Direct activation of PP2A for the treatment of tyrosine kinase inhibitor-resistant lung adenocarcinoma

Abstract

Although tyrosine kinase inhibitors (TKIs) have demonstrated significant efficacy in advanced lung adenocarcinoma (LUAD) patients with pathogenic alterations in EGFR, most patients develop acquired resistance to these agents via mechanisms enabling the sustained activation of the PI3K and MAPK oncogenic pathways downstream of EGFR. The tumor suppressor protein phosphatase 2A (PP2A) acts as a negative regulator of these pathways. We hypothesize that activation of PP2A simultaneously inhibits the PI3K and MAPK pathways and represents a promising therapeutic strategy for the treatment of TKI-resistant LUAD. After establishing the efficacy of small molecule activators of PP2A (SMAPs) in a transgenic EGFRL858R model and TKI-sensitive cell lines, we evaluated their therapeutic potential in vitro and in vivo in TKI-resistant models. PP2A activation resulted in apoptosis, significant tumor growth inhibition, and downregulation of PI3K and MAPK pathways. Combination of SMAPs and TKI afatinib resulted in an enhanced effect on the downregulation of the PI3K pathway via degradation of the PP2A endogenous inhibitor CIP2A. An improved effect on tumor growth inhibition was observed in a TKI-resistant xenograft mouse model treated with a combination of both agents. These collective data support the development of PP2A activators for the treatment of TKI-resistant LUAD.

Keywords: Lung cancer; Oncology; Phosphoprotein phosphatases; Therapeutics; Tumor suppressors.

Figure 1. PP2A activation inhibits lung tumor development in an EGFR-driven TKI-sensitive non–small cell lung carcinoma transgenic model.

(A) Expression of TRE-EGFR^L858R was induced with doxycycline, and mice were administered either vehicle control or 100 mg/kg of SMAP every 48 hours. (B) Axial images obtained using MRI before and after treatment with vehicle control or SMAP. (C) H&E-stained sections of lung samples. (D) Quantification of H&E results. (E) Quantification of MRI results. (F) Immunohistochemical staining to detect apoptosis (TUNEL) and proliferation. Scale bar: 100 µm. (G) Quantification of TUNEL. (H) Quantification of PCNA. (I) Immunohistochemical staining of pERK and pAKT. Scale bar: 20 µm. Respective quantifications are represented as mean ± SD. *P < 0.05; ***P < 0.001.

Figure 2. PP2A activation induces apoptosis in TKI-resistant LUAD cell lines.

(A) Mechanisms of acquired resistance in LUAD. The most common mechanism of acquired resistance to TKIs is through the gatekeeper mutation T790M (tyrosine amino acid changed into a methionine at position 790), which alters the affinity of the drug to EGFR and leads to a sustained activation of both PI3K and MAPK pathways. Another common mechanism of resistance is through bypassing EGFR and sustainably activating the PI3K and MAPK pathways via modifications at the level of the downstream effectors themselves or by inactivating regulators of the pathways, such as PTEN. RAF, rapidly accelerated fibrosarcoma. (B) Proposed model for PP2A activation in EGFR-driven LUAD. SMAP treatment activates PP2A and leads to the downregulation of both PI3K and MAPK pathways that are commonly upregulated in TKI-resistant LUAD. (C) H1650 and H1975 were treated with DMSO vehicle control or increasing concentrations of SMAP DT-061 for 48 hours. Drug treatment resulted in decreased cell viability in both cell lines, with IC₅₀ of 10.6 μM. (D) The ability of H1975 and H1650 cell lines to form colonies when treated with vehicle control or 5, 7.5, or 10 μM SMAP DT-061 every 72 hours for 14 days. (E) Quantification of colony formation assay for H1975. (F) Quantification of colony formation assay for H1650. (G) PARP cleavage at 24 hours in H1975 and H1650 cells treated with indicated concentrations of DT-061. Annexin positivity at 24 hours in (H) H1975 and (I) H1650 cells treated with indicated concentrations of DT-061. Three independent experiments represented as mean ± SD are shown. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3. PP2A activation inhibits tumor growth in a TKI-resistant PDX model.

(A) Tumor volume (mm³) in function of time in a PDX mouse model treated with vehicle control (n = 9), a combination of AZD6244 (24 mg/kg) and MK2206 (6 mg/kg) (n = 5), or SMAP2 (5 mg/kg) (n = 9). The data are represented as mean ± SEM, with *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Gray asterisks compare the combination of AZD6244 (24 mg/kg) and MK2206 (6 mg/kg) group to the vehicle control group, while purple asterisks compare the SMAP2 (5 mg/ kg) group with the vehicle control group. The P values were calculated using a 2-tailed t test. (B) Body weights of mice throughout the treatment. (C) IHC of pERK and pAKT 2 hours after the final treatment. Scale bar: 20 µm.

Figure 4. SMAP DT-061 in combination with TKI has an enhanced effect on apoptosis in vitro and tumor growth inhibition in vivo.

(A) H1975 and (B) H1650 cell lines were treated with vehicle control, 20 μM SMAP DT-061, 1 μM erlotinib, 100 nM afatinib, a combination of SMAP DT-061 and erlotinib, or a combination of SMAP DT-061 and afatinib. Annexin positivity at 24 hours in H1975 and H1650 cells treated with indicated concentrations of DT-061. Three independent experiments are represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. BID: twice daily. (C and D) Western blot analysis of major PP2A and afatinib targets in H1975 treated with 20 μM SMAP DT-061, 100 nM afatinib, or a combination of SMAP DT-061 and afatinib at 24 hours. Results represent 3 independent experiments. (E) H1975 cells (5 million cells per injection) were subcutaneously injected in the right flank of nude mice. Once the tumor volumes reached approximately 100 mm³, mice were randomized and treated with vehicle control (n = 7), SMAP DT-061 (5 mg/kg) (n = 7) twice daily or afatinib (5 mg/kg) (n = 7) twice daily, or a combination of both SMAP DT-061 and afatinib (n = 7). The results are represented as mean ± SEM, with *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The P values were calculated using a 2-tailed t test. (F) Body weights of mice throughout treatment. (G) Proposed model for enhanced effect of SMAP and TKI treatment of EGFR-driven LUAD. The black asterisks compare the different treatments with the vehicle control. The green asterisks compare the combination of Afatinib and DT-061 group with the vehicle control group, while the purple asterisks compare the DT-061 (5 mg/kg) group with the vehicle control group.