Phosphatidylinositol 3-kinase-δ inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals

Abstract

Targeted therapy with imatinib in chronic myeloid leukemia (CML) prompted a new treatment paradigm. Unlike CML, chronic lymphocytic leukemia (CLL) lacks an aberrant fusion protein kinase but instead displays increased phosphatidylinositol 3-kinase (PI3K) activity. To date, PI3K inhibitor development has been limited because of the requirement of this pathway for many essential cellular functions. Identification of the hematopoietic-selective isoform PI3K-δ unlocks a new therapeutic potential for B-cell malignancies. Herein, we demonstrate that PI3K has increased enzymatic activity and that PI3K-δ is expressed in CLL cells. A PI3K-δ selective inhibitor CAL-101 promoted apoptosis in primary CLL cells ex vivo in a dose- and time-dependent fashion that was independent of common prognostic markers. CAL-101-mediated cytotoxicity was caspase dependent and was not diminished by coculture on stromal cells. In addition, CAL-101 abrogated protection from spontaneous apoptosis induced by B cell-activating factors CD40L, TNF-α, and fibronectin. In contrast to malignant cells, CAL-101 does not promote apoptosis in normal T cells or natural killer cells, nor does it diminish antibody-dependent cellular cytotoxicity. However, CAL-101 did decrease activated T-cell production of various inflammatory and antiapoptotic cytokines. Collectively, these studies provide rationale for the clinical development of CAL-101 as a first-in-class targeted therapy for CLL and related B-cell lymphoproliferative disorders.

Figure 1

p110δ is expressed abundantly in CLL cells. (A) CD19⁺ cells from patients with CLL (n = 20) were examined for p110δ expression by immunoblot. (B) CD19⁺ normal B cells, CD3⁺ normal T cells, CD56⁺ normal NK cells, and CD19⁺ cells from patients with CLL (n = 6 each) were examined for p110δ expression by immunoblot. (C) CD19⁺ cells from patients with CLL (n = 12) and from normal donors (n = 7) were examined for PI3K activity. Horizontal lines represent the mean. Results were calculated relative to microgram of protein.

Figure 2

CAL-101 induces selective cytotoxicity in CLL cells independent of IgV_H mutational status or interphase cytogenetics. (A) CD19⁺ cells from patients with CLL (n = 16) were incubated with or without CAL-101 (0.01-100μM) for 48 hours. Viability was determined by MTT assay and was calculated relative to time-matched untreated controls. (B) CD19⁺ cells from patients with CLL (n = 40) were incubated with or without CAL-101 (0.1-10μM) for 48 hours. Viability was determined by annexin/PI flow cytometry. (C) CD19⁺ cells from patients with CLL (n = 40) were incubated with or without 10μM CAL-101 for 12 to 96 hours. Horizontal lines represent the mean. (D) CD19⁺ cells from patients with CLL (n = 40; 10 per group) were incubated with or without 10μM CAL-101 for 48 hours. Cytogenetics was determined independently of our laboratory. (E) CD19⁺ cells from patients with CLL (n = 30; 15 per group) were incubated with or without 10μM CAL-101 for 48 hours. Mutational status was determined independently of our laboratory. Error bars represent the SD from the mean. (F) CD19⁺ cells from CLL patient cells (n = 40) and CD19⁺ cells from normal B cells (n = 9) were incubated with 10μM CAL-101 for 48 hours. (B-F) Viability was determined by annexin/PI flow cytometry and was calculated relative to time-matched untreated controls. Horizontal lines represent the mean.

Figure 3

CAL-101 cytotoxicity against CLL cells is partially dependent on caspase activity. (A) CD19⁺ cells from patients with CLL (n = 4) were incubated with or without 1 or 10μM CAL-101 or 25μM LY294002 (pan-PI3K inhibitor) for 12 hours, and caspase-3 and PARP were assessed by immunoblot. Results are shown from 1 of 4 experiments. (B) CD19⁺ cells from patients with CLL (n = 7) were incubated with or without 1 or 10μM CAL-101 for 12 hours. Cells were lysed, and caspase activity was determined by the amino trifluoromethyl coumarin assay. Results were calculated relative to microgram of protein. Each symbol represents an individual patient. (C) CD19⁺ cells from patients with CLL (n = 6) were incubated with or without 1 or 10μM CAL-101 and 100μM z-VAD-fmk for 48 hours. Viability was determined by annexin/PI flow cytometry and is shown relative to time-matched untreated controls. Each symbol represents an individual patient. Horizontal lines represent the mean. (D) CD19⁺ cells from patients with CLL (n = 4) were incubated with or without CAL-101 (1-10μM) and 100μM z-VAD-fmk for 12 hours. PARP cleavage was assessed by immunoblot. Results are shown from 1 of 4 experiments.

