Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome

Abstract

A somatic activating mutation in AKT1, c.49G>A, pGlu17Lys, that results in elevated AKT signaling in mutation-positive cells, is responsible for the mosaic overgrowth condition, Proteus syndrome. ARQ 092 is an allosteric pan-AKT inhibitor under development for treatment in cancer. We tested the efficacy of this drug for suppressing AKT signaling in cells and tissues from patients with Proteus syndrome. ARQ 092 reduced phosphorylation of AKT and downstream targets of AKT in a concentration-dependent manner in as little as two hours. While AKT signaling was suppressed with ARQ 092 treatment, cells retained their ability to respond to growth factor stimulation by increasing pAKT levels proportionally to untreated cells. At concentrations sufficient to decrease AKT signaling, little reduction in cell viability was seen. These results indicate that ARQ 092 can suppress AKT signaling and warrants further development as a therapeutic option for patients with Proteus syndrome.

Conflict of interest statement

L.G.B. receives Royalties from Amgen and Genentech Corp and receives honoraria from Wiley-Blackwell.

Figures

Figure 1. AKT remained active in mutation-positive cells in the absence of growth factors.

Single cell clones isolated from fibroblasts cultured from an epidermal nevus located on the dorsum of the right hand of patient PS134 were grown with and without serum (indicated above gel images) followed by lysis and western analyses. Clones 1 and 3 (blue bars) were positive for the AKT1 E17K mutation; clones 2 and 4 (red bars) were negative. Histograms show ratios of the infrared signals from each antibody pair as described in the methods. Shaded bars represent ratios in cells grown in serum-free medium. Levels of pAKT and pPRAS40 were elevated in the absence of serum in mutation-positive cells compared to mutation-negative cells.

Figure 2. ARQ 092 decreased the level of pAKT and downstream targets in a concentration-dependent manner.

Clones 3 (mutation-positive) and 4 (mutation-negative) were cultured with or without serum and in the presence of increasing concentrations of ARQ 092 as indicated. (a) Infrared images of western hybridizations of each clone grown using the indicated antibodies. (b) Histograms of the ratios of the infrared signals from each antibody pair. Levels of AKT and PRAS40 phosphorylation were reduced with increasing concentrations of ARQ 092 in both mutation-positive and negative SCC when grown with or without serum compared to levels in untreated cells. Levels of pGSK3α and pGSK3β were reduced in mutation-positive cells with ARQ 092 treatment compared to untreated cells, but were similar in all mutation-negative cells. pBAD was not detected in mutation-negative cells, but was reduced with drug treatment in mutation-positive cells compared to untreated cells. pFOXO3a was only detected in untreated mutation-positive and mutation-negative cells grown with serum and with low concentrations of ARQ 092. pFOXO1 was only detected in mutation-positive cells grown without serum and was not measured. Levels of pS6 were not consistently reduced. Results of additional experiments are shown in Supplementary Fig. 1. CL3, clone 3 (blue bars); CL4, clone 4 (red bars); ser, grown with serum (solid bars); NS, grown without serum (shaded bars).

Figure 3. Levels of pAKT and pPRAS40 were reduced within 2 hours of treating with ARQ 092 and remained low for 72 hours.

Mutation-positive (clone 1) and mutation-negative (clone 2) SCCs were cultured in the presence or absence of serum with 125 nM ARQ 092 and lysed at the times indicated. The media was not changed during the course of the experiment. (a) Infrared images of western hybridizations using the indicated antibodies. (b) Histograms of the ratios of the infrared signals for each antibody pair. The inset in the pAKT histogram shows the ratios from the ARQ 092-treated cells using a smaller scale. Note the increased signal from CL1 lysates grown in serum-free medium at 8, 48 and 72 hours. Results of additional experiments are shown in Supplementary Fig. 4. CL1, clone 1 (blue bars); CL2, clone 2 (red bars), ser, grown with serum (solid bars); NS, grown without serum (shaded bars).

Figure 4. ARQ 092 decreased phosphorylation of AKT and PRAS40 in tissues from a patient with Proteus syndrome.

(a) The location and incubation conditions of punch biopsies taken from two affected toes from patient PS95 are indicated on the photo. Proteins and DNA were extracted as described in Methods. (b) Infrared images of western hybridizations using the indicated antibodies. Histograms below each lane represent the ratio of indicated antibody pair in each lysate. Symbols above images indicate the presence or absence of ARQ 092. AKT1 E17K mutation levels for each biopsy are shown below the graphs.

Figure 5. Platelet-derived growth factor-BB (PDGF-BB) stimulation in cells treated with ARQ 092 resulted in elevated levels of pAKT and pPRAS40.

Mutation-positive (clone 1) and negative (clone 2) SCC were serum starved, treated +/−ARQ 092 and stimulated with PDFG-BB as described in the Methods. Cells were collected at 10 minutes, 2 hours, and 24 hours after PDGF-BB addition. (a) Infrared images of western hybridizations using the indicated antibodies. (b) Histograms of the ratios of the infrared signals for each antibody pair. PDGF-BB stimulation resulted in a 12- to 70-fold increase in pAKT in ARQ 092-treated clone 1 and 24- to 50-fold increase in ARQ 092-treated clone 2 compared to un-stimulated cells. In untreated SCC, pAKT levels increased four- to sevenfold in clone 1 and 30– to >100 fold for clone 2 with PDGF-BB stimulation. For pPRAS40, PDGF-BB stimulation resulted in a 6– to 12-fold increase ARQ 092-treated clone 1 and 7– to 50-fold increase in ARQ 092-treated clone 2 compared to un-stimulated cells. In untreated SCC, pPRAS40 levels increased 8- to 20-fold in clone 2 and showed a 30% decrease or twofold increase in clone 1 upon PDGF-BB stimulation. Taken together, these results showed that Proteus SCCs are able to respond to PDGF-BB stimulation by increasing pAKT and pPRAS40 levels even when AKT signaling is suppressed by ARQ 092. Results of an additional experiment are shown in Supplementary Fig. 4. NS, grown without serum; PDGF, PDGF-BB stimulated

Figure 6. High concentrations of ARQ 092 were needed to reduce viability of cells from patients with Proteus syndrome.

ATP levels were measured using a luminescent assay in mutation-positive and mutation-negative SCCs and patient fibroblasts grown in high (10%) or low (0.5%) serum. The percentage of viable cells at a given dose was calculated as described in the Methods. (a) Luminescence ratios of SCCs grown in high or low serum with increasing concentrations of ARQ 092. Clones 1 and 3 are mutation-positive; clones 2 and 4 are mutation-negative. (b) Luminescence ratios of mutation-positive and mutation-negative fibroblasts from patients PS53 and PS75 grown in high or low serum with increasing concentrations of ARQ 092. The level of the AKT1 E17K mutation in cells from patients PS53 and PS75 were 37–42% and zero in the PS53 and PS75 mutation-negative cells. Mutation-positive clones and cells had lower viability and were clearly separated from mutation-negative clones and cells in both high and low serum. At 1.25 uM, levels 10-fold higher than necessary to effectively reduce AKT signaling, 40–70% of cells were still viable. CL1, clone 1; CL2, clone 2; CL3, clone 3; CL4, clone 4; pos, mutation-positive cells; neg, mutation negative cells V-numbers in the legend indicate the experiment number for the data represented by that line.