Combined Inhibition of AKT and KIT Restores Expression of Programmed Cell Death 4 (PDCD4) in Gastrointestinal Stromal Tumor

Abstract

The majority of gastrointestinal stromal tumor (GIST) patients develop resistance to the first-line KIT inhibitor, imatinib mesylate (IM), through acquisition of secondary mutations in KIT or bypass signaling pathway activation. In addition to KIT, AKT is a relevant target for inhibition, since the PI3K/AKT pathway is crucial for IM-resistant GIST survival. We evaluated the activity of a novel pan-AKT inhibitor, MK-4440 (formerly ARQ 751), as monotherapy and in combination with IM in GIST cell lines and preclinical models with varying IM sensitivities. Dual inhibition of KIT and AKT demonstrated synergistic effects in IM-sensitive and -resistant GIST cell lines. Proteomic analyses revealed upregulation of the tumor suppressor, PDCD4, in combination treated cells. Enhanced PDCD4 expression correlated to increased cell death. In vivo studies revealed superior efficacy of MK-4440/IM combination in an IM-sensitive preclinical model of GIST compared with either single agent. The combination demonstrated limited efficacy in two IM-resistant models, including a GIST patient-derived xenograft model possessing an exon 9 KIT mutation. These studies provide strong rationale for further use of AKT inhibition in combination with IM in primary GIST; however, alternative agents will need to be tested in combination with AKT inhibition in the resistant setting.

Keywords: AKT; GIST; PDCD4; gastrointestinal stromal tumor; imatinib mesylate.

Figure 1

MK-4440 and IM have enhanced combination on in vitro GIST cell growth. Panels 1 & 2, dose-response curves for single agents (IM, MK-4440) in GIST-T1 (A), GIST430 (B) and GIST-T1/829 (C) cell lines. Values represent the mean of survival compared to control cells (n = 6). Panel 3, dose-response curve representing increasing series of combinations in GIST-T1 (A) GIST430 (B) and GIST-T1/829 (C) cell lines. Red box indicates estimation of LD50 (GIST-T1, (A)) or LD30 (GIST430, (B) and GIST-T1/829, (C)) concentration for combination of drugs. Panel 4, single point (blue) on isobole curve for 50% kill (GIST-T1, (A)) or 30% kill (GIST430, (B) and GIST-T1/829, (C)). Red line indicates 50% (GIST-T1) isobole or 30% isobole for GIST430 and GIST-T1/829 for strictly additive effect. CILD50 in GIST-T1 is 0.171 and CILD30 in GIST430 and GIST-T1/829 are 0.079 and 0.0595, respectively, all found within the synergistic triangle.

Figure 2

The combination of IM and MK-4440 inhibits constitutive activation of effectors downstream of KIT. Immunoblot assays of WCEs from GIST-T1 treated with 40 nM IM, 120 nM MK-4440 or the combination; and GIST430 and GIST-T1/829 treated with 1 μM IM, 3 μM MK-4440, or the combination. All treatments were twenty hours in duration. Equal quantities of WCE from each sample were subjected to immunoblotting with specific antibodies, as indicated. GAPDH served as loading control.

Figure 3

Total proteome changes upon IM/MK-4440 treatment. Volcano plot comparisons of total proteome in GIST-T1 (A) and GIST-T1/829 (B) cells treated with IM/MK-4440 combination versus vehicle (control) for 20 h. Differences in log2 LFQ intensities among cell lysates from three experiments determined by paired t-test at FDR of <0.05 using Perseus software. Venn diagram comparisons of downregulated (C) and upregulated (D) proteins between GIST-T1 and GIST-T1/829.

Figure 4

IM and MK-4440 combination induces cell cycle arrest and apoptosis. Immunoblot assays of WCEs from GIST-T1 treated with 40 nM IM, 120 nM MK-4440 or the combination; and GIST-T1/829 and GIST430 treated with 1 μM IM, 3 μM MK-4440, or the combination. All treatments were 20 h in duration. Equal quantities of WCE from each sample were subjected to immunoblotting with specific antibodies, as indicated. GAPDH served as loading control (A). Representative flow cytometry plots (B) and quantification (E) of BrdU incorporation in GIST-T1 treated with 40 nM IM, 120 nM MK-4440 and combination for 72 h; k = 1 × 10³. For comparisons between the combination (IM/ MK-4440) treatment vs. all monotherapies (Vehicle, IM, MK-4440) statistically significant reduction in S-phase, an increase in cell death (**** p < 0.00007), G1 arrest for comparison between the combination (IM/ MK-4440) treatment vs. Vehicle (p < 0.02) and G2 arrest between the combination (IM/ MK-4440) treatment vs. IM, MK-4440 monotherapies (p = 0.01) were observed. Representative flow cytometry plots (C) and quantification (F) of BrdU incorporation in GIST430 treated with 1 μM IM, 3 μM MK-4440 and combination for 72 h. For comparisons between the combination (IM/ MK-4440) treatment vs. all monotherapies (Vehicle, IM, MK-4440) statistically significant reduction in S-phase, an increase in cell death (*** p < 0.0002), and G1 arrest for comparison between the combination (IM/ MK-4440) treatment vs. Vehicle and IM (p < 0.009), and G2 arrest between the combination (IM/ MK-4440) vs. IM treatment (p < 0.008) were observed. Representative flow cytometry plots (D) and quantification (G) of BrdU incorporation in GIST-T1/829 treated with 1 μM IM, 3 μM MK-4440 and combination for 72 h. For comparisons between the combination (IM/ MK-4440) treatment vs. Vehicle and IM treatments statistically significant reduction in S-phase (** p < 0.002), an increase in cell death (p < 0.02), and G1 arrest for comparison between the combination (IM/ MK-4440) treatment vs. all monotherapies (Vehicle, IM, MK-4440) (p < 0.0004) were observed. Data represent mean of three experiments + SD. Ns- no significance.

Figure 5

Immunohistochemical and mutational analysis of a primary GIST and matched PDX model. (A) H&E and c-KIT (CD117) staining for primary GIST (top) and matched GIST PDX9.1 (bottom). (B) Sanger sequencing analysis identified the A502_Y503 duplication in exon 9 of KIT in both primary GIST (middle) and matched GIST PDX9.1 (bottom), but not in a non-tumor specimen (top). Scale bar: 10 μm.

Figure 6

Combination of IM and MK-4440 inhibits GIST growth in vivo. Smoothed tumor growth curves (tumor volume vs. time) were computed for each treatment using the lowess smoother in the R statistical language. (A) A statistically significant decrease in the rate of GIST-T1 xenograft tumor growth was observed for IM/MK-4440 combination (blue) vs. standard monotherapy with IM (red) (p < 0.0006). Tendency to decrease in the rate of tumor growth in PDX9.1 (B) and GIST430 xenograft (C) models under treatment with IM/MK-4440 combination (blue) were observed with no statistical significance in comparison with IM monotherapy (red).

Figure 7

Proposed mechanism of dual inhibition of KIT and AKT in GIST. Dual inhibition of KIT and AKT in GIST cells leads to decreased activation of pS6, associated PDCD4 upregulation, increased apoptosis and cell cycle arrest.