega) after 72-96 h incubation with idelalisib and GS-4059. Synergy for anti-proliferative effects was assessed using the Bliss Model of Independence (Meletiadis et al., Med Mycol, 2005), using MacSynergy II (Prichard et al., MacSynergyTM II, Version 1.0, 1993) or the Chalice software (Horizon Discovery, Inc., Lehar et al., Nature Biotech, 2009). Lysates were analyzed by Simple Western (Protein Simple) or Western blot. Ibrutinib resistance was established by continuous passaging of a clonal isolate of TMD8 in the presence of 10-20 nM ibrutinib. Resistance mutations were identified by whole exon sequencing (WES, GeneWiz). Results: GS-4059 potently inhibited growth (EC50<26 nM) of 3 ABC-DLBCL cell lines (OCI-LY10, Ri-1, and TMD8) that were also sensitive to idelalisib (EC50<210 nM). The combination showed synergistic growth inhibition in OCI-LY10 and TMD8 and increased apoptosis above the level observed with single agents (Table 1). Idelalisib and GS-4059 synergistically inhibited growth in 2 MCL cell lines (Rec-1 and JMV-2). The combination was additive in the other lymphoma cell lines sensitive to these agents. Two mechanisms of resistance to BTK inhibitors were identified in TMD8: an inactivating mutation in the NF-kB inhibitor A20 (TNFAIP3 Q143*), and a BTK mutation (C481F). TMD8 cells with the BTK (C481F) mutation only were less sensitive to idelalisib (Emax = 14% at 1 uM vs. 86% in parental, Figure 1A). Addition of GS-4059 did not enhance growth inhibition in those clones. A20 mutant only TMD8 cells were resistant to GS-4059 (EC50>10 μM), but were sensitive to idelalisib, albeit less than parental (EC50 ≥ 4300 nM vs. 54 nM). Addition of 50 nM GS-4059 to idelalisib provided further growth inhibition, consistent with the presence of wild-type BTK, and increased the potency of idelalisib to a level comparable to parental TMD8 (EC50 ≥ 99 nM, n=5 clones, Figure 1B). Conclusion: Idelalisib and GS-4059 synergistically inhibited the growth of a subset of DLBCL and MCL cell lines. A20 mutation and loss-of -function was identified as a novel mechanism of resistance to BTK inhibitors. Idelalisib less potently inhibited the growth of A20 mutant TMD8 but the combination with GS-4059 provided additional benefit. TMD8 with a BTK-C481F mutation, were resistant to idelalisib and to the combination with GS-4059. These data suggest that the combination of idelalisib and GS-4059 may overcome some mechanisms of resistance to BTK. Table 1. Synergistic inhibition of ABC-DLBCL cell viability by GS-4059 and idelalisib GS-4059 (nM) EC50 of idelalisib (nM) when combined with GS-4059 TMD-8 OCI-LY-10 Ri-1 Pfeiffer 0 254 440 442 174 5 130 38 372 NTc 15 32 22 372 NT 45 24 5 372 174 EC50 shift (fold) 10.6 88 12 1 Synergy Score 65 65 0 0 Figure 1. Growth inhibition of ibrutinib resistant TMD8 with (A) BTK C481F mutation or (B) A20 Q143* mutation A. B. Figure 1. Growth inhibition of ibrutinib resistant TMD8 with (A) BTK C481F mutation or (B) A20 Q143* mutation. / A. / B. Figure 2. Figure 2. Disclosures Tannheimer: Gilead Sciences: Employment, Other: Share holder. Sorensen:Gilead Sciences: Employment, Other: Share holder. Yahiaoui:Gilead Sciences: Employment, Other: Share holder. Meadows:Gilead Sciences: Employment, Other: Share holder. Li:Gilead Sciences: Employment, Other: Share holder. Yue:Gilead Sciences: Employment, Other: Share holder. Tumas:Gilead Sciences: Employment, Equity Ownership. Queva:Gilead Sciences: Other: Share holder.

Bruton tyrosine kinase inhibitor ONO/GS-4059: from bench to bedside

“…ONO/GS-4059 was also evaluated in combination with other agents. Combination of idelalisib, a phophotidylinositol 3 kinase (PI3K) inhibitor [ 69 ], showed synergistic activity in inhibiting the growth of a subset of DLBCL and MCL cell lines, including 3 ABC-DLBCL cell lines (OCI-LY10, Ri-1, and TMD8) and 2 MCL cell lines (Rec-1 and JMV-2) [ 67 ]. Two mechanisms of resistance to BTK inhibitors were identified in the TMD8 cell line: a NF-kB inhibitor A20 mutation (TNFAIP3 Q143*), and a BTK mutation (C481F).…”

Second-generation inhibitors of Bruton tyrosine kinase

“…Further investigations of ONO/GS-4059 are ongoing to ascertain its advantages in combination therapies. Combination of idelalisib and ONO/GS-4059 synergistically inhibited the growth of a subset of DLBCL and MCL cell lines [ 71 ]. This combination led to more significant growth inhibition of the A20 mutant TMD8 cells than single agent idelalisib.…”

Responses to the Selective Bruton’s Tyrosine Kinase (BTK) Inhibitor Tirabrutinib (ONO/GS-4059) in Diffuse Large B-cell Lymphoma Cell Lines

“…Six showed a response to tirabrutinib when concentrations up to 10,000 nM were used; four of these were derived from ABC-type DLBCL (TMD8 with EC50 of 4.5 nM; OCI-LY10, U2932, and HBL1 each with an EC50 of approximately 3000 nM). The other responding cell lines were Pfeiffer, a GCB-DLBCL with an EC50 of around 3000 nM, and REC1, an MCL line with an EC50 of 33 nM [ 10 ]. The cell line most sensitive to tirabrutinib was the CD79B mutant cell line TMD8.…”

“…In the present study, tirabrutinib-resistant TMD8 which had PLCγ2 R665W was examined by combination therapy. It has been reported that tirabrutinib in combination with idelalisib, entospletinib, and the BCL2 inhibitor ABT-199 synergistically increased apoptosis in primary CLL cells, a subset of DLBCL and MCL cell lines [ 10 , 18 , 19 ]. Interestingly, the BTK Cys481S mutation activated cell-cycle reprogramming besides AKT activation [ 20 ], while the CDK4/6 inhibitor palbociclib, in combination with tirabrutinib, showed strong synergy in our resistant model, TMD8R, which does not have BTK Cys481S.…”