Differential regulation of mTOR signaling determines sensitivity to AKT inhibition in diffuse large B cell lymphoma

Abstract

Agents that target components of the PI3K/AKT/mTOR pathway are under investigation for the treatment of diffuse large B cell lymphoma (DLBCL). Given the highly heterogeneous nature of DLBCL, it is not clear whether all subtypes of DLBCL will be susceptible to PI3K pathway inhibition, or which kinase within this pathway is the most favorable target. Pharmacological profiling of a panel of DLBCL cell lines revealed a subset of DLBCL that was resistant to AKT inhibition. Strikingly, sensitivity to AKT inhibitors correlated with the ability of these inhibitors to block phosphorylation of S6K1 and ribosomal protein S6. Cell lines resistant to AKT inhibition activated S6K1 independent of AKT either through upregulation of PIM2 or through activation by B cell receptor (BCR) signaling components. Finally, combined inhibition of AKT and BTK, PIM2, or S6K1 proved to be an effective strategy to overcome resistance to AKT inhibition in DLBCL.

Keywords: AKT; DLBCL; Ibrutinib; S6K1; mTOR.

Figure 1. DLBCL subtypes have different sensitivities to AKT inhibitors

A. Cell lines were sorted according to drug sensitivity (pGI₅₀) by unsupervised hierarchical clustering. Sensitivity was determined using a 72h Alamar Blue assay. B. Dose response curves were generated for the indicated compounds using a 72h CellTiterGlo assay (n = 3). C. DLBCL lines were treated with GSK690693 (5μM) for 1h and 24h. ABC cells are colored in red. GCB are colored in blue.

Figure 2. Distinct regulation of S6K1 signaling in DLBCL subtypes

A. DLBCL lines were treated with GSK690693 (5μM) and MK2206 (5μM) for 1h or 24h before Western blotting. B. Cell lines were treated with PF-4708671 (10μM) or GSK690693 (5μM) and cell viability was measured after 72h by trypan blue staining followed by Cellometer reading (n = 3). Asterisk indicates p < 0.05 C. Cell lines were treated with the indicated compounds for 24h. ABC cells are colored in red. GCB are colored in blue. Intermediate cells are colored in gray.

Figure 3. mTOR signalling is activated independently of AKT in ABC-DLBCL

A. TMD8 cells were treated for 24h hours with ibrutinib (10nM), AZD2014 (200nM), everolimus (100nM) or GSK690693 (5μM). B. Cell lines were treated for 24 hours with inhibitors of BTK (ibrutinib, 10nM), PKCβ (LY-333531, 4μM), SYK (GS-9973, 1μM) C. TMD8 and OCILY10 cells stably expressing pGreenFire NF-κB-Luc were treated for 18h with inhibitors at previously indicated concentrations and IKKβ (Bayer inhibitor C₂₁H₂₅ClN₄O₂, 3μM). Luciferase activity was then assayed using the Dual-Glo (n = 6). Luminescence was normalized to cell number from viable cell counting by Cellometer. D. Cells were treated for 1h or 24h with Bayer IKKβ inhibitor (3μM) before Western blotting. ABC cells are colored in red. GCB are colored in blue. Intermediate cells are colored in gray.

Figure 4. PIM can regulate AKT-independent mTOR signalling

A. PIM2 protein expression was assayed by Western blot. GI₅₀ values for AZD1208 were derived from 72h day Alamar Blue assay. B. Net growth was measured over 72h by viable cell counting using the Cellometer system (n = 3) C. Cell lines were treated for 24h with AZD1208 (1μM) before Western blotting. ABC cells are colored in red. GCB are colored in blue. Intermediate cells are colored in gray.

Figure 5. Combination therapy more effectively inhibits mTOR signalling

A. RI1 cells were treated with GSK690693 (1μM) and AZD1208 (1μM) and proliferation was measured over 72h by CellTiterGlo. B. As in A. but cell were treated for 24h before Western blotting. C. TMD8 cells were treated with GSK690693 (1μM) and ibrutinib (10nM) for 24 hours before Western blotting. D. 72 hour viability was measured in TMD8 by Alamar Blue assay after treatment with ibrutinib and GSK690693 at the indicated concentrations.