Figure 4

CAL-101 does not show cytotoxicity toward other normal immune cells but alters cytokine production. (A) CD3⁺ T cells and CD56⁺ NK cells (n = 9; each) from healthy volunteers were incubated with or without CAL-101 (0.1-10μM) for 48 hours. Viability was determined by annexin/PI flow cytometry and was calculated relative to time-matched untreated controls. (B) CD3⁺ T cells (n = 12) from healthy volunteers were incubated with or without CAL-101 (0.1-10μM) for 48 hours. Cells were stimulated with an anti-CD3 T-cell activation plate for 24 hours, and IL-6, IL-10, and TNF-α production was measured by ELISA. For CD40L mRNA assay, CD4⁺ T cells from healthy volunteers (n = 4) were incubated with and without various doses of CAL-101 and 5 μg/mL CD28. Cells were then stimulated with an anti-CD3 T-cell activation plate for 48 hours. Real-time polymerase chain reaction analysis was done to determine quantities of CD40L mRNA. (C) CD56⁺ NK cells (n = 8) from healthy volunteers were incubated with or without alemtuzumab, CAL-101, or the combination for 4 hours. IFN-γ production was determined by ELISA. (D) CD56⁺ NK cells (n = 3) from healthy volunteers were used as effector cells for a CLL-cell ADCC assay. NK cells were left untreated or treated with 10μM CAL-101; whereas CLL effector cells were treated with alemtuzumab. IgG indicates immunoglobulin G; and DMSO, dimethyl sulfoxide. Error bars represent the SD from the mean.

Figure 5

CAL-101 induces apoptosis more selectively than pan-PI3K inhibitors. (A) CD19⁺ cells from patients with CLL (n = 49) were incubated with or without 10μM CAL-101 or 25μM LY294002 for 48 hours. Viability was determined by annexin/PI flow cytometry and is shown relative to time-matched untreated controls. Horizontal lines represent the mean. (B) CD19⁺ B cells, CD3⁺ T cells, and CD56⁺ NK cells (n = 10; each) were incubated with or without 10μM CAL-101 and 25μM LY294002 for 48 hours. Viability was determined by annexin/PI flow cytometry and is shown relative to time-matched untreated controls. Error bars represent the SD from the mean.

Figure 6

CAL-101 antagonizes CD40-CD40L–mediated CLL cell survival. (A) CD19⁺ cells from patients with CLL (n = 4) were incubated with 10μM CAL-101 and 1μg/mL CD40L for 2 hours. AKT phosphorylation at Ser473 was assessed by immunoblot. Results are shown from 1 of 4 experiments. (B) CD19⁺ cells from patients with CLL (n = 5-18) were incubated with or without various doses of CAL-101 and 1μg/mL CD40L for 48 hours. Viability was determined by annexin/PI flow cytometry and is shown relative to time-matched untreated controls. Horizontal lines represent the mean. (C) CD19⁺ cells from CLL patients (n = 3) were incubated with various concentrations of CAL-101 and 1μg/mL CD40L for 2 hours. AKT phosphorylation at Ser473 was assessed by immunoblot. Results are shown from 1 of 3 experiments. Quantification was done with the Alpha Innotech FluorChemQ MultiImage III system. (D) CD19⁺ cells from patients with CLL (n = 20) were incubated with or without 10μM CAL-101 and 800U/mL IL-4 for 48 hours. Viability was determined by annexin/PI flow cytometry and is shown relative to time-matched untreated controls. Horizontal lines represent the mean. (E) CD19⁺ cells from patients with CLL (n = 4) were incubated with or without 10μM CAL-101 (or 25μM LY294002) and 800U IL-4 for 2 hours. Western blot analysis was done to detect activation of AKT (phosphorylation at Ser473) or STAT3 (phosphorylation at Tyr705). Results are shown from 1 of 4 experiments. (F) CD19⁺ cells from patients with CLL (n = 3) were incubated with 10μM CAL-101 and 1μg/mL CD40L for 2 hours. GSK3β phosphorylation at Ser9 was assessed by immunoblot. Results are shown from 1 of 3 experiments. (G) CD19⁺ cells from patients with CLL (n = 3) were incubated with 10μM CAL-101 and 1μg/mL CD40L for 2 hours. Mcl-1 expression was assessed by immunoblot. Results are shown from 1 of 3 experiments.

Figure 7

CAL-101 antagonizes alternative microenvironment stimuli activated by PI3K pathway. (A) CD19⁺ cells from patients with CLL (n = 5-10) were incubated with or without various doses of CAL-101 and 50 ng/mL BAFF for 48 hours. (B) CD19⁺ cells from patients with CLL (n = 5) were incubated with or without various doses of CAL-101 and 20ng/mL TNF-α for 48 hours. (C) CD19⁺ cells from patients with CLL (n = 5-10) were incubated with or without various doses of CAL-101 on and off fibronectin-coated plates for 48 hours. (D) CD19⁺ cells from patients with CLL (n = 7) were isolated from peripheral blood and incubated with or without 1 or 10μM CAL-101 in suspension or on an HS-5 cell layer for 48 hours. Viability was determined by annexin/PI flow cytometry and is shown relative to time-matched untreated controls for each group. Horizontal lines represent the mean. (E) CD19⁺ cells from patients with CLL (n = 4) were incubated with 1 μg/mL CD40L, 50 ng/mL BAFF, and 20 ng/mL TNF-α for 2 hours. AKT phosphorylation at Ser473 was assessed by immunoblot. Results are shown from 1 of 4 experiments.