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., 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 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. | GS-4059 (nM) | EC50 of idelalisib (nM) when combined with GS-4059 | | ----------------- | -------------------------------------------------- | ---- | | TMD-8 | OCI-LY-10 | Ri-1 | Pfeiffer | | | 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 | | | Table 1. Synergistic inhibition of ABC-DLBCL cell viability by GS-4059 and idelalisib ![Figure 1.][1] Figure 1. Growth inhibition of ibrutinib resistant TMD8 with (A) BTK C481F mutation or (B) A20 Q143* mutation A. B. ![Figure 2.][1] 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. [1]: pending:yes