Differential Role of the B-Cell Receptor Pathway in Diffuse Large Cell B Cell Lymphoma: Temsirolimus Has Additive Effects in Combination with the BTK Inhibitor PCI-32765 and PI3K Inhibitor Cal101 but Antagonizes Bortezomib in GCB Subtype

Diffuse large cell B cell lymphoma (DLBCL) is the most common malignant lymphoma, with the molecular subtypes activated b cell-like (ABC) and germinal center b cell-like (GCB) lymphoma showing distinct biological and clinical characteristics. The B-cell receptor signal pathway including the kinases BTK and PI3K, both upstream of mTOR, play an important role in the pathogenesis of DLBCL. In addition, our previous analyses showed that the proteasome inhibitor Bortezomib also interacts with this pathway.

Established GCB (DB, WILL-2, SU-DHL-4, SU-DHL-5, HT, ULA) and ABC (U2932, HBL-1) cell lines were cultivated under standard cell culture conditions. In all cell lines dose finding experiments with various concentrations of temsirolimus, BTK inhibitor PCI-32765, PI3K delta inhibitor Cal101 and bortezomib were performed in triplicates. Viable cell lines were counted with an automated cell viability analyzer using trypan blue exclusion test after 0, 24, 48 and 72 hours.

Interestingly, all cell lines showed significantly reduced cell growth (in comparison to untreated cell lines) after treatment with temsirolimus (10nM) 72 hours after treatment [53,1%, range 44,4%-69.4%]. In contrast, PCI-32765 (2,5nM) significantly reduced cell growth only in the ABC cell lines HBL-1 [50,2% ± 6,6%] and U2932 [69,7% ± 7,3%] but none of the GCB cell lines. Finally, Cal101 5μM reduced cell growth in both ABC cell lines [U2932 63,1% ± 9,6%; HBL-1 63,2% ± 6,6%] whereas heterogeneous growth reduction was detected in GCB cell lines [ULA 45,6% ± 3,9%; HT 51,9% ± 6,1%; SU-DHL-5 35, 6% ± 3,0%; Will-2 76,5% ±9,3%; DB 74,8% ± 8,7%].

Nonetheless, combination of temsirolimus with PCI-32765 showed synergistic effects in the GCB cell lines DHL-5 [27,2% ± 3,4%] and Will-2[53,4% ± 2,5%] and additive effects in ABC cell lines [HBL-1 28,8% ± 2,4%; U2932 24,9% ± 1,6%]. Combination of temsirolimus with Cal101 also showed additional effects in both, ABC and GCB cell lines. Furthermore the combination of PCI-32765 with Cal101 showed synergistic effects in the GCB cell lines SUDHL-5 [14,0% ± 7,1% and Will-2 [57,8% ± 3,0%] and additive effects in ABC cell lines [HBL-1 28,9% ± 4,7%; U2932 36,6% ± 11,2%]. Cell cycle analysis detected a BTK and PI3K induced G1 phase arrest.

In contrast, temsirolimus [T] combined with bortezomib [B] induced antagonistic effects in the GCB cell lines ULA [TB 62,6% ± 0,7%, T 58,5% ±,9%, B 53,9% ± 1,1%] and SU-DHL-5 [TB 51,9% ± 2,67%, T 53,4% ± 1,7%, B 57,1% ± 0,5%]. Interestingly, in the ABC cell lines HBL-1 [TB 37,4% ± 2,8%,T 52,3% ± 2,8%, B 62,7% ± 4,0%] and U2932 [TB 29,3% ± 3,6%,T 61,8% ± 1,8%, B 43,3% ± 3,7%] additive effects were seen. Furthermore, combination of bortezomib with Cal101 [C] showed antagonistic effects in U2932 [BC 95,8% ± 4,1%], ULA [BC 42,8% ± 7,7%] and SU-DHL5 [BC 47,9% ± 4,0%].

In contrast, combination of bortezomib with PCI-32765 additively impaired cell growth in HBL-1, ULA, SU-DHL5 and DB cell lines.

ABC-DLBCL and GCB-DLBCL show significantly reduced cell growth after treatment with the mTOR inhibitor temsirolimus. In consistency with reported data (Davis 2010) GCB cell lines were not sensitive towards PCI-32765. However, combinations with temsirolimus showed additional or synergistic effects also in GCB cell lines.

In contrast, combination of bortezomib with temsirolimus or CAL101 was antagonistic in some GCB cell lines.

Different genetic mutations involving CD79b and regulators of NF-kB which have been identified in the majority of ABC and some GCB cell lines (HBL-1, U2923 and SUDHL-5) may be responsible for the heterogeneous response towards inhibitors of the B-cell receptor pathway.

Hess:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Dreyling:Pfizer: Research Funding, Speakers Bureau, scientific advisory.