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

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., MacSynergy^TM 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 (E_max = 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.