Simultaneous targeting of PI3Kδ and a PI3Kδ-dependent MEK1/2-Erk1/2 pathway for therapy in pediatric B-cell acute lymphoblastic leukemia

Abstract

B cell acute lymphoblastic leukemia (B-ALL) is the most common hematological malignancy diagnosed in children, and blockade of the abnormally activated PI3Kδ displayed promising outcomes in B cell acute or chronic leukemias, but the mechanisms are not well understood. Here we report a novel PI3Kδ selective inhibitor X-370, which displays distinct binding mode with p110δ and blocks constitutively active or stimulus-induced PI3Kδ signaling. X-370 significantly inhibited survival of human B cell leukemia cells in vitro, with associated induction of G1 phase arrest and apoptosis. X-370 abrogated both Akt and Erk1/2 signaling via blockade of PDK1 binding to and/or phosphorylation of MEK1/2. Forced expression of a constitutively active MEK1 attenuated the antiproliferative activity of X-370. X-370 preferentially inhibited the survival of primary pediatric B-ALL cells displaying PI3Kδ-dependent Erk1/2 phosphorylation, while combined inhibition of PI3Kδ and MEK1/2 displayed enhanced activity. We conclude that PI3Kδ inhibition led to abrogation of both Akt and Erk1/2 signaling via a novel PI3K-PDK1/MEK1/2-Erk1/2 signaling cascade, which contributed to its efficacy against B-ALL. These findings support the rationale for clinical testing of PI3Kδ inhibitors in pediatric B-ALL and provide insights needed to optimize the therapeutic strategy.

Figure 1. Structure of X-370 and its inhibitory activity against lipid kinases

(A) Chemical structure of X-370. (B) X-370 docked to the crystal structure of PI3Kδ. The model was generated based on the co-crystal structure of PI3Kδ and ZSTK474 (PDB ID: 2WXL). (C) IC₅₀ of X-370 against PI3Kδ increased along with ATP concentration. (D) X-370 displayed high selective activity against PI3Kδ isoforms among representative lipid kinases. Data was obtained from Kinase Profiler Services (Millipore, United Kingdom). Briefly, X-370 was solved in DMSO at a concentration of 10 mM, 10-point kinase inhibitory activity was measured over concentrations in a half-log dilution series started at 1000 nM with ATP at a concentration consistent with Km of each isoform of class I PI3K and IC₅₀s were calculated. Relative kinase activity in the presence of 10 μM X-370 compared to DMSO control was measured for the rest of lipid kinases.

Figure 2. X-370 is highly selective against PI3Kδ-mediated signaling at cellular level

(A) The panel of isogenic Rh30-Myr-p110α, Rh30-Myr-p110β, Rh30-Myr-p110γ and Rh30-Myr-p110δ cells were cultured in serum-free medium for 12 h and then incubated with serially diluted X-370 for an additional 1 h. Cell lysates were probed with indicated antibodies. The IC₅₀ values were calculated based on relative band intensity measured with ImageQuant™ TL (GE Healthcare Life Sciences, Piscataway, NJ) (B). Data shown are mean values from three independent experiments. (C, D, E, F) X-370 blocked stimuli-triggered PI3Kδ activation. MEF cells and THP-1 were starved in serum-free medium for 12 h or 4 h respectively and X-370 were added for an additional 1 h treatment following stimulation of IGF-1 at 100 ng/ml for MEF cells (C), LPA at 10 μM for MEF cells (D), MCP-1 at 100ng/ml for THP-1 (E) and M-CSF at 50 ng/ml for THP-1 (F). Similar results were derived from 3 independent experiments.

Figure 3. PI3Kδ inhibition led to blockade of B cell leukemia cells proliferation via induction of cell cycle arrest and apoptosis

Raji cells treated with X-370 for indicated times and at serial diluted concentrations were subjected to Annexin V-FITC/PI staining (A) and PI staining (B) and FACS analysis to show cell apoptosis and cell cycle distribution respectively. (C) X-370 inhibited proliferation of human leukemia primary cells. Freshly isolated human primary mononuclear cells from bone marrow samples of patients were treated with X-370 for 72 h and cell viability was assayed using CCK-8 assay. IC₅₀s of each sample were plotted where 10 μM was assigned to specimens with IC₅₀ >10 μM. Each symbol represents an individual patient sample.

