IPI-145 antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia cells

Abstract

Chronic lymphocytic leukemia (CLL) displays constitutive phosphatidylinositol 3-kinase (PI3K) activation resulting from aberrant regulation of B-cell receptor (BCR) signaling. Previous studies have shown that an oral PI3K p110δ inhibitor idelalisib exhibits promising activity in CLL. Here, we demonstrate that a dual PI3K p110δ and p110γ inhibitor, IPI-145, antagonizes BCR crosslinking activated prosurvival signals in primary CLL cells. IPI-145 causes direct killing in primary CLL cells in a dose- and time-dependent fashion, but does not generate direct cytotoxicity to normal B cells. However, IPI-145 does reduce the viability of normal T and natural killer cells and decrease activated T-cell production of various inflammatory and antiapoptotic cytokines. Furthermore, IPI-145 overcomes the ibrutinib resistance resulting from treatment-induced BTK C481S mutation. Collectively, these studies provide rationale for ongoing clinical evaluation of IPI-145 as a targeted therapy for CLL and related B-cell lymphoproliferative disorders.

Figure 1

IPI-145 inhibits constitutive PI3K signaling, induces selective cytotoxicity in CLL cells, and does not cause direct cytotoxicity toward other normal immune cells but alters cytokine production. (A) B cells were enriched from peripheral blood of patients with CLL and stimulated with plate-bound anti-IgM (n = 4 of 6) and incubated with or without IPI-145 (0.01 to 1 μM) for 1 hour. Cell lysates were immunoblotted for pAKT^S473, total AKT, and GAPDH. (Upper) Representative blots from 4 independent experiments. (Lower) Quantification results. All data were normalized to unstimulated control. Stimulated samples from 4 patients were included for statistical analysis. CLL cells were significantly stimulated compared with untreated control *P < .005. After anti-IgM stimulation, pAKT^S473/AKT were significantly inhibited by IPI-145 compared with vehicle (veh)-treated control **P < .05. Paired t tests were used. (B) B cells from patients with CLL (n = 4-6) were stimulated with plate-bound anti-IgM and incubated with or without IPI-145 (0.01 to 1 μM) for 1 hour. Cell lysates were immunoblotted for pAKT^T308, total AKT, pERK1/2^T202/Y204, total ERK1/2, and GAPDH. The representative blots are from 4 (pAKT^T308, total AKT) and 6 (pERK1/2^T202/Y204, total ERK1/2) independent experiments. (C) B cells from patients with CLL (n = 12) were incubated with or without IPI-145 (0.25 to 5 μM) for 24 to 72 hours. Viability was determined by annexin/PI flow cytometry. A dose-dependent graph from the 48-hour time point is shown on the left (P = .005 for dose-dependent). Each icon represents individual patients. Horizontal bars represent averages. IPI-145 causes significant linear and quadratic cytotoxicity over both time and dose. P < .01 for statistical analysis for linear trend for time. (D) Whole blood from CLL patients (n = 7) was incubated with IPI-145 (0.25 to 5 μM) for 48 hours. Absolute count of live CD19⁺ B cells, CD3⁺ T cells, and CD56⁺ NK cells were measured by flow cytometry. Different icons represent different cell types. For all cell types, there was no statistically significant killing at 0.25 μM. Average viability of CD19⁺ B cells, CD3⁺ T cells, and CD56⁺ NK cells with 0.5 to 5 μM IPI-145 is significant lower than untreated. *, **, ***P < .001. CD19⁺ cytotoxicity to CD19⁺ B cells is significantly more than CD3⁺ T cells (#P < .001) or CD56⁺ NK cells (##P = .001) at 0.5 to 5 μM. (E) CD19⁺ B cells (n = 3) from healthy volunteers were incubated with or without IPI-145 (1 μM) for 48 hours. Viability was determined by annexin/PI flow cytometry and was normalized to time-matched untreated controls. Horizontal bars represent averages. IPI-145 caused significantly more cytotoxicity to CLL cells vs normal B cells *P < .05. (F) CD3⁺ T cells (n = 4-7) from healthy volunteers were stimulated with plate-bound anti-CD3 and soluble anti-CD28 and incubated with or without IPI-145 (0.01 to 5 μM). Cytokine levels were measured by BD Cytometric Bead Array. 0.5 to 5 μM IPI-145 significantly inhibited IL-2 and a trend for inhibition for both IFN-γ and TNF-α. *P < .05.

Figure 2

IPI-145 abrogates the prosurvival AKT signal in XLA-BTK C481S cells. The inducible XLA-BTK wild-type (WT) and XLA-BTK C481S cell lines were induced with 1 μg/mL of doxycycline for 48 hours. Both cells were left untreated or treated with IPI-145 at various concentrations (0.01 to 1 μM) for 1 hour or with ibrutinib (0.5 μM and 1 μM) for 30 minutes followed by washout and incubated in media for another 30 minutes. Cell lysates were immunoblotted for pBTK^Y223, total BTK, pATK^S473, total AKT, and GAPDH. The blots are representative of 3 independent experiments.