1. Volume 31, Issue 1, Pages 64-78 (January 2017)
Simultaneous Inhibition of PI3Kδ and PI3Kα Induces ABC-DLBCL Regression by Blocking BCR-Dependent and -Independent Activation of NF-κB and AKT 
Juliane Paul, Maurice Soujon, Antje M. Wengner, Sabine Zitzmann-Kolbe, Andrea Sturz, Katja Haike, Koh Hui Keng Magdalene, Sze Huey Tan, Martin Lange, Soo Yong Tan, Dominik Mumberg, Soon Thye Lim, Karl Ziegelbauer, Ningshu Liu 
Cancer Cell 
Volume 31, Issue 1, Pages (January 2017)
DOI: /j.ccell
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2. Cancer Cell 2017 31, 64-78DOI: (10.1016/j.ccell.2016.12.003)
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3. Figure 1
Expression and Functional Activity of PI3K Isoforms and PTEN in NHL
(A–C) The expression of PI3K isoforms and PTEN was characterized by immunohistochemistry (IHC) analysis using antibodies selectively against PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ. IHC was conducted using formalin-fixed paraffin-embedded samples from 45 FL (A) and 45 DLBCL (B) patients. The overall incidence (Aa and Ba) and individual relative expression (Ab and Bb) of each PI3K isoform and PTEN in FL and DLBCL was determined using an intensity grading of 0, 1+, 2+ and 3+ for negative, low, moderate, and high expression, respectively. (C) IHC staining of PI3Kα, PI3Kδ, and PTEN in representative ABC-DLBCL samples.
(D) Western blot analysis of PI3K isoforms, PTEN and BTK, in five ABC-DLBCL cell lines and their corresponding genetic mutation status.
(E) Cell viability was assessed using a CellTiter-Glo Luminescent Assay (Promega). The effect of ibrutinib, copanlisib, idelalisib, and BYL-719 was determined at 72 hr after compound treatment versus DMSO treatment (0% inhibition) and the value at 0 hr was used as 100% inhibition. The IC50 (Ea) and IC90 (Eb) of each compound in the tumor cell lines with a low, medium, and high ratio of PI3Kα/PI3Kδ expression were generated from one representative experiment with triplicates on each data point. ∗Greater than indicated maximum concentration tested.
(F) Inhibition of the PI3K pathway was assessed by p-AKT (S473) and p-4E-BP1 (S65) and apoptosis induction by cleaved PARP using western blot in TMD-8 cells 24 hr after BYL-719 and/or idelalisib treatment.
(G) Apoptosis induction was measured by cleaved PARP using western blot in TMD-8 cells at 24 hr after copanlisib or ibrutinib treatment. See also Figure S1, Tables S1 and S2.
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4. Figure 2
Regulation of Nuclear NF-κB Activity by PI3K and BTK Inhibitors in ABC-DLBCL Cell Lines
(A) Effect of PI3K and BTK inhibitors on nuclear activation of NF-κB was assessed using cell lines with stably transfected NF-κB-luciferase reporter constructs. Cells were treated with a series of dilutions of each compound in triplicate and luciferase activity was determined at 24 hr after treatment. IC50 (Aa) and IC90 (Ab) values were determined. ∗Greater than the maximum concentration tested.
(B) Effect of PI3K and BTK inhibitors on NF-κB target gene expression measured by qRT-PCR analysis of NF-κB target gene expression in U2932 cells.
(C) Effect of PI3K and BTK inhibitors on NF-κB-regulated CCL4 production in TMD-8 and U2932 assessed using an ELISA assay at 24 hr after treatment.
(D and E) Regulation of nuclear translocation of p65 NF-κB by copanlisib, idelalisib, and ibrutinib in OCI-Ly3 (D) and TMD-8 cells (E). IHC staining with a primary antibody against p65 NF-κB (red) and DAPI to visualize nuclear DNA (blue) was performed at the indicated time points after treatment.
(F) IC50 isobolograms and combination indices (CIs) for idelalisib and BYL-719 combination in TMD-8 (Fa), HBL-1 (Fb), and WSU-DLCL2 (Fc) cell lines. See also Figure S2 and Table S3.
Cancer Cell  , 64-78DOI: ( /j.ccell )
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5. Figure 3
In vivo Anti-tumor Activity and Mechanism of Action of PI3K and BTK Inhibitors in Ibrutinib-Responsive CD79Bmut/MYD88mut ABC-DLBCL Tumor Models
(A) SCID mice bearing TMD-8 or LY2298 PDX were treated with copanlisib (intravenously) and ibrutinib (orally) at the indicated doses and dosing schedules. Tumor volume was measured twice weekly and reported as the mean volume ± SEM (a and c). NS, non-significant, ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ compared with vehicle. Relative tumor volume is defined as the percentage of the final tumor volume versus the initial tumor volume (at the time point of starting treatment) of each individual animal (b and d). CR, complete response; PR, partial response; SD, stable disease; and PD, progressive disease.
(B) Tumors from the LY2298 study were collected at the indicated time point after compound treatment and subjected to western blot analysis to evaluate BTK, MAPK, AKT, and NF-κB pathway regulation. See also Figure S3.
