The Dual PI3K/mTOR Inhibitor PF-04691502 Induces Substantial Apoptosis in Chronic Lymphocytic Leukemia Cells in Vitro and Prolongs Survival in the Eµ-TCL1 Mouse Model
Authors: Blunt Matthew D , Carter Matthew J , Larrayoz Marta , Aguilar Maria Montserrat , Murphy Sarah , Reynolds Mark , Tipton Thomas , Cox Kerry , Strefford Jonathan C , Stevenson Freda K , Forconi Francesco , Packham Graham , Cragg Mark , Steele Andrew J 
Published: October 2019

Abstract
Pharmacological inhibition of specific B cell receptor signalling pathways within chronic lymphocytic leukemia (CLL) cells offers the potential for improved therapeutic options with reduced off target toxicity. Idelalisib, the PI3Kδ selective inhibitor, has been approved for CLL and significantly improved overall survival among patients with relapsed CLL in combination with rituximab compared to rituximab alone. In addition to PI3Kδ however, there are three other Class I PI3K isoforms, PI3Kα, PI3Kβ and PI3Kγ, with PI3Kα known to have a role in CLL survival and chemotaxis. In neutrophils, functional redundancy between PI3K isoforms is evident, with inhibition of at least three PI3K isoforms required for maximal apoptosis. Inhibition of mTOR is known to induce cell cycle arrest and apoptosis in CLL cells, however prolonged inhibition of mTOR results in activation of a positive feedback loop resulting in PI3K\Akt reactivation. To overcome these caveats, pan PI3K inhibition alongside mTOR inhibition may achieve superior cytotoxicity against CLL cells compared to PI3Kδ or mTOR inhibition alone. We therefore sought to investigate the effect of a dual pan PI3K and mTOR inhibitor, PF-04691502, on primary CLL cells and in the Eµ-TCL1 mouse model of CLL.
Twenty five primary CLL samples were treated with PF-04691502 and downstream signalling and subsequent apoptosis assessed by immunoblotting and Annexin V/PI staining respectively. In primary CLL cells, PF-04691502 induced 80% apoptosis after 24 hours at 10µM concentration. PF-04691502 had an IC50 value of 1µM as assessed by Annexin V/PI staining, with minimal toxicity to normal human B or T cells and a trend towards more specific killing in unmutated CLL (p=0.09) compared to mutated CLL. PF-04691502 inhibited both soluble and immobilised anti-IgM induced signalling and overcame anti-IgM induced survival signals. PF-04691502 abrogated CXCL12 induced signalling and impaired subsequent CLL migration towards the chemokine in a transwell chemotaxis assay. Finally, PF-04691502 was able to overcome protection from co-culture with stroma inducing significant apoptosis of CLL cells when added continuously and in wash out experiments.
To assess the effect of PF-04691502 in vivo, twenty Eµ-TCL1 mice were inoculated with tumour cells and after day 21 dosed once daily with vehicle, 5mg/kg or 10mg/kg PF-04691502 for a further 14 days. Mice were assessed for leukemic cell counts, spleen size and white blood cell count throughout the duration of the experiment. The effect of PF-04691502 on B cell receptor and chemokine receptor induced signalling in ex vivo Eµ-TCL1 cells was assessed by immunoblotting.
Near identical results to the CLL data above were observed. Using Eµ-TCL1 cells isolated from the spleen, PF-04691502 was shown to profoundly inhibit anti-IgM and CXCL12 induced signalling and chemotaxis as well as induce substantial apoptosis as measured by Annexin V/PI staining. Therefore, we subsequently assessed the effect of PF-04691502 in vivo using this model. Mice treated with PF-04691502 displayed a transient increase in leukemic cell numbers in the blood persisting for 1-4 days, followed by a reduction to levels significantly below that of the vehicle control. The whole white blood cell count remained stable in the PF-04691502 treated mice during the course of treatment, whilst the vehicle treated mice showed significant increases in tumour load up to 40×106 cells/ml 33 days post inoculation. Spleen sizes increased steadily over time in the control mice, whilst PF-04691502 treatment prevented this. After 33 days, PF-04691502 treated mice had spleen sizes comparable to non tumor-recipient control animals. In addition, CD5+ B220+ Eµ-TCL1 cells were significantly reduced in the bone marrow, spleen and lymph nodes following PF-04691502 treatment compared to control mice (p=0.0198; p< 0.0001; p=0.0151 respectively). These results demonstrate that PF-04691502 induces substantial apoptosis of primary CLL cells in vitro and significantly prolongs survival in an in vivo murine model of CLL. Such data indicate that dual pan PI3K/mTOR inhibitors may prove efficacious in the treatment of CLL patients.

Disclosures
No relevant conflicts of interest to declare.