Figure 4. X-370 inhibited both Akt and Erk1/2 signaling in Raji and SU-DHL-6 cells

(A) X-370 concentration- and time-dependently inhibited the phosphorylation of Akt and Erk1/2. Raji and SU-DHL-6 cells were treated with X-370 for 1 h (left panel) or treated with 0.5 μM X-370 for indicated times (middle panel). Right panel, Raji cells were incubated in fresh medium following treatment with 0.5 μM X-370 for 1 h. Indicated proteins were detected. (B) Raji-R (the rituximab-resistant derivatives of Raji cells) cells were resistant to X-370. The activity of X-370 on the proliferation of Raji parent and Raji-R cells was determined by CCK-8 assay as described in the methods section. (C) X-370 blocked phosphorylation of Akt but not Erk1/2 in Raji-R cells. Raji-R cells were treated with X-370 for 1h and indicated proteins were detected. Data shown are representative from three independent experiments.

Figure 5. Inhibition of Erk1/2 phosphorylation by X-370 contributed to its antiproliferative activity

(A-C) X-370 inhibited Erk1/2 phosphorylation via PI3K-PDK1-MEK1/2 cascade but not the PI3K-Gab1 positive feedback loop. (A) Raji and SU-DHL-6 cells were exposed to 1 μM X370 for 3 h and protein fraction from cellular membrane was separated using Native Membrane Protein Extraction Kit and probed. WCL: whole cell lysate. (B) Inhibition of PDK1 or MEK1/2 abrogated Erk1/2 phosphorylation. Cells were treated with pharmacological inhibitors of Raf (AZ 628, PLX4032), MEK1/2 (AZD6244), p90RSK (BI-D1870), PI3Kδ (X-370), PDK1 (BX-912), Akt (MK-2206), mTOR (AZD8055) or mTORC1 (Rapamycin) respectively and indicated proteins were detected. (C) X-370 disrupted the association between PDK1 and MEK1/2. Raji cells were treated with 1 μM X370 for 1 h and cell lysates were immunoprecipitated with anti-PDK1 antibody and indicated proteins were detected. WCL: whole cell lysate. (D-F) Inhibition of Erk1/2 phosphorylation was required for the anti-proliferative activity of X-370. (D) Raji cells ectopically expressing a constitutively activated phospho-mimic MEK1 mutant (MEK1 S202D/S204D or MEK DD) were incubated with X-370 or AZD6244 for 1 h and then subjected to Western blot. (E) Raji and Raji MEK DD cells were treated with X-370 for 72 h and cell proliferation was detected by CCK-8 assay. (F) Raji MEK DD cells were treated with X-370 and AZD6244 and cell proliferation was detected by CCK-8 assay. Data were show as mean ± SD of three independent experiments. *: p < 0.05 determined by t-tests at each data point.

Figure 6. X-370-sensitive human primary B-ALL cells contained PI3K-dependent Erk1/2 phosphorylation and combination of AZD6244 and X-370 enhanced inhibitory activity against resistant specimens

(A) X-370-sensitive human primary B-ALL cells contained PI3K-dependent Erk1/2 phosphorylation. Primary B-ALL cells were treated with series diluted X-370 for 72 h. Phosphorylation of Akt and Erk1/2 were detected. (B). Combination of AZD6244 and X-370 enhanced inhibitory activity against resistant specimens. X-370 resistant primary B-ALL cells were treated with 1 μM X-370 alone or cocurently with MEK1/2 inhibitor AZD6244 (1 μM) for 72 h and cell viability were tested by CCK-8 assay. Cell viability of each treated group was compared with unpaired t-tests. *: P < 0.05. (C) X-370-resistant primary B-ALL cells were treated with X-370 in the presence of 1 μM AZD6244 or not for 72 h and phosphorylated Akt and Erk1/2 were then detected by Western blot.