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. 2013 Nov 1;19(21):5940-51.
doi: 10.1158/1078-0432.CCR-13-0850. Epub 2013 Aug 5.

Dual blockade of the PI3K/AKT/mTOR (AZD8055) and RAS/MEK/ERK (AZD6244) pathways synergistically inhibits rhabdomyosarcoma cell growth in vitro and in vivo

Jane Renshaw  1 , Kathryn R Taylor, Ryan Bishop, Melanie Valenti, Alexis De Haven Brandon, Sharon Gowan, Suzanne A Eccles, Ruth R Ruddle, Louise D Johnson, Florence I Raynaud, Joanna L Selfe, Khin Thway, Torsten Pietsch, Andrew D Pearson, Janet Shipley
Affiliations

Dual blockade of the PI3K/AKT/mTOR (AZD8055) and RAS/MEK/ERK (AZD6244) pathways synergistically inhibits rhabdomyosarcoma cell growth in vitro and in vivo

Jane Renshaw et al. Clin Cancer Res. .

Abstract

Purpose: To provide rationale for using phosphoinositide 3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathway inhibitors to treat rhabdomyosarcomas, a major cause of pediatric and adolescent cancer deaths.

Experimental design: The prevalence of PI3K/MAPK pathway activation in rhabdomyosarcoma clinical samples was assessed using immunohistochemistry. Compensatory signaling and cross-talk between PI3K/MAPK pathways was determined in rhabdomyosarcoma cell lines following p110α short hairpin RNA-mediated depletion. Pharmacologic inhibition of reprogrammed signaling in stable p110α knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2 (AZD8055), MEK (AZD6244), and P13K/mTOR (NVP-BEZ235) was evaluated alone and in pairwise combinations.

Results: PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with coactivated MAPK in 36% and 46% of alveolar and embryonal subtypes, respectively. p110α knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway, and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MAP-ERK kinase (MEK) inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug-drug interaction with both combinations.

Conclusions: Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predict a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor.

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Figures

Figure 1
Figure 1. PIK3CA shRNA-mediated knockdown induces cell line-specific compensatory signaling and reciprocal cross-talk between the MAPK and PI3K pathways
A. Representative growth curves following PIK3CA knockdown: Two ARMS (RH30 and RMS-1) and two ERMS (RD and RMS-YM) cell lines were transduced with control (CONSH) or PIK3CA targeted shRNA (PIK3CA KD) on d0 or treated with polybrene only (CONUT). On d2, d5 and d8 cells were counted and on d2 and d5, an appropriate dilution was reseeded in fresh plates enabling accumulative cell counts to be calculated and plotted for d5 and d8. Puromycin (2.5μg/ml) selection was introduced on d2 and maintained for the entire experiment, the difference between the CONUT and CONSH growth curves being a reflection of viral transduction efficiency. B. Western immunoblots of Class 1A PI3K isoforms, confirming selective p110α KD over 8 days in the PIK3CA shRNA transduced cell lines, along with a marker for apoptosis (PARP cleavage) and autophagy (LCBI/II expression). The PARP cleavage seen on d5 is a reflection of puromycin selection between d2-d5. C. Western immunoblots of PI3K pathway biomarkers showing cell line-specific disruption of PTEN, pAKT, pS6, IRS2, pERK and pAMPK in the above cells. * pS6 levels in RMS-1 cells were below the PhosphorImager detection levels. This image was therefore collected following 10mins exposure to X-ray film. D. Quantitation of pERK and total ERK immunoblots shown in C. using ImageQuant software, expressed as the ratio pERK:total ERK. Numbers above each bar: In each cell line the ratio of pERK:total ERK in the CONUT cells was set as 1.0 and the relative fold ratio change calculated for each sample.
Figure 2
Figure 2. Reprogramming of the PI3K and MAPK signaling pathways induces increased sensitivity to mTOR inhibition in the stable p110α KD lines with deregulated IRS2 expression, but increased sensitivity to MEK inhibition only in RMS-1 KD cells with reduced p110α and δ expression
A. Western Immunoblots of Class 1A PI3K isoforms confirming p110α KD in the stable KD lines (PI3K KD) along with upregulation of p110δ expression in the ERMS KD stable lines compared to their CONSH counterparts. Also shown are selected elements of the PI3K and MAPK pathways in the stable p110α KD lines showing cell line-specific upregulation of pThr308AKT (but not pSer473AKT) and IRS2, upregulated pERK and pAMPK levels in all the KD lines, and increased autophagy (LCBI/II), particularly in the ERMS KD lines. B. and C. Representative growth inhibition curves, (MTS assays), following treatment of the paired CONSH and KD lines with NVP-BEZ235 and AZD8055 respectively, showing increased sensitivity to mTOR inhibition in the KD lines with deregulated IRS2 expression. D. Representative growth inhibition curves following treatment with the AZD6244 showing increased sensitivity to MEK inhibition only in RMS-1 cells lacking p110α and δ.
Figure 3
Figure 3. Dual blockade of both the PI3K and MAPK pathways is synergistic in RMS cell lines in vitro
A., B., and C. Combination isobolograms using the combinations: AZD8055/AZD6244, ZSTK474/AZD6244, and NVP-BEZ235/AZD6244 respectively. The GI50 values of compound A and B are plotted on the × and y axes along with the GI50 values of compound B obtained in the presence of various fixed concentrations of compound A. The diagonal line drawn between the GI50 values for the two compounds on the y and × axes is the theoretical line of additivity. All GI50 values to the left of this line indicate synergy. D. Levels of pERK1/2, pSer473AKT, and pSer240/244S6 in RD cells following treatment with AZD8055, AZD6244 and AZD8055/AZD6244 in combination at 0.5×, and 1.0× GI50 concentrations. Proteins from control and drug treated cells were extracted at the indicated time points following treatment. Control and treated samples from each time point were loaded side by side for western immunoblots and quantitated using ImageQuant software. Drug treated/control phosphorylated biomarker levels at each time point are expressed as % of levels at time 0. NB. Levels of pERK in control samples were below consistently quantifiable levels at 48h, when the cells were reaching confluence.
Figure 4
Figure 4. Significantly enhanced antitumor efficacy following treatment of RD xenografts with AZD8055, but not NVP-BEZ235, in combination with AZD6244
A. and B. Head-to-head therapeutic study of RD xenografts treated with AZD6244 and either AZD8055 or NVP-BEZ235 respectively at the indicated doses alone, and in combination. Tumor volumes are expressed as a % volume of each tumor on day 0. Final tumor weights (g) show significantly increased efficacy of the combination AZD8055/AZD6244 compared with AZD6244 alone (**p=0.015) or AZD8055 alone (*p=0.038) and no significant difference in efficacy of the combination NVP-BEZ235/ AZD6244 compared with AZD6244 alone (p=0.13) or BEZ235 alone (p=0.82) (Mann Whitney t test). C. Plasma AZD6244 concentrations 3hrs following the final dose in mice treated with AZD6244 alone or in combination with AZD8055 or NVP-BEZ235 and plasma AZD8055 and NVP-BEZ235 concentrations from the same mice as above. D. Tumor PD biomarkers pERK: phospho(T/Y:202/204:185/187)ERK/total ERK1/2, pAKT: phospho(Ser473)AKT/total AKT and pS6: phospho(240/244)S6/total S6, as determined by MSD immunoassay.
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