MTORC1/2 Inhibition as a Therapeutic Strategy for PIK3CA Mutant Cancers

Abstract

PIK3CA mutations are common in clinical molecular profiling, yet an effective means to target these cancers has yet to be developed. MTORC1 inhibitors are often used off-label for patients with PIK3CA mutant cancers with only limited data to support this approach. Here we describe a cohort of patients treated with cancers possessing mutations activating the PI3K signaling cascade with minimal benefit to treatment with the MTORC1 inhibitor everolimus. Previously, we demonstrated that dual PI3K/mTOR inhibition could decrease proliferation, induce differentiation, and result in a treatment response in APC and PIK3CA mutant colorectal cancer. However, reactivation of AKT was identified, indicating that the majority of the benefit may be secondary to MTORC1/2 inhibition. TAK-228, an MTORC1/2 inhibitor, was compared with dual PI3K/mTOR inhibition using BEZ235 in murine colorectal cancer spheroids. A reduction in spheroid size was observed with TAK-228 and BEZ235 (-13% and -14%, respectively) compared with an increase of >200% in control (P < 0.001). These spheroids were resistant to MTORC1 inhibition. In transgenic mice possessing Pik3ca and Apc mutations, BEZ235 and TAK-228 resulted in a median reduction in colon tumor size of 19% and 20%, respectively, with control tumors having a median increase of 18% (P = 0.02 and 0.004, respectively). This response correlated with a decrease in the phosphorylation of 4EBP1 and RPS6. MTORC1/2 inhibition is sufficient to overcome resistance to everolimus and induce a treatment response in PIK3CA mutant colorectal cancers and deserves investigation in clinical trials and in future combination regimens.

Figure 1.. Patients with activating alterations in phosphoinositide-3 kinase (PI3K) signaling are commonly identified and have minimal benefit from MTORC1 inhibition

Fifteen patients with cancers possessing activating alterations within the PI3K pathway were identified in the University of Wisconsin Carbone Cancer Center Precision Medicine Molecular Tumor Board Registry. These mutations were identified in diverse cancers. Of these patients 6 of them were treated with everolimus. One patient with breast cancer received this therapy in combination with a hormonal agent resulting in stable disease for 226 days. The other five patients had progressive disease with most discontinuing therapy within 60 days secondary to clinical progression. Testing method: 1 = Foundation One; 2 = Foundation ICE; 3 = University of Wisconsin Cancer Gene Mutation Panel; 4 = Foundation T5a; 5 = NCI-MATCH. NA = not applicable.

Figure 2.. MTORC1/2 induces significant responses in Fc¹ Apc^fl/+ Pik3ca^p110* CRC spheroids.

Fc¹ Apc^fl/+ Pik3ca^p110* CRC spheroids possess a lox-stop-lox sequence prior to the p110* transgene, which results in a constitutively active PI3K (A and B). These spheroids were allowed to mature for 24–96 hours prior to treatment with BEZ235, TAK-228 or control (C and D). Baseline and 48 hours post-treatment brightfield images were obtained and change in spheroid diameter measured for individual spheres. A similar median sphere size following treatment with BEZ235 and TAK-228 were observed and both significantly reduced from control (p < 0.001) (E). Reduced phosphorylation of AKT (ser473), RPS6 (Ser235/236), and 4EBP1 (Thr37/46) was observed after treatment of these spheres with BEZ235 and TAK-228 compared to control (F). Population distribution histograms demonstrating variation in change in sphere size for Fc¹ Apc^fl/+ Pik3ca^p110* CRC spheroids after 48 hours of treatment with BEZ235, TAK-228 or control (G). Size bar in panel A = 1 mm.

Figure 3.. Fc¹ Apc^fl/+ Pik3ca^H1047R mice develop invasive colon adenocarcinomas and can be utilized for translational investigations.

