Dual targeting of mTOR and aurora-A kinase for the treatment of uterine Leiomyosarcoma

Abstract

Purpose: The significance of mTOR activation in uterine leiomyosarcoma (ULMS) and its potential as a therapeutic target were investigated. Furthermore, given that effective therapies likely require combination mTOR blockade with inhibition of other targets, coupled with recent observations suggesting that Aurora-A kinase (Aurk-A) deregulations commonly occur in ULMS, the preclinical impact of dually targeting both pathways was evaluated.

Experimental design: Immunohistochemical staining was used to evaluate expression of activated mTOR components in a large (>200 samples) ULMS tissue microarray. Effects of mTOR blockade (using rapamycin) and Aurk-A inhibition (using MLN8237) alone and in combination on human ULMS cell growth, cell-cycle progression, and apoptosis were assessed in cellular assays. Drug interactions were determined via combination index analyses. The antitumor effects of inhibitors alone or in combination were evaluated in vivo.

Results: Enhanced mTOR activation was seen in human ULMS samples. Increased pS6RP and p4EBP1 expression correlated with disease progression; p4EBP1 was found to be an independent prognosticator of patient outcome. Rapamycin inhibited growth and cell-cycle progression of ULMS cell strains/lines in culture. However, only a cytostatic effect on tumor growth was found in vivo. Combining rapamycin with MLN8237 profoundly (and synergistically) abrogated ULMS cells' growth in culture; interestingly, these effects were seen only when MLN8237 was preadministered. This novel therapeutic combination and scheduling regimen resulted in marked tumor growth inhibition in vivo.

Conclusions: mTOR and Aurk-A pathways are commonly deregulated in ULMS. Preclinical data support further exploration of dual mTOR and Aurk-A therapeutic blockade for human ULMS.

Figure 1. mTOR pathway is commonly activated in ULMS

A) PI3K/AKT/mTOR pathway is more active in ULMS, based on gene expression profiling data. Three previously described gene transcription signatures for the PI3K/AKT/mTOR pathway were applied to expression profiles of ULMS samples; each sample was scored for relative signature activity (heatmap is depicted: yellow = more active, blue = less active); B) Representative photographs of ULMS tissue microarray pS6RP, p4EBP1, and pAKT immunostaining (original images were captured at 400× magnification) depicting differences in expression between tumor and normal smooth muscle. Staining for PTEN is also shown and includes an example of PTEN expression loss as compared to positive staining – the later was observed in most evaluable samples; C) Western blot (WB) analyses demonstrate increased phosphorylation of the mTOR downstream effectors, S6K, S6RP, and 4EBP1, and the mTOR upstream regulator, AKT, in protein extracts of human ULMS cell strains/lines as compared to expression noted in normal smooth muscle cells (NSMC). Only Mes-Sa cells exhibited loss of PTEN expression.

Figure 2. mTOR blockade using rapamycin inhibit ULMS cell growth and induces G1 cell cycle arrest

A) Rapamycin (0.1–50nM/4h) blocks the activation of the mTOR downstream targets S6K, pS6RP, and 4EBP1 (WB analyses); B) MTS assays demonstrating a rapamycin (96h) dose-dependent decrease in ULMS cell growth (upper graph). In addition, rapamycin (both as pre- and continuous treatments) inhibits the colony formation capacity of ULMS cells (lower panel); C) Rapamycin treatment (1nM/48h) results in a G1 cell cycle arrest in ULMS cells. WB analyses demonstrate decrease in cyclin D1 and increased p21 expression in treated cells, independent of p53 mutational status. Increased p53 protein expression was found in cells harboring wild-type p53. [Graphs represent the average of at least two repeated experiments ±SD; * denotes statistically significant effects (p<0.05)]

Figure 3. Rapamycin treatment delays the growth of ULMS xenografts

A) Treatment with rapamycin (3.75mg/kg/d, 5 days per week) resulted in SKLMS1 xenograft tumor growth delay compared to control vehicle-treated tumors. Two separate experiments are shown depicting effect of treatment on growth and tumor weight at study termination: B) Immunohistochemical (IHC) staining confirmed decreased p4EBP1 and pS6RP expression in rapamycin treated tumors (original images were captured at 400× magnification).

Figure 4. The Aurk-A inhibitor, MLN8237, inhibits ULMS cell growth inducing G2/M cell cycle arrest and apoptosis

A) WB analysis demonstrating increased Aurk-A protein expression in a panel of ULMS cell strains/lines as compared to normal smooth muscle cells (NSMC); B) MTS assays demonstrating marked MLN8237 dose-dependent (0–100nM/96h) ULMS cell growth inhibition (upper graph). In addition, MLN8237 (both as pre- and continuous-treatment) abrogates the colony formation capacity of ULMS cells (lower panel); C) MLN8237 treatment (75nM/48h) results in a G2/M cell cycle arrest in ULMS cells. Furthermore, increased sub-G1 fraction is observed; D) An increase (~2–4 fold) in apoptosis was observed in MLN8237 treated cells compared to vehicle treated controls (Annexin-V/PI staining FACS analysis). WB analyses further demonstrate increased cleaved PARP in response to treatment. [Graphs represent the average of at least two repeated experiments ±SD; * denotes statistically significant effects (p<0.05)]

Figure 5. Combined mTOR and Aurk-A targeting results in superior (synergistic) anti-ULMS effects in vitro

A) MTS assays were conducted using increasing doses of both rapamycin and MLN8237, three different scheduling regimens were used: 1. simultaneous co-administration of rapamycin (increasing doses; 0–1nM) and MLN8237 (increasing doses; 0–100nM) for 96h (upper graphs), 2. 24h pre-treatment with rapamycin followed by co-treatment with and MLN 8237 for 72 hours (middle graphs), 3. 24h MLN8237 pre-treatment followed by 72hr co-treatment with rapamycin (bottom graphs). Isobologram analyses revealed that growth-inhibitory effects of the drug combination were synergistic when administered per the third schedule (CI<0.9; graphs represent three separate experiments; individual assay results can be found in Fig S2); B) Similarly, superior anti-growth effects are observed in Leio285 and Mes-Sa cells in response to dual mTOR/Aurk-A inhibition (administered in low doses as per the aforementioned schedule) compared to either agent alone (upper panel). Furthermore, combination therapy induces a superior inhibitory effect on colony formation compared to either agent alone (lower panel). [All graphs represent the average of three repeated experiments ±SD; * denotes statistically significant effects (p<0.05)]

Figure 6. Combined mTOR and Aurk-A targeting results in superior anti-ULMS effects in vivo

A) The impact of combined therapy (Rapamycin: 3.75mg/kg/d, five days a week and MLN8237: 15mg/kg/bid, every day. Of note MLN8237 was administered alone on day 1) was assessed in vivo using SKLMS1 xenografts growing in hairless SCID mice. MLN8237 as a single agent significantly inhibited tumor growth as compared to control. Most importantly, combination therapy resulted in significant growth abrogation as compared to rapamycin, MLN8237, or vehicle. Combination treated mice exhibited the most significant decrease in tumor weight as compared to all other therapeutic groups. B) IHC analyses demonstrated decreased Ki67 positive staining cells in all treatment groups, the most pronounced was in combination treatment tumors. An increase in TUNEL positive cells was noted in all treated tumors. Moreover, combination treated tumors exhibited the greatest decrease in CD31 positivity. [* denotes statistically significant effects (p<0.05)].