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. 2017 Jun 1;127(6):2081-2090.
doi: 10.1172/JCI89548. Epub 2017 May 15.

Activation of Tumor Suppressor Protein PP2A Inhibits KRAS-driven Tumor Growth

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Free PMC article

Activation of Tumor Suppressor Protein PP2A Inhibits KRAS-driven Tumor Growth

Jaya Sangodkar et al. J Clin Invest. .
Free PMC article

Abstract

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.

Conflict of interest statement

Conflict of interest: The Icahn School of Medicine at Mount Sinai, on behalf of G. Narla, M. Ohlmeyer, N.S. Dhawan, and D.B. Kastrinsky, has filed patents covering composition of matter on the small molecules disclosed herein for the treatment of human cancer and other diseases (International Application Numbers: PCT/US15/19770, PCT/US15/19764; and US Patent: US 9,540,358 B2). Dual Therapeutics LLC has licensed this intellectual property for the clinical and commercial development of this series of small molecule PP2A activators. G. Narla, M. Ohlmeyer, Y.A. Ioannou, M.D. Galsky, N.S. Dhawan, and D.B. Kastrinsky have an ownership interest in Dual Therapeutics LLC. G. Narla and M. Ohlmeyer are consultants for Dual Therapeutics LLC.

Figures

Figure 1
Figure 1. SMAPs decrease cell viability and inhibit MAPK signaling.
(A) Clonogenic assay of KRAS mutant cell lines (A549, H441, H358) treated with SMAPs for 3 weeks. (B) MTT assay in A549, H441, and H358 cells treated with increasing doses of SMAP at 24 hours. (C) Western blots for p-ERK and ERK normalized to GAPDH in KRAS mutant cell lines treated with SMAP. Data represent mean ± SEM from 3 experiments.
Figure 2
Figure 2. SMAPs promote tumor growth inhibition and inhibit MAPK signaling in KRAS mutant lung cancer.
(A) 1 × 107 H358 cells were subcutaneously injected into nude mice and allowed to grow to an average of 100 mm3. Mice were treated with vehicle control (n = 10), a combination of 6 mg/kg MK2206 and 24 mg/kg AZD6244 (n = 10), or 5 mg/kg SMAP (n = 10) twice a day for 4 weeks. Tumor volume over course of treatment is shown. (B) Tumors were evaluated by sacrificing the mice 2 hours after final treatment. Representative TUNEL staining and p-ERK staining of treated tumors. Scale bars: 20 μM. Original magnification: ×40. (C) Quantification of TUNEL positivity. (D) Quantification of p-ERK levels in xenograft tumors as performed by immunoblotting and densitometry. (E) KRASLA2 mice were randomized into treatment groups after reaching 16 weeks of age. Mice were treated with vehicle control (n = 3) or 15 mg/kg SMAP (n = 3) orally twice a day for 3 weeks. Mice were sacrificed and lungs were extracted 2 hours after final treatment. Representative images of lungs. Scale bar: 5 mM. (F) Percentage of tumor volume was evaluated by ImageJ using 3 sections of H&E for each mouse. (G) Representative TUNEL staining. Scale bars: 50 μM. (H) Quantification of TUNEL positivity in all tumors treated. (I) Immunohistochemistry of p-ERK in treated animals. Scale bars: 20 μM. Original magnification: ×40. (J) KRAS mutant PDX tumor fragments were surgically reimplanted in the right flank of NSG mice. Mice were treated with vehicle control (n = 7) and 5 mg/kg SMAP (n = 6) twice a day for 4 weeks. Tumor volume over course of treatment. (K) Tumors were evaluated by sacrificing mice 2 hours after final treatment. Representative TUNEL staining. Scale bar: 20 μM. Original magnification: ×40. (L) Quantification of TUNEL positivity in all tumors treated. (M) Immunohistochemistry of p-ERK in treated animals. Scale bar: 20 μM. Original magnification: ×40. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test.
Figure 3
Figure 3. Expression of the small T antigen confers resistance to SMAPs.