Fc¹ Apc^fl/+ Pik3ca^H1047R mice possess a lox-stop-lox sequence prior to the human PIK3CA H1047R hotspot mutation (A and B). These mice were allowed to age until moribund. These mice became moribund at an average age of 121 days (C). At necropsy tumors were identified within the small intestine and colon. Upon histologic sectioning, these tumors were found to be well to moderately differentiated adenocarcinomas (D, upper panels). These cancers possessed nuclear CTNNB1 (β-catenin), increased Ki67 and phosphorylation of RPS6 (D, lower panels). Spheroid cultures were able to be generated from these cancers and demonstrated a similar histology (E) and proliferating cells as measured by Ki67 (F). Nuclear CTNNB1 was also observed in the spheroids (G). The diameter of these spheres can be followed over time as a marker of proliferation (H). It was observed that larger spheroids at baseline would typically grow at a slower rate. To standardize experiments between cohorts, a standard change-point analysis was utilized (I). A change point at 164 pixels (373 μm) was identified and only those spheroids below that cut-off were utilized in further investigations. Size bars: D = 500 μm, E = 100 μm, G = 1 mm. The square panels in D are 4x enlargements of the outlined area.

Figure 4.. Apc and Pik3ca^H1047R mutant CRC spheroids are sensitive to MTORC1/2, but not MTORC1 inhibition alone.

Spheroids from Fc¹ Apc^fl/+ Pik3ca^H1047R cancers were allowed to mature for 24–96 hours. Brightfield images were obtained at baseline and following treatment with BEZ235, TAK-228, everolimus or control (A-C). Similar reductions in median relative spheroid size were observed with BEZ235 and TAK-228 compared to control (p < 0.001, A, B and D). Everolimus, an MTORC1 inhibitor, did not significantly decrease spheroid growth compared to control (C and D). Decreased phosphorylation of AKT (Ser473), RPS6 (Ser235/236), and 4EBP1 (Thr37/46) was observed at 6 hours following treatment with TAK-228, but not with BEZ235 or control (E). After 24 hours, similar reductions in the phosphorylation of AKT, RPS6 and 4EBP1 were observed (E). Everolimus did not reduce the phosphorylation of 4EBP1 (D). Population distribution histograms demonstrating variation in change in sphere size for Fc¹ Apc^fl/+ Pik3ca^H1047R CRC spheroids after 48 hours of treatment with BEZ235, TAK-228, everolimus, or control (F). Optical metabolic imaging was also utilized to confirm the therapeutic response. Representative images of NAD(P)H and FAD fluorescence intensities and optical redox ratio (G). The optical redox ratio (NAD(P)H intensity/FAD intensity) is decreased in spheroids treated with TAK-228 and BEZ235 compared to untreated spheroids (G and H). Subpopulation analysis of the redox ratio calculated from fluorescence images of untreated, BEZ235-treated, and TAK-228-treated CRC spheroids (I). The area under the curve for each treatment group was normalized to equal 1. Peaks represent high frequency of cells with similar optical redox ratios. Size bar in panel A-C = 1 mm, G = 100 μm. ****p < 0.0001.

Figure 5.. MTORC1/2 inhibition suppresses growth and MTOR signaling in isogenic human colon cancer cells with and without the PIK3CA^H1047R hotspot mutation.

SW48 human colon cancer cells were treated with increasing concentrations of BEZ235, TAK-228 or control (A). The WST-1 cell proliferation assay was utilized to measure cell viability. Similarly, SW48 cells possessing the PIK3CA^H1047R hotspot mutation (SW48PK) were treated with BEZ235, TAK-228, or control with response measured with the WST-1 assay (B). Inhibition of the phosphorylation of RPS6 and 4EBP1 were greater with TAK-228 (10nM) than BEZ235 (10nM) or control after 24 hours of treatment (C).

Figure 6.. MTORC1/2 inhibition results in a robust treatment response in Fc¹ Apc^fl/+ Pik3ca^H1047R mice.

These mice were allowed to age to ~100 days. At this time-point, the majority of these tumors are invasive cancers. Mice were treated with BEZ235, TAK-228 or control daily by oral gavage. Murine endoscopy was performed at baseline and following 14 days of treatment (A). Percent lumen occlusion was measured for each tumor and the change in percent lumen occlusion shown in a waterfall plot (B). A significant reduction in median change in lumen occlusion was observed with those tumors treated with BEZ235 (−19%) and TAK-228 (−20%) compared to control (+17%; p = 0.02 and p = 0.004, respectively) (B). A similar reduction in phosphorylation of RPS6 was seen after 14 days of treatment with BEZ235 and TAK-228, however a greater reduction in phosphorylation of AKT and 4EBP1 were observed with TAK-228 treatment (C).