Identifying genotype-dependent efficacy of single and combined PI3K- and MAPK-pathway inhibition in cancer

Abstract

In cancer, genetically activated proto-oncogenes often induce "upstream" dependency on the activity of the mutant oncoprotein. Therapeutic inhibition of these activated oncoproteins can induce massive apoptosis of tumor cells, leading to sometimes dramatic tumor regressions in patients. The PI3K and MAPK signaling pathways are central regulators of oncogenic transformation and tumor maintenance. We hypothesized that upstream dependency engages either one of these pathways preferentially to induce "downstream" dependency. Therefore, we analyzed whether downstream pathway dependency segregates by genetic aberrations upstream in lung cancer cell lines. Here, we show by systematically linking drug response to genomic aberrations in non-small-cell lung cancer, as well as in cell lines of other tumor types and in a series of in vivo cancer models, that tumors with genetically activated receptor tyrosine kinases depend on PI3K signaling, whereas tumors with mutations in the RAS/RAF axis depend on MAPK signaling. However, efficacy of downstream pathway inhibition was limited by release of negative feedback loops on the reciprocal pathway. By contrast, combined blockade of both pathways was able to overcome the reciprocal pathway activation induced by inhibitor-mediated release of negative feedback loops and resulted in a significant increase in apoptosis and tumor shrinkage. Thus, by using a systematic chemo-genomics approach, we identify genetic lesions connected to PI3K and MAPK pathway activation and provide a rationale for combined inhibition of both pathways. Our findings may have implications for patient stratification in clinical trials.

Conflict of interest statement

Conflict of interest statement: R.K.T. has received research support from AstraZeneca and Novartis. W.R.S. is an employee of Novartis. C.O., K.P.H., and L.F. are employees of Genentech.

Figures

Fig. 1.

Inhibition of PI3K signaling in cancer. (A) All cell lines were screened for induction of apoptosis using Annexin-V/PI staining after 72-h treatment with PI-103. Bars represent the fraction of apoptotic cells and are sorted from the most sensitive cell line (Left) to the most resistant cell line (Right), and grouped according to the presence of RTK- (EGFR, ERBB2, MET) or RAS-lesions (KRAS, NRAS, BRAF). A two-by-two table highlights the distribution of apoptotic cell lines in the two different genetically defined groups (RTK, RAS). (B) Two PI-103 sensitive (H1975, HCC827) and two resistant cell lines (H441, H460) were treated with PI-103 either in a dilution series (Left) or over time at 1 μM (Right). Pharmacodynamic markers (pAKT, AKT, pS6K, S6K, pERK, ERK, p4EBP1) were assessed by immunoblotting for all cell lines and all conditions. Black bars indicate splicing of noncontiguous bands run on the same gel. (C) Nude mice were s.c. engrafted with H1975, HCC827, AN3CA, and MKN45 cells, and tumors were either treated with vehicle control or the PI3K inhibitor GDC-0941 at 75 to 150 mg/kg. The tumor volume change relative to the tumor volume at day 0 (y axis) are plotted over time (x axis). (D) Growth of lung tumors was induced in the ERBB2YVMA transgenic mice. Tumors detectable by MRI were treated with either vehicle (n = 4) or GDC-0941 (n = 3) at 75–150 mg/kg for 2–4 weeks. Tumor growth was measured by serial MRI (Lower) and tumor volumes were calculated using Image-J (SI Methods).

Fig. 2.

Inhibition of MAPK signaling in cancer. (A) All cell lines were screened for induction of apoptosis using Annexin-V/PI staining after 72 h of treatment with PD0325901. Bars represent the fraction of apoptotic cells and are sorted from the most sensitive cell line (Left) to the most resistant cell line (Right), and grouped according to the presence of RTK- (EGFR, ERBB2, MET) or RAS-lesions (KRAS, NRAS, BRAF). Two-by-two table highlights the distribution of apoptotic cell lines in the two different genetically defined groups (RTK, RAS). (B) Two PD0325901 sensitive (HCC364, Calu6) and two less sensitive cell lines (A549, H441) were treated with PD0325901 either in a dilution series (Left) or over time at a fixed concentration (0.5 μM; Right). Pharmacodynamic markers (pAKT, AKT, pS6K, S6K, pERK, ERK) were assessed by immunoblotting for all cell lines and all conditions. (C) Nude mice were s.c. engrafted with H2122 and A549 cells, and tumors were treated with either the vehicle control, GDC-0941 or PD0325901 at the indicated concentration. Both compounds were administered every other day in the case of H2122 or every day in the case of A549. Similar results were obtained with other doses and other schedules (Fig. 4 D and E). The mean tumor volumes (y axis) are plotted over time (x axis).

