Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers

Abstract

Somatic mutations that activate phosphoinositide 3-kinase (PI3K) have been identified in the p110-alpha catalytic subunit (encoded by PIK3CA). They are most frequently observed in two hotspots: the helical domain (E545K and E542K) and the kinase domain (H1047R). Although the p110-alpha mutants are transforming in vitro, their oncogenic potential has not been assessed in genetically engineered mouse models. Furthermore, clinical trials with PI3K inhibitors have recently been initiated, and it is unknown if their efficacy will be restricted to specific, genetically defined malignancies. In this study, we engineered a mouse model of lung adenocarcinomas initiated and maintained by expression of p110-alpha H1047R. Treatment of these tumors with NVP-BEZ235, a dual pan-PI3K and mammalian target of rapamycin (mTOR) inhibitor in clinical development, led to marked tumor regression as shown by positron emission tomography-computed tomography, magnetic resonance imaging and microscopic examination. In contrast, mouse lung cancers driven by mutant Kras did not substantially respond to single-agent NVP-BEZ235. However, when NVP-BEZ235 was combined with a mitogen-activated protein kinase kinase (MEK) inhibitor, ARRY-142886, there was marked synergy in shrinking these Kras-mutant cancers. These in vivo studies suggest that inhibitors of the PI3K-mTOR pathway may be active in cancers with PIK3CA mutations and, when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers.

Figure 1. Development of a Tet-inducible PIK3CA H1047R mouse model of lung tumorigenesis

(a) Histological analyses of lungs derived from the bitransgenic inducible Tet-op-PIK3CA H1047R /CCSP-rtTA (line #13) mice. Lungs from mice not induced with doxycycline, or those from mice induced for 6 and 14 weeks are shown. Adenocarcinoma is present in the lungs of mice induced with doxycycline after 6 and 14 weeks, respectively. Scale is 200μM and 50μM for upper and lower panels respectively. (b) Rapid disappearance of lung tumors following withdrawal of doxycycline. PIK3CA H1047R /CCSP-rtTA mice were placed on a doxycycline diet for 12 weeks to induce tumor formation, and tumors were assessed by MRI. The same mice were then taken off doxycycline and re-imaged 1, 2 and 3 weeks later. A representative example is shown. Scale is 4.5 mm. (c) Histological analysis of lungs after doxycycline withdrawal. PIK3CA H1047R /CCSP-rtTA mice were placed on a doxy diet until tumors were confirmed by MR imaging. Doxycycline was then withdrawn from their diets, the mice were sacrificed, and their lungs were examined histologically. Shown are the histology sections from two different mice after doxy withdrawal for 1 and 3 weeks respectively. Scale is 200μM and 50μM for upper and lower panels respectively.

Figure 2. NVP-BEZ235 downregulates PI3K signaling in p110-α H1047R induced lung tumors and leads to rapid tumor regression

(a) PIK3CA H1047R /CCSP-rtTA umors were induced in mice by feeding a doxy diet (verified by MR imaging). Mice with established tumors were treated with one dose of NVP-BEZ235 (35mg/kg) and the lungs were harvested 8 hours later. Sections were stained with the indicated antibodies. No primary was used as a control. Scale is 50 μM. (b) Tet-op PIK3CA H1047R /CCSP-rtTA mice were treated with doxycycline until tumors developed. These tumors were imaged by both PET and CT scans (top and lower panels respectively). The mice were then treated with NVP-BEZ235 35mg/kg per day for four days and underwent repeat imaging. Red arrows on the CT scans indicate tumor, and H: Heart. Scale is 5 mm. (c) PIK3CA H1047R /CCSP-rtTA mice were treated with doxy until they developed tumors (confirmed by MRI). Mice with established tumors were treated with NVP-BEZ235 35mg/kg for 3 days (left and middle) or 2 days (right) and the lungs were examined histologically. Scale is 200μM and 50μM for upper and lower panels respectively. (d,e) PIK3CA H1047R /CCSP-rtTA mice with established tumors were treated with either placebo, NVP-BEZ235 35mg/kg or rapamycin 6 mg/kg daily for 2 weeks. (d) A representative MRI is shown before and after treatment for each group. Scales is 4.5 mm. (e) The average tumor volumes of three mice in each treatment group after 2 weeks are shown relative to pretreatment tumor volumes.

Figure 3. PI3K signaling is needed for K-Ras induced tumorigenesis but not tumor maintenance

(a) Genetic deletion of the PI3K regulatory subunit blocks K-Ras-induced tumorigenesis. LSL K-Ras; Pik3r2 -/- mice with either Pik3r1 +/+ (n=7), Pik3r1 F/+ (n=9), or Pik3r1 F/F (n=5) were treated with adenoviral Cre to induce lung tumors development. 15 weeks after adenoviral Cre inhalation, mice were sacrificed and tumor numbers were determined. The average number of tumors per mouse is shown for each genotype. (b,c) Tet-op K-Ras mice were induced to develop lung tumors with doxy, and then treated with NVP-BEZ235 35mg/kg for 2 weeks. (b) A PET-CT scan before and after one week of therapy is shown. Scale is 5 mm. (c) Axial MR images from two representative mice are displayed. Scale is 4.5 mm. (d) Tet-op K-RasG12D/CCSP-rtTA were induced to develop lung tumors on doxy and then were treated with one dose of NVP-BEZ235 35mg/kg. Lungs were harvested 6 hours later and probed for P-Akt (Ser473) and Total Akt.

Figure 4. Combined PI3K and MEK inhibition dramatically shrinks K-Ras G12D induced lung tumors

(a,b) LSL K-Ras mice were induced to develop tumors by adenoviral Cre inhalation. After the establishment of sizeable tumors (determined by MRI), mice were treated with either placebo, NVP-BEZ235 35mg/kg once daily, ARRY-142886 25mg/kg twice daily, or NVP-BEZ235 35mg/kg once daily and ARRY-142886 25mg/kg once daily for two weeks. (a) Representative axial MRIs of the chest are shown. Scales is 4.5 mm. (b) The average tumor volumes of three mice in each treatment group after 2 weeks are shown relative to pretreatment tumor volumes. (c,d) Mice were treated as in (a) for 1.5 days. Six hours after their dose on day two of treatment, the animals were sacrificed. (c) One lung was snap-frozen in liquid nitrogen and assessed by western blotting using the indicated antibodies. (d) The other lung was fixed in formalin and assessed by immunohistochemistry (IHC) with the indicated antibodies. Microscopy was performed at two magnifications for the P-Akt IHC. The scale for the high magnification P-Akt IHC images is 25μM. The scale is 100μM for the other images. Hematoxylin and Eosin stains of the nodules examined by IHC are shown in the Supplemental Material (Supplementary Fig. 6).