(A and B) 1 × 107 H358 control (A) and H358 small T antigen (ST) (B) cells were subcutaneously injected into nude mice and allowed to grow to an average of 100 mm3. Mice were treated with vehicle control (n = 9 for H358 control; n = 8 for H358 ST), a combination of 6 mg/kg MK2206 and 24 mg/kg AZD6244 (n = 8 for H358 control; n = 7 for H358 ST), and 5 mg/kg SMAP (n = 9 for H358 control; n = 7 for H358 ST) twice a day for 4 weeks. Tumor volume over course of treatment. (C) Representative TUNEL staining of treated tumors and immunohistochemistry of SV40 T antigen and p-ERK in treated animals. Scale bars: 20 μM. Original magnification: ×40. (D) Quantification of TUNEL positivity. (E) Quantification of SV40 T antigen levels. (F) Quantification of p-ERK levels. Quantification of SV40 T antigen and p-ERK levels in the xenograft tumors was performed by immunoblotting and densitometry. TUNEL, anti-SV40 T, and ERK signaling in the tumors were evaluated by sacrificing the mice 2 hours after final treatment. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test.
Figure 4
Figure 4. SMAPs binding to protein phosphatase PP2A.
(A) Binding studies of radiolabeled SMAPs with PP2A AC dimer, lysozyme, and PP1 using equilibrium dialysis. (B) Binding of radiolabeled SMAPs to lysozyme as negative control and PP2A (A–C) subunits using equilibrium dialysis. (C) Quantification of the KD and binding maximum (Bmax) for SMAP against the PP2A trimer. Data represent mean ± SD of 3 experiments. (D) Projection of changes in solvent exposure based on hydroxylradical modification in the A subunit of PP2A AC upon SMAP ligand addition. The structure of the A subunit (in light gray) is taken from 2A (PP2A) holoenzyme (PDB 2IAE). Modified amino acids are represented by colored side chains. The color codes indicate the changes in rates of modification for each specific site upon SMAP binding to the A subunit of PP2A AC. Purple indicates the residues that showed change in modification of less than 0.5-fold, blue indicates the residues that show minimal to no change (< 2-fold) in modification, green indicates decreases of more than 2-fold and less than 4-fold, yellow indicates decreases of more than 4-fold and less than 6-fold, orange indicates decreases of more than 6-fold and less than 9-fold, and red indicates decreases of more than 9-fold in the modification rate upon SMAP binding. Data represent mean ± SEM. *P < 0.05, Student’s t test.
Figure 5
Figure 5. Effects of mutations in putative drug-binding site.
(A) Male nude mice were subcutaneously injected (1 × 107 cells per injection) in the right flank with the different isogenic cell lines (control EGFP and putative drug-binding mutant K194R). Once the tumors reached a volume of 100 mm3, the mice were randomly enrolled in vehicle control (n = 6 for EGFP; n = 9 for K194R), a combination of 6 mg/kg MK2206 and 24 mg/kg AZD6244 (n = 8 for EGFP; n = 7 for K194R), or 5 mg/kg SMAP (n = 7 for EGFP; n = 9 for K194R) twice a day for 4 weeks. Mouse tumor volume for control EGFP-expressing H358 xenograft over course of treatment. (B) Tumors were evaluated by sacrificing the mice 2 hours after final treatment. Representative TUNEL staining and p-ERK IHC of treated tumors. Scale bars: 20 μM. Original magnification: ×40. (C) Quantification of TUNEL positivity in tumor. (D) Quantification of p-ERK levels in the xenograft tumors as performed by immunoblotting and densitometry. (E) Mouse tumor volume for drug-binding mutant K194R expressing H358 xenograft over course of treatment. Tumor volume over course of treatment. (F) Tumors were evaluated by sacrificing the mice 2 hours after final treatment. Representative TUNEL staining and p-ERK IHC of treated tumors. Scale bar: 20 μM. Original magnification: ×40. (G) Quantification of TUNEL positivity in tumors treated. (H) Quantification of p-ERK levels in xenograft tumors as performed by immunoblotting and densitometry. Data represent mean ± SEM. **P < 0.01; ***P < 0.001, Student’s t test.

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