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6. Figure 4
In vivo Anti-tumor Activity and Mechanism of Action of PI3K and BTK Inhibitors in Ibrutinib-Resistant MYD88mut and/or CARD11mut ABC-DLBCL Xenograft Tumor Models
(A) SCID mice bearing LY0257 PDX (MYD88mut) and OCI-Ly3 (MYD88mut/CARD11mut) xenografts were treated with copanlisib (intravenously) and ibrutinib (orally) at the indicated doses and dosing schedules. Tumor volume was measured twice weekly and reported as the mean volume ± SEM (a and c). Relative tumor volume (b and d) is defined as the percentage of the final tumor volume versus the initial tumor volume (at the time point of starting treatment) of each individual animal. CR, complete tumor response; PR, partial response; SD, stable disease; and PD, progressive disease. ∗p ≤ 0.05, ∗∗∗p ≤ compared with vehicle.
(B) NF-κB activity was assessed by IL10 gene expression analysis in LY0257 (a) and OCI-Ly3 tumors (b) by RT-PCR.
(C) Western blot analysis of BTK, AKT, MAPK, and NF-κB pathway regulation in LY0257 tumors (a) 3 or 24 hr after treatment and in OCI-Ly3 tumors (b) 1, 7, or 24 hr after treatment with copanlisib or ibrutinib. See also Figure S4.
Cancer Cell  , 64-78DOI: ( /j.ccell )
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7. Figure 5
In Vitro Combination Effects of PI3K Inhibitor Copanlisib and BTK Inhibitor Ibrutinib in ABC-DLBCL Models
(A) IC50 and IC90 isobolograms of copanlisib and ibrutinib combination were generated using a 72-hr CellTiter-Glo proliferation assay. Combination effects were assessed using IC50 and/or IC90 isobolograms for CD79Bmut/MYD88mut TMD-8 (a and b) and TAK1mut/TNFAIP3mut U2932 (c and d) cells and antagonistic combination in MYD88mut/CARD11mut OCI-Ly3 cells (e and f).
(B and C) Mechanistic combination effects on inhibition of NF-κB were measured using a luciferase reporter assay (a and b), and inhibition of p-AKT, p-ERK, and p-BTK was assessed by western blot (c) and in TMD-8 (B) and U2932 (C) cells, as well as the functional consequence on tumor cell survival by measuring apoptotic protein cleaved PARP (Bc) and Mcl-1 (Cc).
(D) Functional and mechanistic combination effects in OCI-Ly3 cells were assessed by combination of copanlisib with the MEK inhibitor refametinib in a CTG assay (a and b) and by western blot analysis of p-AKT, p-BTK, and p-ERK (c). See also Figure S5.
Cancer Cell  , 64-78DOI: ( /j.ccell )
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8. Figure 6
In Vivo Combination Effects of the PI3K Inhibitor Copanlisib and the BTK Inhibitor Ibrutinib
(A) SCID mice bearing TMD-8 tumors were treated with copanlisib (14 mg/kg, intravenously), ibrutinib (20 mg/kg, orally), and their combination at the indicated dosing schedules shown in the legend). Tumor volume was measured twice weekly and reported as the mean ± SEM (a). ∗∗∗p ≤ compared with vehicle and ###p ≤ compared with ibrutinib monotherapy. Relative tumor volume (b) is defined as the percentage of the final tumor volume versus the initial tumor volume (at the time point of starting treatment) of each individual animal. CR, complete tumor response; PR, partial response; SD, stable disease; and PD, progressive disease. Ibrut refers to ibrutinib and copan refers to copanlisib.
(B) SCID mice bearing LY2298 PDX were treated with copanlisib (14 mg/kg, intravenously), ibrutinib (20 mg/kg orally), and their combination at the indicated dosing schedule (red bar indicates copanlisib schedule and gray bar indicates ibrutinib schedule) in the treatment groups 2A, 2B, 3, and 4 (c, d, e, and f, respectively). Relative body weight change during the treatment was calculated by comparison with the initial body weight on day 0 (a). Tumor size was reported as the mean ± SEM during the treatment (b–f). Models resistant to combination therapy are marked as §. Complete response (CR) rate during the treatment is indicated at the bottom of each graph. See also Figure S6.
Cancer Cell  , 64-78DOI: ( /j.ccell )
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9. Figure 7 Targeting PI3Kα/δ for the Treatment of ABC-DLBCL
(A) PI3K-mediated survival signaling pathways in ABC-DLBCL. In addition to activating the AKT pathway, PI3Kα/δ, the two isoforms predominantly expressed in ABC-DLBCL, also regulate BCR-dependent and -independent activation of the NF-κB pathway via cIAPs and p-IκB. The STAT3 signaling pathway is indirectly modulated by PI3K via NF-κB target genes IL10 and IL6. Furthermore, negative crosstalk between PI3K and BTK results in a rebound activation of p-BTK and p-AKT upon inhibition of PI3K and BTK, respectively.
(B) Combination of PI3Kα/δ and BTK inhibitors is proposed for the treatment of ABC-DLBCL with CD79mut or WT CD79 and the NF-κB pathway, while PI3Kα/δ inhibition in combination with SoC, e.g. R-CHOP, is proposed for the treatment of ABC-DLBCL with BCR-independent activation of NF-κB.
Cancer Cell  , 64-78DOI: ( /j.ccell )
Copyright © 2017 Elsevier Inc. Terms and Conditions