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. 2016 Apr 7;11(4):e0152584.
doi: 10.1371/journal.pone.0152584. eCollection 2016.

Activation of the PI3K/mTOR Pathway following PARP Inhibition in Small Cell Lung Cancer

Robert J Cardnell  1 , Ying Feng  2 , Seema Mukherjee  1 , Lixia Diao  3 , Pan Tong  3 , C Allison Stewart  1 , Fatemeh Masrorpour  1 , YouHong Fan  1 , Monique Nilsson  1 , Yuqiao Shen  2 , John V Heymach  1 , Jing Wang  3 , Lauren A Byers  1
Affiliations

Activation of the PI3K/mTOR Pathway following PARP Inhibition in Small Cell Lung Cancer

Robert J Cardnell et al. PLoS One. .

Abstract

Small cell lung cancer (SCLC) is an aggressive malignancy with limited treatment options. We previously found that PARP is overexpressed in SCLC and that targeting PARP reduces cell line and tumor growth in preclinical models. However, SCLC cell lines with PI3K/mTOR pathway activation were relatively less sensitive to PARP inhibition. In this study, we investigated the proteomic changes in PI3K/mTOR and other pathways that occur following PAPR inhibition and/or knockdown in vitro and in vivo. Using reverse-phase protein array, we found the proteins most significantly upregulated following treatment with the PARP inhibitors olaparib and rucaparib were in the PI3K/mTOR pathway (p-mTOR, p-AKT, and pS6) (p≤0.02). Furthermore, amongst the most significantly down-regulated proteins were LKB1 and its targets AMPK and TSC, which negatively regulate the PI3K pathway (p≤0.042). Following PARP knockdown in cell lines, phosphorylated mTOR, AKT and S6 were elevated and LKB1 signaling was diminished. Global ATP concentrations increased following PARP inhibition (p≤0.02) leading us to hypothesize that the observed increased PI3K/mTOR pathway activation following PARP inhibition results from decreased ATP usage and a subsequent decrease in stress response signaling via LKB1. Based on these results, we then investigated whether co-targeting with a PARP and PI3K inhibitor (BKM-120) would work better than either single agent alone. A majority of SCLC cell lines were sensitive to BKM-120 at clinically achievable doses, and cMYC expression was the strongest biomarker of response. At clinically achievable doses of talazoparib (the most potent PARP inhibitor in SCLC clinical testing) and BKM-120, an additive effect was observed in vitro. When tested in two SCLC animal models, a greater than additive interaction was seen (p≤0.008). The data presented here suggest that combining PARP and PI3K inhibitors enhances the effect of either agent alone in preclinical models of SCLC, warranting further investigation of such combinations in SCLC patients.

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Conflict of interest statement

Competing Interests: YF and YS are employees of BioMarin Pharmaceutical Inc. JVH serves on the advisory boards of AstraZenica, AbbVie, Novartis and Genentech. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. PARP inhibition increases PI3K/mTOR pathway activity through LKB1 inactivation in vitro.
(A) Hierarchical clustering of proteins identified in lysates from SCLC cell lines (H69, H82, H841) treated with vehicle or olaparib for 24 hours revealed increased activity of the PI3K/mTOR pathway following PARP inhibition (FDR≤0.1, equivalent p-value≤0.037). (B) Individual phosphorylated proteins in the PI3K/mTOR pathway (p-mTor, p-AKT and p-S6 kinase) are increased following treatment with either olaparib or rucaparib (p≤0.02). (C) Lysates from cell lines treated with olaparib or rucaparib versus vehicle for 24 hours show inactivation of the LKB1 pathway (LKB1, pAMPKα, and pTSC2; p≤0.042).
Fig 2
Fig 2. PARP inhibition inactivates the LKB1 pathway through increased ATP.
(A) Lysates from PARP1 knockdown by shRNA in SCLC cell lines (H69, H1048, H209) results in increased activity of the PI3K/mTOR pathway relative to scramble (Scr) shRNA as determined by western blot. (B) PARP1 knockdown shRNA cells (KD1 and KD2) had lower LKB1 and pAMPKα expression than scramble shRNA cells. (C) H69 and H1048 cells treated with olaparib showed increased [ATP] as measured by ATPlite assay. Data are presented as mean ± SEM; ** p<0.02, *** p<0.01. (D) A proposed model of PI3K pathway activation following PARP inhibition.
Fig 3
Fig 3. BKM-120 inhibits SCLC growth in vitro.
(A) Proliferation assays showed a range of sensitivities to BKM-120 across the SCLC cell line panel. Cell lines with PTEN mutations/deletions are colored red and those with PIK3CA mutations green; clinical Cmax is indicated with a dashed horizontal line. # indicates IC50 not reached at doses tested. (B) Summary of PI3K/mTOR mutation and MYC amplification status of the cell lines (see S3 Fig for cell line names). (C) Cell lines with either a mutation in the PI3K/mTOR pathway (MT) or a MYC amplification (AMP) were more sensitive to BKM-120 than cells without a mutation or amplification (WT and NON-AMP; ** p<0.02.). (D) Biomarker discovery using the RPPA profiles of 47 SCLC cell lines correlated protein expression with BKM-120 IC50 to identify markers predictive of response. Proliferation data are presented as mean ± SEM.
Fig 4
Fig 4. Talazoparib and BKM-120 act additively to inhibit SCLC growth in vitro.
(A) Proliferation assays using clinically achievable doses of talazoparib (6 doses, range 0.1–30 nM) and BKM-120 (3 doses, range 0.1–1 μM) showed an additive interaction in most of the 50 SCLC cell lines tested. Examples show additive responses (H211, DMS-79), and a greater-than-additive response (H1930). (B) Degree (percentage) of inhibition above (green) or below (pink) the predicted additive effect (purple) for each cell line across all clinically achievable doses. (C) Observed proliferation relative to predicted additive effect at individual doses of BKM-120.
Fig 5
Fig 5. Talazoparib and BKM-120 interact synergistically to inhibit SCLC growth in vivo.
Animals bearing an H209 or H1048 xenograft on their flank were treated with talazoparib, BKM-120, or the combination once tumors reached an average volume of 150mm3; effects of the treatments on tumor growth are shown. Each treatment delayed tumor growth, but the combination treatment had a greater effect than the predicted additive effect of each agent alone at day 18 and 25 in the H209 model (Day 18: predicted ΔT/ΔC = 0.33, observed = 0.30; Day 25: predicted ΔT/ΔC = 0.55, observed = 0.40). Data are presented as mean ± SEM, *p<0.05, ***p<0.01.
Fig 6
Fig 6. Talazoparib and BKM-120 have different, complementary modes of action.
(A) Lysates prepared from H1048 and H209 xenografts (3 per treatment group) harvested 2 hours after treatment on day 3 showed increased p-AKT (S473) and p-S6 (S240,244) with talazoparib treatment alone but reduced signaling with BKM-120 treatment alone or the combination (quantification of western blots shown in S6 and S7 Figs). (B) PARP activity, as measured by PAR levels, was decreased in both xenograft models following talazoparib treatment. (C) H1048 xenograft lysates treated with talazoparib and analyzed by RPPA in an independent experiment show increased p-mTOR (S2448) and p-AKT (S473) following treatment.
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