Fig. 3.

Combined inhibition of PI3K- and MAPK-signaling in cancer. (A) All cell lines were screened for synergistic response using seven different combinations of PI103 and PD0325901 (C1 = 0.025 μM PI103 + 0.025 μM PD0325901; C2 = 0.25 μM PI103 + 0.25 μM PD0325901; C3 = 0.1 μM PI103 + 0.1 μM PD0325901; C4 = 0.1 μM PI103 + 0.5 μM PD0325901; C5 = 0.5 μM PI103 + 0.1 μM PD0325901; C6 = 1.0 μM PI103 + 1.0 μM PD0325901; C7 = 0.5 μM PI103 + 0.5 μM PD0325901) in a viability assay (SI Methods). Positive values (red colors) of the synergy strength metric indicate synergy whereas negative values (blue colors) indicate antagonistic drug response. (B) The induction of apoptosis in all NSCLC cell lines (x axis) after 72 h of single PD0325901 (0.25 μM) treatment, single PI-103 (0.5 μM) treatment or combinatorial treatment with both inhibitors is displayed. Apoptosis was assessed by flow cytometry using Annexin-V/PI staining. Bars represent the fraction of apoptotic cells and are sorted from the most sensitive cell line (Left) to the most resistant cell line (Right) for the combined PI3K and MEK treatment. (C) All cell lines were screened for induction of apoptosis using Annexin-V/PI staining after 72-h treatment with either PD0325901 (0.25 μM), PI-103 (0.5 μM), or a combination of both compounds (0.25 μM PD0325901 + 0.5 μM PI-103; x axis) and the resulting fractions of apoptotic cells (y axis) were plotted as box-plots according to the presence of RAS- (Left) or RTK-lesions (Right).

Fig. 4.

Suppression of feedback loops by dual PI3K/MAPK-inhibition enhances tumor shrinkage in vivo. (A) Four NSCLC cell lines (H1975, HCC2429, HCC364, A549) with different genetic lesions were treated for 24 h with PI-103, PIK90, PD0325901 and rapamycin, in various combinations at fixed concentrations (PI-103, 1 μM; PIK90, 5 μM; PD0325901, 0.5 μM; rapamycin, 0.01 μM). Pharmacodynamic markers (pAKT, AKT, pS6K, S6K, pERK, ERK, p4EBP1) were assessed by immunoblotting. (B) Three different cell lines of non-NSCLC cancer type (AN3CA, BT474, MKN45) with different genetic lesions were treated for 6 h with PI-103, PIK90, PD0325901 and rapamycin, in different combinations at fixed concentrations (PI-103 1 μM; PIK90 5 μM, PD0325901 0.5 μM; rapamycin 0.01 μM). Pharmacodynamic markers (pAKT, AKT, pS6K, S6K, pERK, ERK, p4EBP1) were assessed by immunoblotting. The biochemical response to specific inhibitors targeting the primary genetic lesion in the respective cell line is shown as a reference (PD173074 targets FGFR, PD168393 targets ERBB2, PHA665752 targets MET). Black bars indicate splicing of noncontiguous bands run on the same gel. (C and D) Nude mice were s.c. engrafted with AN3CA or H2122 cells, and tumors were treated daily with vehicle control, GDC-0941, PD0325901, or a combination of both at the indicated dose. The tumor volumes (y axis) are plotted over time (x axis). (E) H2122 tumors were grown on nude mice as in D, and mice were treated with an intermittent schedule of the combination of GDC-0941 and PD0325901, both dosed at their MTD (the combination was administered every fourth day; GDC-0941 dose, 150 mg/kg; PD0325901 dose, 25 mg/kg).